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Salmonella enterica serovar Typhimurium (S. Typhimurium), one of the causative agents of salmonellosis, remains worldwide an important public health burden, in part due to its ability to survive, persist and spread in non-host environments. A major difficulty in the control of Salmonella infections is indeed the fact that this pathogen can form biofilms on biotic and abiotic surfaces. In these multicellular assemblages bacteria are better protected against environmental stress factors such as antibiotics, disinfectants and the immune system, as compared to their planktonic counterparts. These species-dependent and biofilm-associated phenotypes are caused by changes in gene regulation and expression profiles, together with changing metabolic fluxes, at the transition from free-living to sessile lifestyle. Insight in these processes is thus highly needed to develop novel anti-Salmonella therapeutics, also effective against biofilms. Therefore, the main objective of this doctoral thesis was to get a better understanding of the genetic processes underlying S. Typhimurium biofilm formation. Hereto, different complementary approaches were explored. Since biofilms inherently represent a high degree of heterogeneity, originating from the different physiological cellular states encountered in these multicellular consortia, average expression profiling techniques such as microarray analysis might not provide sufficient insight into biofilm specific gene expression. In an attempt to address this problem and to refine expression analysis in biofilm studies, we optimized the single-cell Differential Fluorescence Induction (DFI) technique to study S. Typhimurium biofilm-specific gene expression. In addition, we screened a recently constructed, targeted S. Typhimurium mutant library for altered biofilm behaviour. Results from both high-throughput screening methods allowed the identification of some interesting, new genetic determinants involved in Salmonella biofilm formation. Moreover, low-throughput confirmation of these results, such as an elaboration on the role of polyamines during Salmonella biofilm growth, allowed us to confirm the generated high-throughput data and illustrate the hypothesis-generating power of the applied techniques. Through a combination of the generated wet lab data with S. Typhimurium expression compendia using a recently developed in silico approach, additional insights into the highly complex biofilm process were obtained. Moreover, indications were presented that biofilm formation largely relies on quantitative gene expression differences of gene sets that are active in both planktonic and biofilm cells, rather than on biofilm-specific pathways. Simultaneously with these open approaches, it was shown that yijC, encoding a regulator controlling unsaturated fatty acid biosynthesis, in silico predicted to be implicated in S. Typhimurium biofilm growth, was indeed involved in this process.A combinatorial high-throughput technique using chromatin immunoprecipitation microarray assays (ChIP-chip) and transcriptomics allowed the genome-wide delineation of the YijC regulon. Although requiring further investigation, the presented data also suggested the existence of some intriguing small regulatory modules and potential mechanisms on how this regulator impacts on the Salmonella biofilm growth. In summary, in this PhD research the applicability of a cross-platform integration of complementary wet lab, both low- and high-throughput, and in silico techniques to study complex phenotypes such as Salmonella biofilm formation was illustrated. The presented new insights are yet another step towards a better understanding of this predominant bacterial lifestyle, urgently needed in the combat against pathogenic infections.

Salmonella typhimurium --- Contamination biologique --- Biological contamination --- PCR --- Expression des gènes --- gene expression --- Academic collection --- Theses

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PROTEIN ENGINEERING Principles and PracticeEdited by JEFFREY L. CLELAND CHARLES S. CRAIKProteins are involved in every aspect of life-structure, motion, catalysis, recognition and regulation. Protein Engineering: Principles and Practice provides a basic framework for understanding both proteins and protein engineering. This comprehensive book covers general, yet essential knowledge required for successful protein engineering, including everything from the fundamentals to modifying existing proteins and developing new proteins.The book begins by introducing the main concepts of protein engineering, including: understanding protein conformation, comprehending the relationship between protein composition and structure, and potential methods for predicting a protein's conformation.Other major subjects addressed are:* Using different host cell expression systems to produce specific proteins* Protein folding* Structure and function of proteins in relation to drug design* Construction of synthetic metal binding sites in proteins* Manufacture of tissue plasminogen activator* Generation of therapeutic antibodiesThis broad range of topics provides a solid foundation in protein engineering and supplies readers with knowledge essential to the design and production of proteins.Of primary interest to protein scientists-both students and researchers, in academia as well as industry-Protein Engineering is also extremely useful to chemical engineers, protein chemists, biochemists, and pharmaceutical chemists.

Protéine --- proteins --- Structure chimique --- chemical structure --- Expression des gènes --- gene expression --- Génie génétique --- genetic engineering --- Protein engineering.

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Bacteria --- Agent pathogène --- Pathogens --- Identification --- identification --- diagnosis --- Relation hôte parasite --- Host parasite relations --- Plante --- plants --- Animal --- animals --- Expression des gènes --- gene expression --- Contrôle de maladies --- Disease control --- Bacterial diseases --- Pathogenesis --- identification.

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Biologie moléculaire --- Molecular biology --- ADN --- DNA --- ARN --- RNA --- Génie génétique --- genetic engineering --- chromosomes --- Biochimie --- biochemistry --- Mutation --- mutation --- Expression des gènes --- gene expression --- Clonage --- cloning --- Biologie moléculaire --- DNA. --- RNA. --- mutation.

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Protéine --- proteins --- Expression des gènes --- gene expression --- Génie génétique --- genetic engineering --- Escherichia coli --- Saccharomyces cerevisiae --- Technique des traceurs --- Tracer techniques --- Technique de culture --- Culture techniques --- Technique immunochimioluminescente --- Chemiluminescence immunoassays --- Proteins --- Protein engineering. --- Recombinant proteins. --- Protein folding. --- Gene expression. --- Synthesis.