Discovery of broad spectrum compounds against Trypanosoma cruzi throughout high content screening

A tecnologia de high content screening (HCS) permite a triagem de uma grande quantidade de moléculas dentro de um período relativamente curto de forma eficiente e robusta. Sabendo que o Trypanosoma cruzi é promíscuo em relação a infecção de células hospedeiras de mamíferos para ensaios in vitro, praticamente qualquer célula nucleada pode ser usada para triagem fenotípica. No entanto, a dinâmica celular e as interações parasito-hospedeiro podem desempenhar um papel importante nos resultados de triagem e seleção de compostos. Além disso, T. cruzi é uma espécie heterogênea subdividida em seis grupos filogenéticos distintos que também podem apresentar variabilidade na susceptibilidade aos diferentes compostos testados in vitro e in vivo. Um ensaio de HCS para a forma intracelular de diferentes cepas de T. cruzi e em tipos de células hospedeiras distintas foi desenvolvido e validado. Em seguida uma biblioteca contendo compostos foi submetida ao processo de triagem no qual foi identificado e confirmado um composto análogo a auraquina D, com atividade inédita anti-T. cruzi. Em relação ao ensaio com diferentes cepas, a biblioteca Pathogen Box foi triada contra as cepas de T cruzi Sylvio X10/1, Y clH10 e CL Brener, apresentando um perfil distinto de atividade para cada uma. Das 400 amostras testadas, os compostos MMV689028, MMV689029 MMV688958 e MMV688795 foram os únicos selecionados contra as três cepas. Apesar de conhecidas como agentes anti-kineoplastídeos, o amplo espectro de ação dessas moléculas contra as cepas de T. cruzi nunca havia sido explorado. A fim de verificar o efeito da célula hospedeira utilizada no ensaio de alto conteúdo, e sua consequência na seleção de hits, 1.280 compostos foram testados contra amastigotas infectando células das linhagens U2OS, THP-1, VERO e L6. Observamos que existe uma sobreposição muito baixa de compostos selecionados quando células hospedeiras distintas são utilizadas em screening de compostos contra T. cruzi. Os ensaios confirmatórios destacaram os compostos CB1954, FPL64176, AEG3482 e entecavir pela potência e seletividade satisfatórias. Uma outra abordagem na qual empregamos a técnica de high content screening foi o estudo de resistência de T. cruzi aos compostos azólicos e inibidores de CYP51, proposto por muitos como um alvo validado para a quimioterapia de doença de Chagas. Um clone da cepa Y foi cultivado sob concentrações crescentes de ravuconazol, até atingir estabilidade in vitro sob 500 nM. O clone resistente exibiu uma taxa de infecção reduzida e um baixo número de amastigotas intracelulares, quando comparado ao clone parental, in vitro. O clone resistente também apresentou um processo de epimastigogênese lento e diminuiu o tempo de duplicação de população de epimastigota. Nos ensaios de atividade de fármacos, o ravuconazol foi pelo menos 1.000 vezes menos potente no clone resistente e o fenótipo foi mantido mesmo após várias semanas sem a pressão do composto e também após submeter o parasita a um ciclo de vida completo in vitro. O clone resistente ao ravuconazol também mostrou resistência cruzada a outros inibidores azólicos e não azólicos da CYP51, mas não aos nitroheterocíclicos benznidazol e nifurtimox, que agem sob outro mecanismo de ação. Uma nova mutação na TcCYP51 foi identificada no clone resistente. Espera-se que estes resultados auxiliem no entendimento da resistência natural de algumas cepas de T. cruzi aos inibidores de CYP51 e permita um maior entendimento do papel das isoformas de CYP51 na biologia do parasita. Os resultados obtidos nesse estudo apontam para novas perspectivas na descoberta e desenvolvimento de fármacos para doença de Chagas.The high content screening (HCS) technology allows the evaluation of a large number of molecules within a relatively short period of time in an efficient and robust manner. It is well known that Trypanosoma cruzi is promiscuous in terms of mammalian cells infection for in vitro assays, therefore, basically any nucleated cell can be used for phenotypic screening against this parasite. However, cellular dynamics and the specific host-parasite interactions may play important role in the screening results and in hit compound selection. Besides, T. cruzi is a heterogeneous species subdivided into six distinct ephylogenetic groups which may present variable susceptibility to different compounds tested both in vitro and in vivo. A HCS assay was developed and validated using the intracellular stage of different T. cruzi strains and several host cell types. Next, a 1,604-sample library was screened and an aurachin D analogue compound was identified and confirmed with a previously undescribed anti-T. cruzi activity. Regarding the different strains assay, the Pathogen Box library was screened against T. cruzi strains Sylvio X10/1, Y clH10 e CL Brener, presenting a distinct activity profile for each infected cell lineage. From the 400 samples tested, compounds MMV689028, MMV689029 MMV688958 e MMV688795 were the only hits selected against all three strains. Although these molecules are known anti-kinetoplastid agents, the broad-spectrum profile against different T. cruzi strains had not been explored yet. In order to evaluate the host cell type in T. cruzi high content assays, and the implications with compound selection, 1,280 compounds were tested against T. cruzi amastigotes infecting four host cell lineages: U2OS, THP-1, VERO e L6. We observed a low overlap of selected compounds within distinct host cells used in anti-T. cruzi compound screening. After confirmatory assays, compounds CB1954, FPL64176, AEG3482 and entecavir stood out in terms of potency, efficacy and selectivity. Another study in which we applied the high content assay methodology was on T. cruzi drug-induced resistance against azoles and other CYP51 inhibitors, considered by many as a well-validated antichagasic chemotherapeutical target. A Y strain clone was cultivated under increasing ravuconazole concentrations until reaching in vitro stability at 500 nM. The resistant clone exhibited a reduced infection ratio profile and low number of intracellular amastigotes, when compared to susceptible parental clone, in vitro. The resistant parasites also presented a slower epimastigogenesis process and decreased the epimastigote doubling time. In drug activity assays, ravuconazole was at least 1,000 times less potent against the resistant clone and this phenotype was maintained after several weeks, even without drug pressure and also after submitting the parasite into a complete in vitro life cycle. Ravuconazole resistant clones also presented cross-resistance to other azole and non-azoles CYP51 inhibitors, but not to nitroheterocyclic compounds benznidazole and nifurtimox, which act via different mechanism of action. A novel TcCYP51 mutation was identified in the resistant parasites. We hope these results will aid the understanding of natural-resistance occurrence in some T. cruzi strains to CYP51 inhibitors, allowing also a better elucidation of the role of TcCYP51 different isoforms in the parasite biology. The results obtained from this study point towards new perspectives in Chagas disease drug discoveryConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Estudo sobre o aumento da eficiência de combustão de um motor foguete híbrido utilizando parafina como combustível

Trabalho de Conclusão de Curso (graduação)—Universidade de Brasília, Faculdade UnB Gama, 2019.Este trabalho tem como objetivo apresentar uma solução que aumente a eficiência de sistemas híbridos que utilizam parafina como combustível. Com o uso da tecnologia de impressão 3-D foi possível produzir diferentes designs de grãos utilizando o material Polietileno tereftalato de etileno glicol (PETG). A geometria desses grãos conta com células ocas em seu interior onde a parafina é alocada, produzindo assim um grão híbrido. Testes estáticos foram realizados em uma bancada apropriada a fim de coletar dados como pressão, temperatura e vazão mássica, que são necessários para se calcular a eficiência da solução. Modificações na configuração dos grãos e no sistema de injeção de oxidante produziram resultados satisfatórios, com até 89% de eficiência para o par propelente parafina - óxido nitroso. A quantidade de testes realizadas nesta pesquisa ainda não é suficiente para validar a solução, mas já há uma indicativa de possíveis estratégias para aumentar a eficiência de queima da parafina como combustível híbrido.This work aims to present a solution that increases the efficiency of hybrid systems that use paraffin as fuel. Applying 3-D printing technology, it was possible to produce different grain designs using paraffin in structures of polyethylene terephthalate glycol (PETG), in order to increase the combustion efficiency of propellants. The geometry of these grains have hollow cells where the paraffin is allocated, thus producing a hybrid grain. Firing tests were performed on an appropriate bench in order to collect data such as pressure, temperature and mass flow rate, which are required to calculate the efficiency of the solution. Modifications in grain configuration and injection schem produced 89% of efficiency for the paraffin-nitrous oxide pair. The number of tests performed for this paper is not enough to validate the solution, but there is already an indicative of some ways to increase the efficiency of hybrid motors using paraffin as fuel

A New Derivation of the CPT and Spin-Statistics Theorems in Non-Commutative Field Theories

We propose an alternative axiomatic description for non-commutative field theories (NCFT) based on some ideas by Soloviev to nonlocal quantum fields. The local commutativity axiom is replaced by the weaker condition that the fields commute at sufficiently large spatial separations, called asymptotic commutativity, formulated in terms of the theory of analytic functionals. The question of a possible violation of the CPT and Spin-Statistics theorems caused by nonlocality of the commutation relations $[\hat{x}_\mu,\hat{x}_\nu]=i\theta_{\mu\nu}$ is investigated. In spite of this inherent nonlocality, we show that the modification aforementioned is sufficient to ensure the validity of these theorems for NCFT. We restrict ourselves to the simplest model of a scalar field in the case of only space-space non-commutativity.Comment: The title is new, and the analysis in the manuscript has been made more precise. This revised version is to be published in J.Math.Phy

Combustão de mistura óleo-carvão: viabilidade técnica e econômica para carvões brasileiros

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-graduação em Engenharia Mecânica

CONTRIBUIÇÕES DA CROMATOGRAFIA CIRCULAR DE PFEIFFER (CCP) PARA CARACTERIZAÇÃO DE SAÚDE DO SOLO EM DIFERENTES FORMAS DE USO E OCUPAÇÃO

To characterize and compare the behavior of different soil substrates, Pfeiffer's Circular Chromatography technique (CPP) was employed. For this purpose, six different points were demarcated and georeferenced for sampling, respecting the limits of the Sorocaba campus (SP) of the Federal University of São Carlos - UFSCar, in order to obtain heterogeneous results in view of the different forms of use and occupation and different soil classifications. Following the sampling work, the samples were appropriately separated according to soil classification, which varies depending on the dynamics of flow and overlap along the western slope of the Córrego do Ipaneminha (Ipaneminha Stream) valley, where the campus is located. Subsequently, after sieving the substrates, chromatograms were generated, following the proposal by EMBRAPA. It was observed that these chromatograms differ in color, peak shape, plume shape, and integration between the zones along the flow, indicating differences in the composition of the collected samples, considering the environmental variables.Para caracterizar y comparar el comportamiento de diferentes sustratos de suelo, se empleó la técnica de Cromatografía Circular de Pfeiffer (CCP). Para ello, se demarcaron y georreferenciaron seis puntos distintos para la recolección, respetando los límites del campus de Sorocaba (SP) de la Universidad Federal de São Carlos – UFSCar, con el fin de obtener resultados heterogéneos considerando las diferentes formas de uso y ocupación y diferentes clasificaciones de suelos. Después del trabajo de recolección, se separaron las muestras adecuadamente según la clasificación del suelo, la cual varía en función de la dinámica de flujo y superposición a lo largo del escarpe occidental del valle del Córrego do Ipaneminha (Arroyo del Ipaneminha), donde se encuentra el campus. A continuación, luego de tamizar los sustratos, se generaron cromatogramas, siguiendo la propuesta de EMBRAPA. Se observó que estos cromatogramas difieren en color, forma de los picos, forma de las plumas e integración entre las zonas a lo largo del flujo, lo que indica diferencias en la composición de las muestras recolectadas, considerando las variables del entorno.Para caracterizar e comparar o comportamento de diferentes substratos de solo, foi utilizada a técnica da Cromatografia Circular de Pfeiffer. Para tanto, foram demarcados e georreferenciados seis pontos distintos para coleta, respeitando os limites do campus de Sorocaba (SP) da Universidade Federal de São Carlos – UFSCar, a fim de obter resultados heterogêneos tendo em vista as diferentes formas de uso e ocupação e diferentes classificações do solo. Posteriormente ao trabalho de coleta, as amostras foram devidamente separadas conforme a classificação do solo, esta, variando de acordo com a dinâmica de escoamento e sobreposição ao longo da escarpa oeste do vale do Córrego do Ipaneminha, onde o campus se localiza. Na sequência, após o peneiramento dos substratos, foram gerados os cromatogramas, seguindo o proposto pela EMBRAPA. Verificou-se que estes cromatogramas se diferenciam em cor, formato dos picos, formato das plumas e integração entre as zonas ao longo do corrimento, o que denota as dessemelhanças na composição das amostras coletadas, considerando as variáveis do ambiente

Desenvolvimento de uma aplicação web para organização e distribuição de comunicados para a universidade

TCC(graduação) - Universidade Federal de Santa Catarina. Centro Tecnológico. Sistemas de Informação.O presente trabalho tem o intuito de apresentar uma ferramenta de comunicados voltada para toda a comunidade acadêmica da UFSC. Tal ferramenta, que se pretende um sistema agregador de conteúdo voltado à realização de anúncios institucionais, poderá ser usado tanto como um canal de veiculação de mensagens e comunicados entre coordenadores de curso e professores, professores e alunos e entre os estudantes entre si, quanto como um espaço de divulgação de ofertas de emprego, estágio etc, e de compartilhamento de informações gerais relativas à vida cotidiana, como moradia, eventos etc. Ela ambiciona ser um canal unificado, centralizado, pois seu uso pode ser integrado via API a outros sistemas da UFSC.The present work aims to present a communication tool focused on the entire academic community of UFSC. This tool, which is intended to be a content aggregator system mainly aimed at carrying out institutional communiqués, can be used both as a channel for broadcasting messages and announcements between course coordinators and professors, professors and students and among students themselves, as well as a space for publicizing job offers, internships, etc., and sharing general information related to daily life, such as housing, events, etc. Its intention is to be a unified, centralized channel, as its use can be integrated via API with other UFSC systems

Ontology-based question answering systems over knowledge bases: a survey

Searching relevant, specific information in big data volumes is quite a challenging task. Despite the numerous strategies in the literature to tackle this problem, this task is usually carried out by resorting to a Question Answering (QA) systems. There are many ways to build a QA system, such as heuristic approaches, machine learning, and ontologies. Recent research focused their efforts on ontology-based methods since the resulting QA systems can benefit from knowledge modeling. In this paper, we present a systematic literature survey on ontology-based QA systems regarding any questions. We also detail the evaluation process carried out in these systems and discuss how each approach differs from the others in terms of the challenges faced and strategies employed. Finally, we present the most prominent research issues still open in the field

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. in this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors -posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compoundand strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.DNDiInstitut Pasteur Korea (IPK)Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF)/GermanyMedecins Sans Frontieres (Doctors without Borders)/InternationalKorean government (MSIP), Gyeonggi-doKISTIInst Pasteur Korea, Ctr Neglected Dis Drug Discovery CND3, Songnam, South KoreaCtr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias LNBio, Campinas, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, BrazilDrugs Neglected Dis Initiat DNDi, Geneva, SwitzerlandUniversidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, BrazilKorean government (MSIP), Gyeonggi-do: 2007-00559Web of Scienc

Fibrinogen as independent risk factor for ischemic stroke

We have studied fibrinogen levels (Clauss technique) in atherothrombotic ischemic stroke patients, in order to determine its role as a thombogenic risk factor. Twenty nine patients (20 men and 9 women) between 25 and 79 years old were studied; they all have had a atherothrombotic stroke. They were classified into two groups according to the result of their carotid doppler ultrasonography: g1 - without carotid flow reduction (n=l 9) and g2 - with carotid flow reduction (n=10). The fibrinogen mean value was 269mg/dl in gl and 353 mg/dl in g2. There were 47% of patients in gl and 80% of patients in g2 who presented levels >300 mg/dl. The proportions of the groups were significantly different (p300 mg/dl. As diferenças obtidas entre os grupos neste estudo foram significantes. Conclusão- Considerando o nível de risco epidemiológico de 300 mg/dl, nossos resultados sugerem que o fibrinogênio é um fator de risco independente para AVC aterotrombótico, especialmente naqueles com alteração de fluxo carotídeo.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUNIFESP, EPM, Depto. de MedicinaSciEL

Supersymmetric Field-Theoretic Models on a Supermanifold

We propose the extension of some structural aspects that have successfully been applied in the development of the theory of quantum fields propagating on a general spacetime manifold so as to include superfield models on a supermanifold. We only deal with the limited class of supermanifolds which admit the existence of a smooth body manifold structure. Our considerations are based on the Catenacci-Reina-Teofillatto-Bryant approach to supermanifolds. In particular, we show that the class of supermanifolds constructed by Bonora-Pasti-Tonin satisfies the criterions which guarantee that a supermanifold admits a Hausdorff body manifold. This construction is the closest to the physicist's intuitive view of superspace as a manifold with some anticommuting coordinates, where the odd sector is topologically trivial. The paper also contains a new construction of superdistributions and useful results on the wavefront set of such objects. Moreover, a generalization of the spectral condition is formulated using the notion of the wavefront set of superdistributions, which is equivalent to the requirement that all of the component fields satisfy, on the body manifold, a microlocal spectral condition proposed by Brunetti-Fredenhagen-K\"ohler.Comment: Final version to appear in J.Math.Phy