Made available in DSpace on 2014-12-17T14:05:19Z (GMT). No. of bitstreams: 1 DelanneCSSF_TESE.pdf: 5733269 bytes, checksum: db8ba3b543c1bd1c178d56cb6e6335bb (MD5) Previous issue date: 2011-09-08 / The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III ? subunit). In this way, we propose that the C. violaceum ? subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., ?S subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress. / O seq?enciamento do genoma da esp?cie Chromobacterium violaceum identificou um cromossomo simples circular de 4.8 Mb, no qual aproximadamente 40% das ORFs encontradas s?o classificadas como hipot?ticas conservadas ou hipot?ticas. Algumas regi?es g?nicas de interesse biotecnol?gico e biol?gico v?m sendo caracterizadas, como por exemplo, genes de detoxifica??o ambiental e genes de reparo de DNA, respectivamente. Diante disso, o objetivo deste trabalho foi identificar genes de C. violaceum envolvidos com a resposta ao estresse, como por exemplo, mecanismos de reparo de DNA e/ou manuten??o da integridade gen?mica. Para tanto, foi constru?da uma biblioteca gen?mica de C. violaceum na cepa DH10B de Escherichia coli (RecA-), a qual foi testada para clones resistentes a UVC, resultando na sele??o de cinco clones candidatos. Foram identificadas quatro ORFs (CV_3721 a 3724) no clone PLH6A. Das quais, as ORFs CV_3722 e CV_3724, foram subclonadas e uma atividade sin?rgica de complementa??o foi observada. A ocorr?ncia de um operon foi confirmada usando cDNA de C. violaceum em ensaio de RT-PCR. Adicionalmente, foi observada a indu??o do operon ap?s tratamento com UVC, dessa forma, esse operon foi relacionado ? resposta ao estresse em C. violaceum. Ensaios de mutag?nese com rifampicina ap?s tratamento com luz UVC mostraram alta freq??ncia de mutagenicidade para a ORF CV_3722, subunidade ? da Polimerase III. Assim, propomos que esta subunidade de C. violaceum pode agir em DH10B na s?ntese transles?o utilizando Pol IV em uma via RecA independente. Em ensaios de viabilidade outros quatro clones (PLE1G, PLE7B, PLE10B e PLE12H) foram capazes de complementar a fun??o na dose de 5 J/m2. E em ensaios de mutagenicidade PLE7B, PLE10B e PLE12H apresentaram freq??ncias de muta??o com diferen?as significativas em rela??o ao controle (DH10B), demonstrando que de alguma forma eles est?o envolvidos na resposta ao estresse em C. violaceum. Estes clones parecem estar inter-relacionados, provavelmente, regulados por mol?cula mensageira (como o nucleot?deo c-di-GMP) e/ou mol?cula regulat?ria global (como a subunidade ?S da RNA Polimerase). Os resultados obtidos contribuem para um melhor conhecimento da gen?tica desta esp?cie e de seus mecanismos de resposta ao estresse ambiental. / 2020-01-01

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:19:50Z No. of bitstreams: 1 TES_VIRGINIA_CARLA_DE_ALMEIDA_FALCAO_COMPLETO.pdf: 3588046 bytes, checksum: 337be6be89fea4ea58787c070a072a51 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:19:59Z (GMT) No. of bitstreams: 1 TES_VIRGINIA_CARLA_DE_ALMEIDA_FALCAO_COMPLETO.pdf: 3588046 bytes, checksum: 337be6be89fea4ea58787c070a072a51 (MD5) / Made available in DSpace on 2017-06-30T13:20:08Z (GMT). No. of bitstreams: 1 TES_VIRGINIA_CARLA_DE_ALMEIDA_FALCAO_COMPLETO.pdf: 3588046 bytes, checksum: 337be6be89fea4ea58787c070a072a51 (MD5) Previous issue date: 2017-03-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Funda??o de Amparo ? Pesquisa do Estado do Rio Grande do Sul (FAPERGS) / Tuberculosis (TB) has become the leading global cause of death from infectious diseases. In 2015, according to WHO, 10.4 million new cases of tuberculosis worldwide have emerged. Currently the commonly used treatments are not effective against the forms of disease resistant to the most effective anti-TB drugs, and drugs with new mechanisms of action are needed. Mycobacterium tuberculosis dihydroneopterin aldolase (MtDHNA /FolB) is a folate enzyme encoded by the folB gene, which has important properties that make it a potential target for the synthesis of new antimicrobial agents. As a first step for target validation in the antimicrobial drug development pipeline, it is important to prove that the gene encoding a putative target is essential for pathogen?s viability. In this study, using site directed mutagenesis, biochemical analyzes and gene knockout experiments, we demonstrated that the folB gene is essential for the survival of Mtb, and furthermore we prove that this essentiality depends on the aldolase/epimerase activities of the MtFolB protein. The wild-type gene (wt) and the point mutants K99A and Y54F were cloned and expressed, and the corresponding recombinant proteins were purified and monitored for the activities of aldolase, epimerase and oxygenase using HPLC. In contrast to the wild-type MtFolB (wt) enzyme, both mutants had neither aldolase nor epimerase activities under the conditions tested. The Y54F mutant maintained oxygenase activity, whereas for the K99A mutant it was possible to detect oxygenase activity only in the presence of HP and GA as substrates. Knockout experiments showed that the folB gene is essential for the survival of Mtb under the conditions tested. However, unlike the wild-type copy, when the sequences encoding the K99A or Y54F mutants were used for complementation, no viable colonies were obtained, indicating that these point mutants could not rescue the cells after the folB knockout. These results indicate that aldolase and/or epimerase activities are crucial for the survival of Mtb. The construction of Mycobacterium tuberculosis folB-GFP fusion (Mtb) strains containing wild-type folB gene sequence or a deleted C-terminal mutant (folB?C), devoid of the sequence presumably necessary for anchoring the enzyme within nanocage compartments, were performed and together with other cell biology methods described in this work will be used for a better understanding of MtDHNA/FolB cellular functions and for the validation of this enzyme as a therapeutic target. / A tuberculose (TB) tornou-se a principal causa mundial de morte por doen?as infecciosas. Em 2015, de acordo com a OMS, surgiram 10,4 milh?es de novos casos de tuberculose no mundo. Atualmente os tratamentos comumente utilizados n?o s?o eficientes contra as formas da doen?a resistentes aos f?rmacos anti-TB mais eficazes, sendo necess?rios f?rmacos com novos mecanismos de a??o. A di-hidroneopterina aldolase de Mycobacterium tuberculosis (MtDHNA/FolB) ? uma enzima da via do folato, codificada pelo gene folB, que apresenta caracter?sticas importantes que a tornam um potencial alvo para s?ntese de novos agentes antimicrobianos. Neste estudo, por meio de mutag?nese s?tio-direcionada, an?lises bioqu?micas e experimentos de nocaute g?nico, demostramos que o gene folB ? essencial para a sobreviv?ncia de Mtb, e al?m disso provamos que essa essencialidade depende das atividades de aldolase/epimerase da prote?na MtFolB. O gene do tipo selvagem (wt) e os mutantes pontuais K99A e Y54F foram clonados e expressos, e as prote?nas recombinantes correspondentes foram purificadas e monitoradas para as atividades de aldolase, epimerase e oxigenase utilizando HPLC. Em contraste com a enzima MtFolB selvagem (wt), ambas as mutantes n?o apresentaram atividade de aldolase nem de epimerase nas condi??es testadas. A mutante Y54F manteve a atividade da oxigenase, enquanto que para a mutante K99A foi poss?vel detectar a atividade de oxigenase apenas na presen?a de HP e GA como substratos. Os experimentos de nocaute mostraram que o gene folB ? essencial para a sobreviv?ncia de Mtb sob as condi??es testadas. Entretanto, diferentemente da c?pia selvagem, quando as sequ?ncias que codificam os mutantes K99A ou Y54F foram utilizadas para complementa??o, n?o foram obtidas col?nias vi?veis, indicando que estes mutantes pontuais n?o poderiam resgatar as c?lulas ap?s o nocaute do gene folB. Esses resultados indicam que as atividades de aldolase e/ou epimerase s?o cruciais para a sobreviv?ncia de Mtb. A constru??o de cepas com fus?o folB-GFP de Mycobacterium tuberculosis (Mtb) que cont?m a sequ?ncia do tipo selvagem do gene folB ou um mutante com o C-terminal deletado (folB?C), desprovida da sequ?ncia supostamente necess?ria para a ancoragem da enzima dentro dos compartimentos de nanocargas, foram realizadas e juntamente com outros m?todos de biologia celular descritos neste trabalho tamb?m poder?o ser utilizados para um melhor entendimento das fun??es celulares apresentadas por MtDHNA/FolB e para valida??o dessa enzima como potencial alvo terap?utico.