Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-01T13:59:43Z No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-07T15:09:24Z (GMT) No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) / Made available in DSpace on 2017-08-07T15:09:24Z (GMT). No. of bitstreams: 1 JessykaFernandaSantiagoMonte_DISSERT.pdf: 1592894 bytes, checksum: 2e8d0cdaf53e251db5deb8479566e9dc (MD5) Previous issue date: 2017-05-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Estudos envolvendo estabilidade de plasm?deos tiveram in?cio h? pelo menos duas d?cadas e vem crescendo nos ?ltimos anos, desde que os pDNAs apresentaram um enorme potencial como vetores em terapia g?nica. O uso terap?utico desses vetores tem sido dificultado por quest?es de estabilidade, principalmente no que se refere ao processo de produ??o e purifica??o, armazenamento por longos per?odos e serem suscept?veis ? degrada??o por nucleases. Assim, ensaios que permitam analisar estes processos que levam a instabilidade do pDNA podem ser ferramentas importantes para sua compreens?o, associado a outras vari?veis, tais como temperaturas, tempo de armazenamento, tamanho do pDNA, presen?a de sequ?ncias procari?ticas e a??o nucle?sica, assim E. coli DH5-? competente foi produzida, transformada com os pDNAs estudados (pVAX1, pVAX1lacZ e MSPpVAX1), purificados e armazenados em diferentes temperaturas por um intervalo de tempo pr?-determinado e para estabelecer uma rela??o entre a estabilidade dos diferentes pDNAs e sua fun??o biol?gica enquanto vetores, estudou-se a resist?ncia da isoforma super-enrolada ? a??o da nucleases de soro em diferentes concentra??es e ao longo do tempo. Para tal, eletroforese em gel de agarose e transforma??o em E. coli com c?lculo da efici?ncia de transforma??o celular foram realizados. Foi observado ao longo do tempo que a integridade do pDNA super-enrolado foi perdida em fun??o do tempo e da temperatura de armazenamento, al?m disso, os pDNAs contendo sequ?ncias apenas procari?ticas se mostraram mais resistente a esses fatores quando comparado ao que possu?a sequ?ncia procari?tica, mostrando que h? influ?ncia desses fatores na estabilidade do pDNA. Em rela??o ? a??o nucle?sica, o maior plasm?deo foi mais acometido pela atividade dessas enzimas. Quanto a fun??o biol?gica, os ensaios de efici?ncia de transforma??o em E. coli indicaram que houve uma maior percentagem de c?lulas transformadas quando era utilizado plasm?deo na conforma??o super-enrolada. Verificou-se em todos os ensaios, que a isoforma super-enrolada era sempre mais eficiente que as demais isoformas, devendo-se esse fato possivelmente ? sua maior estabilidade citoplasm?tica e a difus?o mais r?pida desta isoforma em dire??o ao n?cleo. Assim esse trabalho mostrou a cin?tica de degrada??o, passo a passo, dos pDNAs estudados, mostrando que a perda da forma super-enrolada compromete a estabilidade dos pDNAs, afetando dessa forma a fun??o biol?gica dos mesmos, comprometendo sua utiliza??o em terapia g?nica e vacinas de DNA. / Studies involving plasmid stability have started for at least two decades and have been growing in recent years, since pDNAs have had enormous potential as vectors in gene therapy, however the therapeutic use of these vectors has been hampered by stability issues, especially in Refers to the process of production and purification, storage for long periods and being susceptible to degradation by nucleases. Thus, assays that allow the analysis of this degradation process can be important tools for its understanding, associated to other variables, such as temperature, storage time, pDNA size and nucleoside action. The competent E. coli DH5-? was produced, transformed with the pDNAs studied (pVAX1, pVAX1lacZ and MSPpVAX1), purified and stored at different temperatures for a predetermined time and to establish a relationship between the stability of the different pDNAs and their Biological function as vectors, the resistance of the supercoiled isoform to the action of serum nucleases at different concentrations and over time was studied. For this purpose, agarose gel electrophoresis and transformation in E. coli with calculation of cell transformation efficiency were performed. It was observed over time that the integrity of the supercoiled pDNA was lost as a function of time and storage temperature, in addition, pDNAs containing only prokaryotic sequences proved to be more resistant to these factors when compared to that having procaryotic sequence pDNA, showing that these factors influence the stability of pDNA. In relation to the nuclease action, the bigger plasmid was more affected by the activity of these enzymes. Regarding biological function, transformation efficiency assays in E. coli indicated that there was a higher percentage of transformed cells when plasmid was used in the supercoiled conformation. It was verified in all the tests that the supercoiled isoform was always more efficient than the other isoforms, possibly due to its greater cytoplasmic stability and the faster diffusion of this isoform towards the nucleus. Thus, this work showed the degradation kinetics, step by step, of the studied pDNAs, showing that the loss of supercoiled form compromises the stability of the pDNAs, thus affecting the biological function of the same, compromising their use in gene therapy and vaccines DNA.