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Educação Financeira tem se tornado cada vez mais relevante para o crescimento econômico dos países, principalmente àqueles que apresentam um baixo nível de alfabetização financeira. Este é o caso do Brasil, que mesmo com a implementação da ENEF em 2010, o país ainda está longe do nível ideal de Alfabetização Financeira, isso se reflete no alto nível de endividamento das famílias brasileiras e no baixo número de pesquisas científicas na área. Nesse sentido, visando contribuir com estudos científicos sobre o tema no Brasil, esta pesquisa tem como objetivo mapear os principais assuntos, autores e fontes relacionados à educação financeira, por meio de revisão bibliométrica, com enfoque nos construtos atitude e comportamento financeiros. Para tanto, foram selecionados 423 artigos, indexados na base de dados Web of Science (WoS). Utilizando-se do software R para as análises bibliométricas, por meio da interface Shiny do pacote Bibliometrix, foram identificados os principais autores, periódicos e termos relevantes da temática Educação Financeira no último triênio (2019-2021). Os resultados indicam, em consonância com a premissa da Lei de Lotka, que um número limitado de pesquisadores produz muito (0,2%), enquanto a maioria dos pesquisadores produz muito pouco (87,40%). Além disso, confirmou-se que no Brasil este tema é incipiente.&nbsp

Angiogenesis is considered to mediate the beneficial effects of mesenchymal cell therapy in spinal cord injury. After a moderate balloon-compression injury in rats, injections of either human adipose tissue-derived stromal/stem cells (hADSCs) or their conditioned culture media (CM-hADSC) elicited angiogenesis around the lesion site. Both therapies increased vascular density, but the presence of hADSCs in the tissue was required for the full maturation of new blood vessels. Only animals that received hADSC significantly improved their open field locomotion, assessed by the BBB score. Animals that received CM-hADSC only, presented haemorrhagic areas and lack pericytes. Proteomic analyses of human angiogenesis-related factors produced by hADSCs showed that both pro- and anti-angiogenic factors were produced by hADSCs in vitro, but only those related to vessel maturation were detectable in vivo. hADSCs produced PDGF-AA only after insertion into the injured spinal cord. hADSCs attracted resident pericytes expressing NG2, α-SMA, PDGF-Rβ and nestin to the lesion, potentially contributing to blood vessel maturation. We conclude that the presence of hADSCs in the injured spinal cord is essential for tissue repair.

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

Este trabalho pretendeu compreender a percepção de residentes em Enfermagem Obstétrica sobre violência obstétrica em uma maternidade referência do município de Belo Horizonte, estado de Minas Gerais, Brasil. Trata-se de estudo descritivo e exploratório de abordagem qualitativa. A coleta dos dados foi realizada por meio de grupo focal. Para análise dos dados foi utilizada a análise de conteúdo de Bardin, da qual emergiram três categorias: condutas inadequadas de assistência ao parto; procedimentos desnecessários com finalidades didáticas e/ou iatrogênicas; e preconceito de gênero, raça/etnia e de classe socioeconômica. O estudo aponta que as residentes reconhecem a prática da violência obstétrica no processo de formação e suas repercussões para a mulher e, ainda, evidencia a necessidade premente de investimento institucional em espaços que promovam discussões sobre a violência obstétrica. This article aims to understand the obstetric nursing residents’ perception of obstetric violence in a reference maternity hospital in the Brazilian city of Belo Horizonte. It is a descriptive, exploratory, and qualitative study. Data was collected through a focus group. Bardin’s content analysis was used to analyze data, from which three categories emerged: inadequate birth assistance conducts; unnecessary procedures with educational and/or iatrogenic purposes; and gender, race/ethnicity, and socioeconomic class bias. The study indicates that residents acknowledge obstetric violence in the educational process and its repercussions to women. It also evidences the pressing need for institutional investment in spaces that foster discussions on obstetric violence. El objetivo de este artículo es comprender la percepción de los residentes en Enfermería Obstétrica sobre la violencia obstétrica en una maternidad referencia del municipio de Belo Horizonte, Brasil. Se trata de un estudio descriptivo, exploratorio de abordaje cualitativo. La colecta de datos se realizó por medio de un grupo focal. Para el análisis de datos se utilizó el análisis de contenido de Bardín, del cual surgieron tres categorías: conductas inadecuadas de asistencia al parto; procedimientos innecesarios con finalidades didácticas y/o iatrogénicas, prejuicio de género, raza/etnia y de clase socioeconómica. El estudio señala que las residentes reconocen la práctica de la violencia obstétrica en el proceso de formación y sus repercusiones para la mujer y, también, muestra la necesidad urgente de inversión institucional en espacios que promuevan discusiones sobre la violencia obstétrica.

Tissue engineering demands the development of scaffolds that mimic natural extracellular matrices (ECM). Despite the success in obtaining synthetic interstitial ECM, the production of an artificial basement membrane (BM), the specialized thin sheet of ECM that is pivotal for the functional organization of most tissues and internal organs, is still not achieved. With the long-term aim of developing a flat BM-like structure here we investigated the behavior of acid-soluble Col IV during simultaneous assembly with laminin (LM) in acidic conditions. The underlying rationale was the previously observed phenomenon of acid-triggered LM polymerization, giving rise to biomimetic polylaminin (polyLM) that can be adsorbed on the substrate. Unexpectedly, we found that Col IV (that does not polymerize in acidic conditions) readily incorporated into the polyLM layer, forming a network that mimics to a great extent the characteristic polygonal morphology of single polyLM observable at micrometric scale. Scanning calorimetry and light scattering measurements supported the notion that polyLM and Col IV could directly interact. The biological properties of the proposed artificial BM-like structure were characterized using human keratinocytes (HACAT) and umbilical vein endothelial cells (HUVEC). HACAT formed stratified cell layers on the hybrid polyLM/Col IV layer, but not on Matrigel, nor on LM or Col IV alone, while HUVEC improved cortical F-actin and tight juctions organization on polyLM/Col IV. Thus, the proposed artificial BM reproduces not only morphological but also some functional properties of the natural BM. STATEMENT OF SIGNIFICANCE: Basement membranes (BMs) are flat biological matrices separating tissue compartments in the body. Their peculiar sheet-like structure is thought to result from the association of two independent protein networks of laminin and collagen IV. While pursuing the development of an artificial BM, we found that, when mixed with acid-induced polymerized laminin, collagen IV immediately conformed to the laminin shape. This implies that the protein networks may not be independently assembled as believed so far, but instead that laminin may command the assembly of collagen IV. Our hybrid matrix was structurally more stable than the commercial BM extract Matrigel and, unlike the latter, supported in vitro formation of a stratified layer of keratinocytes that approximated the organization of the natural epidermis.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Resumo Objetivo: Analisar a trajetória de cuidado de mulheres de um Centro de Referência a Gestantes Privadas de Liberdade (CRGPL), no que concerne às práticas dos trabalhadores no reconhecimento do direito à saúde e integralidade do cuidado. Metodologia: Adotaram-se a fenomenologia sociológica e a teoria do reconhecimento proposta por Alex Honneth (2003). Entrevistaram-se mulheres, agentes penitenciárias, diretores do CRGPL e do Hospital Sofia Feldman e profissionais de saúde, sendo realizada observação direta. Resultados e discussão: Identificaram-se três planos de reconhecimento, sendo que a autoconfiança foi verificada nas relações de vínculo e acolhimento dessas mulheres por gestores e trabalhadores. O autorrespeito foi identificado no entendimento, de profissionais e gestores, de que essas mulheres perderam seus direitos civis, mas que a saúde como um direito deve ser garantida. Verificou-se a autoestima na rede de solidariedade e compromisso construída de forma que resolutividade, referência e contrarreferência dessas mulheres sejam capazes de garantir o direito e a integralidade do cuidado. Conclusões: O estudo busca dar visibilidade a formas de cuidado mais solidárias, em que a garantia à saúde e ao cuidado integral não sejam apenas o cumprimento de um direito individual, mas uma nova forma de tratar essa questão na sociedade. Abstract Objective: To analyze the care trajectory of women in a Reference Center to Pregnant Women Deprived of Liberty (CRGPL), regarding the practices of workers in the recognition of the right to health and integral care to women and newborns. Methodology: As reference, the sociological phenomenology and the theory of recognition proposed by Alex Honneth were adopted. Women, prison officers, CRGPL directors, Sofia Feldman Hospital managers and health professionals of both institutions were interviewed. Also, it was held direct observation of meetings between the two directors. Results and Discussion: Three recognition plans proposed by Honneth were identified. Self-confidence was observed in bond and acceptance relationships of these women by managers and workers. Self-respect was identified in the awareness, by professionals and managers, that these women lost their civil rights, but that health as a right should be guaranteed. There was self-esteem in the network of solidarity and commitment, so that resolution, reference and counter-reference for these women and their children ensure the right to health and integral care. Conclusions: The study seeks to give visibility to forms of more supportive care, that ensuring health and comprehensive care is not only the fulfillment of an individual right, but a new way to handle this issue in society.

Resumo Este estudo teve como objetivo analisar o conceito de qualidade a partir da demanda dos beneficiários da Saúde Suplementar, na perspectiva da integralidade, considerando as relações entre satisfação, assimetria de informação e cuidado em saúde. Foi realizada a análise de uma amostragem de 1.013 e-mails enviados para a Central de Relacionamento da Agência Nacional de Saúde Suplementar de beneficiários da região sudeste, o que resultou na categorização Acesso aos Serviços e Continuidade do Cuidado, com as seguintes subcategorias: Negação; Dificuldade e/ou Imposição de Condicionantes; e Rede de Prestadores Restrita. Os beneficiários enfatizaram aspectos relacionados ao acesso aos serviços e à continuidade do cuidado que explicitam que as estratégias de redução de custos das operadoras têm afetado a qualidade e a integralidade do cuidado. Os relatos apontam que, para além da negação do acesso aos serviços e procedimentos, os beneficiários se vêem diante da restrição da continuidade do cuidado. Os resultados nos colocam o desafio de questionar a estruturação desse setor, cujos contratos se baseiam na definição de um rol de procedimentos e as estratégias de redução de custos se configuram como instrumento de restrição ao próprio direito à saúde, para além do respeito necessário ao direito do consumidor. Abstract This study aimed to analyze the concept of quality from the beneficiaries demands of the Supplementary Health, whose main bias the issue of integral care, considering the relationship between satisfaction, information asymmetry and health care. We analyzed a sample of 1,013 emails of 19,969 that were sent to the Supplemental Health National Agency Relationship Center, between March 18, 2014 and March 19, 2015, relating to complaints of beneficiaries who reside in Southeastern Brazil, resulting in the following categorization: Access to Health Services and Continuity of Care, with the following subcategories: Denial; Difficulty and / or Conditioning Constraints; and Restricted Providers Network. Beneficiaries emphasized aspects related to access to services and continuity of care, noting that in addition to the denial of access to services and procedures, they are faced with the restriction of continuity of care. The results place us the challenge to question the structure of the supplemental health sector, whose contracts are based on the definition of a list of procedures and cost reduction strategies are configured as a restraint instrument to the right to health itself, in addition to the necessary respect to consumer's right.

Cell encapsulation coats cells with an artificial membrane to preserve their physical and functional integrity. Different approaches try to develop more functional and biocompatible materials to avoid cell loss after transplantation due to inflammatory reaction, one of the main causes for graft failure. In this study, the LN-Biodritin biomaterial, based on alginate, chondroitin sulfate, and laminin, previously developed by our group, was further improved by replacing laminin by polylaminin, an artificial laminin polymer with anti-inflammatory properties, generating the new biomaterial polyLN-Biodritin. Capsules containing polylaminin are stable, do not induce macrophage activation in vitro, and are also able to prevent macrophage activation by encapsulated human pancreatic islets in vitro, preserving their glucose-stimulated insulin secretion potential. In addition, when empty capsules containing polylaminin were implanted into immunocompetent mice, the inflammatory response towards the implant was attenuated, when compared with capsules without polylaminin. The results indicate that polylaminin incorporation leads to lower levels of pericapsular growth on the capsules surface, lower infiltration of cells into the peritoneal cavity, and lower production of proinflammatory cytokines, both at the implant site (interleukin-12p70 (IL-12p70), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ (IFN-γ)) and systemically (IL-12p70 and TNF-α). Therefore, polylaminin incorporation into the microcapsules polymer attenuates the host posttransplantation immune response against implanted microcapsules, being likely to favor maintenance of engrafted encapsulated cells.

Fractones are extracellular matrix structures in the neural stem cell niche of the subventricular zone (SVZ), where they appear as round deposits named bulbs or thin branching lines called stems. Their cellular origin and what determines their localization at this site is poorly studied and it remains unclear whether they influence neural stem and progenitor cells formation, proliferation and/or maintenance. To address these questions, we analyzed whole mount preparations of the lateral ventricle by confocal microscopy using different extracellular matrix and cell markers. We found that bulbs are rarely connected to stems and that they contain laminin α5 and α2 chains, respectively. Fractone bulbs were profusely distributed throughout the SVZ and appeared associated with the center of pinwheels, a critical site for adult neurogenesis. We demonstrate that bulbs appear at the apical membrane of ependymal cells at the end of the first week after birth. The use of transgenic mice lacking laminin α5 gene expression (Lama5) in endothelium and in FoxJ1-expressing ependymal cells, revealed ependymal cells as the source of laminin α5-containing fractone bulbs. Loss of laminin α5 from bulbs correlated with a 60% increase in cell proliferation, as determined by PH3 staining, and with a selective reduction in the number of quiescent neural stem cells in the SVZ. These results indicate that fractones are a key component of the SVZ and suggest that laminin α5 modulates the physiology of the neural stem cell niche.Significance StatementOur work unveils key aspects of fractones, extracellular matrix structures present in the SVZ that still lack a comprehensive characterization. We show that fractones extensively interact with neural stem cells, whereas some of them are located precisely at pinwheel centers, which are hotspots for adult neurogenesis. Our results also demonstrate that fractones increase in size during aging and that their interactions with NSPCs become more complex in old mice. Lastly, we show that fractone bulbs are produced by ependymal cells and that their laminin content regulates neural stem cells.

Polylaminin (polyLM) is a flat biomimetic polymer of laminin capable of promoting axonal growth both in vitro and in vivo. It is assembled in a cell-free system when laminin 111 is incubated in acidic pH, whereas incubation in neutral buffer leads to the formation of bulky and irregular polymers (LM). In the present work, we compared the behaviors of cells isolated from the P1 rat retina on polyLM and LM. PolyLM induced cellular spreading and the outgrowth of neurites in contact with the substrate, whereas LM led to the formation of large clusters of cells, with neurites growing only inward. After 24 hr in culture, the number of cells on polyLM increased threefold, and this increase was inhibited by 60% in the presence of the PKA inhibitor H89 and by 41% in the presence of the PKC inhibitor chelerythrine chloride, whereas both inhibitors abolished neuritogenesis. Neither the cell number nor the outgrowth of neurites was affected by the ERK1/2 inhibitor PD98059 on polyLM. On the other hand, PD98059 was able to reduce the cell number on LM, whereas the other inhibitors were not. Immunostaining of P1 retina with an antilaminin antibody revealed that the protein was expressed not only at its inner surface but also within the neuroblast layer in close contact with individual cells. Our results indicate that, when provided in its active polymerized form, laminin can influence both neuritogenesis and proliferation of retinal cells.

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.

[EN] Mimicking the complex intricacies of the extra cellular matrix including 3D configurations and aligned fibrous structures were traditionally perused for producing cartilage tissue from stem cells. This study shows that human adipose derived mesenchymal stem cells (hADMSCs) establishes significant chondrogenic differentiation and may generate quality cartilage when cultured on 2D and randomly oriented fibrinogen/poly-lactic acid nanofibers compared to 3D sandwich-like environments. The adhering cells show well-developed focal adhesion complexes and actin cytoskeleton arrangements confirming the proper cellular interaction with either random or aligned nanofibers. However, quantitative reverse transcription-polymerase chain reaction analysis for Collagen 2 and Collagen 10 genes expression confirms favorable chondrogenic response of hADMSCs on random nanofibers and shows substantially higher efficacy of their differentiation in 2D configuration versus 3D constructs. These findings introduce a new direction for cartilage tissue engineering through providing a simple platform for the routine generation of transplantable stem cells derived articular cartilage replacement that might improve joint function., This work was supported by CIBER-BBN Spain (project BIOSURFACES), and the European Commission through the FP7 Industry-Academia Partnerships and Pathways (IAPP) project FIBROGELNET. The valuable support of the project MAT 2015-69315-C3 MYOHEAL, funded by Spanish Ministry of Science and Innovation is also acknowledged.

BACKGROUND: An inflammatory component consisting of cells and chemical mediators may influence the proliferation and dissemination of the oral squamous cell carcinoma (OSCC). In the present study, we evaluated the possible relationship between Ki-67, tumor-associated macrophages (TAMs), and COX-2 in OSCCs. In addition, the immunodetection of these proteins was associated with different histological grades of malignancy, including invasive and in situ tumors. METHODS: Twenty-seven OSCC cases were examined by light microscopy using criteria adopted WHO, and immunohistochemistry for Ki-67, CD68, and COX-2 using EnVision System in invasive and in situ lesions. Immunohistochemical detection of these proteins was assessed and scored for COX-2, and results were compared with their histological grades of malignancy. RESULTS: A correlation between Ki-67, COX-2, and CD68 was not found. Histological grade of malignancy (HDM) was associated with the Ki-67 immunostaining (P = 0.00), but this was not observed regarding both CD68 (P = 0.51) and COX-2 (P = 0.89). Furthermore, there was a COX-2 overexpression in 62.96% of the sample, and a high density of TAMs in both OSCCs and in situ carcinomas. CONCLUSIONS: Imunolabeling for Ki-67 was directly correlated with less-differentiated tumors, suggesting that this marker may contribute to understand the biological behavior of OSCC, and help to distinguish risk groups of OSCC. Furthermore, the lack of correlation between Ki-67, COX-2, and CD68 indicates that the latter two markers may play a pivotal role in oral carcinogenesis. However, further studies are needed to clarify their contribution for cell proliferation and tumor differentiation.

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four α-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

In the developing nervous system migrating neurons and growing axons are guided by diffusible and/or substrate-bound cues, such as extracellular matrix-associated laminin. In a previous work we demonstrated that laminin molecules could self-assemble in two different manners, giving rise to matrices that could favor either neuritogenesis or proliferation of cortical precursor cells. We investigated whether the ability of astrocytes to promote neuritogenesis of co-cultivated neurons was modulated by the assembling mode of the laminin matrix secreted by them. We compared the morphologies and neuritogenic potentials of laminin deposited by in vitro-differentiated astrocytes obtained from embryonic or neonatal rat brain cortices. We showed that, while permissive astrocytes derived from embryonic brain produced a flat laminin matrix that remained associated to the cell surface, astrocytes derived from newborn brain secreted a laminin matrix resembling a fibrillar web that protruded from the cell plane. The average neurite lengths obtained for E16 neurons cultured on each astrocyte layer were 198±22 and 123±13 μm, respectively. Analyses of surface-associated electrostatic potentials revealed that embryonic astrocytes presented a pI of -2.8, while in newborn cells this value was -3.8. Removal of the sialic acid groups on the embryonic monolayer by neuraminidase treatment led to the immediate release of matrix-associated laminin. Interestingly, laminin reassembled 1 hour after neuraminidase removal converted to the features of the newborn matrix. Alternatively, treatment of astrocytes with the cholesterol-solubilizing detergent methyl-β-cyclodextrin also resulted in release of the extracellular laminin. To test the hypothesis that sialic-acid-containing lipids localized at cholesterol-rich membrane domains could affect the process of laminin assembly, we devised a cell-free assay where laminin polymerization was carried out over artificial lipid films. Films of either a mixture of gangliosides or pure ganglioside GT1b induced formation of matrices of morpho-functional features similar to the matrices deposited by embryonic astrocytes. Conversely, films of phosphatidylcholine or ganglioside GM1 led to the formation of bulky laminin aggregates that lacked a defined structure. We propose that the expression of negative lipids on astrocytes can control the extracellular polymerization of laminin and, consequently, the permissivity to neuritogenesis of astrocytes during development.

The supramolecular architecture of the basement membrane is provided by two enmeshed networks of collagen IV and laminin. The laminin network is maintained exclusively by interactions among individual laminin molecules and does not depend on the presence of other extracellular matrix components. Laminin polymers can be obtained in vitro either in solution or in association with the surface of bilayers containing acidic lipids. In this work, we have tested the hypothesis that the negative charges present on acidic lipids establish an acid microenvironment that is directly responsible for inducing laminin aggregation. Using light-scattering measurements, we show that laminin does not aggregate on vesicles of neutral lipids, whereas instantaneous aggregation occurs to progressively greater extents as the proportion of acidic phospholipids in the vesicles is increased. Aggregation of laminin induced by vesicles containing acidic phospholipids occurs very rapidly, so that maximal aggregation for each condition is reached within 1 min after laminin dilution. Aggregation depends on the presence of Ca(2+) ions, is reversed by increasing ionic strength, and can be detected at laminin concentrations as low as 6 nM. In addition, we show that, in the absence of vesicles, acidification of the bulk solution can also induce laminin self-polymerization through a process that exhibits the same properties as lipid-induced polymerization. The fact that there is a correspondence between the processes of self-polymerization of laminin in acidic medium and in neutral medium but in the presence of vesicles containing negatively charged lipids leads us to propose that the microenvironment of an acidic surface may trigger the assembly of laminin networks. In vivo, such an acidic microenvironment would be provided by negatively charged sialic acid and sulfate groups present in the glycocalyx surrounding the cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitro studies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.

Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent. The resulting conjugate was 98% self-quenched due to fluorescence resonance energy transfer (homotransfer) between neighboring Bodipy molecules. In vitro proteolytic cleavage of the conjugate led to relaxation of self-quenching and to a significant increase in fluorescence. Flow cytometry and fluorescence microscopy indicated that Bodipy-labeled BSA was readily internalized by J774 macrophages and accumulated in intracellular compartments. The kinetics of intracellular degradation of Bodipy-BSA was linear for up to 2 hours and was completely inhibited by a combination of protease inhibitors. Future applications of the methodology reported here may comprise studies of antigen processing and presentation, as well as the investigation of cellular events related to processing and disassembly of intracellular pathogens such as parasites, bacteria and viruses.

We examined the distribution, abundance and density of the kelp gull, at Admiralty Bay during three austral summers from 2002/03 to 2004/05 and compared our results with previously published data. All ice free areas within the bay were surveyed, and nests were individually marked with a hand-held GPS receiver and subsequently plotted on a geographical chart using ArcView (ESRI 2001). Statistical analysis demonstrated that the means are similar for all time periods, suggesting that the population of kelp gulls has remained unchanged in Admiralty Bay since summer of 1978/79 (Jablonski 1986).

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Applications of intrinsic fluorescence measurements in the study of Ca2+-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca2+ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca2+-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180–200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC 10 BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.

Polylaminin (polyLM) is a flat biomimetic polymer of laminin capable of promoting axonal growth both in vitro and in vivo. It is assembled in a cell-free system when laminin 111 is incubated in acidic pH, whereas incubation in neutral buffer leads to the formation of bulky and irregular polymers (LM). In the present work, we compared the behaviors of cells isolated from the P1 rat retina on polyLM and LM. PolyLM induced cellular spreading and the outgrowth of neurites in contact with the substrate, whereas LM led to the formation of large clusters of cells, with neurites growing only inward. After 24 hr in culture, the number of cells on polyLM increased threefold, and this increase was inhibited by 60% in the presence of the PKA inhibitor H89 and by 41% in the presence of the PKC inhibitor chelerythrine chloride, whereas both inhibitors abolished neuritogenesis. Neither the cell number nor the outgrowth of neurites was affected by the ERK1/2 inhibitor PD98059 on polyLM. On the other hand, PD98059 was able to reduce the cell number on LM, whereas the other inhibitors were not. Immunostaining of P1 retina with an antilaminin antibody revealed that the protein was expressed not only at its inner surface but also within the neuroblast layer in close contact with individual cells. Our results indicate that, when provided in its active polymerized form, laminin can influence both neuritogenesis and proliferation of retinal cells.

In this study, we propose to understand teamwork based on the practice of the professionals working in a health institution. A review was performed on the material produced by the Extended Research Community, comprised of clients, professionals, and managers of the Sofia Feldman Hospital. After collecting and transcribing the material, it was grouped according to similarity, which resulted in three empirical categories. It was evidenced that teamwork in health care expressed the values of the institution, the existence of room for dialogue, which promotes collective decision-making and autonomy, and the liability of the clients, professionals and managers in the health care process. It was also verified that the care is organized in a way that contributes to the process of professional development. In conclusion, teamwork is a consequence and determinant of practices permeated with patient-centered care and relationships that promote dialogue, strengthened by formative processes and favored by a collegial management practice. Descriptors: Comprehensive Health Care; Patient Care Team; Interpersonal Relations; Health Manpower. RESUMEN Se propone aprender el trabajo en equipo a partir de la práctica del profesional en institución de salud. Se utilizó material generado por la Comunidad Ampliada de Investigación, compuesta por pacientes, profesionales y administradores del Hospital Sofia Feldman (HSF). El material recolectado, transcripto y agrupado según similaridad, determinó tres categorías empíricas analizadas. Se evidenció que el trabajo en equipo en la producción de atención de salud expresa los valores institucionales, la existencia de espacios dialógicos, posibilitando decisiones compartidas, ejercicio de la autonomía y responsabilización de pacientes, profesionales y administradores en producción de la atención. Se verifica también contribución de la organización del cuidado para el proceso formativo de profesionales. Se concluye en que el trabajo en equipo es consecuencia y determinante en prácticas de salud impregnadas por el enfoque en el paciente y relaciones dialógicas, potencializado por los procesos formativos y favorecido por una práctica de administración colegiada. Descriptores: Atención Integral de Salud; Grupo de Atención al Paciente; Relaciones Interpersonales; Recursos Humanos en Salud. RESUMO Neste estudo, propomos apreender o trabalho em equipe a partir da prática dos profissionais em uma instituição de saúde. Utilizou-se, para tanto, do material produzido pela Comunidade Ampliada de Pesquisa, composta por usuários, profissionais e gestores do Hospital Sofia Feldman (HSF). O material coletado, após transcrito e agrupado de acordo com sua similaridade, originou três categorias empíricas, as quais foram analisadas. Evidenciou-se que o trabalho em equipe, na produção do cuidado em saúde, expressa os valores institucionais, a existência de espaços dialógicos possibilitando as decisões compartilhadas, o exercício da autonomia e a responsabilização de usuários, profissionais e gestores na produção do cuidado. Verificou-se também a contribuição da organização do cuidado para o processo de formação dos profissionais. Concluiu-se que o trabalho em equipe é consequência determinante nas práticas em saúde, permeadas pela centralidade no usuário e relações dialógicas, potencializado pelos processos formativos e favorecido por uma gestão colegiada. Descritores: Assistência Integral à Saúde; Equipe de Assistência ao Paciente; Relações Interpessoais, Recursos Humanos em Saúde.

The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5-DNS-dT6) served as energy donor to Fl or eosin isothiocyanate coupled to the 5' end of an oligothymidylate polymer of 6 residues length (eosin-dT6) served as energy acceptor from Fl. Labeling of dT6 with 2,5-DNS or eosin did not significantly interfere with recognition by the anti-ssDNA binding site. With the 2,5-DNS/Fl energy transfer pair, the calculated values of R(k2 = 2/3), R(min), and R(max) were 44, 37, and 54 A, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 A, respectively, were obtained. Considering the sizes of the two SCA domains and the length of the interdomain polypeptide linker, an R value of approximately 140 A would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 A indicated that BiSCA was not an extended and rigid molecule. The efficiency of energy transfer increased with increasing temperature in the range of 10-30 degrees C, suggesting that conformational fluctuations of the protein resulted in decreased average distance between BiSCA active sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Polylaminin (polyLM) is a polymerized form of the extracellular matrix protein laminin obtained upon pH acidification. Here microscopy and spectroscopic tools are used to study the cell compatibility and the structural stability of polyLM, aiming at establishing its robustness as a biopolymer for therapeutic use. PolyLM is cell compatible as judged by the efficiency of attachment and neuritogenesis. It is resistant to low temperature. Addition of urea or an increase in hydrostatic pressure leads to polymer disassembly. PolyLM biofilms remain stable for 48 h in contact with cell culture medium. The sedimented polymer recovered after centrifugation and adsorbed on a glass coverslip preserved its original structure and its biological properties.

Regeneration of spinal cord injury (SCI) is a major topic of biomedical research. Laminin is an extracellular matrix protein implicated in neural development and regeneration, but despite that, there are no reports of exogenous laminin contributing to improve the outcome of experimental SCI. Here we investigated whether a biomimetic polymer of laminin assembled on pH acidification, henceforth called polylaminin, could be used to treat SCI in rats. Acute local injection of polylaminin, but not of nonpolymerized laminin, improved motor function after thoracic compression, partial or complete transection. In the latter case, the BBB score for open field locomotion 8 wk after lesion increased from 4.2 ± 0.48 to 8.8 ± 1.14 in animals treated with polylaminin of human origin. Accordingly, neurons retrogradely labeled from the sublesion stump were detected in the spinal cord and brain stem, indicating regrowth of short and long fibers across a complete transection. Polylaminin also played an unsuspected anti-inflammatory role, which underlies the early onset of its positive effects on locomotion from the first week after treatment. The beneficial effects of polylaminin were not observed in animals treated with the nonpolymerized protein or vehicle only. We propose that polylaminin is a promising therapeutic agent to treat human SCI.