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A metodologia de superfície de resposta foi utilizada para otimizar as condições e obter maiores produção de etanol e viabilidade para a linhagem de S. cerevisiae 63M em processo descontínuo, resultando nas condições: 200 g.L-1 de sacarose, 40 g.L-1 de inóculo a 30 °C. Diferentes tipos de processos foram comparados e o processo que apresentou maiores viabilidade, produtividade e rendimento foi o descontínuo alimentado por pulsos de volumes decrescentes de sacarose a 30 °C. A redução da concentração de sacarose foi uma estratégia que permitiu aumentar a temperatura até 37 °C sem perdas em viabilidades. Uma linhagem utilizada nas destilarias brasileiras foi comparada com a linhagem 63M em temperaturas elevadas e observou-se que a 63M produziu maior produtividade e rendimento. Oito ciclos sucessivos de fermentação com reutilização de células da linhagem 63M foram realizados em meio sintético em processo descontínuo alimentado por pulsos de sacarose a 37 °C e uma perda gradual de viabilidade foi observada, mas o etanol final permaneceu constante nos oitos ciclos. / Surface response methodology was used to optimize the conditions and to obtain higher ethanol production and viability to strain 63M of S. cerevisiae in batch culture, resulting in the conditions: 200 g.L-1 sucrose, 40 g.L-1 inoculum at 30 °C. Different types of processes were compared and the process that presented higher viability, productivity and yield was pulse fed-batch using five decreasing pulses of sucrose at 30 °C. The reduction of the sucrose concentration was a strategy that allowed increasing the temperature up to 37 °C without losses in viabilities. An industrial strain used in Brazilian distilleries was compared with strain 63M at high temperatures and it was observed that strain 63M produced higher productivity and yield. Eight successive cycles of fermentation with reuse of cells of strain 63M were carried out in synthetic medium in fed-batch process using sucrose pulses at 37 °C and a gradual loss of viability was observed, but the final ethanol was kept constant in the eight fermentation cycles.

Chez les eucaryotes, les pores nucléaires (NPCs), ancrés dans l’enveloppe nucléaire (EN), régulent les échanges nucléocytoplasmiques. Ces complexes, très conservés, sont composés d’une trentaine de protéines appelées nucléoporines (Nups) présentes en multiples copies au sein de chaque NPC. Chez la levure S. cerevisiae, seules quatre Nups, dont la protéine Pom33, possèdent des domaines transmembranaires. Une étude réalisée en amont de ce projet a permis de caractériser Pom33 et de montrer que le mutant pom33∆ est viable et ne présente pas de défaut apparent de transport nucléocytoplasmique mais se caractérise par un défaut de distribution des NPCs. Pom33 joue également un rôle dans l’assemblage des pores nucléaires au sein de l’EN (biogenèse de novo des NPCs). POM33 appartient à une famille de gènes très conservés. Il possède un paralogue chez S. cerevisiae, PER33, qui code pour une protéine localisée majoritairement au réticulum endoplasmique et minoritairement aux NPCs et qui n’est pas impliquée dans la biogenèse des NPCs. Chez les mammifères, il n’existe qu’un homologue de Pom33/Per33, TMEM33. Dans le cadre de ce doctorat, nous nous sommes demandés quels étaient les déterminants contribuant à l’adressage spécifique de Pom33 au niveau des NPCs et à sa fonction dans la biogenèse de ces structures. La purification de Pom33-ProtA, suivie de spectrométrie de masse, nous a permis d’identifier un nouveau partenaire de Pom33, le facteur d’import Kap123. Des approches in vitro ont montré une interaction directe entre Kap123 et le domaine C-terminal (CTD) de Pom33, qui est perturbée en présence de RanGTP. Par ailleurs, des prédictions in silico ont révélé la présence dans ce domaine CTD de deux hélices amphipathiques, conservées chez l’humain. Des analyses par dichroïsme circulaire et flottaisons ont confirmé la capacité du CTD à s’organiser en hélice en présence de membranes lipidiques et à interagir préférentiellement avec les membranes très courbées. L’expression d’une version mutée de Pom33-CTD, incapable de se lier aux membranes et couplée à la GFP, a révélé la capacité de ce domaine à agir comme un NLS, importé spécifiquement dans le noyau par Kap123. Alors que la délétion du domaine CTD affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs, la mutation des résidus basiques impliqués dans l’interaction avec Kap123 ou des résidus permettant sa liaison aux membranes lipidiques ne récapitule pas ce phénotype. En revanche, la perte combinée de ces deux déterminants affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs ainsi qu'une interaction génétique avec le mutant nup133∆, impliqué dans la biogenèse de novo des NPCs. Les résultats obtenus lors de cette étude indiquent donc que l’adressage de Pom33 est un mécanisme actif et multifactoriel, qui met en jeu au moins deux déterminants dans son domaine CTD. Ces données indiquent également un rôle de ce domaine dans la biogenèse de novo des NPCs, qui pourrait néanmoins n’être qu’un effet indirect de son rôle dans l’adressage de Pom33 aux NPCs. Au cours de cette étude, nous avons également mis en évidence d’autres partenaires potentiels de Pom33, en particulier Myo2, une localisation de Pom33 au niveau du bourgeon lors de la division et une interaction génétique entre POM33 et KAP123. Ces observations préliminaires ouvrent de nouvelles pistes de réflexion quant au rôle de Pom33 lors de la division cellulaire. / In eukaryotic cells, nucleocytoplasmic exchanges take place through the nuclear pores complexes (NPCs). These conserved macromolecular assemblies are embedded in the nuclear envelope (NE) and composed of ~30 distinct proteins called nucleoporins (Nups), each presents in multiple copies. In the budding yeast Sacharomyces cerevisiae, there are only four transmembrane Nups, including Pom33. A previous study leds to the characterization of Pom33 and revealed that pom33∆ mutant cells, although viable and without apparent alteration in nucleocytoplasmic transport, display NPCs distribution defect. Pom33 also contributes to the biogenesis of NPCs into the intact NE (de novo biogenesis). Pom33 is highly conserved among species and has a paralogue in S. cerevisiae, Per33, which can associate with NPCs but is mainly localized at the endoplasmic reticulum (ER) and NE. Unlike Pom33, Per33 is not involved in NPCs distribution and biogenesis. In mammalian cells, there is a unique homologue of Pom33/Per33, named TMEM33. In the context of this thesis, we aimed to identify the determinants involved in the specific targeting of Pom33 to NPCs and in its function in pore biogenesis. To characterize these determinants, we first performed affinity-purification experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123 that involves positively-charged residues within Pom33-CTD and is altered in the presence of Ran-GTP. Moreover, in silico analyses predicted the presence of two evolutionarily-conserved amphipathic ~-helices within Pom33-CTD. Circular dichroism studies and liposome co-floatation assays confirmed that this CTD domain is able to fold into ~-helices in the presence of liposomes and revealed its preferential binding to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, Pom33-CTD behaves as a Kap123-dependent nuclear localization domain. While deletion of Pom33 C-terminal domain (Pom33-∆CTD-GFP) impairs Pom33 NPC targeting and stability and leads to a NPC distribution phenotype, mutants affecting either Kap123 binding or the amphipathic properties of the ~-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs and leads to an altered NPC distribution and a genetic interaction with the deletion of NUP133, a gene coding for a nucleoporin involved in NPCs biogenesis. Together, these results indicate that Pom33 targeting to NPCs is an active and multifactorial process that requires at least two determinants within its CTD. They also suggest a role of Pom33-CTD in the de novo NPCs biogenesis process, which could however only be an indirect consequence of its requirement for Pom33 targeting to NPCs. Our mass spectrometry analysis also identified other partners of Pom33, in particular Myo2, a molecular motor required for the cell cycle-regulated transport of various organelles and proteins and for correct alignment of the spindle during mitosis. Our studies also revealed a specific localization of Pom33 at the bud tip during mitosis and a genetic interaction between POM33 and KAP123. Taken together, these preliminary observations open new perspectives regarding additional functions of Pom33 during cell division.

The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible. / Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą]

Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą] / The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible.

A metodologia de superfície de resposta foi utilizada para otimizar as condições e obter maiores produção de etanol e viabilidade para a linhagem de S. cerevisiae 63M em processo descontínuo, resultando nas condições: 200 g.L-1 de sacarose, 40 g.L-1 de inóculo a 30 °C. Diferentes tipos de processos foram comparados e o processo que apresentou maiores viabilidade, produtividade e rendimento foi o descontínuo alimentado por pulsos de volumes decrescentes de sacarose a 30 °C. A redução da concentração de sacarose foi uma estratégia que permitiu aumentar a temperatura até 37 °C sem perdas em viabilidades. Uma linhagem utilizada nas destilarias brasileiras foi comparada com a linhagem 63M em temperaturas elevadas e observou-se que a 63M produziu maior produtividade e rendimento. Oito ciclos sucessivos de fermentação com reutilização de células da linhagem 63M foram realizados em meio sintético em processo descontínuo alimentado por pulsos de sacarose a 37 °C e uma perda gradual de viabilidade foi observada, mas o etanol final permaneceu constante nos oitos ciclos. / Surface response methodology was used to optimize the conditions and to obtain higher ethanol production and viability to strain 63M of S. cerevisiae in batch culture, resulting in the conditions: 200 g.L-1 sucrose, 40 g.L-1 inoculum at 30 °C. Different types of processes were compared and the process that presented higher viability, productivity and yield was pulse fed-batch using five decreasing pulses of sucrose at 30 °C. The reduction of the sucrose concentration was a strategy that allowed increasing the temperature up to 37 °C without losses in viabilities. An industrial strain used in Brazilian distilleries was compared with strain 63M at high temperatures and it was observed that strain 63M produced higher productivity and yield. Eight successive cycles of fermentation with reuse of cells of strain 63M were carried out in synthetic medium in fed-batch process using sucrose pulses at 37 °C and a gradual loss of viability was observed, but the final ethanol was kept constant in the eight fermentation cycles.

Le vieillissement est un processus complexe déterminé par des facteurs génétiques et environnementaux qui varie d’un individu à l’autre. Bien que le vieillissement soit la cause principale de nombreuses maladies, nos connaissances sur le sujet sont relativement limitées. Tout au long de ce travail, j’ai utilisé la levure bourgeonnante Saccharomyces cerevisiae pour identifier les facteurs génétiques et environnementaux influant sur le vieillissement et pour comprendre les interactions qu’ils entretiennent entre eux. Jusqu’à présent, les approches classiques de génétique ont permis de découvrir un certain nombre de gènes impliqués dans la régulation du vieillissement chronologique de la levure (CLS), basé sur la longévité de celle-ci en conditions non-prolifératives. Or, ces approches se sont essentiellement centrées sur des souches de laboratoire et n’ont que très peu exploité les richesses de la biodiversité. Dans une première partie, j’ai utilisé une large cohorte de levures composée de plus de 1000 souches naturelles de S. cerevisiae afin d’estimer la variabilité de longévité existant au sein de l’espèce. Leur longévité a été étudiée dans différentes conditions connues pour freiner le vieillissement : sous restriction calorique ou en présence d’un agoniste de la restriction calorique, la molécule rapamycine, qui inhibe directement la voie de signalisation TOR. Les microorganismes passent la majeure partie de leur vie dans des environnements défavorables, pauvres en ressources nutritives. Leur capacité à survivre à ces périodes de restriction (CLS) est donc primordiale. J’ai observé que les souches sauvages ont tendance à spontanément initier le programme de méiose aboutissant à la formation de spores lorsque les conditions environnementales deviennent restreintes. En revanche, les souches domestiques préfèrent entrer en quiescence, ce qui leur confère une viabilité et une résistance accrues. De plus, en ayant recours à une approche basée sur des gènes présélectionnés et à une étude d’association pangénomique, j’ai observé que la variabilité de longévité entre les différentes souches est déterminée par un large spectre de polymorphismes génétiques, tels que des mutations non-synonymes ou non-sens, et par l’absence ou la présence de certains gènes. Toutes ces composantes génétiques interagissent pleinement avec l’environnement. Dans une deuxième partie, j’ai réalisé une analyse de liaison génétique grâce à 1056 souches descendantes de deux souches parentales. La longévité (CLS) de ces 1056 souches a été mesurée dans le but d’identifier des locus de caractères quantitatifs (QTLs). Le vieillissement chronologique a été déterminé à la fois à partir d’un milieu riche, d’un milieu restreint en calories, ou en présence de rapamycine. J’ai identifié 30 QTLs distincts, certains d’entre eux sont communs et récurrents dans plusieurs environnements, tandis que d’autres sont plus spécifiques et occasionnels. Les deux QTLs principaux, associés aux gènes HPF1 et FLO11, codent tous deux des protéines du mur cellulaire, et sont jusqu’à présent non reconnus comme régulateurs du vieillissement. Etonnement, ces deux gènes contiennent des répétitions d’ADN en tandem qui s’avèrent être massivement amplifiées dans une des deux souches parentales d’origine. Alors que les allèles courts de HPF1 et FLO11 n’ont pas d’effet sur le vieillissement, les allèles longs sont relativement délétères, hormis en présence de rapamycine. Après investigation, il semble que la forme allongée de HPF1 provoque la flottaison des cellules de levure au cours de la phase de croissance, les exposants à des taux plus élevés d’oxygène. / Aging is a classical complex trait varying quantitatively among individuals and affected by both the genetic background and the environment. While aging is the highest risk factor for a large number of diseases, little is known about the underlying molecular mechanisms. Identifying the causal genetic variants underlying natural variation in longevity and understanding their interaction with the genetic background and the environment remains a major challenge. In this work, I used the budding yeast, Saccharomyces cerevisiae, to identify environmental and genetic factors contributing to aging. While extensive classical genetic studies discovered several genes involved in the regulation of chronological lifespan (CLS), which measures cell viability dynamic in non-dividing condition, using laboratory strains in standard conditions, there are only few studies exploiting variations in natural populations. In the first part, I used a large cohort of more than 1000 sequenced natural S. cerevisiae strains to provide a species-wide overview of CLS variability. Longevity was measured in different environments, including calorie restriction (CR), a natural intervention known to increase lifespan, and in the presence of rapamycin (RM), a drug that mimics CR by downregulating the TOR pathway. Unicellular microorganisms spend most of their lifetime in harsh restricted environments interrupted by short windows of growth, making CLS an important and likely adaptive trait. I found that wild strains subjected to CLS tend to trigger the meiotic developmental process leading to the formation of gametes wrapped into a very resistant cell wall. In contrast, domesticated strains tend to enter quiescence state when starved and display a tremendous variability in their survival capacity. Moreover, using both candidate gene approach and genome-wide association studies (GWAS), I demonstrated that variability in CLS is determined by a full spectrum of genetic variant that include gene presence/absence, copy number variation, non-synonymous SNPs and loss of function. All these genetic features were strongly regulated by the environment. In the second part, I performed linkage analysis using 1056 diploid segregants derived from a two parent advanced intercross. These 1056 diploid segregants were phenotyped for CLS to map quantitative trait loci (QTLs). The CLS was measured in complete media, CR and RM environments across multiple time points. I mapped 30 distinct QTLs, with some shared across different environments and time points, while others were unique to a specific condition. The two major effect size QTLs were linked with natural variation in the cell wall glycoproteins FLO11 and HPF1, previously unknown to regulate CLS. Interestingly, both genes presented massive intragenic tandem repeat expansions in one of the founder strain used in the crossing scheme. While the short versions of FLO11 and HPF1 alleles did not impact CLS, tandem repeat expansions within those genes were sufficient to confer a dominant detrimental effect that was partially buffered by rapamycin treatment. Further investigation revealed that the extended form of HPF1 makes cells floating during exponential phase, exposing them to higher oxygen rates, and leading to perturbation of redox homeostasis, activation of misfolded protein response, and alteration of multiple genes involved in methionine, ribosome and lipid biosynthesis, eventually contributing to CLS shortening. Taken together, my work provided an unprecedented overview of natural variation in CLS in a genetic model system and revealed multiple genetic and environmental factors that shape the species phenotypic variation.

É esperado que a produção de combustíveis a partir de resíduos lignocelulósicos alcance representatividade na matriz energética mundial. Neste cenário, leveduras desempenharão importante papel como plataformas microbianas para os processos de conversão dos açúcares derivados da biomassa em etanol (de segunda geração) e em outros produtos de interesse. Apesar dos avanços consideráveis na área, a fermentação de hidrolisados lignocelulósicos ainda apresenta alguns desafios científicos e tecnológicos, como por exemplo, os problemas enfrentados na fermentação devido à presença de diversos inibidores oriundos dos processos de pré-tratamento e hidrólise da biomassa (tais como furaldeídos, compostos fenólicos e ácidos orgânicos). A geração destes inibidores reduz consideravelmente a eficiência da etapa fermentativa, e muitas vezes, inviabiliza o processo como um todo. Neste contexto, o presente trabalho estudou a identificação dos principais compostos lignocelulósicos inibidores na fermentação por leveduras presentes em cinco amostras de hidrolisados industriais. Ácido glicólico e ácido acético, dentre os ácidos orgânicos foram identificados em todas as amostras analisadas. Em relação aos furaldeídos, foram detectados furfural e 5- hidroximetil-furufural (HMF) em todas as amostras analisadas. Os principais e mais abundantes compostos fenólicos identificados pelo sistema foram ácido p-cumárico (em 4 de 5), ácido ferúlico (em 3 de 5) e ácido vanílico (em 3 de 5). Tendo os principais compostos quantificados e identificados, realizou-se um estudo de mínimas e máximas concentrações de alguns destes inibidores em quatro diferentes linhagens laboratoriais e industriais (CEN.PK113-7D, CEN.PK112, SA-1 e JAY270), a fim de verificar a toxicidade dos mesmos frente aos principais parâmetros cinéticos da fermentação (fase lag, µmáx, produção de biomassa e etanol). De forma geral, a linhagem industrial S. cerevisiae SA-1 se mostrou mais tolerante frente às demais linhagens, em praticamente todas as condições analisadas. Visto que a presença de algumas bactérias nas linhas de produção de etanol é sabida, procurou-se verificar a resistência destas na presença dos compostos inibitórios. Ao comparar a resistência frente aos inibidores entre bactérias lácticas e leveduras laboratoriais e industriais, notou-se uma potencial robustez das bactérias frente às leveduras, uma vez que as últimas foram incapazes de crescer no meio de cultivo acrescido de um coquetel de inibidores, enquanto que as bactérias apresentaram crescimento. Por fim, investigou-se a influência conjunta de três compostos inibitórios (ácido acético, HMF e ácido p-cumárico) sobre os principais parâmetros fisiológicos da linhagem SA-1, a mais tolerante das linhagens avaliadas. Assim, através de um delineamento composto central rotacional (DCCR), pode-se analisar estatisticamente os efeitos dos três inibidores, com o qual verificou-se que a maior toxicidade (redução da velocidade específica de crescimento) é exercida pelo HMF. De uma maneira geral, a linhagem SA-1 pode ser considerada uma plataforma microbiana com grande potencial de aplicação na produção de etanol de segunda geração, bem como no estudo das bases moleculares para tolerância aos inibidores oriundos desses processos. / It is expected that the production of fuels from lignocellulosic residues will be representative in the world energy matrix. In this scenario, yeasts will play an important role as microbial platforms for the conversion processes of sugars derived from biomass into ethanol and other products of interest. Despite the considerable advances in the area, the fermentation of lignocellulosic hydrolysates still presents some scientific and technological challenges, such as the problems faced in the fermentation due to the presence of several inhibitors from the processes of pretreatment and hydrolysis of biomass (such as furaldehydes, phenolic compounds and organic acids). The presence of these inhibitors considerably reduces the efficiency of the fermentation step, and often hampers the process itself. In this context, the present work aimed to identify major inhibitory lignocellulosic compounds for yeast fermentation present in five samples of industrial hydrolysates. Glycolic acid and acetic acids were identified in all samples analysed. In relation to furaldehydes, furfural and 5-hydroxymethyl-furfural (HMF) were detected in all samples as well. The major and most abundant phenolic compounds identified were p-coumaric acid (in 4 out of 5 samples), ferulic acid (in 3 out of 5) and vanillic acid (in 3 out of 5). After quantification of these compounds, minimum and maximum concentrations of the major ones were studied in four different laboratory and industrial strains (CEN.PK113-7D, CEN.PK112, SA-1 and JAY270), in order to verify their toxicity against physiological parameters such as elongation of lag phase and growth rate. In general, the S. cerevisiae SA-1 industrial strain was more tolerant in comparison to the other 3 strains for virtually all compounds investigated. Since bacteria is present during fuel ethanol production, , the resistance of these bacteria for the same inhibitory compounds was also evaluated. When comparing the resistance against inhibitors between lactic acid bacteria and yeast strains, a higher robustness of bacteria in relation to yeasts was observed, since yeasts were unable to grow in a medium supplemented with a cocktail of inhibitors, whereas bacteria did grow. Finally, the influence of three inhibitory compounds (acetic acid, HMF and p-coumaric acid) on the main physiological parameters of the strain SA-1, the most tolerant among the strains evaluated, was investigated. Thus, using a rotatable central composite design (RCCD), the effects of the three inhibitors were analysed, and it was found that the highest toxicity (reduction in specific growth rate) is caused by HMF. Overall, SA-1 is a promising platform yeast strain for second generation ethanol production and for understanding strain robustness toward lignocellulosic inhibitory compounds.