Metabólitos secundários produzidos por Streptomyces com atividade antibiótica apresentam relevante importância biotecnológica para as indústrias farmacêuticas e agroquímicas. Dentre estes metabólitos, podemos destacar a nigericina, um antibiótico poliéter usado como aditivo em ração animal atuando como promotor de crescimento e no tratamento de algumas doenças, como a malária, em carcinoma nasofaríngeo, a vaccínia, entre outras. Neste trabalho foram avaliadas duas cepas de actinobactérias potenciais produtoras de nigericina, a EUCAL 26 e a EUCAL 74. As duas actinobatérias foram fermentadas em cinco meios de cultivo diferentes (BD, Czapek, ISP2, M29 e TSB). A cepa EUCAL 26 foi a mais promissora na produção de nigericina em meio Czapeck. A partir da EUCAL 26, foi feito um estudo da máxima produção de nigericina em meio Czapek variando o pH do meio, temperatura de fermentação, e período de fermentação. As melhores condições encontradas foram em pH 7,0 a 25 °C por 27 dias. Foi realizado também um estudo de otimização de aumento de escala de fermentação, de um volume de meio Czapeck de 50 mL, para um volume de 4 L. Também foram avaliados dois resíduos agroindustriais (Farmal e Melaço de Soja) para a produção de nigericina. O meio de Melaço de Soja aumento a produção em aproximadamente 300x quando comparado com o meio Czapeck padrão. Os efeitos dos nutrientes do meio Czapeck também foram avaliados. A retida do K2HPO4 do meio produziu um aumento de 50x na produção de nigericina, quando comparado com o meio Czapeck controle. Também foi avaliado o efeito da adição de -butirolactonas sintéticas, moléculas de sinalização hormonal, para a produção de nigericina. Das 15 -butirolactonas testadas, a DP21A foi a mais eficiente, pois além de aumentar a produção de nigericina em 23x, também diminui o período máximo de sua produção. Todas as analises realizadas neste trabalho para o monitoramento da produção de nigericina, foram feitas empregando a espectrometria de massas sequencial acoplada à cromatografia liquida de ultra eficiência. / Secondary metabolites produced by Streptomyces with antibiotic activity have significant biotechnological importance for the pharmaceutical and agrochemical industries. Among them, nigericin stands out as an antibiotic polyether used as growth promoter in animal feed and for treatment of some diseases such as malaria, nasopharyngeal carcinoma, and vaccinia. In this study, two actinobacteria strains considered potential producers of nigericin named EUCAL 26 and 74 were tested. The two actinobacteria were fermented in five different culture media (BD, Czapek, ISP2, M29 and TSB). EUCAL 26 strain was the most promising in producing nigericin in amid Czapeck media. For EUCAL 26, a study of maximum production of nigericin in Czapek medium at varying the pH, fermentation temperature and fermentation period have been performed. As a result, the best conditions were pH 7.0, at 25 °C for 27 days. In addition, an optimization study for scale-up fermentation have been done, where a volume of 50 mL Czapeck medium have been expanded to 4 L, in order to obtain the highest production of nigericin. Two agroindustrial residues (FARMAL and Honey Soy) have also been evaluated for nigericin production. The honey soy medium increased nigericin production in the rate of 300 when compared with standard Czapeck medium. The effects of nutrients from Czapeck medium have also been evaluated. Removal of K2HPO4 from culture medium resulted in an increase of 50 times when compared with the control Czapeck medium. The effect of adding synthetics -butyrolactones (hormone signaling molecules) for the production of nigericin have also been evaluated. From 15 tested -butyrolactones, DP21A was the most efficient. In addition to increase nigericin yield in 23x it also reduced the period for its maximum production. All analyzes performed in this study to monitor the nigericin production were performed using tandem mass spectrometry coupled to ultra high performance liquid chromatography.

Metabólitos secundários produzidos por Streptomyces com atividade antibiótica apresentam relevante importância biotecnológica para as indústrias farmacêuticas e agroquímicas. Dentre estes metabólitos, podemos destacar a nigericina, um antibiótico poliéter usado como aditivo em ração animal atuando como promotor de crescimento e no tratamento de algumas doenças, como a malária, em carcinoma nasofaríngeo, a vaccínia, entre outras. Neste trabalho foram avaliadas duas cepas de actinobactérias potenciais produtoras de nigericina, a EUCAL 26 e a EUCAL 74. As duas actinobatérias foram fermentadas em cinco meios de cultivo diferentes (BD, Czapek, ISP2, M29 e TSB). A cepa EUCAL 26 foi a mais promissora na produção de nigericina em meio Czapeck. A partir da EUCAL 26, foi feito um estudo da máxima produção de nigericina em meio Czapek variando o pH do meio, temperatura de fermentação, e período de fermentação. As melhores condições encontradas foram em pH 7,0 a 25 °C por 27 dias. Foi realizado também um estudo de otimização de aumento de escala de fermentação, de um volume de meio Czapeck de 50 mL, para um volume de 4 L. Também foram avaliados dois resíduos agroindustriais (Farmal e Melaço de Soja) para a produção de nigericina. O meio de Melaço de Soja aumento a produção em aproximadamente 300x quando comparado com o meio Czapeck padrão. Os efeitos dos nutrientes do meio Czapeck também foram avaliados. A retida do K2HPO4 do meio produziu um aumento de 50x na produção de nigericina, quando comparado com o meio Czapeck controle. Também foi avaliado o efeito da adição de -butirolactonas sintéticas, moléculas de sinalização hormonal, para a produção de nigericina. Das 15 -butirolactonas testadas, a DP21A foi a mais eficiente, pois além de aumentar a produção de nigericina em 23x, também diminui o período máximo de sua produção. Todas as analises realizadas neste trabalho para o monitoramento da produção de nigericina, foram feitas empregando a espectrometria de massas sequencial acoplada à cromatografia liquida de ultra eficiência. / Secondary metabolites produced by Streptomyces with antibiotic activity have significant biotechnological importance for the pharmaceutical and agrochemical industries. Among them, nigericin stands out as an antibiotic polyether used as growth promoter in animal feed and for treatment of some diseases such as malaria, nasopharyngeal carcinoma, and vaccinia. In this study, two actinobacteria strains considered potential producers of nigericin named EUCAL 26 and 74 were tested. The two actinobacteria were fermented in five different culture media (BD, Czapek, ISP2, M29 and TSB). EUCAL 26 strain was the most promising in producing nigericin in amid Czapeck media. For EUCAL 26, a study of maximum production of nigericin in Czapek medium at varying the pH, fermentation temperature and fermentation period have been performed. As a result, the best conditions were pH 7.0, at 25 °C for 27 days. In addition, an optimization study for scale-up fermentation have been done, where a volume of 50 mL Czapeck medium have been expanded to 4 L, in order to obtain the highest production of nigericin. Two agroindustrial residues (FARMAL and Honey Soy) have also been evaluated for nigericin production. The honey soy medium increased nigericin production in the rate of 300 when compared with standard Czapeck medium. The effects of nutrients from Czapeck medium have also been evaluated. Removal of K2HPO4 from culture medium resulted in an increase of 50 times when compared with the control Czapeck medium. The effect of adding synthetics -butyrolactones (hormone signaling molecules) for the production of nigericin have also been evaluated. From 15 tested -butyrolactones, DP21A was the most efficient. In addition to increase nigericin yield in 23x it also reduced the period for its maximum production. All analyzes performed in this study to monitor the nigericin production were performed using tandem mass spectrometry coupled to ultra high performance liquid chromatography.

The innate immune system is the first line of defence against infection. It is comprised of physicochemical barriers and a variety of cell types including macrophages and dendritic cells. Pathogens express specific pathogen associated molecular patterns (PAMP) which are recognised by pattern recognition receptors (PRR) on macrophages to initiate an innate immune response. Gram-negative bacteria such as Salmonella enterica serovar Typhimurium express a range of bacterial PAMPs recognised by Toll-like receptors (TLRs) including lipopolysaccharides (LPS) recognised by TLR-4 and lipoproteins by TLR-2. The activation of TLRs results in activation of nuclear factor κB (NF-κB) to drive transcription of mRNA coding for pro-inflammatory proteins such as tumor necrosis factor α (TNF-α) and pro-interleukin (IL) 1β. Myeloid cells also possess intracellular PRRs including the nucleotide-binding domain and leucine-rich repeat (NLR) family. NLR family CARD domain- containing protein 4 (NLRC4) and NLR family pyrin domain-containing protein 3 (NLRP3) are the main NLRs engaged in recognising S. Typhimurium infection, leading to formation of the inflammasome. The inflammasome is a macromolecular complex assembled in the cytoplasm, and usually contains a NLR, the structural protein apoptosis-associated speck-like protein containing a CARD (ASC) and effector enzymes such as cysteine-dependent aspartate-directed protease (caspase) -1 and caspase-8. This structure is responsible for processing the cytokines pro- IL-1β and pro-IL-18 to their mature form and is involved in triggering a pro-inflammatory process of cell death termed pyroptosis. The formation of the inflammasome therefore results in cell death and secretion of proinflammatory cytokines which play important roles in controlling infections. Inflammasome activity must be tightly coordinated, as its dysregulation is associated with a variety of auto-inflammatory and auto-immune diseases. The signalling events leading to inflammasome assembly are poorly understood and the molecules involved in fine-tuning its activity are only beginning to be discovered. The aim of this thesis was to discover new molecules involved in inflammasome activation and/or in keeping its activity in check. To achieve this goal, I performed S. Typhimurium infection assays in primary bone marrow derived macrophages (BMDM) derived from C57BL/6 mice wild type (WT) and compared the resulting cellular viability, intracellular bacteria counts and IL-1β production to that of BMDMs derived from C57BL/6 mice lacking proteins involved with, or suspected to be involved with, innate immune activity. Amongst the proteins I studied, caspase recruitment domain 9 (CARD9) inhibited inflammasome-mediated IL-1β production. Multiple independent genome-wide association studies link this protein to inflammatory pathologies such as Crohn's disease, but its role in canonical inflammasomes was largely unexplored. To investigate how CARD9 inhibits inflammasome-mediated IL-1β production I have conducted assays in WT and Card9-/- BMDMs, including stimulation of specific NLRs with their purified ligands, infection with bacterial strains deficient in NLRC4 activation, and infection assays in presence of pharmacological inhibitors. By employing these approaches, I observed that CARD9 has a negative role on NLRP3-dependent IL-1β production. Specifically, in response to activation of the NLRP3 by Salmonella infection, CARD9 negatively regulates pro-IL-1β transcription, and decreases IL-1β processing by inhibiting spleen tyrosine kinase (SYK)-mediated NLRP3 activation and represses caspase-8 activity in the inflammasome. CARD9 expression is suppressed in the course of S. Typhimurium infection which may act as a mechanism to increase IL-1β production during the infection. In conclusion, I have established a connection between CARD9 and IL-1β production by the canonical NLRP3 inflammasome and elucidated some of the mechanisms involved in this process. I have also found evidence that other proteins are likely to be involved in inflammasome regulation and the elucidation of their roles will be addressed in future studies.

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