L’information génétique contenue dans le noyau de la cellule doit être dupliquée fidèlement afin d’être transmise aux cellules filles pendant la division cellulaire. Pour organiser leur division, les cellules suivent un cycle reproductible composé de quatre étapes appelé cycle cellulaire. La préparation et l’exécution du programme de réplication de l’ADN ont lieu pendant des phases spécifiques du cycle grâce à l’intervention de multiples partenaires protéiques et de régulateurs structuraux. En particulier, la réplication de l’ADN s’effectue sur une matrice complexe constituée d’ADN associé à des protéines appelée chromatine. Cette dernière influence et est influencée par la réplication de l’ADN. Le travail présenté ici a pour objectif de faire le lien entre la conformation de la chromatine et la cinétique de réplication de l’ADN. Pour ce faire, nous combinons plusieurs techniques. La cytométrie de flux nous permet de suivre la quantité d’ADN présent dans une population de cellules et, à l’aide d’une méthode développée dans notre laboratoire, d’extraire le programme de réplication moyen d’une population de cellules. La technique de SAXS fournit des informations sur l’organisation locale des protéines et de l’ADN in vivo. Nos données peuvent être interprétées comme un cristal liquide avec un ordre nématique et une faible longueur de corrélation, ce qui suggère que la chromatine de la levure est majoritairement dépourvue d’une organisation en fibre de 30nm in vivo. Par ailleurs, par la méthode de peignage d’ADN, nous reproduisons les résultats précédemment obtenus montrant que la distance entre zones répliquées est d’environ ~60kb qui correspond à la distance entre des origines de réplication identifiées. Cependant, d’après l’étude du comportement dynamique de l’initiation, nous proposons que les initiations sont plus fréquentes que ce qui a été mesuré précédemment et correspondent à la distance entre les protéines MCM disposées sur le génome. / Genetic information carried in the cell nucleus must be faithfully duplicated to be transmitted to daughter cells during cell division. In order to orchestrate their division, cells go through a reproducible 4 stages cycle called «cell cycle». The preparation and execution of the DNA replication program is restricted to specific phases and implies many proteic and structural regulators. In particular, DNA replication occurs on a complex template of DNA associated with proteins. The latter is both influencing and influenced by DNA replication. This work aims at investigating the link between chromatin conformation and the kinetics of DNA replication. In order to do so, we combine several techniques. Using flow cytometry, we follow the evolution of a cell population with regards to their DNA content and, with a method developed in our laboratory, decipher the population averaged temporal program of DNA replication. SAXS data provide information on the local organisation of protein and DNA in vivo. Our data can be interpreted as a liquid crystal with a nematic order and a short correlation length, which suggest that yeast chromatin in vivo is predominantly devoid of 30 nm fibres organisation. On the other hand, we performed DNA combing to study the replication program in single cells. We reproduce previously obtained result showing that distance between replicated tracks is of ~60kb which corresponds to the distance between known origins of replication. However, studying the behaviour of initiation, we propose that the initiation events are more frequent than previously measured and correspond to distances between MCMs proteins loaded on the genome.

Les télomères garantissent la stabilité des extrémités chromosomiques. Une défaillance de protection entraine l’apparition de chromosomes dicentriques (c-à-d. possédant deux centromères) instables en mitose. La présence de chromosomes dicentriques est donc une source de mutagénèse et une menace pour la viabilité des cellules. Chez Saccharomyces cerevisiae, les dicentriques issus d’une fusion de télomères cassent préférentiellement à la fusion. Ce processus inexpliqué permet la régénération d’un caryotype normal et protège les chromosomes des conséquences néfastes d’une fusion accidentelle de leurs extrémités. Ce manuscrit explore les mécanismes moléculaires de cette voie de secours. La haute affinité de Rap1, pour ses sites consensus en tandem ou pour des séquences télomériques est suffisantes pour former un point chaud de cassure des chromosomes dicentriques. Une protéine hétérologue ayant aussi une haute affinité fixation pour sa séquence mime la présence de fusions de télomères, montrant que la forte affinité d’une protéine pour ses sites en tandem suffit à créer un point chaud. En l’absence de séquence télomérique interne, les chromosomes dicentriques cassent plutôt aux régions péricentromériques. Ces positions de cassure dépendent d’une force générée par les Condensines capable de relocaliser rapidement les centromères des chromosomes dicentriques au site de cytodiérèse avant leur cassure. De plus, le repliement des chromosomes dicentriques dépendant des Condensines est également nécessaire à une cassure préférentielle aux séquences fixant Rap1. En anaphase, ces séquences forment aussi un isolateur capable de séparer deux domaines chromosomiques. Ainsi, les télomères fusionnés sont secourus par un mécanisme qui favorise une capture des fusions et des régions péricentromériques par le septum dépendant de la conformation des chromosomes dirigées par les Condensines et par Rap1, Ces résultats suggèrent que les séquences télomériques fixant Rap1 bloquent l’extrusion de boucles par Condensine. De plus ce travail propose un nouvel outil pour l’étude de la condensation in vivo. Il montre également que la cassure des chromosomes dicentriques survient pendant la septation et que cytodiérèse n’est pas ralentie par la présence d’un pont de chromatine. / Telomeres ensure chromosome end stability. Failure to do so would lead to chromosome end fusions and the formation dicentric chromosomes (i.e. chromosomes with two centromeres) that are unstable in mitosis. Dicentrics are a threat to cell viability and a source of extensive mutagenesis. In Saccharomyces cerevisiae, dicentrics formed by telomere fusion preferentially break at the fusion. This unexplained process allows the recovery of a normal karyotype and protects the genome from the detrimental consequences of accidental telomere fusions. Here, I address the molecular basis of this rescue pathway. Simple tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. I also adress the mechanism allowing breakage at pericentromeric regions when dicentric do not bear telomeric sequences. During anaphase, Condensins generate forces sufficient to rapidly relocalize the centromeres to the bud neck and refold dicentrics prior their breakage by cytokinesis. This relocalisation is essential for breakage at pericentromeres. Moreover Condensin-dependent refolding is essential to the preferential breakage at telomere fusions, more generally at Rap1-bound arrays and which delimit insulated chromosomal domains. Thus, the rescue of fused telomeres results from a Condensin- and Rap1-driven chromosome conformation that favours fusion entrapment where the septum closes. These results suggest that Rap1-bound telomere sequences stall loop-extrusion by Condensins. In addition, this work provides a new and direct way to monitor Condensin activity on chromatin in live cells. It also shows that dicentric chromosomes are broken during septation and that cytokinesis is not delayed by chromatin bridges.

Fungal infections are caused by a variety of fungi, and with a variety of clinical manifestations. Antifungal treatments are limited due to host toxicity and fungi gaining resistance. By utilizing the model organism Saccharomyces cerevisiae, we hope to elucidate the molecular mechanisms of fungal pathogenesis that we can then validate in the human pathogen Candida albicans, as well as explore options for novel therapies. Small molecule signaling is a method by which single-cell organisms can communicate with one another, enabling them to coordinate gene expression. This is a useful tool because it allows microbes to turn on phenotypes that are only valuable when done in large numbers, such as bioluminescence, or virulence traits. We have previously shown that the yeast Saccharomyces cerevisiae synthesizes the secondary metabolite indole-3-acetic acid (IAA) from tryptophan. IAA is secreted into the environment, where it acts as a signal. At low concentrations, the IAA signals yeast to induce virulence traits, while at high concentrations, it is lethal. The purpose of this thesis was to investigate the molecular mechanism of IAA (plant hormone auxin) regulation in fungi, specifically, Saccharomyces cerevisiae and the human pathogen Candida albicans. Towards this end, I first focused my efforts on evaluating the role of S. cerevisiae Grr1, as a putative IAA receptor. By evaluating the IAA response of several Grr1 mutants, I was able to show that the leucine-rich repeat region, while not required for function, likely plays a significant role in maintaining the structural integrity of the protein. Next, I evaluated IAA associated phenotypes, such as filamentation, surface adhesion and IAA uptake of the grr1 null mutant in the human pathogen Candida albicans. Together, these data support the hypothesis that GRR1 regulates IAA response, probably by regulating the IAA uptake carriers.

No description available.

No description available.

DNA damage tolerance (DDT) is a process utilized by cells to bypass replication blocking lesions in the DNA, preventing replication fork collapse and maintaining genomic stability and cell viability. In Saccharomyces cerevisiae DDT consists of two branched pathways. One branch allows direct replication past lesions in the DNA utilizing specific error-prone polymerases, a process known as translesion DNA synthesis (TLS). The other branch utilizes homologous recombination and template switch to replicate past damaged DNA in an error-free manner. RAD5 has traditionally been characterized as belonging to the error-free pathway of DNA damage tolerance. The protein is multi-functional, with several specific activities identified and classified to the error-free branch of DDT. However, there is also evidence for additional uncharacterized activities of the protein. The goal of this research was to determine which branches of DNA damage tolerance the uncharacterized activities of Rad5 are involved in. A two-pronged approach was utilized, elucidation of the physical interactions of the protein, and examination of the genetic interactions between RAD5 and other DDT genes. The evidence indicates that Rad5 plays a partial role in TLS and the protein is known to physically interact with Rev1, a member of the TLS pathway. We assumed this physical interaction mediates the TLS activity of Rad5. The yeast two-hybrid assay was utilized to examine the interaction between Rev1 and truncated Rad5 fragments, and the N-terminal 30 amino acids of Rad5 proved sufficient to maintain the interaction. This research sets the stage to identify key residues in Rad5 for the interaction with Rev1, and the creation of a TLS deficient rad5 mutant by targeting those key residues. Genetic interactions between RAD5 and genes required for the initiation of DDT in the cell were examined based on sensitivity to killing by various DNA damaging agents. We determined that the functions of Rad5 rely on PCNA modification, and thus do not function in a cellular process unrelated to Rad5. Potential uncharacterized functions are discussed on the basis of these results and the results of the interaction studies. Future structural and functional studies are proposed to better understand the role of Rad5 in the cell.

<p>Many cells are remarkably proficient at tracking even shallow chemical gradients, despite tiny differences in receptor occupancy across the cell. Stochastic receptor-ligand interactions introduce considerable noise in instantaneous receptor occupancy, so it is thought that spatial information must be integrated over time to allow noise filtering. The mechanism of temporal integration is unknown. We used the mating response of the budding yeast, <italic>Saccharomyces cerevisiae</italic>, as a model to study eukaryotic gradient tracking. </p><p>During mating, yeast cells polarize and grow up a gradient of pheromone to find and fuse with opposite-sex partners. Exposure to pheromone causes polarity regulators to cluster into a tight "patch" at the cortex, directing growth toward that site. Timelapse microscopy of fluorescently-labeled polarity proteins revealed that the patch wandered around the cortex during gradient tracking. Mathematical modeling and genetic analysis suggested that fusion of vesicles near the polarization site could perturb the polarity patch and promote wandering. Wandering is decreased due to global effects from pheromone signaling as well as interactions between receptor-activated G&beta; and the exchange factor for the polarity regulator Cdc42. We found that artificially stabilizing patch wandering impaired accurate gradient tracking.</p><p>We suggest that ongoing polarized vesicle traffic causes patch wandering, which is locally reduced by pheromone-bound receptors. Thus, over time, spatial information from the pheromone gradient biases the random wandering of the polarity patch so that growth occurs predominantly up-gradient. Such temporal integration may enable sorting the low signal from stochastic noise when tracking shallow gradients.</p> / Dissertation

The subcellular localisation of mRNA is a widespread mechanism to determine the fate of mRNAs in eukaryotes. Translationally repressed mRNAs localise to P-bodies and stress granules where their decay and storage, respectively, are directed. In a study from the Ashe lab, specific mRNAs were identified to localise, in actively growing S. cerevisiae, to cytoplasmic granules that do not seem to be related to P-bodies or stress granules but appear to be associated with active translation (Lui et al., 2014).It is possible that this might represent a strategy to co-regulate the expression of proteins from the same pathway. In the work of this thesis, microscopy techniques to visualise RNAs in live cells were used to extend the localisation analysis to several mRNAs encoding translation factors. The investigated transcripts were all found to localise to mostly one or two cytoplasmic granules per cell and would sometimes overlap with other transcripts, suggesting that each granule contains a mixture of mRNAs. Granules tend to migrate to the bud tip and may provide the daughter cell with a "start-up kit" of transcripts essential for rapid growth. A similar pattern can be observed in yeast cells growing undergoing filamentous growth, with granules harbouring translation factor transcripts often found in the apical quarter of the elongated cell. Although the mechanism by which the granules form and their protein composition are not yet known, high-throughput genetic screens performed as part of this work offer some insight into factors that might be involved in granule assembly and proteins that partially overlap with the granules. We propose that granules containing translation factor mRNAs might be functioning as a specialised factory for the translation machinery and are possibly being directed to the point in the cell where the rhythm of protein production is highest.

Made available in DSpace on 2016-06-02T19:55:40Z (GMT). No. of bitstreams: 1 6307.pdf: 2700977 bytes, checksum: 1f73d2ff8bc2b699bcb0f74928efc3c9 (MD5) Previous issue date: 2014-04-23 / Financiadora de Estudos e Projetos / Glucose isomerase (GI) was covalently immobilized and co-immobilized with baker yeast in calcium alginate beads for ethanol production by simultaneous isomerization and fermentation of xylose or hemicellulose hydrolysate from bagasse. GI was covalently immobilized on glutaraldehyde-chitosan and high protein loads were immobilized (30-68 support mg.g-1). High immobilization yields (100%) and activity recovery (90%) were achieved. Under typical operation conditions of simultaneous isomerization and fermentation (pH 5, 35°C, medium with nutrients and ethanol concentrations up to 70g.L-1) the derivative remained 90% of initial activity after 120 hours. Calcium carbonate was used to maintain the pH in isomerization/fermentation medium higher than 5.0, ensuring high conversion of xylose at 32- 35°C. Accordingly, concentrations of enzyme and yeast in the reactor of 60-120.103UI.L-1 and 50g.L-1, respectively, resulted in better yields and selectivity for ethanol batch production. After consumption of ~ 65g.L-1 of xylose in about 12 hours, 21g.L-1 of ethanol and 14g.L-1 of xylitol was produced. In a medium without calcium carbonate, partial sugar conversion was observed due to the reduction of pH medium, which decreased up to 4.7, causing the inactivation of GI. The treatment of sugarcane bagasse with diluted acid resulted in hydrolysate with 61.0g. L-1 of xylose, 7.3g.L-1 of arabinose, 7.2g.L-1 of glucose, 10.4g.L-1 of acetic acid, 0.53g.L-1 of furfural, 0.06g.L-1 of hydroxymethylfurfural and 4.3g.L-1 of lignin derivatives. Baker yeast was able to produce ethanol from non-detoxified liquid hydrolysate as efficiently as pure xylose medium. Simultaneous isomerization and fermentation using non-detoxified hydrolysate led to 23 g.L-1 ethanol, yield of 0.34g.g-1 and a productivity of 1.8g.L-1.h-1. Continuous process was conducted for 7 days. The operation for longer periods of time will be possible with pH control and microbial contamination prevention. / Neste estudo, a enzima glicose isomerase e a levedura de panificação Saccharomyces cerevisiae foram coimobilizadas em gel de alginato de cálcio para aplicação no processo contínuo de produção de etanol a partir de xilose e de hidrolisados de hemicelulose do bagaço de cana via simultânea isomerização e fermentação dos açúcares. A imobilização prévia da enzima em suportes porosos antecedeu o processo de coimobilização. Elevadas cargas de proteína foram imobilizadas no suporte glutaraldeído-quitosana (30-68 mg.g-1 de suporte). Neste caso, elevados rendimentos de imobilização (~100 %) e atividade recuperadas superiores a 90 % foram alcançados. Em condições típicas de simultânea isomerização e fermentação de xilose (pH 5, 35 °C, presença de nutrientes e concentrações de etanol até 70 g.L-1) a enzima imobilizada manteve aproximadamente 90% da atividade inicial após 120 horas. Carbonato de cálcio foi utilizado para manutenção do pH do meio de isomerização/fermentação superior a 5,0, garantindo elevada conversão da xilose a 32-35 °C. Nessas condições, concentrações de enzima e levedura no reator da ordem de 60&#61485;120&#61620;103UI.L-1 e 50 g.L-1, respectivamente, resultaram nos melhores rendimentos e seletividade em etanol, em batelada. Após o consumo de 65 g.L-1 de xilose no intervalo em 12 horas, em média, 21 g.L-1 de etanol e 14 g.L-1 de xilitol foram produzidos. Na ausência de carbonato de cálcio, a conversão do açúcar foi parcial devido à redução do pH do meio, que chegava a 4,7, impossibilitando a atividade de GI. O hidrolisado de hemicelulose resultante do tratamento do bagaço de cana com ácido diluído apresentou composição média de 61,0 g.L-1 de xilose; 7,3 g.L-1 de arabinose; 7,2 g.L-1 de glicose; 10,4 g.L- 1 de ácido acético; 0,53 g.L-1 de furfural; 0,06 g.L-1 de hidroximetilfurfural e 4,3 g.L-1 de derivados da lignina. A simultanea isomerização e fermentação de meio preparado com hidrolisado não destoxificado gerou 23 g.L-1 de etanol, com rendimento de 0,34 g.g-1 e produtividade de 1,8 g.L-1.h-1. O processo contínuo de simultanea isomerização e fermentação de xilose foi conduzido com rendimentos equivaventes ao processo descontínuo por um período de 7 dias. Nesse caso, a operação por longos períodos será possível com o controle do pH e prevenção contra contaminações microbianas.

The production of biofuels and in particular bioethanol has increased rapidly since the early 1990’s. The advantages of biofuels include reduced CO2 production, a decrease in fuel importation for many nations (notably the US and Brazil), and comparatively simple blending with fossil fuels. The production of basic fuel ethanol (1st generation) involves the use of an energy crop feedstock (corn in US and sugar cane in Brazil). The feedstock is processed via simple mechanical methods to release the simple carbohydrates, mixed with water and fermented anaerobically via S. cerevisiae yeast into ethanol and CO2. Due to the low market value of fuel ethanol, profit margins are restrictive, and as a result sterilisation and aseptic techniques are not economically viable, and contamination by environmental organisms is commonplace. The current system of biocontrol involves the addition of antibiotics, primarily penicillin and virginiamycin, to the fermentation. While these antibiotics are broad spectrum and highly effective in reducing the impact of contamination, the negative environmental impacts of antibiotic usage are well known. In order to reduce the impact of contamination and reduce reliance on antibiotics an alternative system of biocontrol is required. In this thesis various biocontrol agents are assessed, including bacteriophage, hop acids, chitosan, onion oil extract, copper and silver ions. The effect of these agents on the growth of various contaminant bacteria and a strain of S. cerevisiae is assessed and fermentations are carried out under sterile and controlled contaminated conditions to generate data on the effect of the contaminant and the various methods of biocontrol. Other possibilities investigated include the insertion of plasmids containing heat shock proteins into S. cerevisiae to enhance thermo-tolerance.