Enrichment of IFN- producing cells in different murine adipose tissue depots upon infection with an apicomplexan parasite.

Here we report that lean mice infected with the intracellular parasite Neospora caninum show a fast but sustained increase in the frequency of IFN-γ-producing cells noticeable in distinct adipose tissue depots. Moreover, IFN-γ-mediated immune memory could be evoked in vitro in parasite antigen-stimulated adipose tissue stromal vascular fraction cells collected from mice infected one year before. Innate or innate-like cells such as NK, NK T and TCRγδ(+) cells, but also CD4(+) and CD8(+) TCRβ(+) lymphocytes contributed to the IFN-γ production observed since day one of infection. This early cytokine production was largely abrogated in IL-12/IL23 p40-deficient mice. Moreover, production of IFN-γ by stromal vascular fraction cells isolated from these mice was markedly lower than that of wild-type counterparts upon stimulation with parasite antigen. In wild-type mice the increased IFN-γ production was concomitant with up-regulated expression of genes encoding interferon-inducible GTPases and nitric oxide synthase, which are important effector molecules in controlling intracellular parasite growth. This increased gene expression was markedly impaired in the p40-deficient mice. Overall, these results show that NK cells but also diverse T cell populations mediate a prompt and widespread production of IFN-γ in the adipose tissue of N. caninum infected mice

In vitro and in vivo effects of SerpinA1 on the modulation of Transthyretin proteolysis

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.This research was funded by COMPETE 2020 of PT2020 through the European Regional Development Fund (ERDF), “NETDIAMOND—New Targets in DIAstolic heart failure: from coMOrbidities to persoNalizeD medicine” project financed by the European Structural and Investment Funds (ESIF), through the Programa Operacional Regional (POCI-01-0145-FEDER-016385) and HEALTHUNORTE: Setting-up biobanks and regenerative medicine strategies to boost research in cardiovascular, musculoskeletal, neurological, oncological, immunological, and infectious diseases, NORTE- 01-0145-FEDER-000039. FB was supported by FCT—Fundação para a Ciência e Tecnologia/MEC— Ministério da Educação e Ciência with a PhD fellowship (SFRH/BD/123674/2016)

Selected vs. non-selected under-20 national futsal players: differences between physical performance and training intensity experienced in training camps

The aim of this study was two-fold: (i) analyze the variations in the physical fitness of selected and non-selected under-20 male national futsal players; and (ii) analyze the variations in training intensity monitored during training camps. Thirty-three Taiwan under-20 national futsal players were monitored for training intensity during 18 training camps. They were divided into two groups: selected (n = 14) and non-selected (n = 17) players. The physical assessments included the following measures: body mass, distance covered at Yo-Yo intermittent recovery test Level 1, final velocity at 30-15 Intermittent Fitness Test (30-15 IFT), standing long jump, maximum heart rate (HR), and 1-min sit-up. The training intensity was monitored using the rate of perceived exertion (RPE), HR at different intensity zones, and locomotor demands measured at different speed thresholds. The results revealed that the selected players were significantly faster in the 15-m sprint with ball (p = 0.001) and 30-m sprint (p = 0.001). Additionally, the selected players presented significantly greater HRaverage and time spent above 90% maximum HR during the three-to-six-day training camps (p < 0.05) compared to the non-selected players. Interestingly, the NS demonstrated a greater number of sprints during the training camps (p = 0.001), while the selected players presented greater distance/minute and average speed (p = 0.001). A regression analysis showed that the distance/minute and average speed was a significant predictor of maximum HR in the selected players. As conclusions, the physical fitness outcomes are different between the selected and non-selected national futsal players. The selected players spent more time in high intensity HR demands in training sessionsinfo:eu-repo/semantics/publishedVersio

Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation

Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein–ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR

Os transportadores de monocarboxilatos como mediadores na angiogénese: O seu papel nas interações tumor-endotélio

Dissertação de Mestrado em Ciências da SaúdeDuring the hyperplasic growth of tumors, there is an impairment in both nutrient and oxygen supply to the neoplastic cells located far away from blood vessels, which would influence tumor progression. Thus, tumors have acquired the ability to assemble their own vasculature, mainly through the pre-existing vessels – tumor angiogenesis. However, tumor blood vessels exhibit structural and functional abnormalities, leading to the development of hypoxic regions, which are responsible for the metabolic reprogramming towards glycolysis, regardless of oxygen availability – “Warburg effect”. The end-product of the pathway, lactic acid, is readily released to the tumor milieu through MCTs, contributing to malignant progression. Thus, the aims of the current work are to investigate 1) the role of MCTs on endothelial cell response to hypoxia and 2) the role of MCTs on the angiogenic stimulation by tumor cells. Hence, our experiments demonstrated that MCT1 and MCT4 isoforms, their molecular chaperones, CD147 and CD44, as well as other key metabolic markers are expressed in human brain endothelial cells, mainly under hypoxia, contributing to the increased glycolytic phenotype. Further inhibition of MCT activity, using CHC, as well as MCT downregulation impaired endothelial cell viability and the development of capillary-like structures, which seems to be independent on lactate transport activity, under hypoxic environments. Upon endothelial cell growth in glioma cells’ conditioned media (CM), metabolic adaptations in HBMEC cells were observed, which may contribute to the maintenance of endothelial cell survival, in spite of a decrease in endothelial cell proliferation and, consequently in the development of capillary-like structures that were observed in vitro. In vivo experiments showed a similar phenotypic alteration in chick chorioallantoic membrane vascularization, after exposure to MCT4- and MCT1/4- silenced glioma cells’ CM from both normoxia and hypoxia, relatively to scramble groups. Thus, besides its role in tumor cells, our data point out the importance of MCT1 and, to a lower extent MCT4, on the maintenance of endothelial cell function, under normoxia. Under hypoxia, the absence of these both isoforms seems to be counterbalanced, which may be due to the overexpression of other transporters at the plasma membrane of endothelial cells. In addition, MCTs seem to be players in tumor microenvironment, acting as essential mediators in tumor-endothelial cell interplay.Durante o crescimento hiperplásico dos tumores, há um défice no transporte de nutrientes e de oxigénio para as células distantes dos vasos sanguíneos, comprometendo o desenvolvimento do tumor. Assim, as células tumorais desenvolveram a capacidade de construir a sua própria rede vascular, recorrendo à do tecido - angiogénese tumoral. Todavia, a vasculatura tumoral possui anomalias que promovem o desenvolvimento de regiões de hipóxia. Consequentemente, as células neoplásicas são capazes de reprogramar o seu metabolismo, favorecendo a glicólise, independentemente da disponibilidade de oxigénio – “efeito de Warburg”. O ácido lático resultante é libertado para o microambiente tumoral, via MCTs, promovendo a progressão maligna. Assim, pretende-se avaliar 1) o papel dos MCTs na resposta das células endoteliais à hipóxia, bem como 2) o seu papel na estimulação da angiogénese. Foi demonstrado que MCT1 e MCT4, as suas proteínas auxiliares, bem como marcadores importantes na via glicolítica são expressos em células endoteliais cerebrais, nomeadamente em hipóxia, contribuindo para o aumento do fenótipo glicolítico. A inibição da atividade dos MCTs, bem como a inibição da sua expressão, diminuiu a viabilidade celular e o desenvolvimento de estruturas do tipo capilar que, sob hipóxia, parece ser independente do transporte de lactato. O crescimento de células endoteliais em meio condicionado proveniente de células tumorais, cuja expressão dos MCTs foi inibida, induziu adaptações metabólicas em células endoteliais, contribuindo para a manutenção da viabilidade celular, apesar da diminuição da proliferação celular e do número de estruturas do tipo capilar desenvolvidas. Estudos in vivo demonstraram alterações fenotípicas na vascularização na membrana corioalantóide do embrião de galinha após a adição de meios condicionados, produzidos em normoxia e hipóxia após silenciamento individual do MCT4 ou em combinação com o MCT1. Em suma, além do seu papel em células tumorais, os nossos resultados sugerem a importância do MCT1, e em menor extensão do MCT4, na manutenção da função endotelial, em normoxia. Em hipóxia, a inibição do MCT1 e do MCT4 parece ser compensada, pela sobre expressão de outros transportadores na membrana plasmática de células endoteliais, sob condições de hipóxia. Além disso, os MCTs parecem também desempenhar um papel importante no microambiente tumoral, atuando como proteínas essenciais nas interações tumorendotélio

Estimulação multissensorial junto de pessoa com demência de tipo Alzheimer: estudo de caso clínico

A estimulação multissensorial está presente nas nossas vidas desde que nascemos, até que morremos. A estimulação multissensorial é importante para comunicarmos com o mundo, ambiente e pessoas. Vários estudos e investigações têm demonstrado um particular interesse pelo desenvolvimento de atividades de estimulação multissensorial que proporcionam à pessoa de idade mais avançada benefícios em termos cognitivos, sociais e afetivos. Este estudo clínico tem como título o seguinte tema: “Estimulação Multissensorial em Idoso dependente e institucionalizado com Doença de Alzheimer e respectivo Estudo de Caso”. Assim o objetivo do presente estudo foi conhecer os benefícios da prática de sessões individuais de estimulação multissensorial na pessoa com demência dependente e institucionalizado. Para tal e através de uma metodologia mista com contornos de investigação-ação, foram realizadas oito sessões individuais de estimulação multissensorial a uma pessoa de idade mais avançada institucionalizada. O processo de recolha dos dados foi realizado no Lar de Terceira Idade Quintinha da Conceição. A amostra era composta por um único utente dessa mesma instituição. Os instrumentos de recolha de dados utilizados foram os seguintes testes psicométricos: CSDD (Escala de Cornell para a Depressão na Demência), GDS (Global Deterioration Scale), Escala de Katz e Classificação Funcional da Marcha de Holden, grelhas de observação, registos narrativos das sessões de estimulação multissensorial, pesquisa documental e a observação direta. Os resultados do estudo indicam uma melhoria significativa a nível dos comportamentos disruptivos, ou estabilização do estado de humor, contacto ocular, melhorias a nível da comunicação verbal e da motricidade fina, melhoria significativa no que diz respeito à sua expressão facial, pois aparentou estar mais calma e relaxada

Psychobiological Changes during National Futsal Team Training Camps and Their Relationship with Training Load

The aim of this study was two-fold: (1) to analyze the within-week variations of heart rate, session-rated of perceived exertion (sRPE), total distance, distance in 8.0-11.99 km/h-1, recovery distance in 12.0-17.99 km/h-1, distance in >18.0 km/h-1, maximum speed, number of sprints, heart rate variability, delayed onset muscle soreness (DOMS), and fatigue during training camps of a national futsal team; and (2) to analyze the relationships between load and the well-being. Twenty-eight men from the Chinese Taipei U-20 national futsal team were analyzed. Comparisons of training days revealed that the total distance was significantly smaller on day 1 (d = -1.22) and day 6 (d = -1.95) than on day 3. The sRPE values were significantly lower on day 1 than days 4 (d = -1.53), 5 (d = -2.07), and 6 (d = -2.59). The relationships between training load and recovery parameters revealed moderate correlations between the DOMS and the sRPE recorded one (r = -0.321) and two days before training (r = -0.289). It is possible conclude that first day imposed a smaller external load and internal load, and that the internal load had a greater dependent relationship with reported DOMS and fatigue during the training camps

PITB: A high affinity transthyretin aggregation inhibitor with optimal pharmacokinetic properties

The aggregation of wild-type transthyretin (TTR) and over 130 genetic TTR variants underlies a group of lethal disorders named TTR amyloidosis (ATTR). TTR chemical chaperones are molecules that hold great promise to modify the course of ATTR progression. In previous studies, we combined rational design and molecular dynamics simulations to generate a series of TTR selective kinetic stabilizers displaying exceptionally high affinities. In an effort to endorse the previously developed molecules with optimal pharmacokinetic properties, we conducted structural design optimization, leading to the development of PITB. PITB binds with high affinity to TTR, effectively inhibiting tetramer dissociation and aggregation of both the wild-type protein and the two most prevalent disease-associated TTR variants. Importantly, PITB selectively binds and stabilizes TTR in plasma, outperforming tolcapone, a drug currently undergoing clinical trials for ATTR. Pharmacokinetic studies conducted on mice confirmed that PITB exhibits encouraging pharmacokinetic properties, as originally intended. Furthermore, PITB demonstrates excellent oral bioavailability and lack of toxicity. These combined attributes position PITB as a lead compound for future clinical trials as a disease-modifying therapy for ATTR