Metho of quantification of nucleotides by HPLC-MS/MS and evaluation of the activity of sildenafil analoges in phosphodiesterase

Orientador: Gilberto de NucciTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: No presente trabalho foi padronizado um novo método para a dosagem da atividade de fosfodiesterase in vitro, por HPLC-MS/MS. Este novo método conseguiu apresentar exatidão, precisão, sensibilidade e rapidez nas análises; monitorando os nucleotídeos (AMP, GMP, AMPc e GMPc). O desenvolvimento de novos fármacos derivados de um protótipo aponta para a obtenção de moléculas com um melhor perfil farmacocinético ou uma melhor relação estrutura-atividade. Atualmente o sildenafil é considerado o principal fármaco para o tratamento de disfunção erétil. Neste trabalho, avaliaram-se novos compostos denominados análogos de sildenafil (carbonato de lodenafil, dímero uréia e dímero uretana). Os análogos foram analisados quanto à atividade em PDE5 e agregação plaquetária humana, in vitro. Foi determinada a estabilidade destes compostos, em meio ácido e plasma humano, in vitro, além de seus possíveis metabólitos em microssomas e hepatócitos de rato in vitro, e os seus parâmetros farmacocinéticos via intravenosa e oral, em cão, in vivo. Os resultados mostraram que os análogos de sildenafil inibem a atividade de PDE e não inibem a agregação plaquetária do mesmo modo que o sildenafil in vitro, no entanto potencializam a ação do doador de NO (SNP). Os análogos de sildenafil foram estáveis em meio ácido e em plasma humano. No ensaio de metabolização, os dímeros uréia e uretana não foram metabolizados, entretanto o carbonato de lodenafil foi metabolizado principalmente em lodenafil, in vitro. O carbonato de lodenafil é rapidamente biotransformado em lodenafil, após administração v.i. e v.o. em cão. Concluiu-se que este trabalho apresenta um novo método de dosagem de PDEs e uma nova perspectiva terapêutica para a disfunção erétil, representada pelo carbonato de lodenafil, o qual inibe concentração-dependente a atividade de PDE5Abstract: In the present work a new method for the dosage of the activity of phosphodiesterase was standardized in vitro, for HPLC-MS/MS. This new method obtained to present exactness, precision, sensitivity, and rapidity in the analyses; monitoring the nucleotides (AMP, GMP, cAMP and cGMP). The development of new drug derived from an archetype points with respect to the molecule attainment with one better pharmacokinetic profile or one better relation structure-activity. Currently the sildenafil is considered the main drug for the treatment of erectile dysfunction. In this work, we evaluate new analogous called composites of sildenafil (carbonate of lodenafil, dimer urea and dimer uretana). The analogous ones had been analyzed how much the activity in PDE5 and platelet aggregation human being, in vitro. The stability of these composites was determined, in human acid way and plasma, in vitro, beyond its possible metabolites in microsomes and hepatocytes of rat in vitro, and its pharmacokinetic profile after intravenous and oral, in dog, in vivo. The results had shown that the analogous of sildenafil inhibit the activity of PDE and they do not inhibit the platelet aggregation in a similar way that the sildenafil in vitro, however potencializam the action of the giver of NO (SNP). The analogous ones of sildenafil are presented steady in human acid way and plasma. In the metabolization assay, metabolization of dimer urea and dimer uretana was not observed, however the lodenafil carbonate was metabolizado mainly in lodenafil, in vitro. The lodenafil carbonate quickly is biotransformation in lodenafil, after administration v.i. and v.o. in dog. We conclude that this work presents a new method for analyze activity of PDEs and a new therapeutically perspective for the erectile dysfunction, represented for lodenafil carbonate, which inhibits concentration-dependent the activity of PDE5DoutoradoDoutor em Farmacologi

Uma Perspectiva das Concepções de Calouros Universitários Sobre o Valor Absoluto de Números Reais

Este artigo inicia-se com uma interpretação do papel do conceito de valor absoluto na Matemática universitária e com uma possível descrição de como os alunos aprendem tal conceito. O referencial teórico Ação-Processo-Objeto-Esquema (APOS) utilizado para descrever o desenvolvimento cognitivo é uma extensão das idéias de Piaget sobre abstração reflexiva, e permite-nos classificar as construções mentais presentes no processo de aprendizagem de conceitos matemáticos avançados. Baseados num modelo provisório de aprendizagem do valor absoluto de números reais, os dados de entrevistas com calouros universitários são interpretados. Os resultados sugerem que o nível de abstração dos alunos participantes não lhes permite um entendimento adequado do conceito. Alguns dados também indicam que o trabalho em grupo e as representações gráficas são fatores relevantes em propostas de ações didáticas, pois parecem facilitar a construção de um conhecimento mais eficiente e significativo

NLRP3 inflammasome activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1b that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1b in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL- 1 receptor, we demonstrated that IL-1b signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus

TRAIL-R1 and TRAIL-R2 mediate TRAIL-dependent apoptosis in activated primary human B lymphocytes

The maintenance of B cell homeostasis requires a tight control of B cell generation, survival, activation, and maturation. In lymphocytes upon activation, increased sensitivity to apoptotic signals helps controlling differentiation and proliferation. The death receptor Fas is important in this context because genetic Fas mutations in humans lead to an autoimmune lymphoproliferative syndrome that is similar to lymphoproliferation observed in Fas-deficient mice. In contrast, the physiological role of TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) in humans has been poorly studied so far. Indeed, most studies have focused on tumor cell lines and on mouse models whose results are difficult to transpose to primary human B cells. In the present work, the expression of apoptosis-inducing TRAIL-R1 and TRAIL-R2 and of the decoy receptors TRAIL-R3 and TRAIL-R4 was systematically studied in all developmental stages of peripheral B cells isolated from the blood and secondary lymphoid organs. Expression of TRAIL-Rs is modulated along development, with highest levels observed in germinal center B cells. In addition, T-dependent and T-independent signals elicited induction of TRAIL-Rs with distinct kinetics, which differed among B cell subpopulations: switched memory cells rapidly upregulated TRAIL-R1 and -2 upon activation while naïve B cells only reached similar expression levels at later time points in culture. Increased expression of TRAIL-R1 and -2 coincided with a caspase-3-dependent sensitivity to TRAIL-induced apoptosis in activated B cells but not in freshly isolated resting B cells. Finally, both TRAIL-R1 and TRAIL-R2 could signal actively and both contributed to TRAIL-induced apoptosis. In conclusion, this study provides a systematic analysis of the expression of TRAIL-Rs in human primary B cells and of their capacity to signal and induce apoptosis. This dataset forms a basis to further study and understand the dysregulation of TRAIL-Rs and TRAIL expression observed in autoimmune diseases. Additionally, it will be important to foresee potential bystander immunomodulation when TRAIL-R agonists are used in cancer treatment.Fil: Staniek, Julian. Albert Ludwigs University of Freiburg; AlemaniaFil: Lorenzetti, Raquel. Albert Ludwigs University of Freiburg; AlemaniaFil: Heller, Bianca. Albert Ludwigs University of Freiburg; AlemaniaFil: Janowska, Iga. Albert Ludwigs University of Freiburg; AlemaniaFil: Schneider, Pascal. Universite de Lausanne; SuizaFil: Unger, Susanne. Albert Ludwigs University of Freiburg; AlemaniaFil: Warnatz, Klaus. Albert Ludwigs University of Freiburg; AlemaniaFil: Seidl, Maximilian. Albert Ludwigs University of Freiburg; AlemaniaFil: Venhoff, Nils. Albert Ludwigs University of Freiburg; AlemaniaFil: Thiel, Jens. Albert Ludwigs University of Freiburg; AlemaniaFil: Smulski, Cristian Roberto. Comisión Nacional de Energía Atómica. Centro Atómico Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Rizzi, Marta. Albert Ludwigs University of Freiburg; Alemani

CONTRIBUIÇÕES DA TEMÁTICA FREIRIANA PARA O ENSINO DE CIÊNCIAS: UMA ANÁLISE NAS ATAS DO ENPEC NO PERÍODO 2011-2019

Understanding the relevance of research involving Freirean epistemology, this study aims to analyze the contributions of the Freirean theme to Science Teaching, present in the ENPEC minutes from 2011 to 2019 and its possible consequences for the training process of the subjects. The methodology adopted was based on a “state of the art” type survey, with a total of 5,394 works being mapped. Of these, 44 had the following terms in the title and/or keywords: “Freire”; “Liberating Education” and Democratic Education”. For data analysis, Discursive Textual Analysis was used, with the creation of the following descriptors: year of publication, authors, institutions, keywords, research subjects, educational level and area of knowledge. To analyze the titles, objectives, methodologies and results achieved, three a priori categories were created: Category I: Practices involving the Freirean theme; Category II: Teacher education based on Freire's epistemology and Category III: Conceptions, ways and possibilities in the use of Freire's theme. As a result, it was possible to observe that the use of the Freirean approach in the works allows to assist in the critical and reflective training process of the subjects, directing them towards social (co)existences.Entendiendo la relevancia de la investigación que involucra a la Epistemología Freiriana, este estudio tiene como objetivo analizar los aportes de la Temática Freiriana para la Enseñanza de las Ciencias, presente en las actas de la ENPEC de 2011 a 2019 y sus posibles consecuencias para el proceso de formación de los sujetos. La metodología adoptada se basó en un levantamiento tipo “estado del arte”, con un total de 5.394 obras en mapeo. De estos, 44 tenían los siguientes términos en el título y / o palabras clave: “Freire”; “Educación liberadora” y Educación democrática”. Para el análisis de los datos se utilizó el Análisis Textual Discursivo, con la creación de los siguientes descriptores: año de publicación, autores, instituciones, palabras clave, sujetos de investigación, nivel educativo y área de conocimiento. Para analizar los títulos, objetivos, metodologías y resultados alcanzados, se crearon tres categorías a priori: Categoría I: Prácticas que involucran el Tema Freireano; Categoría II: Formación del profesorado basada en la epistemología freiriana y Categoría III: Concepciones, formas y posibilidades en el uso de la temática freiriana. Como resultado, se pudo observar que el uso del Enfoque Freiriano en los trabajos permite ayudar en el proceso de formación crítica y reflexiva de los sujetos, encaminándolos hacia las (co) existencias sociales.Compreendendo a relevância das pesquisas envolvendo a epistemologia Freiriana, o presente estudo tem o objetivo de analisar as contribuições da temática Freiriana para o ensino de Ciências presentes nas atas do ENPEC no período 2011 até 2019 e seus possíveis reflexos para o processo formativo dos sujeitos. A metodologia adotada pautou-se em uma pesquisa do tipo “estado da arte” sendo mapeado um total de 5.394 trabalhos. Destes, 44 apresentavam no título e/ou palavras-chaves os seguintes termos: “Freire”, “educação libertadora” e educação democrática”. Para a análise dos dados utilizou-se a Análise Textual Discursiva com a criação dos seguintes descritores: ano de publicação, autores, instituições, palavras-chaves, sujeitos da pesquisa, nível de ensino e área do conhecimento. Para analisar os títulos, objetivos, metodologias e resultados alcançados foram criadas três categorias a priori: categoria I: práticas envolvendo a temática Freiriana, categoria II: a formação de professores pautada na epistemologia Freiriana e categoria III: concepções, caminhos e possibilidades no uso da temática Freiriana. Como resultados foi possível observar que o uso da abordagem Freiriana nos trabalhos permite auxiliar no processo formativo crítico e reflexivo dos sujeitos direcionando para as (con)vivências sociais

Hepatotoxicity In Hiv-infected Children And Adolescents On Antiretroviral Therapy.

Adverse drug reactions are a significant problem in patients on antiretroviral therapy (ART). We determined liver enzyme elevation frequencies in HIV-infected children and adolescents receiving ART, and their association with risk factors. Cross-sectional study, at the Pediatrics Immunodeficiency Division, University Hospital, Universidade Estadual de Campinas. Medical records of 152 children and adolescents (54.6% male; median age 7.48 years) were analyzed, with a mean of 2.6 liver enzyme determinations per patient. Clinically, patients were classified in categories N (6), A (29), B (78) and C (39). Serum levels of aspartate aminotransferase and alanine aminotransferase were evaluated. Hepatotoxicity was scored as grade 1 (1.1-4.9 times upper limit of normality, ULN), grade 2 (5.0-9.9 times ULN), grade 3 (10.0-15.0 times ULN) and grade 4 (> 15.0 times ULN). To assess hepatotoxicity risk factors, odds ratios (OR) and adjusted odds ratios (aOR) for age, gender, TCD4+ cell count, viral load and medication usage were calculated. We observed grade 1 hepatotoxicity in 19.7 % (30/152) patients. No cases of grade 2, 3 or 4 were detected. There was a significant association between hepatotoxicity and use of sulfonamides (OR, 3.61; 95% confidence interval (CI), 1.50-8.70; aOR, 3.58; 95% CI, 1.44-8.85) and antituberculous agents (OR, 9.23; 95% CI, 1.60-53.08; aOR, 9.05; 95% CI, 1.48-55.25). No toxicity was associated with ART. One fifth of patients experienced mild hepatotoxicity, attributed to antituberculous agents and sulfonamides. Our results suggest that ART was well tolerated.125205-

Abatacept modulates CD80 and CD86 expression and memory formation in human B-cells

Background: Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits T-cell activation and is expressed on T-regulatory cells. Human CTLA-4 deficiency results in severe immune dysregulation. Abatacept (CTLA-4 Ig) is approved for the treatment of rheumatoid arthritis (RA) and its mechanism of action is attributed to effects on T-cells. It is known that CTLA-4 modulates the expression of its ligands CD80 and CD86 on antigen presenting cells (APC) by transendocytosis. As B-cells express CD80/CD86 and function as APC, we hypothesize that B-cells are a direct target of abatacept. Objectives: To investigate direct effects of abatacept on human B-lymphocytes in vitro and in RA patients. Methods: The effect of abatacept on healthy donor B-cells’ phenotype, activation and CD80/CD86 expression was studied in vitro. Nine abatacept-treated RA patients were studied. Seven of these were followed up to 24 months, and two up to 12 months only and treatment response, immunoglobulins, ACPA, RF concentrations, B-cell phenotype and ACPA-specific switched memory B-cell frequency were assessed. Results: B-cell development was unaffected by abatacept. Abatacept treatment resulted in a dose-dependent decrease of CD80/CD86 expression on B-cells in vitro, which was due to dynamin-dependent internalization. RA patients treated with abatacept showed a progressive decrease in plasmablasts and serum IgG. While ACPA-titers only moderately declined, the frequency of ACPA-specific switched memory B-cells significantly decreased. Conclusions: Abatacept directly targets B-cells by reducing CD80/CD86 expression. Impairment of antigen presentation and T-cell activation may result in altered B-cell selection, providing a new therapeutic mechanism and a base for abatacept use in B-cell mediated autoimmunity.Fil: Lorenzetti, Raquel. Albert Ludwigs University of Freiburg; AlemaniaFil: Janowska, Iga. Albert Ludwigs University of Freiburg; AlemaniaFil: Smulski, Cristian Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Bariloche; Argentina. Comisión Nacional de Energía Atómica. Gerencia del Área de Energía Nuclear. Instituto Balseiro; ArgentinaFil: Frede, Natalie. Albert Ludwigs University of Freiburg; AlemaniaFil: Henneberger, Nadine. Albert Ludwigs University of Freiburg; AlemaniaFil: Walter, Lea. Albert Ludwigs University of Freiburg; AlemaniaFil: Schleyer, Marei-Theresa. Albert Ludwigs University of Freiburg; AlemaniaFil: Hüppe, Janika M.. Albert Ludwigs University of Freiburg; AlemaniaFil: Staniek, Julian. Albert Ludwigs University of Freiburg; AlemaniaFil: Salzer, Ulrich. Albert Ludwigs University of Freiburg; AlemaniaFil: Venhoff, Ana. Albert Ludwigs University of Freiburg; AlemaniaFil: Troilo, Arianna. Albert Ludwigs University of Freiburg; AlemaniaFil: Voll, Reinhard Edmund. Albert Ludwigs University of Freiburg; AlemaniaFil: Venhoff, Nils. Albert Ludwigs University of Freiburg; AlemaniaFil: Thiel, Jens. Albert Ludwigs University of Freiburg; AlemaniaFil: Rizzi, Marta. Albert Ludwigs University of Freiburg; Alemani

Conhecimento de professores do ensino fundamental sobre primeiros socorros na infância

The aim was to identify the knowledge of elementary school teachers regarding first aid in childhood. Non-experimental, descriptive and exploratory field research, with a quantitative approach. A questionnaire with closed questions was elaborated, and this was sent to the target population in the model called “snowball”, where, after identifying the target group of the research, they will indicate other possible interviewees, generating a connection network. It was found that the participants had considerable knowledge on the subject. Some participants report participating in annual lectures, courses, online classes and workshops. On the other hand, some participants declared that they had never participated in a first-aid activity. The research showed the need for the first-aid course in the higher education curriculum, since schools and children's recreation spaces need to have teachers prepared to guarantee students a safe environment. Therefore, through the Health at School Program, nursing can contribute to the training of these teachers through health education.Objetivou-se identificar o conhecimento de professores do ensino fundamental quanto aos primeiros socorros na infância. Pesquisa de campo de caráter não experimental, descritivo e exploratório, de abordagem quantitativa. Foi elaborado um questionário com perguntas fechadas, e este foi enviado para a população-alvo no modelo chamado “bola de neve”, onde, após identificar o grupo alvo da pesquisa, estes indicarão outros possíveis entrevistados, gerando uma rede de conexão. Verificou-se que os participantes apresentaram conhecimento considerável quanto ao assunto. Alguns participantes declaram participar de palestras anualmente, cursos, aulas online e oficinas. Por outro lado, alguns participantes declararam nunca terem participado de atividade sobre primeiros-socorros. A pesquisa mostrou a necessidade do curso de primeiros-socorros no currículo do ensino superior, visto que as escolas e espaços de recreação infantil precisam ter professores preparados para garantir aos alunos um ambiente seguro. Diante disso, através do Programa Saúde na Escola, a enfermagem pode contribuir com a capacitação desses professores através da educação em saúde

TRAIL-R1 and TRAIL-R2 Mediate TRAIL-Dependent Apoptosis in Activated Primary Human B Lymphocytes.

The maintenance of B cell homeostasis requires a tight control of B cell generation, survival, activation, and maturation. In lymphocytes upon activation, increased sensitivity to apoptotic signals helps controlling differentiation and proliferation. The death receptor Fas is important in this context because genetic Fas mutations in humans lead to an autoimmune lymphoproliferative syndrome that is similar to lymphoproliferation observed in Fas-deficient mice. In contrast, the physiological role of TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) in humans has been poorly studied so far. Indeed, most studies have focused on tumor cell lines and on mouse models whose results are difficult to transpose to primary human B cells. In the present work, the expression of apoptosis-inducing TRAIL-R1 and TRAIL-R2 and of the decoy receptors TRAIL-R3 and TRAIL-R4 was systematically studied in all developmental stages of peripheral B cells isolated from the blood and secondary lymphoid organs. Expression of TRAIL-Rs is modulated along development, with highest levels observed in germinal center B cells. In addition, T-dependent and T-independent signals elicited induction of TRAIL-Rs with distinct kinetics, which differed among B cell subpopulations: switched memory cells rapidly upregulated TRAIL-R1 and -2 upon activation while naïve B cells only reached similar expression levels at later time points in culture. Increased expression of TRAIL-R1 and -2 coincided with a caspase-3-dependent sensitivity to TRAIL-induced apoptosis in activated B cells but not in freshly isolated resting B cells. Finally, both TRAIL-R1 and TRAIL-R2 could signal actively and both contributed to TRAIL-induced apoptosis. In conclusion, this study provides a systematic analysis of the expression of TRAIL-Rs in human primary B cells and of their capacity to signal and induce apoptosis. This dataset forms a basis to further study and understand the dysregulation of TRAIL-Rs and TRAIL expression observed in autoimmune diseases. Additionally, it will be important to foresee potential bystander immunomodulation when TRAIL-R agonists are used in cancer treatment

JAK inhibitors differentially modulate B cell activation, maturation and function: A comparative analysis of five JAK inhibitors in an in-vitro B cell differentiation model and in patients with rheumatoid arthritis

BackgroundJanus kinase (JAK) inhibitors have been approved for the treatment of several immune-mediated diseases (IMIDs) including rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other IMIDs. However, detailed studies investigating the effects of different JAK inhibitors on B cells are missing. Within this study, we therefore aimed to characterize the effect of JAK inhibition on the B cell compartment.MethodsTo this end, we investigated the B cell compartment under JAK inhibition and compared the specific effects of the different JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib (JAK1/2) as well as filgotinib (selective JAK1) on in-vitro B cell activation, proliferation, and class switch recombination and involved pathways.ResultsWhile B cell phenotyping of RA patients showed an increase in marginal zone (MZ) B cells under JAK inhibition, comparison with healthy donors revealed that the relative frequency of MZ B cells was still lower compared to healthy controls. In an in-vitro model of T-cell-independent B cell activation we observed that JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. We detected an altered B cell differentiation with a significant increase in MZ-like B cells and an increase in plasmablast differentiation in the first days of culture, most pronounced with the pan-JAK inhibitor tofacitinib, although there was no increase in immunoglobulin secretion in-vitro. Notably, we further observed a profound reduction of switched memory B cell formation, especially with JAK1/2 inhibition. JAK inhibitor treatment led to a dose-dependent reduction of STAT3 expression and phosphorylation as well as STAT3 target gene expression and modulated the secretion of pro- and anti-inflammatory cytokines by B cells.ConclusionJAK inhibition has a major effect on B cell activation and differentiation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis