Microbial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individ- ual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.

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Bisphenol A is an estrogenic compound that is found in polycarbonate plastics and epoxy resins; humans are continuously exposed to the compound and it is believed to possess the same carcinogenic effects as estrogen (Iso, 2006). In this study, I used Saccharomyces cerevisiae as a model organism to identify mechanisms by which BPA acts based on the genomic profiling of kinase genes from a Mat-α haploid deletion library. Kinases regulate many other proteins, so the identification of a single mutant could identify an entire affected pathway of genes. I conducted a systematic screen of these mutants using the phenotype of growth inhibition. Using solid growth assays, I identified 17 BPA sensitive mutants, six of which were related to the high osmolarity growth pathway, which is involved in osmotic stress response and could be a mechanism of defense of S. cerevisiae against BPA. I implemented liquid growth assays, protein analysis, as well as microscopy for a more in depth study of the effects of BPA on these mutants. Bisphenol-A initially inhibits the growth of S. cerevisiae, however, there were some strains that appeared to show adaptation in the presence of the compound. I found that BPA inhibits cell cycle progression, and may affect the phosphorylation regulation of Cdc28, but without affecting the production of the protein. This study provides clues for predicting the effects of BPA on homologous genes in mammals and identifying similar pathways of resistance. By having a better understanding of the effects on BPA on the cell, the compound can be better regulated by the EPA and complications resulting from continuous exposure to BPA can be treated effectively.

Prions are self-perpetuating and, in most cases, aggregation-prone protein isoforms that transmit neurodegenerative diseases in mammals and control heritable traits in yeast. Prion conversion requires a very high level of identity of the interacting protein sequences. Decreased transmission of the prion state between divergent proteins is termed "species barrier" and was thought to occur due to the inability of divergent prion proteins to co-aggregate. Species barrier can be overcome in cross-species infections, for example from "mad cows" to humans. We studied the counterparts of yeast prion protein Sup35, originated from three different species of the Saccharomyces sensu stricto group and exhibiting the range of prion domain divergence that overlaps with the range of divergence observed among distant mammalian species. Heterologous Sup35 proteins co-aggregated in S. cerevisiae cells. However, in vivo cross-species prion conversion was decreased and in vitro polymerization was cross-inhibited in at least some heterologous combinations, thus demonstrating the existence of prion species barrier. Our data suggests that species-specificity of prion transmission is controlled at the level of conformational transition rather than co-aggregation. We have shown the Sup35 prion domain is sufficient for the species barrier among the S. sensu stricto species, and constructed SUP35 chimeric prion domains, combining the subregions of various origins Our data demonstrated in different cross-species combinations, different modules of prion domain play a crucial role in the controlling of species-specificity of prion transmission. One essential amino acid position has been identified in S. cerevisiae and S. paradoxus system. Our data support a model suggesting that identity of the short amyloidogenic sequences is crucial for the species barrier. Sup35 originated from three different species of the S. sensu stricto group were capable of forming a prion in S. cerevisiae. However, it was not known whether they are capable of generating and maintaining the prion state in the homologous cell environment. We have constructed the S. paradoxus and S. bayanus strains with appropriate markers, and we were able to demonstrate de novo [PSI+] formation in S. paradoxus but not in S. bayanus. Our data show that [PSI+] formation is not a unique property of S. cerevisiae.

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L'organisation tridimensionnelle du génome émerge comme un mécanisme de contrôle important dans la fonction génomique. Les études chez S. cerevisiae ont largement contribué à démontrer l'importance fonctionnelle de l'organisation nucléaire. Pendant la fermentation, les 16 chromosomes d'un noyau haploïde de S. cerevisiae sont organisées avec centromères liés à la SPB et télomères regroupés en 3-4 foyers localisés à la périphérie nucléaire. Cet organisation permet la concentration de protéines silencieuse (SIRS) et semble importants pour les fonctions du génome. Le but de mon travail de doctorat était d'étudier la chromatine télomérique silencieuse dans different transitions métaboliques.Nous avons constaté que le génome de cellules quiescente subit une réorganisation spatiale majeure suite à la source de carbone épuisement. Cette modification de l'architecture nucléaire est entraîné par le regroupement des télomères en un foyer unique (hypercluster) localisée au centre du noyau. Nous montrons également que cette réorganisation est un événement programmé déclenché par les espèces réactives de oxigen (ROS) produits lors de la respiration. Enfin, nous déclarons que l'excès d'activité Sir2 contrecarre le regroupement des télomères lors de la quiescence et a un rôle négatif sur la durée de vie en quiescence. Notre travail suggère que la réorganisation drastique du génome en ‘hypercluster’ de télomères favorise la survie lors de quiescence, et dénoue un lien entre le métabolisme, l'organisation nucléaire et le vieillissement. / The tri-dimensional organization of the genome emerges as an important, still poorly understood, control mechanism in genomic function. Studies in S. cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Upon logaritmic growth, the 16 chromosomes of a S. cerevisiae haploid nucleus are organized into the Rabl conformation, with centromeres bound at the SPB and telomeres grouped in 3-4 foci localized at the nuclear periphery. Telomere clusters allow the concentration of silencing proteins (SIRs) and appear important for genome functions. The aim of my doctorate work was to study telomeric silent chromatin upon major metabolic transitions. We found that the genome of long-lived quiescent cells undergoes a major spatial re-organization following carbon source exhaustion. This change in nuclear architecture is driven by the grouping of telomeres into a unique focus (hypercluster) localized in the center of the nucleus. We also show that this reorganization is a programmed event triggered by reactive oxigen species (ROS) produced upon early respiration and involves the DNA damage checkpoint pathway. Finally, we report that excess of Sir2 activity counteracts telomere clustering upon quiescence and has a negative role on chronological life span. Our work suggests that the drastic genome reorganization due to telomere grouping favors survival upon quiescence, and unravels a novel connection between metabolism, nuclear organization and aging.

The membrane potential is one of the most important parameters of the living cell. It can be measured using carbocyanine fluorescent probes. In this thesis we examined parameters of several dyes of this family. For further experiments three of them were chosen - diOC3(3), diIC1(3) a diIC2(5) as a supplement to diSC3(3) and diSC3(5), which represent standard probes used at biophysical department of Institut of Physics. We compared the rates of their accumulation in S. cerevisiae cells to determine if they were MDR pumps' substrates. The other goal of this work was to decide whether the results obtained using different probes are equivalent and to determine if the presence of a probe affects the spectral characteristics of another. For this purpose we have chosen diSC3(3) and diSC3(5). With those dyes we examined the influence of the acidification on membrane potencial of the yeast S. cerevisiae. We showed that the information on depolarization obtained using both probes were matching very well.

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mRNA molecules form an intermediate in the transfer of sequences from DNA to ribosomes in order to guide protein production. Errors can be introduced into mRNA, producing aberrant proteins which place a strain on cellular regulatory machinery, causing increased risks of apoptosis, cancer, and decreased fitness. These errors may be introduced due to decreased transcriptional proofreading capabilities, exposure to chemicals, or mistakes in RNA editing machinery. It is important to investigate these causes of transcription errors to better understand the long-neglected area of mRNA fidelity which has such significant impacts on our cellular functions. In this paper, it was determined that addition of adenine opposite from abasic sites, not genomic uracil pairing with adenine, are a probable cause of G-to-A transcription errors. That exposure to Roundup causes increased levels of transcription errors, potentially due to oxidative stress. And finally, that off-target ADAR gene editing of transcripts occurs at high levels.

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