Salmonella enterica serotype Typhimurium is a rod-shaped, motile, Gram-negative bacterium that causes gastroenteritis in immunocompetent individuals. S. Typhimurium produces an extracellular protein termed curli, a bacterial amyloid with a cross beta-sheet tertiary structure that is common across all amyloids. Curli formation is critical for biofilm formation by enteric pathogens such as S. Typhimurium and E. coli. Curli expression requires the production of multiple proteins, which are encoded by two operons known as csgBAC and csgDEFG. Curli production can be induced in vitro by low temperature and low osmolarity, which is evident by growth on T-medium plates for 72 hours at 28oC. Earlier studies have shown that curli is expressed in sepsis patients with E. coli, as well as in mice after S. Typhimurium infection. This is evidenced by the production of antibodies to CsgA, the major subunit of curli. Our lab has shown that curli fibers are recognized by the TLR2/TLR1 complex of the innate immune system during infection. Infection with curli expressing bacteria causes elevated levels of proinflammatory cytokines, nitric oxide, and autoantibodies. Nonetheless, the details of curli expression in vivo during bacterial infection remain unknown. The focus of these studies was to elucidate the location where bacteria expresses curli in vivo during infection. Initially, we used S. Typhimurium strains carrying plasmids with csgB and csgD promoter regions fused to the gfp gene to study curli expression in vivo by use of flow cytometry. Unfortunately, we were unable to determine curli expression with this model, due to the diminished fluorescence intensity of GFP under anaerobic conditions in the gastrointestinal tract. As the question of curli expression in vivo was left unanswered, we next used a long-term infection model of S. Typhimurium with the goal of determining seroconversion to curli as well as the location and timing of curli expression. Using CBA/J mice infected with wild-type S. Typhimurium or a curli mutant strain, we were able to identify seroconversion to CsgA in the mice infected with the wild-type strain through ELISA and western blot analysis. We were also able to identify autoantibody production in mice infected with the wild-type strain through ELISA. However, we were unable to determine curli expression in the feces of mice either by western blot or qPCR data. We were also able to identify autoantibody production in mice infected with the wild-type strain through an anti-double stranded DNA ELISA. Preliminary findings lead us to hypothesize that curli expression may occur very early on in infection, and may be expressed inside cells such as macrophages. Overall, our results partially elucidate curli expression in vivo, although more research is needed in order to answer our remaining questions regarding location and timing of expression. / Biomedical Sciences

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:09:15Z (GMT). No. of bitstreams: 1 Cordeiro_MelinaAparecida_M.pdf: 3889960 bytes, checksum: 3e92b071b7e108ebb8bff09fe751b180 (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Doctor of Philosophy / Rational Design and Scalable Production of De novo Autogenic Engineered Living Materials Hoda Mohsen Youssef Hammad General Audience Abstract This work introduces a new approach to creating "engineered living materials" that combine biology with advanced protein engineering to form dynamic, self-assembling structures. By harnessing the natural abilities of bacteria, the research demonstrates how these microorganisms can be reprogrammed to produce protein fibers that serve as the foundation for creating larger, customizable biomaterials. The study develops a specialized platform that allows bacteria to secrete and assemble protein building blocks with well-defined structures, enabling control over the physical and mechanical properties of the final material. Key innovations include the design of protein architectures inspired by naturally occurring systems and the use of advanced computational tools, such as molecular dynamics simulations and structural prediction artificial intelligence tools, to fine-tune these designs. By modifying the core structure of these proteins, the research shows how one can systematically influence how the material forms at a larger scale, leading to a diverse range of materials—from soft hydrogels to more robust films and plastics. Moreover, the work addresses the challenge of scaling up production. A novel vesicle-based secretion strategy streamlines the manufacturing process by enabling the simultaneous production and purification of these protein fibers, eliminating many of the complex steps typically involved in biomaterial production. This scalable process promises to bridge the gap between laboratory research and real-world applications, potentially impacting fields such as construction, biomedicine, and sustainable manufacturing. In summary, this research represents a significant step forward in the design and production of engineered living materials, offering a versatile platform that merges synthetic biology with materials science to create eco-friendly, programmable, and scalable materials for future technologies.