Caracterização do papel do sistema de secreção do tipo VI bacteriano sobre a resposta imunológica inata de células de mamíferos infectados por Escherichia coli

Dissertação (mestrado)—Universidade de Brasília, Instituto de Biologia, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2016.Escherichia coli é uma bactéria gram-negativa, que apresenta uma alta diversidade genética e fenotípica. Muitas linhagens de E. coli são inofensivas, mas algumas apresentam fatores de patogenicidade. Muitos produtos, estruturas e propriedades bacterianas podem ser utilizadas para evadir as defesas do hospedeiro através de sistemas de secreção. O sistema de secreção de tipo VI (SST6) é considerado como um potencial fator de virulência que tem a capacidade de translocar substratos efetores para o interior de vários tipos de células, tanto procarióticas quanto eucarióticas. O SST6 tem sido descrito como uma importante maquinaria celular envolvido principalmente na competição entre células procarióticas. No entanto, o papel do SST6 na imunidade da célula hospedeira eucariótica é pouco compreendida. Portanto, o objetivo deste estudo foi investigar o papel do SST6 bacteriano na apresentação de antígenos e sobre a indução da ativação da resposta inflamatória em células de mamíferos infectados por E. coli. Nós avaliamos a modulação de alguns mecanismos da imunidade inata durante a infecção causada pela bactéria E coli SEPT362 e a linhagem mutante para o gene IcmF. Aqui demonstramos que SST6 beneficia a E. coli, promovendo sua sobrevivência no interior das células do hospedeiro participando da adesão, internalização e viabilidade da bactéria no interior de macrófagos. Nossos resultados mostraram que E. coli selvagem inibe a expressão das moléculas CD1 dos grupos I e II e consequentemente, a apresentação de antígenos lipídicos. Diferentemente, a linhagem mutante para IcmF desencadeou uma regulação positiva dessas moléculas, mostrando a participação do SST6 nesse processo. Além disso, SST6 está associada à inibição da expressão de moléculas de MHC II, indicando provável bloqueio na apresentação de antígenos peptídicos e uma possível forma de escape imunológico pelo patógeno. Ademais, os nossos resultados mostraram que, SST6 participa na modulação negativa da expressão de moléculas co-estimulatórias CD40 e CD80 em macrófagos. Também verificamos que, E. coli SEPT362 induz a expressão aumentada do receptor nuclear PPARɣ dependente de SST6 em macrófagos e não induz a translocação do fator de transcrição NF- kB durante a infecção. A infecção por E. coli também induziu o aumento da biogênese de corpúsculos lipídicos de forma dependente de SST6. Nós identificamos que SST6 participa na redução da viabilidade das células de macrófagos, recrutamento de macrófagos para a cavidade peritoneal e favorece o estabelecimento do patógeno no fígado. Finalmente, observamos que SST6 participa na indução da secreção de óxido nítrico e a da citocina IL-6 em macrófagos, no entanto, a regulação positiva da secreção de IL-10 é independente do SST6. Todos estes resultados mostram um novo papel para o papel do SST6 na imunidade inata dos hospedeiros e no progresso da patogênese.Escherichia coli is Gram-negative bacteria that exhibit a high genetic and phenotypic diversity. Many products, structures and bacterial properties can be used to overcome host defenses, mainly through a mechanism mediated by secretion systems. The type VI secretion system (T6SS) is considered a potential virulence factor capable of translocating substrate effectors into largest cell types, both prokaryotic and eukaryotic cells. T6SS has been described as important cell machinery involved mainly in competition among prokaryotic cells. However, the role of T6SS in eukaryotic host cell immunity is poorly understood. Therefore, the aim of this study was investigate the role of bacterial T6SS on the antigen presentation and modulation of inflammatory response in mammalian cells infected by E. coli. We evaluated the modulation of different mechanisms involved in innate immune during the infection caused by E. coli wild type strain (SEPT362) and the knockout strains IcmF-deficient (delta IcmF). Here we demonstrate that, T6SS is beneficial to survival advantage of E. coli into host cells, since it participates in adherence, internalization and viability of E. coli bacteria within macrophages. Our results showed that E. coli wild type inhibits the expression of CD1 group I and group II molecules, and consequently the lipids antigens presentation. In contrast, the knockout strain delta IcmF triggered an upregulation of these molecules, showing the participation of T6SS in this process. Moreover, T6SS is associated with inhibition of expression of MHC II molecules, indicating participation in peptide antigen presentation and a possible form of immune escape by the pathogen. Furthermore, our results showed that T6SS participates in the modulation of expression of co-stimulatory molecules CD40 and CD80 on macrophages. In addition, we also found that E. coli SEPT362 induces an increased expression of the nuclear receptor PPARɣ in macrophage dependent of T6SS and did not induce the translocation of the transcription factor NF-kB during the infection. The infection by E. coli SEPT362 also induced a T6SS dependent increase of lipid droplets biogenesis. Moreover, we identified that T6SS participates in the reduction of macrophage cell viability, macrophages recruitment to mice peritoneal cavity and establishment of the pathogen in the liver. Finally we observed that T6SS participates in the induction of secretion of nitric oxide and IL-6 cytokine in macrophage, however the upregulation of IL-10 secretion is T6SS independent. Taken together, our results present a new T6SS role in innate immune host response and progress of E. coli pathogenesis

Caracterização do impacto da infecção pelo vírus ZIKA no metabolismo energético do testículo e fertilidade masculina

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2020.O Zika vírus (ZIKV) é um vírus transmitido por artrópodes (arbovírus) que também foi identificado como sendo transmitido por outras vias, incluindo transmissão sexual, o que implica em um grande impacto na saúde publica. Já está bastante consolidado em modelo animal que o ZIKV consegue infectar, se reproduzir e causar danos severos na homeostase testicular. Entretanto poucas vias foram descritas e correlacionadas a esse processo de patogênese. Portanto, nesse trabalho buscamos caracterizar algumas vias que possam demonstrar os efeitos da infecção por ZIKV testículo, um órgão de privilégio imunológico. Para avaliar a relação da infecção viral e a modulação molecular do sistema reprodutor masculino, analisamos os testículos de camundongos C57BL/6 infectados ou não com o isolado brasileiro ZIKV (ZIKV BR) por espectrometria de massas com ionização por eletrospray (ESI-MS). Observamos um perfil metabólico diferencial entre os testículos infectados e não infectados pelo ZIKV. Vários metabólitos, especialmente da via glicolítica, foram modulados pelo ZIKV nos testículos, incluindo o aumento da abundância relativa de glicose. Além disso, verificamos por western blot que a infecção por ZIKV causa modulação na expressão de proteínas da via glicolítica. A expressão das proteínas fosfoglicerato quinase (PGK) do tipo 2 e hexoquinase I foi reduzida pela infecção do ZIKV nos testículos. A presença de ZIKV no testículo de camundongos após 14 dias de infecção pelo ZIKV foi confirmada por RT-PCR, mas não foi encontrado material genético viral no plasma ou no cérebro destes animais, sugerindo um tropismo desta cepa pelo testículo. Para investigar as consequências da infecção por ZIKV em testículos humanos, sêmen de homens positivos ou negativos para ZIKV foram coletados e avaliados por proteômica LC-MS/MS Label- free. O sêmen de homens previamente positivos para ZIKV mostrou modulação nas proteínas do metabolismo energético e da espermatogênese. Tomados em conjunto, nossos resultados mostraram que a infecção por ZIKV pode afetar a fisiologia dos testículos pela modulação da via glicolítica. Todos esses dados sugerem a influência da infecção pelo ZIKV no metabolismo celular que poderia apoiar o estabelecimento e a replicação do ZIKV nos testículos. Além disso, esse estudo possibilita a condução de uma investigação aprofundada sobre como o ZIKV pode modular os mecanismos celulares que promovem danos nos testículos para fornecer mais indicações sobre alvos terapêuticos.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Zika virus (ZIKV) was first reported transmitted by arthropods, however it has been identified transmitted by other routes, including sexual transmission which became a major impact on public health. It is already well established in the animal model that ZIKV can infect and cause severe damage to testicular homeostasis. However, only few pathways have been described and correlated with this pathogenesis process. Therefore, in this work we aimed to characterize some pathways that can demonstrate the effects of ZIKV infection in the testicle, an immune privileged organ. To assess the relationship of viral infection and molecular modulation of the male reproductive system, we analyzed the testicle of C57BL/6 mice infected or not with Brazilian ZIKV (ZIKV BR) by Electrospray Ionization Mass Spectrometry (ESI-MS). We observed a differential metabolic profile between testicles infected and not infected by ZIKV. Several metabolites, especially from the glycolytic pathway, were modulated by ZIKV in testis, including an increase in the relative percentage of glucose. In addition, we verified by Western Blot that ZIKV infection causes modulation the expression of proteins in the glycolytic pathway. The expression of protein phosphoglycerate kinase (PGK) type 2 and hexokinase I was reduced by ZIKV infection in the testicles. The presence of ZIKV in the testis of mice after 14 days of ZIKV infection was confirmed by RT-PCR, but no viral genetic material was found in the plasma or brain, suggesting a tropism of this strain for testicle. To investigate the consequences of ZIKV infection in human testicles, semen from ZIKV-positive or negative men were collected and evaluated by LC-MS / MS Label- free proteomics. The semen of men previously positive for ZIKV showed modulation in the proteins of energy metabolism and spermatogenesis. Taken together, our results showed that ZIKV infection can affect the physiology of the testes by modulating the glycolytic pathway. All of these data suggest the influence of ZIKV infection on cell metabolism that could support the establishment and replication of ZIKV in the testicles. In addition, this study makes it possible to conduct an in-depth investigation of how ZIKV can modulate the cellular mechanisms that promote damage to the testicles to provide further indications on therapeutic targets

Static and Fatigue Behaviour of the Main Section of a Fast Patrol Boat

This paper analyses the static resistance, as well as the fatigue resistance, of the main section of a fast patrol boat designed and manufactured in Portugal. The ship under study is a high-speed lightweight craft that was mainly fabricated with two types of aluminium series alloys, namely the 5083-H111 and the 6082-T6 alloys, which are commonly used in shipbuilding. The structural response of the critical section of the ship was obtained using the Finite Element Method (FEM), when the structure was submitted to different loading conditions, such as the hydrodynamic and hydrostatic sea loads, the longitudinal sagging or hogging, or the loads on fuel oil tanks. Results obtained for the load cases considered showed that the Von Mises equivalent stresses do not exceed the Yield Strength of the aluminium alloys used in the manufacturing of the ship. In addition, strain gages were placed on the main ship's bulkhead, near a structural detail in its bottom, and real-time acquisition strain data was collected using a computer code routine written in LabView. The Rainflow cycle counting method was applied to load spectrum gathered in order to obtain the Rainflow matrix and to predict the fatigue life of the critical main section of the ship.publishersversionpublishe

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

Absence of the caspases 1/11 modulates liver global lipid profile and gut microbiota in high-fat-diet-induced obese mice

Obesity is a chronic disease with rising worldwide prevalence and largely associated with several other comorbidities, such as cancer, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Hepatic steatosis, a hallmark of NAFLD, is strongly correlated with obesity and has been correlated with changes in the gut microbiota, which can promote its development through the production of short-chain fatty acids (SCFAs) that regulate insulin resistance, bile acid, choline metabolism, and inflammation. Recent studies have suggested a controversial role for the inflammasome/caspase-1 in the development of obesity and non-alcoholic steatohepatitis (NASH). Here, we evaluated the role of inflammasome NLRP3 and caspases 1/11 in the establishment of obesity and hepatic steatosis in diet-induced obese mice, correlating them with the global lipid profile of the liver and gut microbiota diversity. After feeding wild-type, caspases 1/11, and NLRP3 knockout mice with a standard fat diet (SFD) or a high-fat diet (HFD), we found that the caspases 1/11 knockout mice, but not NLRP3 knockout mice, were more susceptible to HFD-induced obesity, and developed enhanced hepatic steatosis even under SFD conditions. Lipidomics analysis of the liver, assessed by MALDI-MS analysis, revealed that the HFD triggered a significant change in global lipid profile in the liver of WT mice compared to those fed an SFD, and this profile was modified by the lack of caspases 1/11 and NLRP3. The absence of caspases 1/11 was also correlated with an increased presence of triacylglycerol in the liver. Gut microbial diversity analysis, using 16S rRNA gene sequencing, showed that there was also an increase of Proteobacteria and a higher Firmicutes/Bacteroidetes ratio in the gut of caspases 1/11 knockout mice fed an HFD. Overall, mice without caspases 1/11 harbored gut bacterial phyla involved with weight gain, obesity, and hepatic steatosis. Taken together, our data suggest an important role for caspases 1/11 in the lipid composition of the liver and in the modulation of the gut microbial community composition. Our results further suggest that HFD-induced obesity and the absence of caspases 1/11 may regulate both lipid metabolism and gut microbial diversity, and therefore may be associated with NAFLD and obesity10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP312359/2016-02016/22577-6This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq -#312359/2016-0). CM was funded by the Canada Research Chair Program, the Canadian Foundation for Innovation, McGill University, and the Canadian Institutes for Health Research (PJT-149098). ME was funded by the São Paulo Research Foundation (FAPESP) (2016/22577-6)

Potential neuroprotective and antiinfammatory efects provided by omega-3 (DHA) against Zika virus infection in human SH-SY5Y cells

Zika virus (ZIKV) has a strong tropism for the nervous system and has been related to post-infection neurological syndromes. Once neuronal cells are infected, the virus is capable of modulating cell metabolism, leading to neurotoxicity and cellular death. The negative efect of ZIKV in neuron cells has been characterized. However, the description of molecules capable of reversing these cytotoxic efects is still under investigation. In this context, it has been largely demonstrated that docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is highly neuroprotective. Here, we hypothesized that DHA’s neuroprotective proprieties could have an infuence on ZIKV-induced neurotoxicity in SHSY5Y cells. Our data showed that pre-treatment of SH-SY5Y cells with DHA increased the cell viability and proliferation in ZIKV-infected cells. Moreover, DHA triggered an anti-infammatory response in those infected cells. Besides, DHA was capable of restoring mitochondria function and number in ZIKVinfected SH-SY5Y cells. In addition, cells pre-treated with DHA prior to ZIKV infection presented a lower viral load at diferent times of infection. Taking together, these results demonstrated that DHA has a potential anti-infammatory and neuroprotective efect against ZIKV infection in these neuron-like cells and could be a useful tool in the treatment against this virus

Dinâmicas de (investig)ação em contextos de promoção da língua portuguesa: a presença do CIDTFF na rede Camões, I.P.

Sem resumo disponível.publishe