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Previous issue date: 2017-12-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Chromobcterium violaceum (C. violaceum) ? uma bact?ria Gram-negativa, encontrada em regi?es tropicais e subtropicais, considerada organismo modelo de vida livre. Alguns estudos prote?micos realizados com esta bact?ria demonstraram sua capacidade de adapta??o a desafios ambientais, como alta concentra??o de ferro e exposi??o ao estresse oxidativo. No entanto, nenhum estudo foi feito com esta esp?cie submetida ? microgravidade simulada (MGS), ou seja, em condi??es em que a gravidade ? artificialmente reduzida para menos de 1xg. Estudos MGS podem ser importantes para entender as modula??es em n?vel molecular sofridas pelos organismos vivos. Portanto, o objetivo deste estudo foi caracterizar a resposta de C. violaceum, como organismo modelo de vida livre, cultivado em MGS, usando t?cnicas de prote?mica para entender como a bact?ria responde a esse estresse. A MGS foi conseguida por meio da rota??o do vessel ao redor do eixo horizontal perpendicular ao vetor gravitacional nos sistemas de cultura de c?lulas rotativas - Rotating Cell Culture Systems ? (RCCS4). MGS foi conduzido a uma velocidade de 40 rpm por um per?odo de 12 horas para obter a curva de crescimento a cada 2 horas. Prote?nas totais foram extra?das de amostras de cultura de c?lulas bacteriana coletada ?s 5 e 12 horas, correspondendo as fases exponenciais inicial (MG5) e tardia (MG12), respectivamente, tendo a curva de crescimento como refer?ncia. Ap?s a tripsiniza??o, as amostras foram analisadas em espectr?metro de massas Q-TOF. Como resultado um total de 212 prote?nas durante ?s 5h de crescimento e 192 ?s 12h foram detectadas, das quais 144 delas foram comuns em ambos os per?odos. No proteoma de C. violaceum obitido em 5h de crescimento 195 prote?nas foram identificadas em gravidade normal (GN5) e 155 prote?nas foram identificadas em MG5, sendo 18 upreguladas, 19 downreguladas e 17 expressas somente na condi??o de MG5. No proteoma obtido em 12h de crescimento, 165 prote?nas foram identificadas em gravidade normal (GN12) e 173 em MG12, das quais, 17 foram upreguladas, 22 downreguladas e 28 expressas somente na condi??o de MG12 Al?m disso, foi poss?vel identificar 25 prote?nas com fun??o desconhecida em MG5 e MG12. Utilizando ferramentas computacionais foi poss?vel construir redes de intera??es prote?na-prote?na (PPI) e as sub-redes contendo grupo de prote?nas com fun??es correlacionadas, analisar vias metab?licas, processos biol?gicos, contexto gen?mico e busca por dom?nios conservados e sequ?ncias hom?logas de prote?nas com fun??o desconhecida. Como resultado, identificamos sub-redes relacionadas com a bioss?ntese de prote?nas, regula??o da transcri??o e tradu??o, resposta ao estresse e metabolismo energ?tico, conclu?mos que as respostas celulares associadas ? express?o diferencial induzida pela MGS levaram ? diminui??o do crescimento de C. violaceum, acompanhado pela regula??o negativa de prote?nas relacionadas com processos transcricionais, traducionais e libera??o de energia por vias aer?bicas e pela regula??o positiva de prote?nas envolvidas no estresse oxidativo, na via anaer?bica e na sobreviv?ncia celular. / Chromobcterium violaceum (C.violaceum) is a Gram-negative bacteria, which has been found at tropical and subtropical regions, considered a free living model organism. Some proteomic studies performed with this bacterium have demonstrated its ability to adapt to environmental challenges such as high iron concentration and oxidative stress exposure. However, no study was made with this specie submitted to simulated microgravity (SMG), that is, under conditions in which the gravity is artificially reduced to less than 1xg. MGS studies may be important in understanding the molecular-level modulations undergone by living organisms. Therefore, the aim of this study was to characterize the response of C. violaceum, as free-living model organism, cultured at MGS, using proteomics techniques in order to understand how this bacterium response to this stress. The SMG was achieved by rotating the vessel around the horizontal axis perpendicular to the gravitational vector in Rotating Cell Culture Systems (RCCS4). SMG was conducted at a speed of 40 rpm for a period of 12 hours to obtain the growth curve every 2 hours. Total protein extraction was made in two times: 5 and 12 hours, corresponding to early (MG5) and late (MG12) exponential phase, respectively, taking the growth curve as a reference. After trypsinization, samples were analyzed with Q-TOF mass spectrometer. As a result a total of 212 proteins during 5h of growth and 192 at 12h were detected, of which 144 of them were common in both periods. We detected 155 proteins during MG5, from which 18 proteins were upregulated, 19 down-regulated and 17 proteins were exclusive when compared to GN5. In the proteome obtained in 12h of growth, 165 proteins were identified in normal gravity (GN12) and 173 in MG12, of which, 17 were upregulated, 22 were downregulated and 28 expressed only in MG12 condition. In addition, it was possible to identify 25 proteins with unknown function in MG5 and MG12. Using computational tools it was possible to construct networks of protein-protein interactions (PPI) and sub-networks containing group of proteins with correlated functions, to analyze metabolic pathways, biological processes, genomic context and search for conserved domains and homologous sequences of proteins with unknown function . As a result, we identified sub-networks related to protein biosynthesis, transcription regulation and translation, stress response and energetic metabolism, we conclude that the cellular responses associated with differential expression induced by MGS led to a decrease in the growth of C. violaceum, accompanied by the negative regulation of proteins related to transcriptional, translational and energy release by aerobic pathways and by the positive regulation of proteins involved in oxidative stress, anaerobic pathway and cell survival.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/24946 |
| Date | 15 December 2017 |
| Creators | Santos, Jonathas Diego Lima |
| Contributors | 32398336468, Lima, Daniel Chaves de, 06756927418, Santos, Elizeu Antunes dos, 41305655400, Souza, Gustavo Antonio de, 00534185908, Pfenning, Magnolia de Ara?jo Campos, 47532033449, Medeiros, Silvia Regina Batistuzzo de |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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