In this thesis I describe a functional genomics resource in which each yeast gene, with its native promoter and 3’UTR, is cloned on a uniquely barcoded low-copy vector. We refer to this resource as the Molecular Barcoded Yeast ORF (MoBY-ORF) library 1.0. Each gene carried by MoBY-ORF 1.0 should mimic its native expression and thus is best suited for complementation cloning. The vector backbone of MoBY-ORF 1.0 is compatible with the mating-assisted genetically integrated cloning (MAGIC) system for recombination cloning in bacterial cells, which allows the transfer of the ORF fragment and its barcoded cassette to other vector backbones. Taking advantage of the MAGIC system, we created a multi-copy version of the library, which we refer to as MoBY-ORF 2.0. I used MoBY-ORF 1.0 to map drug resistant mutants by complementation cloning with a barcode microarray readout. I investigated several drugs with known targets in my proof-of-principle experiments and showed the feasibility of this method. I identified a single mutation that causes resistance to two different natural products, theopalauamide and stichloroside. By doing so, I was able to link these two chemicals to their cellular target, ergosterol. In fact, theopalauamide represents a new class of sterol binding chemical. I also describe the use of MoBY-ORF 2.0 to clone dosage suppressors of conditional temperature-sensitive mutants. By doing so, and combing our own data with published literature, we showed that dosage suppression interactions often overlap with protein-protein interactions and negative genetic interactions but not positive interactions; however the majority of dosage suppression interactions are unique and thus they represent an unique edge on a global functional interaction map. We also describe the first genome-wide dosage suppressor interaction map of budding yeast.

In this thesis I describe a functional genomics resource in which each yeast gene, with its native promoter and 3’UTR, is cloned on a uniquely barcoded low-copy vector. We refer to this resource as the Molecular Barcoded Yeast ORF (MoBY-ORF) library 1.0. Each gene carried by MoBY-ORF 1.0 should mimic its native expression and thus is best suited for complementation cloning. The vector backbone of MoBY-ORF 1.0 is compatible with the mating-assisted genetically integrated cloning (MAGIC) system for recombination cloning in bacterial cells, which allows the transfer of the ORF fragment and its barcoded cassette to other vector backbones. Taking advantage of the MAGIC system, we created a multi-copy version of the library, which we refer to as MoBY-ORF 2.0. I used MoBY-ORF 1.0 to map drug resistant mutants by complementation cloning with a barcode microarray readout. I investigated several drugs with known targets in my proof-of-principle experiments and showed the feasibility of this method. I identified a single mutation that causes resistance to two different natural products, theopalauamide and stichloroside. By doing so, I was able to link these two chemicals to their cellular target, ergosterol. In fact, theopalauamide represents a new class of sterol binding chemical. I also describe the use of MoBY-ORF 2.0 to clone dosage suppressors of conditional temperature-sensitive mutants. By doing so, and combing our own data with published literature, we showed that dosage suppression interactions often overlap with protein-protein interactions and negative genetic interactions but not positive interactions; however the majority of dosage suppression interactions are unique and thus they represent an unique edge on a global functional interaction map. We also describe the first genome-wide dosage suppressor interaction map of budding yeast.

Resumo: Xanthomonas axonopodis pv. citri (Xac) constitui um dos principais patógenos da citricultura mundial. Essa litobactéria causa cancro em folhas. ramos e frutos de citros em geral. ocasionando grandes perda econômicas anuais. A fim de estudar a interação dessa bactéria com seu hospedeiro empregou-se a análise de mutantes e da expressão gênica global por meio de microarranjos de DNA (transcrissoma). Na primeira parte do estudo foi obtida uma biblioteca de cerca de 10.000 Illutantes do isolado 306 de Xac. Desses. 3.300 foram inoculados em folhas de limoeiro cravo e sua capacidade em causar cancro cítrico foi avaliada aos três dias da inoculação. Após quatro inoculaçàes. 56 mutantes foram selecionados por apresentarem virulência alterada e suas ORFs interrompidas foram identificadas por meio de seqüenciamento. Uma análise dessas ORFs mostrou que havia tanto ORFs quc codificavam para genes relacionados com a patogenicidade em fitobactérias como possihilitou identificar novos genes associados à infecção. além de ORFs hipotéticas. para as quais ainda não foi atrihuída nenhuma função. Dentre os genes previamente implicados na patogênese. pode-se citar o gene "rpB4. o qual participa do sistema de secreção do tipo três. Já o gene hrlA. que contribui na virulência de algumas bactérias patogênicas de animais. até o presente ainda não tinha sido relatado como tendo importância no processo de patogênese em fitobactérias. A ORF XACO~40 é um exemplo de uma proteína hipotética que interfere significativamente no processo infeccioso. uma vez que o mutante para esse locos não induziu sintomas da doença. assim como os demais mutantes citados acima. Na segunda parte deste trabalho. produziu-se um microarranjo de DNA contendo 6.9 I 2 sondas imobilizadas (2.673 ORFs. 87 ORFs repetidas. 312 controles negativos e X4 controles positivos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Xanthomonas axonopodis pv. citri constitutes one of the main and most damaging pathogcn of lhe world-wide citriculture. This phytobacteria causes canker in leaves, branches and fruits of citrus. causing scvcre cconomic losses. In order to study the interaction of this bacterium with its citrus host. two approaches had been used to proceed a post-genomic functional analysis: wholc genol11c mutagcncsis and global expression analysis by cDNA microarrays technique. In the first part of this study a Xac 306 mutants library with '" 10,000 mutants was obtained. From thesc. 3.300 wcrc inoculatcd in mexicam lemon tree leaves and its capacity to cause citrus canker was cvalualcd. Aftcr four inoculation cicIes, 56 mutants had been selected as having modified palhogcnicily. The inlcrruplcd ORFs were identified by DNA sequencing and analysis of these ORFs identificd ORFs coding for genes previously known as related to pathogenicity proccss in phytobactcrilll11 as wcll gcnes not yet described as involved in pathogenicity, incIuding hypothetical gcncs. Al110ng the gencs previously known as implied in pathogens attack, the hrpB4 gene is one that was found, whic participates of the type three secretion system. To the hrtA gene, whose participation in the virulcnce process has been described to some animal pathogenic bacteria but never yet as having a role in to pathogenicity process in phytobacteria, was found to be involved in the pathogenicity of Xac. The ORF XAC0340 is an example of hypothetical protein which presents grcat intcrfcrcncc in thc infcctious process: the mutant for this gene does not induce any diseasc sYl11ploms in citric Icaves...(Complete abstract, click electronic access below) / Orientador: Jesus Aparecido Ferro / Coorientador: Julio Cezar Franco de Oliveira / Banca: Acelino Couto Alfenas / Banca: Shaker Chuck Parah / Banca: Eliana Gertrudes Macedo Lemos / Banca: Maria Helena de Souza Goldman / Doutor

Made available in DSpace on 2014-06-11T19:32:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:21:47Z : No. of bitstreams: 1 laia_ml_dr_jabo_prot.pdf: 9478549 bytes, checksum: f92e822aa9f06920bc2065ca991c0323 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Xanthomonas axonopodis pv. citri (Xac) constitui um dos principais patógenos da citricultura mundial. Essa litobactéria causa cancro em folhas. ramos e frutos de citros em geral. ocasionando grandes perda econômicas anuais. A fim de estudar a interação dessa bactéria com seu hospedeiro empregou-se a análise de mutantes e da expressão gênica global por meio de microarranjos de DNA (transcrissoma). Na primeira parte do estudo foi obtida uma biblioteca de cerca de 10.000 Illutantes do isolado 306 de Xac. Desses. 3.300 foram inoculados em folhas de limoeiro cravo e sua capacidade em causar cancro cítrico foi avaliada aos três dias da inoculação. Após quatro inoculaçàes. 56 mutantes foram selecionados por apresentarem virulência alterada e suas ORFs interrompidas foram identificadas por meio de seqüenciamento. Uma análise dessas ORFs mostrou que havia tanto ORFs quc codificavam para genes relacionados com a patogenicidade em fitobactérias como possihilitou identificar novos genes associados à infecção. além de ORFs hipotéticas. para as quais ainda não foi atrihuída nenhuma função. Dentre os genes previamente implicados na patogênese. pode-se citar o gene rpB4. o qual participa do sistema de secreção do tipo três. Já o gene hrlA. que contribui na virulência de algumas bactérias patogênicas de animais. até o presente ainda não tinha sido relatado como tendo importância no processo de patogênese em fitobactérias. A ORF XACO~40 é um exemplo de uma proteína hipotética que interfere significativamente no processo infeccioso. uma vez que o mutante para esse locos não induziu sintomas da doença. assim como os demais mutantes citados acima. Na segunda parte deste trabalho. produziu-se um microarranjo de DNA contendo 6.9 I 2 sondas imobilizadas (2.673 ORFs. 87 ORFs repetidas. 312 controles negativos e X4 controles positivos... / Xanthomonas axonopodis pv. citri constitutes one of the main and most damaging pathogcn of lhe world-wide citriculture. This phytobacteria causes canker in leaves, branches and fruits of citrus. causing scvcre cconomic losses. In order to study the interaction of this bacterium with its citrus host. two approaches had been used to proceed a post-genomic functional analysis: wholc genol11c mutagcncsis and global expression analysis by cDNA microarrays technique. In the first part of this study a Xac 306 mutants library with ' 10,000 mutants was obtained. From thesc. 3.300 wcrc inoculatcd in mexicam lemon tree leaves and its capacity to cause citrus canker was cvalualcd. Aftcr four inoculation cicIes, 56 mutants had been selected as having modified palhogcnicily. The inlcrruplcd ORFs were identified by DNA sequencing and analysis of these ORFs identificd ORFs coding for genes previously known as related to pathogenicity proccss in phytobactcrilll11 as wcll gcnes not yet described as involved in pathogenicity, incIuding hypothetical gcncs. Al110ng the gencs previously known as implied in pathogens attack, the hrpB4 gene is one that was found, whic participates of the type three secretion system. To the hrtA gene, whose participation in the virulcnce process has been described to some animal pathogenic bacteria but never yet as having a role in to pathogenicity process in phytobacteria, was found to be involved in the pathogenicity of Xac. The ORF XAC0340 is an example of hypothetical protein which presents grcat intcrfcrcncc in thc infcctious process: the mutant for this gene does not induce any diseasc sYl11ploms in citric Icaves...(Complete abstract, click electronic access below)

>Magister Scientiae - MSc / Functional genomics determines the biological functions of genes on a global scale by using large volumes of data obtained through techniques including next-generation sequencing (NGS). The application of NGS in biomedical research is gaining in momentum, and with its adoption becoming more widespread, there is an increasing need for access to customizable computational workflows that can simplify, and offer access to, computer intensive analyses of genomic data. In this study, the Galaxy and Ruffus frameworks were designed and implemented with a view to address the challenges faced in biomedical research. Galaxy, a graphical web-based framework, allows researchers to build a graphical NGS data analysis pipeline for accessible, reproducible, and collaborative data-sharing. Ruffus, a UNIX command-line framework used by bioinformaticians as Python library to write scripts in object-oriented style, allows for building a workflow in terms of task dependencies and execution logic. In this study, a dual data analysis technique was explored which focuses on a comparative evaluation of Galaxy and Ruffus frameworks that are used in composing analysis pipelines. To this end, we developed an analysis pipeline in Galaxy, and Ruffus, for the analysis of Mycobacterium tuberculosis sequence data. Furthermore, this study aimed to compare the Galaxy framework to Ruffus with preliminary analysis revealing that the analysis pipeline in Galaxy displayed a higher percentage of load and store instructions. In comparison, pipelines in Ruffus tended to be CPU bound and memory intensive. The CPU usage, memory utilization, and runtime execution are graphically represented in this study. Our evaluation suggests that workflow frameworks have distinctly different features from ease of use, flexibility, and portability, to architectural designs.

BACKGROUND: The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF. METHODS: Total RNA of peripheral blood mononuclear cells was subjected to microarray profiling in a training (45 IPF individuals) and two independent validation cohorts (21 IPF/10 controls, and 75 IPF individuals, respectively). To identify a gene set predictive of IPF prognosis, we incorporated genomic, clinical, and outcome data from the training cohort. Predictor genes were selected if all the following criteria were met: 1) Present in a gene co-expression module from Weighted Gene Co-expression Network Analysis (WGCNA) that correlated with pulmonary function (p < 0.05); 2) Differentially expressed between observed "good" vs. "poor" prognosis with fold change (FC) >1.5 and false discovery rate (FDR) < 2 %; and 3) Predictive of mortality (p < 0.05) in univariate Cox regression analysis. "Survival risk group prediction" was adopted to construct a functional genomic model that used the IPF prognostic predictor gene set to derive a prognostic index (PI) for each patient into either high or low risk for survival outcomes. Prediction accuracy was assessed with a repeated 10-fold cross-validation algorithm and independently assessed in two validation cohorts through multivariate Cox regression survival analysis. RESULTS: A set of 118 IPF prognostic predictor genes was used to derive the functional genomic model and PI. In the training cohort, high-risk IPF patients predicted by PI had significantly shorter survival compared to those labeled as low-risk patients (log rank p < 0.001). The prediction accuracy was further validated in two independent cohorts (log rank p < 0.001 and 0.002). Functional pathway analysis revealed that the canonical pathways enriched with the IPF prognostic predictor gene set were involved in T-cell biology, including iCOS, T-cell receptor, and CD28 signaling. CONCLUSIONS: Using supervised and unsupervised analyses, we identified a set of IPF prognostic predictor genes and derived a functional genomic model that predicted high and low-risk IPF patients with high accuracy. This genomic model may complement current prognostic tools to deliver more personalized care for IPF patients.

Lactococcus lactis est une espèce appartenant au groupe des bactéries lactiques, largement utilisées dans l’industrie pour leur capacité à produire de l’acide lactique au cours de la fabrication des produits laitiers fermentés. L’étude de la diversité globale de L. lactis ssp. lactis a été entreprise par l’intégration de données biologiques obtenues à partir d’analyses génétiques, génomiques, physiologiques, transcriptomiques et métaboliques. L’accès à la phylogénie de l’espèce par l’étude de la variabilité génétique du génome cœur par MLST (MultiLocus Sequence Typing) a permis de décrire deux groupes de souches : les souches environnementales, génétiquement très diverses, isolées de laits crus, de plantes ou d’animaux et les souches domestiquées, génétiquement très proches, isolées des levains utilisés dans l’industrie laitière. Malgré la perte de diversité génétique observée dans les souches domestiquées probablement associée à un processus de spécialisation à un environnement technologique, l’approche intégrative a permis de montrer que ce groupe présente une diversité génomique et fonctionnelle aussi importante que les souches environnementales. L’investigation des génomes de la sous-espèce lactis par la mesure de la taille des chromosomes et la caractérisation en nombre et en taille du contenu plasmidique, a révélé une variabilité de plus de 300 kb en capacité de codage génétique des souches domestiquées et environnementales. D’autre part, les souches domestiquées appartenant au biovar Diacetylactis ont montré des physiologies et des régulations métaboliques différentes, résultant en une production d’arômes de type diacetyl ou acétoïne variable selon la souche. Enfin, le séquençage du génome de la souche environnementale A12 isolée d’un levain de panification, et sa comparaison avec les 4 génomes actuellement séquencés de L. lactis a révélé un pangénome (ensemble des gènes de l’espèce) étendu, montrant que cette espèce offre un grand réservoir de diversité. Environ 20 % des gènes spécifiques de souches ont été mis en évidence témoignant des grandes capacités adaptatives de la sous-espèce. L’étude approfondie de la souche A12 par une analyse transcriptomique a permis de rendre compte des mécanismes impliqués dans l’adaptation d’une souche à un écosystème complexe / The Lactococcus lactis species belong to lactic acid bacteria group widely used for their ability to produce lactic acid in fermented dairy products. The study of the global diversity of L. lactis ssp. lactis was carry out by the integration of biological data obtained from genetic, genomic, physiological, transcriptomic and metabolic analyses. The genetic variability investigated by MLST (MultiLocus Sequence Typing) describe two strains groups according to their phylogeny : the “environmental” strains, displaying high genetic diversity and isolated from different natural environments such as raw milks, plants and animals and the “domesticated” strains, genetically closely related, isolated from starters in dairy industries. Despite the lost of genetic diversity in domesticated strains, probably associated to a specialisation process, the integrative approach showed a genomic and functional diversity as huge as in environmental strains. The characterization of chromosome size and plasmidic content of the lactis subspecies revealed a variation higher than 300 kb in genetic coding capacity for domesticated and environmental strains. Moreover, the domesticated strains belonging to the biovar Diacetylactis showed different physiologies and metabolic regulations resulting in variable amount of aroma produced according to the strains. Finally, the genome sequencing of the A12 strain isolated from sourdough bread and its comparison with 4 other L. lactis genomes already sequenced revealed a spread pangenome (all the genes of a species). Approximately 20 % of each genome correspond to strain specific genes, showing large adaptive capacities of the subspecies. The in-depth study of the A12 strain by transcriptomic analysis allows to highlight mechanisms involved in the adaptation of a strain to a complex ecosystem

A inativação do cromossomo X (ICX) em fêmeas é um exemplo de regulação epigenética. O silenciamento de um dos cromossomos X leva à formação estável da heterocromatina facultativa através da aquisição de múltiplas modificações na cromatina que são mantidas nas subsequentes divisões celulares. Atualmente, algumas características epigenéticas associadas à manutenção da ICX têm sido descritas, contudo os mecanismos de ação e a identidade dos diferentes fatores envolvidos na manutenção da ICX ainda são desconhecidos ou pouco compreendidos. Nosso laboratório realizou uma triagem funcional genômica por bibliotecas de shRNAs (short harpin RNAs) para encontrar genes envolvidos na manutenção da ICX em humanos. A partir deste estudo foram identificados 20 novos genes candidatos a estarem envolvidos na manutenção da ICX. Assim, o objetivo deste trabalho foi validar o grau de envolvimento de dois destes genes candidatos (H3F3B e ASF1A) no processo de controle epigenético do cromossomo X. Para isto, foi realizado o silenciamento dos genes candidatos através da utilização de partículas lentivirais portando shRNAs específicos em fibroblastos primários femininos heterozigotos para uma mutação no gene HPRT e com desvio total de ICX, onde o único alelo normal do gene HPRT está no Xi. A reativação do Xi nestas células foi avaliada por cultivo das mesmas em meio HAT, que seleciona células HPRT+. Só sobreviveram os fibroblastos que foram silenciados para o gene H3F3B. Nestes, as células transduzidas com o shH3F3B.2 expressam o alelo selvagem do gene, presente no Xi, além do gene mutante. Ensaios de RNA-FISH e trimetilação de histonas foram feitos nessas células para avaliar a perda das marcas de cromatina inativa. Foi observada uma perda da nuvem de XIST nas células transduzidas com o shH3F3B.2 e selecionadas em HAT em passagens altas. Por último, análises de expressão alelo-específica de genes ligados ao X comprovaram que dois genes que são submetidos à ICX apresentaram expressão do alelo inativado (FLNA e FHL1). Porém, também foi observada uma mudança no padrão de expressão alelo-específica em genes autossômicos. Finalmente, as análises de expressão geral do cromossomo X mostraram que as células transduzidas com o shH3F3B.2 e selecionada em HAT tinham uma expressão gênica aumentada em relação ao controle. Em conclusão, nossos resultados sugerem uma descondensação da cromatina no cromossomo Xi e portanto um provável envolvimento do gene H3F3B na manutenção da ICX. / The X chromosome Inactivation (XCI) in females is an example of epigenetic regulation. Silencing of one of the X chromosomes leads to the stable formation of the facultative heterochromatin through the acquisition of multiple modifications in the chromatin that are maintained in the subsequent cell divisions. Currently, some epigenetic features associated with the maintenance of XCI have been described. Nonetheless, the mechanisms of action and the identity of the different factors involved in the maintenance of XCI are still unknown or poorly understood. Our laboratory performed a genomic functional screening by shRNA (short harpin RNAs) libraries to find genes involved in the maintenance of XCI in humans. From this study, we identified 20 new candidate genes to be involved in the maintenance of XCI. Thus, the objective of this work was to validate the degree of involvement of two of these candidate genes (H3F3B and ASF1A) in the epigenetic process control of the X chromosome. For this, the silencing of the candidate genes was performed in female heterozygous primary fibroblasts for a mutation of the HPRT gene and with a total XCI shift through the use of lentiviral particles carrying specific shRNAs, where the only normal allele of the HPRT gene is in the Xi (inactivated X). Xi reactivation was evaluated in these cells by culturing them in HAT medium, which selects HPRT + cells. Only the fibroblasts that were silenced for the H3F3B gene survived. Furthermore, the cells transduced with shH3F3B.2 express the HPRT wild gene allele, present in Xi, in addition to the mutant gene. RNA-FISH and histone trimethylation assays were performed on these cells to evaluate the loss of inactive chromatin marks. A loss of the XIST cloud was observed in cells transduced with shH3F3B.2 and selected in HAT at high passages. Finally, allele-specific expression analyzes of X-linked genes showed that two genes that undergo XCI showed expression of the inactivated allele (FLNA and FHL1). However, a change in allele-specific expression pattern was also observed in autosomal genes. Finally, the X chromosome general expression analyses showed that cells transduced with shH3F3B.2 and selected on HAT had increased gene expression relative to the control. In conclusion, our results suggest a decondensation of the chromatin in the Xi chromosome and therefore a probable involvement of the H3F3B gene in the maintenance of ICX.

Em estudos de proteção ao genoma contendo lesões de C. crescentus, foi feita uma varredura em uma biblioteca clones mutados pelo transposon Tn5 e sensíveis à luz UV-B e MMS. Dos 249 clones selecionados conseguimos identificar mutações em 102 genes, distribuídos em dez categorias funcionais, incluindo metabolismo de DNA. Além de genes já descritos como atuantes na defesa de genoma a lesões, os dados ainda adicionam funções potenciais para outros genes. Investigação mais detalhada indica que vários dos genes identificados podem afetar o estresse e ciclo celular, podendo estar relacionados a respostas do tipo pontos de checagem. Além disso, verificamos que mutantes para genes envolvidos em reparo excisão de nucleotídeos são mais sensíveis à H2O2, indicando que nessa bactéria, este sistema de reparo atua também em lesões oxidativas no DNA. Os dados obtidos são pioneiros no estabelecimento de funções para vários genes de C. crescentus e de outras alfa-proteobactérias. / This work aimed to identify genes related to DNA damage protection in C. crescentus, by means of screening a Tn5 -mutated library for clones sensitive to UV-B light and MMS. Out of 249 selected clones, we were able to identify mutations in 102 genes, classified in ten different functional categories, including DNA metabolism. In addition to genes already described as playing a role in genome defense to lesions, the results provide new potential functions to several other genes. More detailed investigation indicates also that the mutations in some of these genes may also affect cell stress and cell cycle, being potentially involved in check-point responses. Moreover, mutants for nucleotide excision repair genes were also found to be sensitive to H2O2, indicating that this DNA repair system also acts in DNA oxidative damage. These data are the first establishing a role in genome protection for several genes in C. crescentus, as well as other alpha-proteobacteria.

O fungo filamentoso Trichoderma reesei é um dos principais fungos utilizados para a produção em larga escala de enzimas devido a sua grande capacidade de produção e secreção de holocelulases para aplicação em processos de sacarificação da biomassa vegetal lignocelulósica. Embora o T. reesei seja utilizado como um dos principais produtores de celulases a nível industrial, diversos processos e estudos são realizados com o objetivo de aprimorar o entendimento de todo o mecanismo de degradação de biomassa vegetal além de prover o aumento da eficiência tanto da produção quanto da atividade das celulases. No presente trabalho foi realizado a construção de uma linhagem mutante para o gene cel74a (codificando para uma xiloglucanase) e a caracterização funcional de CEL74A na regulação gênica de holocelulases durante o cultivo em bagaço de cana-de-açúcar. Os nossos resultados mostraram que deleção de cel74a possivelmente pode estar envolvida no sinergismo da regulação da expressão de holocelulases durante o cultivo em bagaço de cana-de-açúcar. A partir da análise do perfil de expressão gênica, foi possível observar a redução na expressão de todos os genes de celulases testados (cel7a, cel7b e cel6a) embora não tenha afetado a atividade enzimática, ao passo que as hemicelulases (xyn1 e xyn2) apresentaram aumento tanto na expressão quanto na atividade enzimática. Na linhagem ?cel74a, foi observado redução na liberação de glicose, xilose e galactose após a hidrólise de xiloglucano. Além disso, a atividade de CEL74A foi modulada na presença de cálcio e pode ser necessária para a atuação mais eficiente de outras enzimas envolvidas na degradação de xiloglucano. Desta forma, em T. reesei, CEL74A apresenta um papel importante tanto na regulação de genes holocelulolíticos quanto para a degradação eficiente de bagaço de cana-de-açúcar, contribuindo para a elucidação de mecanismos pelos quais este fungo utiliza para a utilização do bagaço de cana-de-açúcar como fonte de carbono / The filamentous fungus Trichoderma reesei is one of the main fungi used for the large-scale production of enzymes due to their great capacity of production and secretion of holocellulases for application in saccharification processes of lignocellulosic plant biomass. Although T. reesei is used as one of the main producers of cellulases at industrial level, several processes and studies are carried out with the aim of improving the understanding of the whole plant biomass degradation mechanism, as well as increasing the efficiency of both the production and cellulase activity. In the present work the construction of a mutant lineage for the cel74a gene (coding for a xyloglucanase) and the functional characterization of CEL74A in the gene regulation of holocellulases during the cultivation of sugarcane bagasse were carried out. Our results showed that deletion of cel74a may be involved in the regulation of holocellulase expression during sugarcane bagasse cultivation. From the analysis of the gene expression profile, it was possible to observe the reduction in the expression of all tested cellulase genes (cel7a, cel7b and cel6a), although it did not affect the enzymatic activity, whereas the hemicellulases (xyn1 and xyn2) presented increase in both expression and enzymatic activity. In the ?cel74a strain, a reduction in glucose, xylose and galactose release was observed after xyloglucan hydrolysis. In addition, the activity of CEL74A was modulated in the presence of calcium and may be required for the more efficient performance of other enzymes involved in the degradation of xyloglucan. Thus, in T. reesei, CEL74A plays an important role both in the regulation of holocellulolytic genes and in the efficient degradation of sugarcane bagasse, contributing to the elucidation of mechanisms by which this fungus uses for the use of sugarcane bagasse as source of carbon