O sonho: teatro-música experimental

Trabalho de Projeto submetido à Escola Superior de Teatro e Cinema para cumprimento dos requisitos necessários à obtenção do grau de Mestre em TEATRO - especialização em Artes Performativas (Teatro-Música).

Um buraco na boca: edição crítica do romance experimental de António Aragão

Este projecto apresenta uma edição crítca do romance Um buraco na boca, de António Aragão, elaborada a partr dos testemunhos disponíveis na tradição impressa. É feita uma súmula do percurso e obra do autor, com destaque para as suas criações no campo da fcção. Associado a esse trabalho, apresenta-se uma proposta de defnição do conceito de romance experimental e respectvo mapeamento das produções que encaixam no modelo proposto. Faz-se ainda um comentário sobre os actuais desafos da Crítca Textual na era digital, tendo em vista o planeamento de uma edição electrónica de Um buraco na boca

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Endocrine response and perceived stress test during an experimental challenge task in adult survivors of a childhood cancer.

BACKGROUND: Although long-term implications of cancer in childhood or adolescence with regard to medical conditions are well documented, the impact on mental health and on response to stress, which may be an indicator of psychological vulnerability, is not yet well understood. In this study, psychological and physiological responses to stress were examined.¦PROCEDURE: Fifty-three participants aged 18-39 years (n = 25 survivors of childhood or adolescence cancer, n = 28 controls) underwent an experimental stress test, the Trier Social Stress Test (TSST). Participants were asked to provide repeated evaluations of perceived stress on visual-analogical scales and blood samples were collected before and after the TSST to measure plasma cortisol.¦RESULTS: The psychological perception of stress was not different between the two groups. However, the cancer survivors group showed a higher global plasma cortisol level as well as higher amplitude in the response to the TSST. The global cortisol level in cancer survivors was increased when depression symptoms were present. The subjective perception of stress and the plasma cortisol levels were only marginally correlated in both groups.¦CONCLUSIONS: It is suggested that the exposure to a life-threatening experience in childhood/adolescence increases the endocrine response to stress, and that the presence of depressive symptoms is associated with an elevation of plasma cortisol levels. A better knowledge of these mechanisms is important given that the dysregulations of the stress responses may cause psychological vulnerability. Pediatr Blood Cancer 2012; 59: 138-143. © 2011 Wiley Periodicals, Inc.

Two's company, three's a crowd: experimental evaluation of the evolutionary maintenance of trioecy in Mercurialis annua (Euphorbiaceae).

Trioecy is an uncommon sexual system in which males, females, and hermaphrodites co-occur as three clearly different gender classes. The evolutionary stability of trioecy is unclear, but would depend on factors such as hermaphroditic sex allocation and rates of outcrossing vs. selfing. Here, trioecious populations of Mercurialis annua are described for the first time. We examined the frequencies of females, males and hermaphrodites across ten natural populations and evaluated the association between the frequency of females and plant densities. Previous studies have shown that selfing rates in this species are density-dependent and are reduced in the presence of males, which produce substantially more pollen than hermaphrodites. Accordingly, we examined the evolutionary stability of trioecy using an experiment in which we (a) indirectly manipulated selfing rates by altering plant densities and the frequency of males in a fully factorial manner across 20 experimental plots and (b) examined the effect of these manipulations on the frequency of the three sex phenotypes in the next generation of plants. In the parental generation, we measured the seed and pollen allocations of hermaphrodites and compared them with allocations by unisexual plants. In natural populations, females occurred at higher frequencies in denser patches, a finding consistent with our expectations. Under our experimental conditions, however, no combination of plant densities and male frequencies was associated with increased frequencies of females. Our results suggest that the factors that regulate female frequencies in trioecious populations of M. annua are independent of those regulating male frequencies (density), and that the stable co-existence of all three sex phenotypes within populations is unlikely.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts.

Artificial antigen-presenting cells (aAPC) are widely used for both clinical and basic research applications, as cell-based or bead-based scaffolds, combining immune synapse components of interest. Adequate and controlled preparation of aAPCs is crucial for subsequent immunoassays. We reveal that certain proteins such as activatory anti-CD3 antibody can be out-competed by other proteins (e.g. inhibitory receptor ligands such as PDL1:Fc) during the coating of aAPC beads, under the usually performed coating procedures. This may be misleading, as we found that decreased CD8 T cell activity was not due to inhibitory receptor triggering but rather because of unexpectedly low anti-CD3 antibody density on the beads upon co-incubation with inhibitory receptor ligands. We propose an optimized protocol, and emphasize the need to quality-control the coating of proteins on aAPC beads prior to their use in immunoassays.

Comparison of four embolic materials for portal vein embolization: experimental study in pigs.

Different embolic materials for portal vein embolization (PVE) were evaluated. Twenty pigs received left and median PVE. Hydrophilic phosphorylcholine, N-butyl cyanoacrylate, hydrophilic gel, and polyvinyl alcohol (PVA) particles measuring either 50-150 microm or 700-900 microm were used in five pigs each. Portography and portal vein pressure measurement were performed before, immediately after PVE, and before being euthanized at day 7. Tissue wedges from embolized, and non-embolized liver were obtained for pathology. After complete embolization, recanalization occurred at 7 days in one gel and one 700-900 PVA embolization. Post-PVE increase in portal pressure was found in all groups (p = 0.01). The area of the hepatic lobules in non-embolized liver was larger than in the embolized liver in all groups (p = 0.001). The ratios of the areas between non-embolized/embolized livers were 1.65, 2.19, 1.57, and 1.32 for gel, NBCA, 50-150 PVA and 700-900 PVA, respectively; the ratios of fibrosis between the embolized and non-embolized livers were 1.37, 3.01, 3.49, and 2.11 for gel, NBCA, 50-150 PVA and 700-900 PVA, respectively. Hepatic lobules in non-embolized liver were significantly larger with NBCA than in other groups (p = 0.01). Fibrosis in embolized liver was significantly higher for NBCA and 50-150 PVA (p = 0.002). The most severe changes in embolized and non-embolized liver were induced by 50-150 PVA and NCBA PVE.

Epistasis and maternal effects in experimental adaptation to chronic nutritional stress in Drosophila.

Based on ecological and metabolic arguments, some authors predict that adaptation to novel, harsh environments should involve alleles showing negative (diminishing return) epistasis and/or that it should be mediated in part by evolution of maternal effects. Although the first prediction has been supported in microbes, there has been little experimental support for either prediction in multicellular eukaryotes. Here we use a line-cross design to study the genetic architecture of adaptation to chronic larval malnutrition in a population of Drosophila melanogaster that evolved on an extremely nutrient-poor larval food for 84 generations. We assayed three fitness-related traits (developmental rate, adult female weight and egg-to-adult viability) under the malnutrition conditions in 14 crosses between this selected population and a nonadapted control population originally derived from the same base population. All traits showed a pattern of negative epistasis between alleles improving performance under malnutrition. Furthermore, evolutionary changes in maternal traits accounted for half of the 68% increase in viability and for the whole of 8% reduction in adult female body weight in the selected population (relative to unselected controls). These results thus support both of the above predictions and point to the importance of nonadditive effects in adaptive microevolution.

Failure of vancomycin continuous infusion against experimental endocarditis due to vancomycin-intermediate Staphylococcus aureus.

Continuous infusion of vancomycin was evaluated against experimental endocarditis due to heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and VISA. Animals were infected with hVISA PC1 (vancomycin MIC, 2 mg/liter) or VISA PC3 (vancomycin MIC, 8 mg/liter) and treated for 5 days with constant serum levels of 20 or 40 mg/liter. Vancomycin continuous infusion was unsuccessful, as 20 mg/liter was barely active against PC1 (6 of 13 sterile vegetations) and 40 mg/liter failed against PC3 (2 of 9 sterile vegetations).

CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.