Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:05:43Z No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:06:06Z (GMT) No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Made available in DSpace on 2018-04-10T13:06:06Z (GMT). No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) Previous issue date: 2018-02-20 / Protease inhibitors have a broad biotechnological application, which goes since the development of several drugs to your utilization as a bioinsecticide, antifungal and as an antibacterial agent. However, those are found in small quantities in their natural sources, which unfeasible it utilization in industrial scale. Therefore, the heterologous production ends up as a method that allows the increase of scale production of those proteins. The Inga laurina Trypsin Inhibitor (ILTI) previously characterized showed an inhibitory effect in proteases extracted from the midgut of insects, besides reducing its larval developments by up to 84%, therefore, boing a candidate to be used as a potential bioinsecticide. Thus, the present work aimed at the heterologous production of ILTI in Komagataella phaffii (Pichia pastoris), followed by the optimization of the culture modes in a bioreactor with the objective of maximizing the production of the recombinant protein. For this, the gene that codifies ILTI were cloned in the expression vector pPIC9K, followed by your insertion on the strain GS115 by electroporation. PCR analysis showed that the recombinant vector was integrated into the genome of the yeast, and all the clones obtained had MutS genotype. The expression was performed for 96 hours by adding 0.5% methanol. An analysis of the proteins on the supernatant of the recombinant strain culture, by SDS-PAGE, confirmed the production of a protein with a size close to 20 KDa. Data from MALDI-TOF confirmed that the obtained protein is, in fact, the recombinant ILTI. Furthermore, inhibitory assays showed that the produced protein had activity against trypsin. Thus, culture in bioreactors was performed to optimize the production of this heterologous protein. In order to increase its expression, fed-batch was performed where on the batch phase the biomass production was favored, and the feeding phase was programmed to continuously supply methanol, based on the methanol specific consumption and its specific growth velocity, using methanol as carbon source. During the fermentations, 351.27 UIT were obtained in the extract of crude fermentation broth, and a specific activity of 2.07 UIT/mg protein. Although widely used as a host for the production of heterologous proteins, studies of the production of protease inhibitors in K. phaffii are still very limited. Until the moment, there is no report in the optimization of the production of serine protease inhibitors in K. phaffii, making this study pioneering and essential for the beginning of scaling up the process of this technology. / Os inibidores de protease possuem uma ampla aplica????o biotecnol??gica que vai desde o desenvolvimento de diversos f??rmacos at?? sua utiliza????o como bioinseticidas, antif??ngicos e como agentes antibacterianos. Por??m, estes s??o encontrados em pequenas quantidades nas suas fontes naturais, o que inviabiliza sua utiliza????o em escala industrial. Sendo assim, a produ????o heter??loga acaba sendo um m??todo que permite o aumento da escala de produ????o dessas prote??nas. O inibidor de tripsina de Inga laurina (ILTI) foi caracterizado previamente e mostrou possuir efeito inibit??rio em proteases extra??das do trato digestivo de insetos, al??m de diminuir em at?? 84% seu desenvolvimento larval, sendo, portanto, um candidato a ser utilizado como um poss??vel bioinseticida. Dessa forma, o presente trabalho teve como objetivo a produ????o heter??loga do inibidor ILTI, em Komagataella phaffii (Pichia pastoris), seguido da otimiza????o dos modos de cultivo em biorreator com o objetivo de maximizar a produ????o da prote??na recombinante. Para isso, o gene que codifica o ILTI foi clonado no vetor de express??o pPIC9K seguindo de sua inser????o na cepa GS115 por eletropora????o. An??lises de PCR mostraram que o vetor recombinante foi integrado ao genoma da levedura e que todos os clones obtidos possu??am gen??tipo MutS. A indu????o da express??o foi realizada durante 96 horas por meio da adi????o de metanol ?? 0,5%. A an??lise de prote??nas presentes no sobrenadante da cultura da cepa recombinante, por meio de SDS-PAGE, confirmou a produ????o de uma prote??na com tamanho pr??ximo a 20 KDa. Dados obtidos em MALDI-TOF confirmaram que a prote??na obtida ?? de fato ILTI recombinante. Al??m disso, ensaios inibit??rios mostraram que a prote??na produzida possui atividade contra tripsina. Dessa forma, cultivo em biorreatores foram realizados com a finalidade de otimizar a produ????o dessa prote??na heter??loga. A fim de aumentar sua express??o foram realizadas bateladas alimentadas, onde durante a fase de batelada foi favorecida a produ????o de biomassa, e a fase de alimenta????o programada para fornecer metanol de forma cont??nua, com base nos dados de velocidade espec??fica de consumo de metanol e velocidade de crescimento espec??fica nesta fonte de carbono. Ao longo das fermenta????es realizadas foi obtido 351,27 UIT no extrato bruto do caldo fermentado e uma atividade espec??fica de 2,07 UIT/mg de prote??na. Apesar de ser amplamente utilizada como hospedeira para a produ????o de prote??nas heter??logas, estudos da produ????o de inibidores em K. phaffii ainda s??o muito limitados. At?? o momento n??o existem relatos de otimiza????o da produ????o de inibidores de serinoproteases em K. phaffii, sendo esse estudo pioneiro e essencial para o in??cio do processo de escalonamento dessa tecnologia.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:02Z No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:47Z (GMT) No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Made available in DSpace on 2018-04-16T14:40:47Z (GMT). No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) Previous issue date: 2018-03-06 / Klebsiella pneumoniae is one of the main bacterial agents that may cause infections related to health care assistance. K. pneumoniae frequently carries the resistance gene K. pneumoniae carbapenemase (KPC). Currently, tigecycline can be considered one of the last therapeutic options for KPC, but reports of tigecycline-resistant KPC isolates are on the rise, being indicated as most common mechanism the increased AcrAB-TolC efflux pump system expression. However, molecular tigecycline resistance mechanisms associated to AcrAB-TolC still remains obscure. Thus, the main goal of this study was to verify if tigecycline resistance can be related to the presence of mutations in the regulatory genes of AcrAB-TolC, AcrR and RamR. Therefore, 32 K. pneumoniae isolates were used, identification and antibiogram performed using Vitek 2 systems. The minimum inhibitory concentrations were confirmed using E-test. Primers were designed in order to verify mutations within AcrR and RamR genes. PCR analysis showed that the mutations found within these genes were transversions (94% for AcrR and 90% for RamR) and transitions (6% for AcrR and 10% for RamR). Nevertheless among the mutations, no distinction between tigecycline susceptible and resistant isolates was found. Some of the transversions caused change in the amino acid encoding 6 in AcrR and 15 in RamR. Presence of these types of mutations evaluation can be seen as the first bacterial resistance study step, as it may be caused by oxidative damage for bacterial DNA, frequently caused by antibiotic selective pressure. Tigecycline resistance found in this study`s clinical isolates may be associated to alterations in another genes that can trigger mechanisms associated to this antibiotic. / Klebsiella pneumoniae consiste em um dos principais agentes bacterianos causadores de infec????es relacionadas ?? assist??ncia ?? sa??de (IRAS). K. pneumoniae carrega frequentemente o gene de resist??ncia K. pneumoniae carbapenemase (KPC). Atualmente, a tigeciclina pode ser considerada uma das ??ltimas op????es terap??uticas para KPC, mas os relatos de isolados de KPC resistentes a tigecilina est??o em ascens??o, sendo a hiperexpress??o da bomba de efluxo AcrAB-TolC indicado como mecanismo mais comum. No entanto, os mecanismos moleculares de resist??ncia ?? tigeciclina associada ao AcrAB-TolC permanecem obscuros. Desta forma o objetivo deste trabalho foi verificar se a resist??ncia a tigeciclina pode estar relacionada ?? presen??a de muta????es nos genes ArcR e RamR, reguladores de AcrAB-TolC. Para tanto, 32 isolados de K. pneumoniae foram utilizados, sendo a identifica????o e o antibiograma feitos utilizando o sistema Vitek 2. A confirma????o das concentra????es inibit??rias m??nimas (CIMs) foram realizadas por E-test. Iniciadores foram desenhados para verifica????o de muta????es nos genes (AcrR e RamR). As an??lises por PCR mostraram que as muta????es encontradas nos genes AcrR e RamR foram substitui????es por transvers??o (94% e 90% para AcrR e RamR respectivamente) e transi????o (6% e 10% para AcrR e RamR respectivamente), por??m n??o foi identificada distin????o da presen??a de muta????es entre isolados sens??veis e resistentes a tigeciclina. Algumas tranvers??es ocasionaram mudan??a na codifica????o do amino??cido, sendo 6 em AcrR e 15 em RamR. A avalia????o da presen??a desses tipos de muta????es consiste em um primeiro passo para o estudo da resist??ncia bacteriana, j?? que pode ser causada por dano oxidativo ao DNA bacteriano, frequentemente ocasionado por press??o seletiva dos antibi??ticos. A resist??ncia a tigeciclina encontrada nos isolados cl??nicos do presente estudo, provavelmente pode estar associada a altera????es em outros genes desencadeadores de mecanismos de resist??ncia a tigeciclina.

Submitted by Kelson Anthony de Menezes (kelson@ucb.br) on 2016-11-23T11:36:50Z No. of bitstreams: 1 DaivaDomenechTupinambaDissertacao2015.pdf: 12204296 bytes, checksum: db4048c47ac3f9f47171655f6bfb3e91 (MD5) / Made available in DSpace on 2016-11-23T11:36:50Z (GMT). No. of bitstreams: 1 DaivaDomenechTupinambaDissertacao2015.pdf: 12204296 bytes, checksum: db4048c47ac3f9f47171655f6bfb3e91 (MD5) Previous issue date: 2015-03-12 / The Amazon rainforest is home to huge diversity of macro-species. However, little is known about the microbial diversity. The effect of land-use after deforestation is of great importance in the development of public policies. The metagenome were extracted from soils of native forest and an adjacent cultivated area with oil palm and pyrosequencing of 16S rRNA genes of archaea communities present in those soils was used for phylogenetic characterization of the archaeal microbiota, in an unprecedented characterization of native Amazonian soil and soils cultivated with oil palm. All OTUs of the native forest soils and cultivated area with oil palm were classified into two phyla: Euryarchaeota and Thaumarchaeota. Thaumarchaeota phylum was predominant only in native forest. Euryarchaeota, especially methanogenic archaea, were prevalent in cultivated area with oil palm. Various genera involved in biogeochemical cycles, as AOA and methanogenic archaea, were identified in all samples. In native forest the genera with larger representation were Candidatus Nitrosotalea and Candidatus Nitrososphaera, AOAs. In the cultivated area with oil palm the genus with larger representation was Rice Cluster I. There is a direct correlation between levels of organic matter and total carbon and the diversity of archaea in Amazonian soils. In addition, anthropization also showed impact on this diversity. This is the first study to characterize the microbiota of archaea in Amazonian soils using specific primers and high-throughput sequencing. This work also characterize the archaeal communities in soils cultivated with oil palm with and without symptoms of Fatal Yellowing. The growth of world energy demand and concern with climate changes lead to a worldwide increase in the search for alternative sources of energy. Within this scenario, agroenergy presents itself as a viable alternative. However, there are still several limitations to the production of biofuels, such as efficiency and cost of the production process as well as the quality of the energy feedstock available. Palm oil is one of the most promising sources of oil for biodiesel production in Brazil, and the Fatal Yellowing (FY), a disease with unknown etiology, is limiting the use of palm. From the metagenome extracted from soils associated to oil palms with and without symptoms of FY was used pyrosequencing of 16S rRNA genes of archaeal communities for phylogenetic characterization, in an attempt of an association of some microorganism with FY, and an unprecedented characterization of soils cultivated with oil palms with and without FY. In the comparison among oil palms with and without FY symptoms, the three groups were different among then; group 8 showed higher diversity and had lower coverage. All groups presented two phyla: Thaumarchaeota and Euryarchaeota. There was prevalence of the second in all groups, with an increase in abundance of methanogenic archaea with FY. In the analysis of genera, significant differences between the groups were observed, especially for genera Rice Cluster I and Ca. Nitrosotalea, which showed an increase in abundance directly proportional to the increase of the FY symptoms. The genera Ca. Nitrososphera and Methanocella showed the opposite; a decrease in abundance with the increase of FY symptoms. However, it???s not possible to say that these genera are related to FY. This work is complementary to the study of bacterial microbiota of these soils, already performed; and the study of fungal microbiota, in progress. This is an unpublished study, which will contribute to future studies on the Fatal Yellowing. / A floresta Amaz??nica ?? ber??o de enorme diversidade de macroesp??cies. Entretanto, pouco se sabe sobre a diversidade microbiana. O efeito do uso da terra ap??s o desmatamento das florestas ?? de grande import??ncia no desenvolvimento de pol??ticas p??blicas. A partir do metagenoma extra??do do solo de mata nativa Amaz??nica e de uma ??rea adjacente cultivada com dendezeiros foi utilizado o pirosequenciamento do 16S rRNA das comunidades de arqueias presentes nesses solos para caracteriza????o filogen??tica e an??lise comparativa das comunidades de arqueias. Todas as OTUs dos solos de mata nativa e ??rea cultivada com dendezeiros foram classificadas em apenas dois filos: Euryarchaeota e Thaumarchaeota. O filo Thaumarchaeota foi predominante apenas na mata nativa, sendo Euryarchaeota, especialmente arqueias metanog??nicas, predominantes nos solos cultivados com dendezeiros. Diversos g??neros envolvidos com os ciclos biogeoqu??micos, como arqueias oxidadoras de am??nia e metanog??nicas, foram identificados nas duas amostras. Na mata nativa os g??neros classificados que apresentam a maior representa????o foram Candidatus Nitrosotalea e Candidatus Nitrososphaera, AOAs. J?? na ??rea cultivada com dendezeiros o g??nero de maior representa????o foi Rice Cluster I. Foi encontrada um correla????o direta entre os n??veis de mat??ria org??nica e carbono total e a diversidade de arqueias nos solos amaz??nicos. Al??m disso, a antropiza????o tamb??m apresentou impacto sobre essa diversidade. Este ?? o primeiro estudo de caracteriza????o da microbiota de arqueias em solos amaz??nicos usando primers espec??ficos e sequenciamento de alto desempenho. Este trabalho tamb??m caracterizou as comunidades de arqueias em solos cultivados com dendezeiros com e sem sintomas de Amarelecimento Fatal. O crescimento da demanda energ??tica mundial e preocupa????o com as mudan??as clim??ticas levou a um aumento da busca mundial por fontes alternativas de energia, o que est?? levando diversos pa??ses a buscarem na bioenergia uma alternativa. Entretanto, ainda existem diversas limita????es na produ????o de biocombust??veis, seja na efici??ncia e custo do processo produtivo, seja na qualidade das fontes energ??ticas dispon??veis. O dend?? ?? uma das fontes mais promissoras de ??leo para a produ????o de biodiesel no Brasil, sendo o Amarelecimento Fatal, doen??a com fator etiol??gico desconhecido, um limitante no uso do dend??. A partir do metagenoma extra??do dos solos associados a dendezeiros com e sem sintomas de AF foi utilizado o pirosequenciamento do 16S rRNA das comunidades de arqueias para caracteriza????o filogen??tica. Foi realizada uma an??lise comparativa das comunidades de arqueias de solos de dendezeiros com e sem sintomas de AF, numa tentativa de associa????o de algum microrganismo com essa doen??a. Na compara????o entre dendezeiros com e sem sintomas de AF, os tr??s grupos estudados diferiram entre si; o grupo 8 apresentou maior diversidade e obteve menor cobertura. Todos os grupos apresentaram dois filos: Thaumarchaeota e Euryarchaeota. Houve preval??ncia do segundo em todos os grupos, com aumento na abund??ncia de arqueias metanog??nicas com o AF. Na an??lise entre g??neros, foram observadas diferen??as significativas entre os grupos, especialmente para os g??neros Rice Cluster I e Ca. Nitrosotalea, que apresentaram um aumento em suas abund??ncias diretamente proporcional ao aumento dos sintomas do AF. Os g??neros Ca. Nitrososphaera e Methanocella apresentaram uma rela????o inversa; uma queda na abund??ncia com o aumento dos sintomas do AF. Entretanto, n??o se pode afirmar que estes grupos est??o relacionados ao AF. Este trabalho ?? complementar ao estudo da microbiota bacteriana desses solos, j?? realizado; e pelo estudo da microbiota f??ngica, em andamento. Trata-se de um estudo in??dito, que ir?? contribuir para os estudos futuros sobre o Amarelecimento Fatal.