A utilização de sémen fresco na fertilização in vitro de embriões ovinos melhora a qualidade dos blastocistos na raça portuguesa Merino

A produção de embriões em ovinos é uma tarefa difícil, exigindo experiência e condições onerosas, principalmente na produção de embriões in vivo. A recolha sistemática de oócitos em animais de matadouro ou em animais vivos por ovum pick-up, permite a produção in vitro de embriões (IVP), em larga escala e menos dispendiosa, nos pequenos ruminantes. Esta possibilidade é importante não só como fonte de embriões mas também de oócitos e zigotos para fins comerciais ou de investigação, facilitando a sua disponibilidade em tecnologias emergentes tais como a clonagem ou a transgénese. Para IVP foram desenvolvidos vários protocolos de maturação, utilizando fertilização in vitro (IVF) com sémen fresco ou congelado. Em Portugal, a produção de embriões in vitro foi somente realizada com sémen congelado dada a sua disponibilidade em condições de rotina. Contudo, o sémen fresco poderá melhorar a produção de embriões frescos ou criopreservados. Este trabalho teve como objectivo comparar a eficiência da IVP em ovinos usando diferentes protocolos de maturação de oócitos e IVF com sémen fresco ou congelado. Oócitos (n=1768) recolhidos em matadouro foram maturados em meio TCM199 com 100 μM cisteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 e gentamicina (mat A, n=692) ou suplementada com 10 μg mL-1 FSH e 0,3 mM piruvato de sódio (mat B, n=707) a 39 ºC e 5% CO2 durante 22h. O sémen fresco (FS) e congelado/descongelado (TS) de carneiros de raça Merino Branco (n=3) foi lavado ou submetido a swim-up, respectivamente. Após a fertilização (18h p.i.), os presumíveis zigotos foram cultivados em meio de fluido sintético do oviducto (SOF) enriquecido com aminoácidos e BSA a 38,5 ºC, em atmosfera humidificada com 5% O2, 5% CO2 e 90% N2 até ao estadio de 2-4-8 células. Após clivagem, o desenvolvimento embrionário prosseguiu até ao estadio de blastocisto em meio SOF, BSA e 10% FCS. A qualidade foi avaliada no dia 6-7, classificando-se como bons, médios e maus, baseado nos parâmetros IETS. Os dados das taxas de produção embrionária foram analisados utilizando ANOVA. Foi utilizado o teste de Mann-Whitney U para avaliação da qualidade dos embriões. Os diferentes protocolos de maturação não interferiram (p>0,05) quer com as taxas de maturação quer com as taxas de produção de embriões. A qualidade embrionária foi superior (p=0,004) na fertilização com sémen fresco (bom: FS=40,1±8,0% vs TS=32,9±5,6%; média: FS=20,1±4,7% vs TS=35,7±5,8%; má: FS=39,8±9,8% vs TS=31,4±7,6%). Em conclusão, estes resultados preliminares mostram que o sémen fresco de carneiro pode ser facilmente utilizado para fertilização in vitro e melhora a qualidade dos embriões produzidos.#Embryo production in sheep is a difficult task demanding experience and expensive facilities, particularly when dealing with in vivo embryo production. Easy ways to obtain ovine embryos consist of collecting oocytes at slaughterhouses or systematically pick them up from live animals, allowing a large scale and cheaper in vitro embryo production (IVP) for small ruminants. Those are important sources of embryos, oocytes and zygotes for commercial, laboratorial and research proposes, making easier the availability of resources for emerging techniques like cloning or transgenesis. For IVP, several oocyte maturation protocols have been developed using fertilization (IVF) either with fresh or frozen-thawed semen. In Portugal, IVP has been done through IVF using cryopreserved semen because it is easily available for routine use. However, the use of fresh semen could improve embryo production and cryopreservation results. The aim of this work was to compare the efficiency of in vitro embryo production in ovine using different oocyte maturation protocols and fresh or frozen semen for IVF. Abattoir-derived oocytes (n=1768) were matured in TCM199, 10 μM cysteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 and gentamicin (mat A, n=692) or plus 10 μg mL-1 FSH and 0.3 mM sodium piruvate (mat B, n=707) at 39 ºC and 5% CO2 for 22h. Prior to fertilization, either fresh (FS) or frozen/thawed (TS) semen from Merino rams (n=3) was washed or submitted to swim-up respectively. Presumptive zygotes (18h p.i.) were cultured in synthetic oviductal fluid (SOF) enriched with aminoacids and 6 mg mL-1 BSA at 38.5 ºC, under 5% O2, 5% CO2 and 90% N2 in an humidified atmosphere until the stage of 2-4-8 cell embryos. After assessing cleavage, embryo development proceeded until the blastocyst stage in SOF+BSA and 10% FCS. Quality was evaluated on D6-7 by scoring embryos as good, fair and bad based on IETS guidelines. Data from embryo production rates were analysed using ANOVA. Mann-Whitney U test was used for embryo quality evaluation. Different maturation protocols did not interfere (P>0.05) either on maturation or on embryo quality or production rates. Embryo quality was higher (P=0.004) when fertilization was accomplished with fresh semen (good: FS=40.1±8.0% vs TS=32.9±5.6%; fair: FS=20.1±4.7% vs TS=35.7±5.8%; bad: FS=39.8±9.8% vs TS=31.4±7.6%). Preliminar results show that ram fresh semen can be easily used for in vitro fertilization and improves the quality of produced embryos

Productive restructuring and the reallocation of work and employment: a survey of the “new” forms of social inequality

O propósito do presente artigo consiste em questionar a inevitabilidade dos processos de segmentação e precarização das relações de trabalho e emprego, responsáveis pela inscrição de “novas” formas de desigualdade social que alicerçam o actual modelo de desenvolvimento das economias e sociedades. Visa-se criticar os limites da lógica econômica e financeira, de contornos globais, que configuram um “novo espírito do capitalismo”, ou seja, uma espécie de divinização da ordem natural das coisas. Impõe-se fazer, por isso, um périplo analítico pelas transformações em curso no mercado de trabalho, acompanhado pela vigilância epistemológica que permita enquadrar e relativizar as (di)visões neoliberais e teses tecnodeterministas dominantes. A perspectivação de cenários sobre o futuro do trabalho encerrará este périplo, permitindo-nos alertar para os condicionalismos histórico-temporais, para a urgência de se desocultar o que de ideológico e político existe nas actuais lógicas de racionalização e para os processos de ressimbolização do trabalho e emprego enquanto “experiência social central” na contemporaneidade.The scope of this paper is to question the inevitability of the processes of segmentation and increased precariousness of the relations of labor and employment, which are responsible for the introduction of “new” forms of social inequality that underpin the current model of development of economies and societies. It seeks to criticize the limits of global financial and economic logic, which constitute a “new spirit of capitalism,” namely a kind of reverence for the natural order of things. It is therefore necessary to conduct an analytical survey of the ongoing changes in the labor market, accompanied by epistemological vigilance which makes it possible to see neoliberal (di)visions and dominant technodeterministic theses in context. The enunciation of scenarios on the future of work will conclude this survey and will make it possible to draw attention to both the historical and temporal constraints and to the urgent need to unveil what is ideological and political in the prevailing logic of rationalization and processes to reinstate work and employment as a “central social experience” in contemporary times

Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B <em>Streptococcus</em>

ABSTRACT: Several bacterial pathogens decorate their surfaces with sialic acid (Sia) residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic acid-binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, sialoadhesin (Sn) is a unique Siglec with an elongated structure that lacks intracellular signaling motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via sialoadhesin-sialic-acid-mediated recognition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restricted the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in sialoadhesin-deficient mice. Thus, sialoadhesin represents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens. KEY MESSAGE: Sialoadhesin is critical for macrophages to phagocytose and clear GBS. Increased GBS organ dissemination in the sialoadhesin-deficient mice. Reduced anti-GBS IgM production in the sialoadhesin-deficient mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-014-1157-y) contains supplementary material, which is available to authorized users

Trabalho educativo do enfermeiro na Estratégia Saúde da Família: dificuldades e perspectivas de mudanças

Este estudo objetivou conhecer as dificuldades e perspectivas de mudanças que os enfermeiros identificam no desenvolvimento das ações educativas na Estratégia Saúde da Família (ESF). Trata-se de uma pesquisa qualitativa descritivo-exploratória. Os dados foram coletados junto a 20 enfermeiros que atuam na ESF, no âmbito da 10ª Regional de Saúde do Paraná, por meio de entrevistas semiestruturadas, no mês de abril de 2010, as quais foram submetidas à análise de conteúdo. Os resultados demonstraram que os enfermeiros enfrentam dificuldades no desenvolvimento da educação em saúde junto aos usuários, à equipe, aos gestores e quanto à falta de recursos físicos, materiais e financeiros. Mas, a partir das dificuldades sentidas, buscam alternativas diversificadas para superá-las e sugerem modificações visando à melhoria na atenção primária à saúde da população, principalmente, no que tange ao trabalho educativo

Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology

Employing an open-source tool to assess astrocyte tridimensional structure

Astrocytes display important features that allow them to maintain a close dialog with neurons, ultimately impacting brain function. The complex morphological structure of astrocytes is crucial to the role of astrocytes in brain networks. Therefore, assessing morphologic features of astrocytes will help provide insights into their physiological relevance in healthy and pathological conditions. Currently available tools that allow the tridimensional reconstruction of astrocytes present a number of disadvantages, including the need for advanced computational skills and powerful hardware, and are either time-consuming or costly. In this study, we optimized and validated the FIJI-ImageJ, Simple Neurite Tracer (SNT) plugin, an open-source software that aids in the reconstruction of GFAP-stained structure of astrocytes. We describe (1) the loading of confocal microscopy Z-stacks, (2) the selection criteria, (3) the reconstruction process, and (4) the post-reconstruction analysis of morphological features (process length, number, thickness, and arbor complexity). SNT allows the quantification of astrocyte morphometric parameters in a simple, efficient, and semi-automated manner. While SNT is simple to learn, and does not require advanced computational skills, it provides reproducible results, in different brain regions or pathophysiological states.The authors acknowledge funding from national funds through the FCT—Foundation for Science and Technology—project (PTDC/SAU-NSC/118194/2010) to G.T., V.M.S., S.G.G. and J.F.O., and fellowships (SFRH/BD/89714/2012 to V.M.S., SFRH/BPD/97281/2013 to J.F.O., SFRH/BD/101298/2014 to S.G.G., PD/BD/114120/2015 to S.P.N, and PD/BD/127822/2016 to G.T.); Marie Curie Fellowship FP7-PEOPLE-2010-IEF 273936 and BIAL Foundation Grants and 207/14 to J.F.O.; QREN and FEDER funds through Operational program for competitiveness factors—COMPETE, “ON.2 SR&TD Integrated Program—NORTE-07-0124-FEDER-000021”; National and European funds through FCT, and FEDER through COMPETE (PEst-C/SAU/LA0026/2011 and FCOMP-01-0124-FEDER-022724; PEst-C/SAU/LA0026/2013 and FCOMP-01-0124-FEDER-037298, respectively)info:eu-repo/semantics/publishedVersio

Yellow fever epizootics in non-human primates, São Paulo state, Brazil, 2008-2009

Since 2000, the expansion of Sylvatic Yellow Fever (YF) has been observed in the southeast of Brazil, being detected in areas considered silent for decades. Epizootics in non-human primates (NHPs) are considered sentinel events for the detection of human cases. It is important to report epizootic events that could have impact on the conservation status of susceptible species. We describe the epizootics in NHPs, notified in state of São Paulo, Brazil, between September 2008 to August 2009. Ninety-one epizootic events, involving 147 animals, were reported in 36 counties. Samples were obtained from 65 animals (44.2%). Most of the epizootics (46.6%) were reported between March and April, the same period during which human cases of YF occurred in the state. Biological samples were collected from animals found dead and were sent to Instituto Adolfo Lutz, in São Paulo. Two samples, collected in two counties without an indication for YF vaccination, were positive for the virus. Another 48 animals were associated with YF by clinical-epidemiological linkage with laboratory confirmed cases. Because the disease in human and NHPs occurred in the same period, the detection of the virus in NHPs did not work as sentinel, but aided in the delineation of new areas of risk