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Computational methods used and developed in this thesis contribute to the analysis of complex biological data. The aim was to bridge the gap between genotype and phenotype present in different experimental outcomes. The impact of variations in individual proteins and pathways can change the phenotypes. Currently, the understanding of the mechanisms with precise details about compromised phenotypes is far from complete. In this regard, an experimental study to address a stress/disease condition can provide the insights of causal molecules and mechanisms. In fact, the complementarity of computational methods and experimental setups can further enhance the understanding. Over the years, the integration of experimental and computational setups has contributed in explaining the grey areas of phenotypes. To this end, with advancements in technology providing wealth to new data, it is almost impossible to make sense of data without in silico input. Based on this idea several computational methods have been developed to contribute towards “omics” studies. However, functional analyses of many phenotypes need more creative and effectives computational protocols. Therefore, in order to assess the impact of variations on phenotypes more robust tools are required. Motivated about this notion, we analyzed disparate data sets coming from different experiments. The aim of these experiments was to understand the molecular phenotypes by assessing the impact of variations on protein networks, pathways and conserved functional regions. For this, we tailored different computational pipelines. These frameworks specifically map the variations onto the functional regions like modified residues or protein domains. The resulting data with mutations mapped to the functional regions can provide a refined glimpse of pathways that are operating a phenotype. This method can facilitate the understanding of complex phenotypes. The projects conducted during this thesis work can be separated into two parts. In the first part, we performed three projects about yeast computational systems biology. In addition to that, in the second part, we performed a project about viral disease and therapeutic targets prediction. For the first project, we generated new experimental data from yeast under ethanol stress. At one instance, we analyzed the genomic variation data from whole genome sequencing. These variations were mapped onto the functional regions, for further analyses and interpretation. The resulting data suggested that the modified residues suffered the least mutational burden. Whereas the presence of multi-protein domains and pathways show these functional units can contribute to the stress-tolerating behavior of yeast. The second project of this thesis work was to develop a computational protocol. The aim of this project was to facilitate researchers who are working on big data from yeast experiments. Budding yeast as a model organism is very popular in the research community, to understand fundamental biological questions. Yet, researchers lacked a tool facilitating the analysis of mutational data from yeast. We thus created a python based tool, yMap, which can take big data containing the mutations at genetic/proteomic level. This tool maps the mutated residues to several evolutionary conserved and functionally important regions. In the end of a typical analysis, an output contains the information regarding mutated protein regions, mutation-types, pathway enrichment and network visualization. We believe that, in the areas of systems biology, this automated protocol can contribute to and facilitate yeast research. Our third project was based on the data integration of two different types of yeast data. The theme was to understand and explain the protein regulation in yeast under ethanol stress. For this reason we created a data integration computational framework based on the yeast protein-protein interactions. The strategy helped in the probing of causal regulatory proteins for their possible role in protein regulation of stress orientated yeast clones. We could also establish potential involvement of mutations in protein regulation along side with regulatory proteins. Altogether, this project contributed in understanding the mechanisms of protein regulation in ethanol stress via regulatory proteins and mutations. The second part of this thesis was dedicated to human disease. The aim was to create a new computational protocol to contribute to bringing further the present understanding of Ebola virus disease (EVD). Moreover, based on our strategy, we suggested possible therapeutic targets of EVD. In this regard, we mapped the most conserved regions of Ebola virus proteins, and predicted modified residues present on these conserved regions. The phosphorylation was the most abundant type of modification predicted in our analysis. To target Ebola virus proteins, we predicted that the phosphorylation contributing kinases from host genome. These kinases are potentially involved in the protein modifying events of Ebola virus. This project opens a new area of research to analyze the conserved modified residues in order to target possible modifying enzymes. To sum it up, we conducted four different projects to contribute to understanding the gap present between the genotype and phenotype. Each of the projects on yeast genome brought unique insights of the molecular mechanisms present underneath a phenotype. Moreover, the evolutionary insights of Ebola virus proteins can facilitate drug development based on our analysis. Additionally, the methodologies developed in each project can facilitate larger research community to perform their research. samenvatting

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Refrigerated foods are one of the most rapidly growing segments in the food industry. Refrigerated storage is necessary because these foods are perishable and can support growth of microorganisms that can cause spoilage or foodborne disease. Psychrotrophy, i.e. the ability to grow at temperatures below 4 °C, is widespread among bacteria of diverse phylogenetic lineages, but often varies at the genus, species or even strain level. The adaptations that allow bacteria to grow at low temperature are of interest from a basic scientific viewpoint, but also because they may lead to improved strategies to control undesirable psychrotrophs in the food production chain. Studies on the molecular and cellular basis of psychrotrophs have mainly focussed on Gram-positive bacteria, Listeria monocytogenes in particular. Other psychrotrophic food-related bacteria particularly those belonging to the Enterobacteriaceae family, have been much less studied. Therefore, the current work was intended to improve our understanding of the mechanisms that are required in enterobacteria to grow under cold stress. Serratia plymuthica RVH1, a psychrotrophic strain isolated from a catering kitchen that has been studied extensively in our laboratory, was chosen for this work, and a genome-wide mutational approach was chosen as the initial strategy. A transposon-based mutant library of S. plymuthica was constructed and screened for mutants showing impaired growth at low temperature but normal growth at optimal temperature [30 °C]. Four putative psychrotrophy mutants displaying consistent growth impairment at 4 and 10 °C were isolated and genetically analysed. The transposons in these four mutants were localised in open reading frames that were putatively identified as plsC [1-acyl-sn-glycerol-3 phosphate acyltransferase], mnmA [tRNA-specific 2-thiouridylase], an unnamed ORF [polysaccharide pyruvyl transferase], and ubiB [2-octaprenylphenol hydroxylase]. None of these genes, except for fatty acid biosynthesis [plsC], and none of the pathways in which these genes are involved have been previously related to psychrotrophy. Since plsC and mnmA mutants showed almost no and severe impaired growth, respectively, at 10 and 4 °C, these were selected for detailed analysis. The mutant with transposon insertion in the upstream region of plsC gene encoding lysophosphatidic acid acyltansferase involved in biosynthesis of phosphatidic acid [PA, a primary intermediate in membrane glycerolipid biosynthesis], showed a six to sevenfold reduced ratio of palmitoleic acid to oleic acid [C16:1 / C18:1] though the ratio of saturated to unsaturated fatty acid was unaffected. Low temperature growth defect and fatty acid composition were mostly restored by introduction of a complementation plasmid overexpressing plsC. Low temperature growth was also partially restored by supplementation of C16:1 to the growth medium, indicating that a shift from C18:1 to C16:1 was required for psychrotrophy.The mutant was also significantly more susceptible to pressure treatment at 250 MPa but not at higher pressure, and its growth was reduced at low pH but not at elevated NaCl concentration. These results provided novel information on the role of fatty acid composition on bacterial stress tolerance. The impact of the observed fatty acid shift on cold adaptation is in line with the well-known homeoviscous adaptation principle, but how knock-out or modulation of PlsC activity in the mutant leads to this shift remains unclear. This mutant may prove useful in further studies addressing the precise function of PlsC in S. plymuthica. A second mutant that was subjected to a more detail analysis harboured a transposon insertion in mnmA, a gene encoding a tRNA-specific 2-thiouridylase involved in 2-thiouridine modification [s2] at the wobble position in the anticodon stem loop. In a parallel screening in the context of another project in our research group, a mutant of another tRNA modification gene, mnmE, was identified to support growth of E. coli MG1655 in mildly acidic conditions. These observations from independent screenings suggested a more general role for tRNA modification in stress management, and therefore we constructed single knockout of both genes in S. plymuthica, and analysed their tolerance to temperature, osmotic, acid, protein synthesis inhibitors, and oxidative stresses. While S. plymuthica required the MnmA-mediated modification for normal growth at low temperature, this modification affected growth at supra-optimal temperature in E. coli. Interestingly, the effects of MnmA on growth under temperature stress in both bacteria disappeared when the bacteria were grown at an elevated level [2 - 3 % w/v] of sodium chloride in the growth media. The MnmE-mediated modification had no influence on growth under temperature stress in S. plymuthica or E. coli. Nonetheless, both MnmA and MnmE were indispensable for normal growth of E. coli under mildly acid conditions while this role in S. plymuthica was less pronounced. Further, MnmA supported survival of a lethal tert-butyl hydroperoxide challenge. Lastly, the sensitivity to antibiotics inducing translation infidelity was also influenced by MnmA- and MnmE-mediated modification in strain-dependent manner confirming their involvement in protein biosynthesis. Modifications of uridine tRNA at the wobble position of ASL [Anticodon Stem Loop] are important for the efficiency and fidelity of the translation. Lacking these modifications causes translational inefficiency leading to physiological consequences that are magnified during stress conditions. This can be explained by two proposed models of the role of tRNA modification as reported by others. The first model assumes that translation of stress response proteins depends more strongly on modified tRNAs than translation of other proteins. This is because stress response transcripts are rich in codons that are decoded by modified tRNA's. If the demand for such tRNAs cannot be fulfilled, the translation of the stress response transcripts decreases and that may lead to incapability of the cell to overcome stress. Second, the role of tRNA modifications on maintaining proteome homeostasis. In this model, the absence of modified tRNA's in stress conditions induces ribosome pausing eliciting protein aggregates and thus introducing acute proteotoxic insults. Consequently, proteome integrity is perturbed. In conclusion, this work has contributed to a better understanding of some genes and cellular functions required for psychrotrophy in S. plymuthica. Some of these functions had not been previously related to psychrotrophy. Fatty acid composition and tRNA modification have been studied in more detail in particular, and appear to play a role in tolerance to other stresses as well. The knowledge of the cellular process and pathways involved in low temperature growth may be used for discovery of novel preservatives specifically targeting psychrotrophic bacteria in refrigerated foods, thus increasing the safety and stability of the foods.

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