51 research outputs found
Involvement of the caveolae in the permeability of the blood-brain barrier after envenoming by Phoneutria nigriventer in rats
Orientador: Maria Alice da Cruz-HöflingDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Neste trabalho investigamos a permeabilização da barreira hematoencefálica (BHE) pela peçonha da aranha Phoneutria nigriventer (PNV) através da via transcelular no cerebelo de ratos. As cavéolas foram analisadas nas células endoteliais pela expressão de proteínas associadas à sua formação (caveolina-1, Cav-1 e dinamina-2, Din2) e internalização (caveolina-1 fosforilada, pCav-1 e quinase da família Src, SKF), e nos astrócitos com avaliação da caveolina-3 (Cav-3) e da conexina-43 (Cx43) (formadora de junções comunicantes). A ação do PNV sobre o endotélio também foi avaliada pela ativação (acoplamento) ou inativação (desacoplamento) da enzima eNOS, produtora de óxido nítrico (NO). As estruturas que compõe a BHE foram avaliadas através de microscopia eletrônica de transmissão. Inicialmente, o estudo de Cav-1 contemplou sua localização, expressão gênica e proteica após o envenenamento em diferentes idades, ratos neonatos eram mais suceptíveis ao envenenamento do que ratos adultos. Após PNV, a imunomarcação para Cav-1 foi mais evidente na camada granular e molecular e em neurônios de Purkinje. A expressão Cav-1 e Din2 (foramdoras das vesículas e seu gargalo, respectivamente) aumentou em períodos de envenenamento agudo (1 h), de recuperação (5 h) e na ausência de sinais clínicos (24 h); em contrapartida SKF e pCav-1 envolvidas na internalização caveolar foram superexpressas em períodos opostos (às 2 h e 72 h). O PNV induziu aumento da metaloproteinase-9 da matriz (MMP9), importante mediadora de quebra da BHE e aumentou a formação e o tráfego de vesículas no endótelio após envenenamento. A análise de eNOS revelou desacoplamento (aumento de monômeros) nos períodos de envenenamento agudo (1-2 h) com progressivo retorno e super-expressão de dímeros (re-acoplamento) às 72 h; essas alterações foram relacionadas à ação do PNV sobre os níveis intracelulares de cálcio investigado pelo aumento na expressão de calmodulina e confimado pela localização de calbindina-D28. Os dados revelam a interferência do PNV sobre a homeostase endotelial e função vascular ao afetar o sistema eNOS/NO, importante controlador do tônus vascular. Nos astrócitos, as cavéolas são formadas por Cav-3 e sua superexpressão é associada a doenças neurológicas. O PNV aumentou significativamente os níveis basais de Cav-3 em astrócitos GFAP positivos (astrogliose reativa) em períodos de aumento de Cx43 (às 1, 5 e 24 h), e na vigência de edema citotóxico nos pés astrocitários e alterações nos contatos sinápticos axo-dendríticos e axo-somáticos. Em conjunto os resultados revelam que: (a) a quebra da via transcelular da BHE pelo PNV tem aumento da endocitose via cavéolas; (b) componentes da unidade neurovascular, como endotélio, astrócitos e neurônios estão intimamente envolvidos; (c) no endotélio, os efeitos são mediados pelo sistema eNOS/NO; (d) a SKF ativa o sistema endocítico e de transporte vesicular; (e) nos astrócitos, a dinamica expressão de Cx43 e Cav-3 e o retorno aos níveis basais em paralelo com a ausência de sinais de intoxicação nos animais (72 h) dá evidências de que ambas as proteínas interagem na resposta astrocitária. Os dados permitem sugerir que a presença de peptídeos neurotóxicos no veneno de Phoneutria nigriventer estão no centro dos efeitos aqui relatadosAbstract: In this work, we investigated the blood-brain barrier (BBB) permeabilization induced by Phoneutria nigriventer venom (PNV) in the transcellular route of rats¿ cerebellum. Caveolae was analyzed in endothelial cells accessing the expression of proteins involved in caveolae formation (caveolin-1, Cav-1 and dynamin-2, Dyn2) and internalization (phosphorylated Caveolin-1, pCav-1 and Src kinase family, SKF), in astrocytes caveolae role were evaluated with caveolin-3 (Cav-3) and connexin-43 (Cx43) (gap-junction main protein). PNV action on the endothelium was also investigated through activation (coupling) or inactivation (uncoupling) of eNOS enzyme, responsible for nitric oxide (NO) production. BBB components were evaluated using transmission electron microcopy. Initially, Cav-1 study addressed its localization along with Cav-1 protein and gene expression after envenoming in different age animals, neonate rats were more susceptible to envenoming than adult rats. After PNV, Cav-1 labeling was intense in granular and molecular layers and in Purkinje neurons. Cav-1 and Dyn2 (responsible for caveolae vesicles formation and scission, respectively) expression increased in periods of acute envenomation (1 h), recovery (5 h) and in the absence of clinical signals (24 h); in opposition SKF and pCav-1 involved in caveolae internalization were overexpress in opposite periods (at 2 h and 72 h). PNV induced increases in matrix metaloproteinases-9 (MMP9) an important BBB breakdown mediator, and increases in vesicles formation and traffic in the endothelium after envenoming. The study of eNOS activity revealed uncoupling (increasing in eNOS monomers) in acute periods after envenomation (1 h and 2 h) and progressive return followed by overexpression of dimers (re-coupling) at 72 h; those alterations were related to PNV action on calcium intracellular levels confirmed by Calmodulin increased expression and confirmed using Calbindin-D28 localization. Data revealed PNV interference on endothelial homeostasis and vascular function once affects the eNOS/NO system, an important vascular tonus controller. In astrocytes, caveolae are formed by Cav-3 and its overexpression is related to neurological disorders. PNV increased the basal levels of Cav-3 in GFAP-positive astrocytes (reactive astrogliosis) in the same periods as increased Cx43 (at 1, 5 e 24 h), during cytotoxic edema in astrocytes end-feet and alterations in axo-dendrites and axo-somatic synaptic contacts. Together, the results revealed that: (a) the BBB breakdown in transcellular route by PNV involves upregulation of caveolae endocytosis (b) the neurovascular unit components such as the endothelium, astrocytes and neurons are intimal involved (c) in the endothelium the effects are mediated by the eNOS/NO system and (d) SKF activates endocytic system and vesicular transport; (e) in the astrocytes, Cx43 and Cav-3 dynamic expression and their return to basal level in parallel with the absence of toxic signals in the animals (72 h) provides evidence that both protein interacts in astrocytes response. The data allows us to suggest that the neurotoxic peptides presented in Phoneutria nigriventer venom are in the center of the effects reported hereMestradoBiologia TecidualMestra em Biologia Celular e Estrutura
Reduced Graphene Oxide Induces Transient Blood-brain Barrier Opening: An In Vivo Study
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: The blood-brain barrier (BBB) is a complex physical and functional barrier protecting the central nervous system from physical and chemical insults. Nevertheless, it also constitutes a barrier against therapeutics for treating neurological disorders. In this context, nanomaterial-based therapy provides a potential alternative for overcoming this problem. Graphene family has attracted significant interest in nanomedicine because their unique physicochemical properties make them amenable to applications in drug/gene delivery and neural interface. Results: In this study, reduced graphene oxide (rGO) systemically-injected was found mainly located in the thalamus and hippocampus of rats. The entry of rGO involved a transitory decrease in the BBB paracellular tightness, as demonstrated at anatomical (Evans blue dye infusion), subcellular (transmission electron microscopy) and molecular (junctional protein expression) levels. Additionally, we examined the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) as a new imaging method for detecting the temporal distribution of nanomaterials throughout the brain. Conclusions: rGO was able to be detected and monitored in the brain over time provided by a novel application for MALDI-MSI and could be a useful tool for treating a variety of brain disorders that are normally unresponsive to conventional treatment because of BBB impermeability.13Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [11/50400-0, 2012/24782-5]CNPq [305099/2011-6, 486142/2012-4]FAPESP [08/57906-3]CNPq [573913/2008-0
Reduced Graphene Oxide: Nanotoxicological Profile In Rats
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)We have previously demonstrated that reduced graphene oxide (rGO) administered intravenously in rats was detected inside the hippocampus after downregulation of the tight and adherens junction proteins of the blood-brain barrier. While down-regulators of junctional proteins could be useful tools for drug delivery through the paracellular pathway, concerns over toxicity must be investigated before clinical application. Herein, our purpose was to trace whether the rGO inside the hippocampus triggered toxic alterations in this brain region and in target organs (blood, liver and kidney) of rats at various time points (15 min, 1, 3 h and 7 days). Results: The assessed rGO-treated rats (7 mg/kg) were clinically indistinguishable from controls at all the time points. Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO single injection seemed to produce minimal toxicological effects at the time points assessed. Relative to control, the only change was a decrease in the blood urea nitrogen level 3 h post-treatment and increases in superoxide dismutase activity 1 h and 7 days post-treatment. While no alteration in leukocyte parameters was detected between control and rGO-treated animals, time-dependent leukocytosis (rGO-1 h versus rGO-3 h) and leukopenia (rGO-3 h versus rGO-7 days) was observed intra-treated groups. Nevertheless, no inflammatory response was induced in serum and hippocampus at any time. Conclusions: The toxic effects seemed to be peripheral and transitory in the short-term analysis after systemic administration of rGO. The effects were self-limited and non-significant even at 7 days post-rGO administration.14Brazilian funding agency Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/24782-5]Brazilian funding agency Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [305099/2011-6, 486142/2012-4]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
Epidemiological profile of acute kidney injury in critically ill patients admitted to intensive care units : a prospective brazilian cohort
Introdução: A injúria renal aguda (IRA) é uma síndrome frequente em pacientes admitidos em unidades de terapia intensiva (UTI) e está associada a negativos desfechos clínicos. O objetivo do presente estudo foi conhecer o perfil epidemiológico de pacientes com IRA admitidos em UTIs.
Métodos: Estudo de coorte prospectiva, realizado em três UTIs do Distrito Federal, Brasil. Entre o período de outubro/2017 e dezembro/2018, 8.131 pacientes foram incluídos na coorte. A IRA foi definida de acordo com o critério KDIGO. Os principais desfechos avaliados foram o desenvolvimento de IRA e mortalidade dentro de 28 dias de internação.
Resultados: Dos 8.131 pacientes acompanhados, 1.728 desenvolveram IRA (21,3%). Dos 1.728 pacientes com IRA, 1.060 (61,3%) desenvolveram o estágio 1, já os estágios 2 e 3 representaram 154 (8,9%) e 514 (29,7%), respectivamente. Destes, um total de 459 (26,6%) realizou terapia renal substitutiva. A mortalidade observada foi de 25,7% para aqueles com IRA e 4,9% para os não IRA.
Discussão: Os pacientes com IRA, comparados aos não IRA, apresentaram maior mortalidade. Da mesma forma, entre os pacientes com IRA, os estágios superiores estiveram associados à maior ocorrência de óbito. A incidência de IRA (21,3%) e mortalidade (25,7%) em nosso estudo está em consonância com a maior meta-análise já conduzida, na qual foram observadas incidência e mortalidade de 21,6 e 23,9%, respectivamente. Esses achados confirmam a importância de se estabelecer a diretriz KDIGO para definição e manejo da IRA em UTIs brasileiras.Introduction: Acute kidney injury (AKI) is a frequent syndrome affecting patients admitted to intensive care units (ICU), and it is associated with poor clinical outcomes. The aim of the present study was to understand the epidemiological profile of patients with AKI admitted to ICUs.
Methods: Prospective cohort study, carried out in three ICUs in the Federal District, Brazil. Between October/2017 and December/2018, 8,131 patients were included in the cohort. AKI was defined according to the KDIGO criteria. The main outcomes assessed were AKI development and mortality within 28 days of hospitalization.
Results: Of the 8,131 patients followed up, 1,728 developed AKI (21.3%). Of the 1,728 patients with AKI, 1,060 (61.3%) developed stage 1, while stages 2 and 3 represented 154 (8.9%) and 514 (29.7%), respectively. Of these, 459 (26.6%) underwent renal replacement therapy. The mortality was 25.7% for those with AKI, and 4.9% for those without AKI.
Discussion: Patients with AKI had higher mortality rates when compared to those without AKI. Likewise, among patients with AKI, higher disease stages were associated with higher death occurrences. AKI incidence (21.3%) and mortality (25.7%) in our study is in line with the largest meta-analysis ever conducted, in which incidence and mortality of 21.6 and 23.9% were observed, respectively. These findings confirm the importance of establishing the KDIGO guideline for the definition and management of AKI in Brazilian ICUs
The transcribed ultraconserved region uc.160+ enhances processing and A-to-I editing of the miR-376 cluster : hypermethylation improves glioma prognosis
Transcribed ultraconserved regions (T-UCRs) are noncoding RNAs derived from DNA sequences that are entirely conserved across species. Their expression is altered in many tumor types, and, although a role for T-UCRs as regulators of gene expression has been proposed, their functions remain largely unknown. Herein, we describe the epigenetic silencing of the uc.160+ T-UCR in gliomas and mechanistically define a novel RNA-RNA regulatory network in which uc.160+ modulates the biogenesis of several members of the miR-376 cluster. This includes the positive regulation of primary microRNA (pri-miRNA) cleavage and an enhanced A-to-I editing on its mature sequence. As a consequence, the expression of uc.160+ affects the downstream, miR-376-regulated genes, including the transcriptional coregulators RING1 and YY1-binding protein (RYBP) and forkhead box P2 (FOXP2). Finally, we elucidate the clinical impact of our findings, showing that hypermethylation of the uc.160+ CpG island is an independent prognostic factor associated with better overall survival in lower-grade gliomas, highlighting the importance of T-UCRs in cancer pathophysiology.Peer reviewe
O COACHING COMO FERRAMENTA PARA DESENVOLVER COMPETÊNCIAS: Um estudo de caso na Secretaria de Planejamento do Estado de Alagoas - SEPLAG
Este artigo analisa a possibilidade do uso do coaching como auxiliar no processo da gestão por competência. O presente estudo visa responder a seguinte indagação, quais as dificuldades no desenvolvimento e aprimoramento da gestão por competência? E tem como objetivo analisar as possíveis soluções para as dificuldades no desenvolvimento da gestão por competências. Abordando os diversos conceitos de competência, as formas de implantação desse modelo de gestão, as dificuldades encontradas na implantação desse modelo, e as formas de soluciona-las, tendo em vista o coaching como a principal ferramenta de solução. Foi adotado como metodologia deste estudo a pesquisa bibliográfica e estudo de caso, na coleta de dados incluiu livros, periódicos, artigos científicos entre outras fontes. Foi utilizado um questionário contendo 10 itens para avaliar a percepção de pessoas que vivenciam o processo da gestão por competência. A partir da coleta de dados foi possível obter conclusões parciais sobre o uso do coaching como uma ferramenta essencial para a implantação da gestão por competências, auxiliando no desenvolvimento e aprimoramento das competências individuais, e por sua vez, organizacionais
O papel da arquitetura das bromélias e fatores abióticos na ocupação por anuros
Bromeliads are excellent models for the study of biological communities, as their structural complexity simulates highly heterogeneous environments. The present study explored the effect of the structural complexity of terrestrial bromeliads as a refuge site, as well as the influence of macro-spatial variables on the abundance of anurans in the Caatinga biome. This study was carried out in the municipality of Paulo Afonso, state of Bahia, northeast of Brazil, where two sites with a high abundance of bromeliads of the species Aechmea aquilega were selected. The sites were visited twice a month, in a total of 15 visits. The survey of the presence and abundance of anurans in the interior of the plants was performed visually. Bromeliads with anurans inside had their height, leaf number and internal temperature (intrinsic variables) measured. Air temperature, air humidity and precipitation (extrinsic variables) were also measured. Principal Component Analysis and Multiple Regression Model were used to identify the influence of these variables on the abundance of anurans in the interior of the plants. A total of 147 anurans, distributed among the Bufonidae and Hylidae families, were found, with an average of 1.3 individuals per bromeliad and 9.8 individuals per visit. The internal temperature of the bromeliads was 1.6ºC lower than air temperature, however, the intrinsic and extrinsic variables did not have an effect on the abundance of anurans. Anurans preferably used large bromeliads, with 78.2% of individuals using bromeliads taller than 60 cm. The results suggested that the biotic and abiotic factors discussed did not significantly influence the use of bromeliads by anurans. Additional studies addressing other aspects of the structural complexity of bromeliads and environmental factors are necessary to better understand the use of these plants by anurans in the Caatinga biome.Key words: amphibians, community structure, refuge site, semi-arid, intrinsic and extrinsic variables, hydric stress.Bromélias são excelentes modelos para estudo de estrutura de comunidades biológicas, já que sua complexidade estrutural simula ambientes altamente heterogêneos. Este trabalho investigou o efeito da complexidade estrutural de bromélias terrícolas, como sítio de refúgio, e das variáveis macro-espaciais na abundância de anuros na Caatinga. O estudo foi realizado no município de Paulo Afonso, Estado da Bahia, Nordeste do Brasil, onde foram selecionados dois sítios que apresentaram alta abundância de bromélias da espécie Aechmea aquilega. Os sítios foram visitados duas vezes ao mês, totalizando 15 visitas. A investigação da presença e da abundância de anuros no interior dos vegetais se deu por meio de busca visual. Bromélias com anuros em seu interior tiveram registradas sua altura, seu número de folhas e sua temperatura interna (variáveis intrínsecas), além de temperatura do ar, umidade do ar e precipitação (variáveis extrínsecas). Análise dos Componentes Principais e modelo de Regressão Múltipla foram utilizados para identificar a influência destas variáveis na abundância de anuros no interior dos vegetais. Foram encontrados 147 anuros distribuídos entre as famílias Bufonidae e Hylidae, com média de 1,3 indivíduos por bromélia e 9,8 indivíduos por visita. Temperaturas internas das bromélias mostraram-se 1,6ºC mais baixas que as temperaturas do ar, no entanto, as variáveis intrínsecas e extrínsecas não tiveram efeito na abundância de anuros. Anuros utilizaram predominantemente bromélias grandes, com 78,2% dos indivíduos utilizando bromélias acima de 60 cm de altura. Os resultados sugerem que os fatores bióticos e abióticos investigados não apresentam influência significativa no uso de bromélias pelos anuros. Estudos adicionais que abordem outros aspectos da complexidade estrutural das bromélias e fatores ambientais são necessários para melhor compreensão do uso dessas plantas por anuros na Caatinga.Palavras-chave: anfíbios, estrutura de comunidade, sítio de refúgio, semiárido, variá veis intrínsecas e extrínsecas, estresse hídrico
Reduced graphene oxide induces transient blood–brain barrier opening: an in vivo study
Caveolae As A Target For Phoneutria Nigriventer Spider Venom
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)An important transcellular transport mechanism in the blood-brain barrier (BBB) involves caveolae, which are specialized delta-shaped domains of the endothelial plasma membrane that are rich in cholesterol, glycosphingolipids and the scaffolding protein Caveolina-1 (Cav-1). In this work, we investigated whether the increase in endocytosis and transendothelial vesicular trafficking in rat cerebellum after blood-brain barrier breakdown (BBBb) induced by Phoneutria nigriventer spider venom (PNV) was mediated by caveolae. The expression of Cav-1, phosphorylated Cav-1 (pCav-1), dynamin-2 (Dyn2), Src kinase family (SKF) and matrix-metalloproteinase-9 (MMP9), proteins involved in caveolar dynamics and BBB opening, was investigated. Immunofluorescence, western blotting (WB) and transmission electron microscopy were used to assess changes at 1, 2, 5, 24 and 72 h post-venom. WB showed upregulation of Cav-1, Dyn2 and MMP9 at 1, 5 and 72 h (corresponding, respectively, to intervals when intoxication was most evident, when signs of recovery were present, and when no intoxication was detectable). In contrast, pCav-1 and SKF, which are essential for internalization and transport, decreased when Cav-1 and Dyn2, proteins essential for caveolar formation, were increased. Overall, these changes indicated that vesicular trafficking across the endothelium (high pCav/SKF levels) coincided with lower numbers of caveolae (Cav-1/Dyn2 downregulation) and lower expression of MMP9. Thus, the internalization (disassembly) of caveolae alternates with caveolar neoformation (assembly), resulting in changes in caveolar density in the endothelium membrane. These caveolar dynamics imply tensional mechanical stress that is important in triggering key signaling mechanisms. We conclude that PNV-induced breakdown of transcellular transport in the BBB is caused by an increase in caveolae-mediated endocytosis; this effect was correlated with the progression of temporal signs of envenoming. Caveolar dynamics are probably involved in shear stress and BBBb regulatory mechanisms in this experimental model. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).54111118Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2005/53625-1]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [486142/2012-4]CNPq [305099/2011-6]FAPESP [2012/24782-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP
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