Proteômica de Chromobacterium violaceum submetida à microgravidade simulada
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...
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Bactéria Expressão diferencial Proteômica shotgun e rede de interação CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
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Bactéria Expressão diferencial Proteômica shotgun e rede de interação CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA Santos, Jonathas Diego Lima Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
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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. |
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Medeiros, Silvia Regina Batistuzzo de |
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Medeiros, Silvia Regina Batistuzzo de Santos, Jonathas Diego Lima |
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doctoralThesis |
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Santos, Jonathas Diego Lima |
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Santos, Jonathas Diego Lima |
| title |
Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
| title_short |
Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
| title_full |
Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
| title_fullStr |
Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
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Proteômica de Chromobacterium violaceum submetida à microgravidade simulada |
| title_sort |
proteômica de chromobacterium violaceum submetida à microgravidade simulada |
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Brasil |
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2018 |
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https://repositorio.ufrn.br/jspui/handle/123456789/24946 |
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AT santosjonathasdiegolima proteomicadechromobacteriumviolaceumsubmetidaamicrogravidadesimulada |
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1773963405968801792 |
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ri-123456789-249462019-01-30T20:00:15Z Proteômica de Chromobacterium violaceum submetida à microgravidade simulada Santos, Jonathas Diego Lima Medeiros, Silvia Regina Batistuzzo de Lima, Daniel Chaves de Santos, Elizeu Antunes dos Souza, Gustavo Antonio de Pfenning, Magnolia de Araújo Campos Bactéria Expressão diferencial Proteômica shotgun e rede de interação CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA 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. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Chromobcterium violaceum (C. violaceum) é uma bactéria Gram-negativa, encontrada em regiões tropicais e subtropicais, considerada organismo modelo de vida livre. Alguns estudos proteômicos realizados com esta bactéria demonstraram sua capacidade de adaptação a desafios ambientais, como alta concentração de ferro e exposição ao estresse oxidativo. No entanto, nenhum estudo foi feito com esta espécie submetida à microgravidade simulada (MGS), ou seja, em condições em que a gravidade é artificialmente reduzida para menos de 1xg. Estudos MGS podem ser importantes para entender as modulações em nível 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 técnicas de proteômica para entender como a bactéria responde a esse estresse. A MGS foi conseguida por meio da rotação do vessel ao redor do eixo horizontal perpendicular ao vetor gravitacional nos sistemas de cultura de células rotativas - Rotating Cell Culture Systems – (RCCS4). MGS foi conduzido a uma velocidade de 40 rpm por um período de 12 horas para obter a curva de crescimento a cada 2 horas. Proteínas totais foram extraídas de amostras de cultura de células bacteriana coletada às 5 e 12 horas, correspondendo as fases exponenciais inicial (MG5) e tardia (MG12), respectivamente, tendo a curva de crescimento como referência. Após a tripsinização, as amostras foram analisadas em espectrômetro de massas Q-TOF. Como resultado um total de 212 proteínas durante às 5h de crescimento e 192 às 12h foram detectadas, das quais 144 delas foram comuns em ambos os períodos. No proteoma de C. violaceum obitido em 5h de crescimento 195 proteínas foram identificadas em gravidade normal (GN5) e 155 proteínas foram identificadas em MG5, sendo 18 upreguladas, 19 downreguladas e 17 expressas somente na condição de MG5. No proteoma obtido em 12h de crescimento, 165 proteínas foram identificadas em gravidade normal (GN12) e 173 em MG12, das quais, 17 foram upreguladas, 22 downreguladas e 28 expressas somente na condição de MG12 Além disso, foi possível identificar 25 proteínas com função desconhecida em MG5 e MG12. Utilizando ferramentas computacionais foi possível construir redes de interações proteína-proteína (PPI) e as sub-redes contendo grupo de proteínas com funções correlacionadas, analisar vias metabólicas, processos biológicos, contexto genômico e busca por domínios conservados e sequências homólogas de proteínas com função desconhecida. Como resultado, identificamos sub-redes relacionadas com a biossíntese de proteínas, regulação da transcrição e tradução, resposta ao estresse e metabolismo energético, concluímos que as respostas celulares associadas à expressão diferencial induzida pela MGS levaram à diminuição do crescimento de C. violaceum, acompanhado pela regulação negativa de proteínas relacionadas com processos transcricionais, traducionais e liberação de energia por vias aeróbicas e pela regulação positiva de proteínas envolvidas no estresse oxidativo, na via anaeróbica e na sobrevivência celular. 2018-03-23T11:23:51Z 2018-03-23T11:23:51Z 2017-12-15 doctoralThesis SANTOS, Jonathas Diego Lima. Proteômica de Chromobacterium violaceum submetida à microgravidade simulada. 2017. 152f. Tese (Doutorado em Bioquímica) - Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2017. https://repositorio.ufrn.br/jspui/handle/123456789/24946 por Acesso Aberto application/pdf Brasil UFRN PROGRAMA DE PÓS-GRADUAÇÃO EM BIOQUÍMICA |