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Chemotherapeutika stellen nach wie vor eine der wichtigsten Behandlungs-optionen bei Krebs dar. Ihre akuten und chronischen Nebenwirkungen aber limitieren ihre Anwendung. Aktuelle klinische Studien deuten auf einen positiven Effekt von Kurzzeitfasten auf die Nebenwirkungen von Chemotherapeutika hin. Eine Erklärung hierfür könnte das unterschiedliche Ansprechen von normalen und malignen Zellen auf Chemotherapeutika in einer Mangelsituation sein, das als »differential stress resistance« (DSR) bezeichnet wird. Dieses Phänomen lässt nicht-maligne Zellen bei Restriktion von Glukose und Wachstumsfaktoren weniger sensitiv auf Chemotherapeutika reagieren als maligne Zellen. Das Ziel der vorliegenden Arbeit war zu untersuchen, ob ein Mangel an Glukose und Wachstumsfaktoren das Ansprechen nicht transformierten Zellen 5-FU abschwächen kann während das von kolorektalen Karzinomzellen (Colo741, LS174T, HCT116, HT29 und SW620) gleichbleibt. Optimale Kulturbedingungen für Tumorzellen in vitro stellen 11 mmol/l Glukose und 10 % FCS dar, während 3 mmol/l Glukose und 1 % FCS Mangelbedingun¬gen repräsentieren. Glukosewerte von 3 mmol/l werden auch mit Kurzzeitfasten erreicht. Da der Großteil der soliden Tumoren mit Sauerstoff unterversorgt ist, wurden Untersuchungen auch bei tumorphysiologischen Sauerstoffbedingungen von u. a. 5 % durchgeführt. Der antiproliferative Effekt von 5-FU wurde als halbmaximale inhibitorische Konzentration (IC50) für eine Kultur¬dauer von 72 Stunden bestimmt. Im Mangelmedium mit 3 mmol/l Glukose und 1 % FCS verstärkte sich der antiproliferative Effekt von 5-FU bei drei der fünf getesteten kolorektalen Karzinomzelllinien in Gegenwart von 5 % Sauerstoff im Vergleich zum Standard¬medium mit 11 mmol/l Glukose und 10 % FCS. Die Unterschiede in den IC50 Werten für 5-FU bei diesen drei Zelllinien (Colo741, HCT116, HT29) waren signifikant und bei den beiden anderen Zelllinien (LS174T, SW620) zeigten sich Tendenzen. Dagegen nahm bei Fibroblasten der antiproliferative Effekt von 5-FU im Mangelmedium ab, die Zellen waren somit besser vor dem Chemo¬therapeutikum geschützt. Eine Restriktion von Glukose und Wachstumsfaktoren verändert den antiproliferativen Effekt von 5-FU bei kolorektalen Karzinomzellen nicht und verringert den der Fibroblasten. Damit zeigen die Zellen das Phänomen der »differential stress resistance« (DSR), dass bei 5 % und 21 % Sauerstoff beobachtet wurde, nicht aber bei und 1 % Sauerstoff. Bei 5 % Sauerstoff wurde bei 3/5 Zelllinien sogar ein besseres Ansprechen auf 5 FU nachgewiesen. Der Einfluss von Sauerstoff auf die »differential stress resistance« ist bisher wenig untersucht und basiert vermutlich auf HIF-1-abhängige intrazelluläre Signal¬wege. Die Bedeutung von Sauerstoff und seinem Transkriptionsfaktor HIF-1 für DSR ist bisher nicht verstanden und sollte deshalb weiter untersucht werden. / The phenomenon of »differential stress resistance« in human colorectal carcinoma cell lines: augmentation of the antiproliferative effects of 5-fluoruracil under glucose restriction and low oxygen supply

Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...

In order to develop an environmentally friendly seaweed extract treatment which will benefit both pre- and post-harvest qualities of vegetables, the effects of pre-harvest application of the brown algae Ascophyllum nodosum extracts on the nutritional quality and post-harvest storability of spinach (Spinacia oleracea L.) was investigated. Plants treated with A. nodosum extracts accumulated higher concentrations of iron, potassium, total soluble protein, and total phenolics as compared to untreated controls. 1H NMR and LC-MS analysis revealed a roughly 50% enhanced accumulation of the 9 flavonoids identified, which is partially confirmed by the elevated chalcone isomerase activity. A. nodosum extract treatment caused an increase in transcription of the genes related to plant growth, osmolyte accumulation, and antioxidative activities. Post-harvest analysis revealed that A. nodosum extract treatment caused an enhanced storability of spinach leaves in terms of visual quality, weight loss, and senescence. Lipid peroxidation and ascorbate content were correlated with visual quality during storage. Animal experiments using the Caenorhabditis elegans nematode model revealed that spinach extracts prolonged the life span of C. elegans, and A. nodosum extract-enhanced polyphenols exerted improved beneficial effects in C. elegans against oxidative and heat stresses. Taken together, the results suggest that A. nodosum extracts enhance both pre- and post-harvest quality of spinach through stimulation of flavonoid pathways, thus leading to accumulation of flavonoids and promotion of anti-radical capacity in spinach leaves, which may protect the plant tissue against reactive oxygen species and subsequent decay. Furthermore, the increased flavonoid content in spinach exerted beneficial effects in C. elegans against oxidative and heat stresses via different mechanisms.

<p><h1>Abstract</h1></p> <p><strong> </strong></p> <p><strong> </strong> Understanding the adaptive mechanisms of large regulatory networks can provide insight into long-term survival of bacterial populations in nature. The RpoS master stress regulator found in <em>E. coli</em> controls the expression of nearly 10% of the genome when cells enter stationary phase or in response to general stress conditions. Despite its important role in stress protection, mutations in the <em>rpoS </em>gene are frequently selected in laboratory strains, pathogenic strains and natural isolates. Loss-of-function mutations are beneficial in long-term stationary phase cultures and in steady state glucose- limited chemostat cultures. Although these mutants have increased utilization of an extensive set of substrates, selection for loss of RpoS function occurs at the cost of reduced stress resistance. Previous studies have demonstrated that highly reversible mutations occur within the <em>rpoS</em> gene (for example, transversions and nonsense mutations) when selected on succinate minimal media; however, no study has yet identified a natural compound that can select for restoration of RpoS function. In this study we identify a natural compound allowing restoration of RpoS⁺ cells from a succinate selected RpoS^- culture. Using an RpoS-dependent <em>osmY-lacZ</em> fusion reporter strain carrying a loss-of-function point mutation in the <em>rpoS</em> gene, we demonstrate that growth on 6% NaCl results in selection of mutants with restored RpoS function occurring at a mutation frequency of 10^-9 mutants per cell plated. This is confirmed by RpoS protein detection, and <em>rpoS</em> sequencing results show transversion or transition mutations. These results are the first to demonstrate that selection for restoration of RpoS function can be mediated by a single condition/compound and are consistent with the idea that mutations in the <em>rpoS</em> gene may act as a physiological molecular switch to control the expression of the RpoS regulon.</p> <p><strong> </strong></p> / Master of Science (MSc)

Forkhead box (Fox) transcription factors have a conserved function in regulating lifespan and onset of age related disease in organisms from worms to mammals. Key functions in this process are the regulation of the cell cycle, oxidative stress response, and apoptosis. A complex post-translational code from nutrient, growth factor, and stress induced signals regulates Fox activity, target specificity, stability, and subcellular localization; however, many of the Fox mechanisms and targets responsible for regulating lifespan remain elusive. The budding yeast, Saccharomyces cerevisiae, is a powerful model for unravelling the genetic mechanism and pathways. Yeast encodes four Fox transcription factors, Fkh1, Fkh2, Fhl1 and Hcm1, and their roles in aging are only recently being examined. In this study, we utilized the chronological lifespan and oxidative stress assays, to explore evolutionary conservation of lifespan regulation in two of the yeast Fox orthologs, FKH1 and FKH2. We observed that deletion of both FKH genes in S. cerevisiae, impedes normal lifespan and stress resistance. Furthermore, fkh1Δ fkh2Δ cells were found to be non-responsive to caloric restriction, an intervention that extends lifespan from yeast to mammals. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress resistance. Additionally, we show the Anaphase-Promoting Complex (APC) genetically interacts with the FKHs, likely functioning in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival defect is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ beyond either apc5CA or fkh1Δ fkh2Δ. Finally, we observed that both the FKHs and APC genetically interact with nutrient-responsive lifespan-regulating kinase encoding genes SCH9 and TOR1. This study establishes that the yeast FKHs play a role as regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism. We speculate this involves combined regulation of stress response, genomic stability, and cell cycle.

Résumé : Saccharomyces cerevisiae, utilisée depuis des millénaires pour la fermentation du vin du fait de son endurance et de ses qualités inégalables, est de nos jours largement utilisée pour inoculer les mouts de raisin. Néanmoins, lors de l'inoculation, les souches oenologiques doivent faire face à des stress spécifiques qui peuvent compromettre le début de la fermentation. L’objectif de ce travail est d'élucider les bases métaboliques et moléculaires de la résistance multi-stress pendant la phase de latence en conditions oenologiques. Nous avons tout d'abord caractérisé un ensemble de levures oenologiques en mettant l'accent sur des facteurs de stress caractéristiques des vins rouges et des vins blancs. La température et le stress osmotique affectent fortement cette phase pour toutes les souches, alors que le SO2, les lipides et la thiamine ont un effet souche-dépendant. Ces données ont servi de base à deux approches parallèles. Une approche d'évolution expérimentale a permis, en appliquant des pressions sélectives caractéristiques de la phase de latence, de sélectionner des souches évoluées présentant une phase de latence plus courte. Plusieurs mutations de novo potentiellement impliquées dans le phénotype évolué ont été identifiées par séquençage de leur génome. En parallèle, une approche QTL combinant des croisements inter-souches, une étape de propagation industrielle et séchage des descendants, et la sélection de cellules bourgeonnantes par FACS a été développée. Ces deux stratégies ont permis d’identifier plusieurs variants alléliques impliqués dans la paroi cellulaire, le transport du glucose, le cycle cellulaire et la résistance au stress, jouant un rôle potentiellement important pendant la phase de latence. L’ensemble de ces résultats apporte de nouvelles connaissances sur la diversité et les bases génétiques de l'adaptation des levures à la phase de latence oenologique et offre un cadre d’amélioration des propriétés des souches. De plus, nous avons montré que K. marxianus a un potentiel pour des cultures mixtes et des contributions aromatiques positives en conditions oenologiques, ouvrant de nouvelles possibilités pour des études ultérieures.Titre : Résistance au stress lors de la phase de latence en fermentation oenologique et développement de levures optimiséesMots clés : Fermentation oenologique, levure, phase de latence, résistance multi-stress, QTL, évolution adaptative, K. marxianus / Abstract: Saccharomyces cerevisiae has been used for millennia to perform wine fermentation due to its endurance and unmatched qualities and is nowadays widely used as wine yeast starter. Nevertheless, at the moment of inoculation, wine yeasts must cope with specific stress factors that can compromise the fermentation start. The objective of this work was to elucidate the metabolic and molecular bases of multi-stress resistance during wine fermentation lag phase. We first characterized a set of commercialized wine yeast strains by focusing on stress factors typically found at this stage in red wines and in white wines. Temperature and osmotic stress had a drastic impact in lag phase for all strains whereas SO2, low lipids and thiamine had a more strain dependent effect. Based on these data, we developed two parallel approaches. Using an evolutionary engineering approach where selective pressures typically present in lag phase were applied, we obtained evolved strains with a shorter lag phase in winemaking conditions. Whole genome sequencing allowed to identify several de novo mutations potentially involved in the evolved phenotype. In parallel, a QTL mapping approach was conducted, combining an intercross strategy, industrial propagation and drying of the progeny populations and selection of the first budding cells by FACS. Both strategies allowed the identification of several allelic variants involved in cell wall, glucose transport, cell cycle and stress resistance, as important in lag phase phenotype. Overall, these results provide a deeper knowledge of the diversity and the genetic bases of yeast adaptation to wine fermentation lag phase and a framework for improving yeast lag phase. Additionally, we showed that K. marxianus has potential for mixed cultures and positive aromatic contributions under oenological conditions, opening new possibilities for further studies.Title: Stress resistance during the lag phase of wine fermentation and development of optimized yeastsKeywords: Wine fermentation, yeast, lag phase, multi-stress resistance, QTL, adaptive evolution, K. marxianus

Les systèmes moléculaires décrits chez les Archaea mettent en évidence un caractère primitif et une simplicité, comparativement à leurs homologues eucaryotes. Par ailleurs, leur caractère extrêmophile a pour conséquence une hyper-robustesse qui rend leur manipulation in vitro et les études structurales beaucoup plus aisées. Ainsi les Archaea représentent de bons modèles pour comprendre les fonctions cellulaires complexes, particulièrement celles qui mettent en jeu de grandes machineries moléculaires, comme celles impliquées dans la protéolyse. Mon travail de thèse a consisté à comprendre les mécanismes de résistance et l'importance des systèmes de protéolyse dans l'adaptation des Archaea halophiles aux stress environnementaux. Les Archaea halophiles accumulent des concentrations multi-molaires de KCl/NaCl dans leur cytosol (3.4M KCl / 1.1M NaCl chez Halobacterium salinarum). Ainsi, les protéines de ces organismes sont elles-mêmes halophiles et ne sont solubles et repliées que dans des conditions de salinité extrêmes (de 2 à 5M).Nous avons étudié la réponse de l'Archaea halophile stricte H. salinarum à des stress, dont les stress à basse salinité, en se focalisant en particulier sur les modifications de la dynamique moléculaire du protéome in vivo (diffusion neutronique). Au cours de ce travail, il a été mis en évidence un phénomène de survie à la basse salinité associé à des modifications morphologiques.Un autre objectif de ma thèse a été de contribuer à la compréhension du rôle dans la protéolyse intracellulaire du complexe aminopeptidasique TET, dans les conditions de stress mises en place dans les études précédentes. / Molecular systems described for Archaea show primitive and simple characteristics, compared to their homologous eukaryotes. In addition, extremophilic characteristic results in an hyper-robust which makes in vitro manipulation and structural studies much easier. Thus, Archaea represent good models for understanding complex cellular functions, particularly those that involve large molecular machines, such as those involved in proteolysis. My thesis consisted in understanding the resistance mechanisms and the importance of proteolytic systems in the adaptation of halophilic Archaea to environmental stresses. Halophilic Archaea accumulate multi-molar concentrations of KCl / NaCl in their cytosol (3.4M KCl / NaCl 1.1M for Halobacterium Salinarum). This requires a very special biochemistry that allows operation in solvents where free water is scarce. Thus, the proteins of these organisms are themselves halophilic and are soluble and folded only in extreme salinity conditions (2 to 5 M). This particular biochemistry partly explain the extraordinary ability of halophilic Archaea to resist physical and chemical stress (temperature, radiation, dehydration). We study the response of the halophilic Archaea strict H. salinarum at low-salinity stress. Indeed, beyond the osmotic shock, the fall of the environment salt concentration causes a decrease in the intracellular KCl concentration, which should have a direct effect on the folding state of intracellular protein, as in case of heat stress. In the first part of this thesis, a study was conduct to determine viability limits and cytosolic modifications, associated with a salinity decrease. These studies involve intracellular salt dosages, viability studies (microscopic counts, color live / dead), induction of chaperone proteins linked to stress response and biophysical neutron experiments, to evaluate the effect of stress on proteins folding. In this work, a phenomenon of survival at low salinity linked to morphological changes was revealed. To describe this phenomenon, this second study involves confocal microscopy experiences, fluorescence microscopy, viability tests, counting on box, scanning electron microscopy, electron microscopy by negative staining, salt intracellular dosages and proteins separation experiments, to study the overall proteome composition during low salinity stress. In this study, a fall of the intracellular K $^+$ concentration and the proteome clarification during stress was revealed. Low salt concentrations causes halophiles proteins denaturation, the accumulation of misfolded proteins in the cytoplasm involves chaperones systems and intracellular proteolysis machinery. In this context, another objective of my thesis was to contribute to the understanding of the intracellular proteolysis role in the PAN-proteasome system and in the aminopeptidase TET complex, in stress conditions established in previous studies. This part of the thesis involves experiments of endopeptidase activity assay, aminopeptidase activity assay, quantification of mRNA genic expression by Northern blot, immunoprecipitation, proteins separation by sucrose gradient and proteasome chemical inhibition (drug). We show the role of the PAN-proteasome system in stress response and we deepen our understanding of the aminopeptidase TET role in vivo. This protease appears to have an independent role of the proteasome complex. The protease TET seems to participate at the amino acids treatment in cells to maintain the metabolic activities in nutritional deficiencies.

Les parodontites sont des maladies chroniques inflammatoires causées par un biofilm bactérien. Elles sont la première cause de perte des dents dans les pays industrialisés et représentent donc un coût important pour la société. Le biofilm buccal est composé de plus de 500 espèces différentes, parmi lesquelles Porphyromonas gingivalis est reconnue comme une cause majeure du développement des symptômes. Cette bactérie à Gram négatif est considérée comme anaérobie bien qu’elle tolère des concentrations faibles en oxygène, ce qui favorise la colonisation de la cavité orale. Notre objectif était de mettre en évidence les processus biologiques conférant à P. gingivalis sa résistance à l’oxygène et au stress oxydant, mais également ceux impliqués dans la transition métabolique en concentrations variables d’oxygène. Des analyses in silico des génomes de souches de P. gingivalis ont révélé la présence d’un système respiratoire dépendant de l’oxygène, impliquant une cytochrome bd oxydase CydAB. Nous avons construit un mutant de P. gingivalis ATCC 33277 par délétion des gènes cydAB. Nos travaux ont montré que ce mutant était plus sensible que la souche parentale aux espèces réactives de l’oxygène (ROS) dont le peroxyde d’hydrogène et le générateur d’anion superoxyde paraquat. De plus, nous avons démontré que CydAB était impliquée dans le phénotype aérotolérant de P. gingivalis, et que cette enzyme consommait effectivement l’oxygène grâce à une étude par oxygraphie à haute résolution. Les mécanismes de régulations en réponse aux ROS et à l’oxygène sont encore mal connus, notamment en ce qui concerne la régulation positive de l’expression des gènes cydAB en présence d’oxygène. Deux gènes codant des régulateurs de type FNR ont été identifiés dans le génome de P. gingivalis, l’un d’entre eux codant un régulateur de la réponse au stress nitrosant, HcpR. Le second gène PGN_1569 a fait l’objet de notre étude. Par mutation et par analyses transcriptomiques, nous avons démontré que ce régulateur s’autorégulait négativement et activait l’expression de 4 groupes de gènes en anaérobie, n’incluant pas les gènes cydAB. L’expression de ces gènes est par ailleurs contrôlée par d’autres régulateurs redox, OxyR et/ou SigH et/ ou RprY. Cette étude met donc en évidence une connexion entre FNR et les autres régulateurs de la réponse au stress oxydant chez P. gingivalis. Des études complémentaires permettront de caractériser la fonction encore hypothétique des protéines codées par le régulon FNR. Il est intéressant de noter que l’absence de FNR confère à P. gingivalis une plus grande capacité à former un biofilm en anaérobie / Periodontal diseases are chronic inflammatory infections caused by bacteria in oral biofilm they are the first cause of loss of tooth in industrial countries with an important cost for the society. The biofilm comprises more than 500 bacterial species. Amongst them, Porphyromonas gingivalis, a Gram-negative bacterium, is well known as a major causative agent of periodontitis. Although considered as mainly anaerobe, P. gingivalis tolerates low oxygen concentration, therefore enhancing its ability to colonize the oral cavity. Our aim was to decipher the biological processes underpinning the resistance of P. gingivalis to oxygen and reactive oxygen species (ROS) and to characterise the transition from anaerobiosis to hypoxia. In silico studies of P. gingivalis genomes have revealed the presence of a putative oxygen-dependent respiratory system involving a cytochrome bd oxidase CydAB. We constructed a mutant deleted for cydAB genes in the P. gingivalis ATCC 33277 strain. Our study showed that cydAB mutation increased the sensibility of the mutant to reactive oxygen species such as the anion-superoxide generator paraquat and hydrogen peroxide. Moreover we demonstrated that CydAB is involved in the aerotolerance of P. gingivalis, and in oxygen consumption, as demonstrated by high resolution respirometry assay. Many regulations in response to ROS and oxygen are still unexplained in P. gingivalis, including the activation of cydAB expression by oxygen exposure. Two genes encoding FNR-like regulators were identified in the genome of P. gingivalis. One of them encodes the HcpR regulator which controls part of the nitrosative stress response. The second gene PGN_1569 was the focus of our study. By mutation and transcriptome analysis, we demonstrated that this FNR-like regulator repressed its own transcription and activated the expression of 4 gene clusters in anaerobiosis, but not including cydAB genes. The expression of these 4 gene clusters is also controlled by other redox regulators, OxyR and/or SigH and/or RprY. Therefore, this study pointed out the interplay between FNR and known oxidative stress response regulators of P. gingivalis. Further work will study the functions of the hypothetical proteins encoded by the FNR regulon. Interestingly, the fnr mutant displayed higher ability than the wild-type strain to form biofilm in anaerobiosis.