Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:22:30Z No. of bitstreams: 2 tiago_jose_silva.pdf: 1811358 bytes, checksum: 1654fd6d60ba2571e51302a48d7407c9 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:25:31Z (GMT) No. of bitstreams: 2 tiago_jose_silva.pdf: 1811358 bytes, checksum: 1654fd6d60ba2571e51302a48d7407c9 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:25:33Z (GMT) No. of bitstreams: 2 tiago_jose_silva.pdf: 1811358 bytes, checksum: 1654fd6d60ba2571e51302a48d7407c9 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:26:09Z (GMT) No. of bitstreams: 2 tiago_jose_silva.pdf: 1811358 bytes, checksum: 1654fd6d60ba2571e51302a48d7407c9 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-01-07T13:26:09Z (GMT). No. of bitstreams: 2 tiago_jose_silva.pdf: 1811358 bytes, checksum: 1654fd6d60ba2571e51302a48d7407c9 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2014 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / A produ??o biotecnol?gica de enzimas por fungos filamentosos requer o conhecimento das suas caracter?sticas de crescimento, fisiologia e metabolismo porque a compreens?o das respostas biol?gicas e nutricionais contribui para o ajuste dos bioprocessos. Neste trabalho foram analisados o crescimento apical e alguns par?metros bioqu?micos e fisiol?gicos da resposta das linhagens Aspergillus tubingensis AN1257 e Talaromyces trachyspermus T10.5 a fontes de carbono complexas indutoras de carboidrases. Foi utilizada uma abordagem polif?sica de identifica??o baseada em caracter?sticas fenot?picas e moleculares. As linhagens foram cultivadas em meio suplementado com glicose (controle), amido e carboximetilcelulose (CMC). O crescimento apical foi avaliado quanto aos eventos iniciais, hidrata??o, polariza??o e extens?o do tubo germinativo, bem como foi investigado o papel da via de PKC nestes processos. As linhagens diferiram quanto ao potencial de produ??o de carboidrases sendo o T. trachyspermus T10.5 o mais eficiente para a express?o de amilase. A produ??o de celulase foi verificada apenas em meio s?lido. Os eventos iniciais do crescimento apical de T. trachyspermus T10.5 foram atrasados pelos carboidratos polim?ricos em meio s?lido: ap?s 14h de cultivo a propor??o de con?dios apolares, polares e com tubo germinativo foi de 31,0 ? 5,7%; 34,0 ? 0,0% e 35,0 ? 5,7% (glicose) contra 40,0 ? 0,0%; 45,0 ? 0,8% e 14,0 ? 1,6% (amido) ou 90,0 ? 4,9%; 6,0 ? 1,6% e 4,0 ? 3,3% (CMC). A biomassa de T. trachyspermus T10.5 foi formada exponencialmente no per?odo de 24 a 48h em cultivo submerso e n?o foi diminu?da por nenhuma fonte de carbono. Nas culturas submersas, a fase exponencial de crescimento foi simult?nea ao consumo exponencial do substrato; o esgotamento das fontes de carbono precedeu o in?cio da fase estacion?ria; a atividade de ?-amilase (239,2 ? 11 U/min.mL) induzida por amido coincidiu com o crescimento exponencial, permaneceu est?vel durante a fase estacion?ria e foi reprimida por glicose. O crescimento apical de A. tubingensis AN1257 respondeu mais rapidamente ? fonte de carbono: 36,0 ? 4,3% dos con?dios cultivados em meio suplementado com glicose estavam polarizados ap?s 6h, contra 1,3 ? 0,9% (amido) e 2,7 ? 1,9% (CMC). A extens?o do tubo germinativo da linhagem AN1257 tamb?m foi atrasada e reduzida por amido e CMC. A forma??o de biomassa de A. tubingensis AN1257 nas culturas submersas foi fortemente reduzida por CMC, que apresentou um efeito tamponante no meio. Nas demais culturas submersas, o crescimento exponencial de A. tubingensis AN1257 ocorreu entre 0 a 24h (glicose) ou 12 a 24h (amido) coincidindo com forte acidifica??o do meio e consumo acentuado de substrato. O efeito da ativa??o de PKC foi distinto nas duas linhagens: a ativa??o dessa enzima n?o parece modular a germina??o em A tubingensis AN1257, mas inibe fortemente a germina??o de T. trachyspermus T10.5. Assim, as duas esp?cies apresentaram respostas diferentes ? fonte de carbono e ? ativa??o de PKC, com fisiologia, germina??o e crescimento apical peculiares, cuja compreens?o permitir? direcionar a aplica??o biotecnol?gica das linhagens AN1257 e T10.5 de forma mais eficiente. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT The biotechnological production of enzymes by filamentous fungi requires the knowledge of its characteristics of growth, physiology and metabolism because the comprehension of the biological and nutritional responses contributes to the adjustment of the bioprocesses. In this work, it were analyzed the apical growth, and some biochemical and physiological parameters of the response of the strains Aspergillus tubingensis AN1257 and Talaromyces trachyspermus T10.5 to complex carbon sources that induce carbohydrases. It was utilized a polyphasic approach of identification based on phenotypic and molecular characteristics. The strains were cultivated in media supplemented with glucose (control), starch and carboxy-methylcellulose (CMC). The apical growth was evaluated regarding the initial events, hydration, polarization and germtube extension, and the role of the PKC pathway in these processes was investigated as well. The strains differed according to their potential for carbohydrase production and T. trachyspermus T10.5 was the more efficient for amylase expression. Cellulase production was verified only in solid media. The initial events of T. trachyspermus T10.5 apical growth were delayed by the polymeric carbohydrates in solid media: after 14h of cultivation, the proportion of non-polar, polar and germtube-containing conidia was 31.0 ? 5.7%; 34.0 ? 0.0% and 35.0 ? 5.7% (glucose) versus 40.0 ? 0.0%; 45.0 ? 0.8% and 14.0 ? 1.6% (starch) or 90.0 ? 4.9%; 6.0 ? 1.6% and 4.0 ? 3.3% (CMC). The biomass of T. trachyspermus T10.5 was formed exponentially from 24 to 48h during submerged cultivation, and it was not decreased by any carbon source. In the submerged cultures, the exponential growth phase was simultaneous to the exponential consumption of the substrate; depletion of the carbon sources preceded the stationary phase; the ?-amylase activity (239.2 ? 11 U/min. mL) induced by starch coincided with the exponential growth, remained stable during the stationary phase and was repressed by glucose. A. tubingensis AN1257 apical growth responded more promptly to the carbon source: 36.0 ? 4.3% of the conidia cultivated in medium supplemented with glucose were polarized after 6h, versus 1.3 ? 0.9% (starch) and 2.7 ? 1.9% (CMC). Germtube extension by strain AN1257 was also delayed and reduced by starch and CMC. Biomass formation by A. tubingensis AN1257 in submerged cultures was strongly reduced by CMC, which presented a buffering effect in the medium. In other submerged cultures, the exponential growth of A. tubingensis AN1257 occurred from 0 to 24h (glucose) or 12 to 24h (starch), concomitant to a strong acidification of the medium and to intense substrate consumption. The effect of PKC activation was different in the two strains: activation of this enzyme does not seem to modulate germination in A. tubingensis AN1257, but it strongly inhibits T. trachyspermus T10.5 germination. Thus, the two species presented different responses to the carbon source and to PKC activation, with peculiar physiology, germination and apical growth, whose comprehension will allow direct the biotechnological application of strains AN1257 and T10.5 more efficiently.

Os fungos filamentosos passam por um crescimento polarizado, desde a germinação ao alongamento das hifas, até formar um complexo micélio. A região apical do crescimento polarizado do fungo apresenta dois tipos diferentes de vesículas, entre elas, as microvesículas. As ADP-ribosylation factors (ARFs), são proteínas monoméricas ligadoras de GTP e pertence ao grupo de proteínas da superfamília Ras. Essas proteínas são divididas em cinco famílias: ARF, RAB, RAN, RAS e RHO que formam um conjunto de sub-sistemas que são responsáveis, entre outras funções, pela regulação do transporte de vesículas no interior da célula fúngica, entre outras funções, como transduções de sinais e regulação do tráfego vesicular na região de crescimento apical, o spitzenkörper. São proteínas de ancoramento e de marcação de vesículas, envolvidas no tráfego, catálise e fusão por meio de sinalização de membrana-alvo para as vesículas de transporte transmembrana. As ARF são importantes para o crescimento das hifas, além de participar da montagem de vesículas por meio de endocitoses, do transporte destas vesículas entre as organelas e na exocitose. Adicionalmente, as ARFs sofrem o processo de N-miristoilação, uma irreversível lipidação proteica em que o miristato do miristoil CoA é covalentemente ligado a uma glicina secundária da proteína alvo, aumentando a sua hidrofobicidade. Além desta regulação, as ARFs são moduladas pela ação das ARF-GAP (GTPase Activating Protein) e ARF-GEFs (Guanine nucleotide Exchange Factor). Neste trabalho foi proposta a deleção de três ARFs preditivamente miristoiladas (arfA, arfB and arlA), além de dupla-deleção com ?gcsA (ARF-GAP) e a caracterização genotípica e fenotípica das ADP ribosylation fator no fungo filamentoso patogênico Aspergillus fumigatus. Como caracterização das linhagens deletadas, notou-se que arfA demonstra ser essencial para A. fumigatus, enquanto que o fungo foi capaz de se desenvolver na ausência de arfB, arlA e duplo mutantes com ?gcsA. Porém, de forma alternada nas linhagens mutantes, houve redução do diâmetro da colônia, desestruturação de conidióforos, polarização dicotômica e redução de corpos lipídicos na região de crescimento apical. Além das alterações da parede celular que implicou em altações na carga de superfície, formação de biofilme e virulência. Testes de sensibilidades, bem como as análises de níveis de expressão gênica frente a a compostos danosos a eucariotos e antifúngicos evidenciaram que as ARFs e GcsA estão envolvidas em reparos a danos frente a diferentes alvos citoplasmáticos. Ainda, a localização das ARFs fusionadas com GFP (Green Flourescence Protein) em A. fumigatus evidenciou que ArfB está nas regiões apicais das hifas e conidióforos, enquanto ArlA está distribuído em todo citoplasma. Portanto as ARFs em A. fumigatus estão envolvidas nos processos básicos do fungo, como: o crescimento, a virulência e a reprodução / The filamentous fungi undergo polarized growth, from germination to hyphae elongation, to form a mycelial complex. The apical region of the polarized growth of the fungus presents two different types of vesicles, among them, the microvesicles. ADP-ribosylation factors (ARFs) are monomeric GTP-binding proteins and belong to a group of superfamily Ras proteins. These proteins are divided into five families: ARF, RAB, RAN, RAS and RHO that form a set of subsystems that are responsible, over others things, for the regulation of vesicle transport within the fungal cell, among other functions, such as signal transduction and regulation of the vesicular traffic in the apical growth region, the Spitzenkörper. They are anchoring and vesicle marking proteins involved in trafficking, catalysis and fusion by means of target membrane signaling to the transmembrane transport vesicles. ARFs are important for the growth of hyphae, besides participating in vesicle assembly through endocytosis, the transport of these vesicles between the organelles and exocytosis. In addition, the ARFs undergo the N-myristoylation process, an irreversible protein lipidation in which the myristoyl CoA myristate is covalently linked to a secondary glycine of the target protein, increasing its hydrophobicity. In addition to this regulation, the Arfs are modulated by the action of Arf-GAP (GTPase Activating Protein) and ARF-GEFs (Guanine nucleotide Exchange Factor). In this work, the deletion of three myristoylated ARFs (arfA, arfB and arlA), as well as double-deletion with ?gcsA (ARF-GAP) and phenotypic and genotypic characterization of ADP ribosylation fator in the pathogenic fungus Aspergillus fumigatus was proposed. As a characterization of the deleted strains, arfA shown to be essential for A. fumigatus, whereas the fungus was able to develop in the absence of arfB, arlA and double mutants with ?gcsA. However, in the mutant strains, there was a decrease in colony diameter, deconjugation of conidiophores, dichotomous polarization and reduction of lipid bodies in the apical growth region. In addition, cell wall changes were registered that implied in surface charge elevations, biofilm formation and virulence. In tests of sensitivities, as well as the analysis of levels of gene expression against compounds harmful to eukaryotes and antifungals showed that ARFs and GcsA (Arf-GAP) are involved in damage repair against different cytoplasmic targets. Furthermore, the location of the GFP-fused GFPs (Green Flourescence Protein) in A. fumigatus evidenced that ArfB is in the apical regions of the hyphae and conidiophores, while ArlA is diffuse in every cytoplasm. Therefore, the ARFs in A. fumigatus are involved in the basic processes of the fungus, such as growth, virulence and reproduction