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.

Made available in DSpace on 2014-12-17T14:05:18Z (GMT). No. of bitstreams: 1 LourenaMF_TESE_Capa_ate_pag79.pdf: 4572586 bytes, checksum: 2630047160ff526cc964f166fb86ab33 (MD5) Previous issue date: 2011-03-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex? 355; Tween? 80; Spam? 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q?). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way / A terapia g?nica ? um dos maiores desafios propostos pela pesquisa p?s-gen?mica e se baseia na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local de a??o. Assim, ? necess?rio criar ve?culos, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi avaliar dois diferentes vetores n?o virais, lipossomas e nanoemuls?es cati?nicos, e sua poss?vel utiliza??o na terapia g?nica. Para isso, foram utilizados lip?deos cati?nicos e co-tensoativos na produ??o dos dois sistemas. As nanoemuls?es foram produzidas pelo m?todo de sonica??o e compostas por Captex? 355; Tween? 80; Spam? 80; lip?deo cati?nico, Estearilamina (EA) ou N-[1-(2,3-Dioleoiloxi)propil]-N,N,Ntrimetilamonio metilsulfato (DOTAP); e ?gua ultra-pura (Milli-Q?). Estes sistemas foram caracterizados quanto ao tamanho m?dio de got?cula, ?ndice de polidispers?o (PI) e potencial zeta. Avaliou-se ainda a estabilidade dos sistemas e suas capacidades de compacta??o do material gen?tico. Os lipossomas foram preparados a partir do m?todo de hidrata??o do filme e compostos por DOTAP, Dioleilfosfatidiletanolamina (DOPE), na presen?a ou aus?ncia de Rodaminafosfatidiletanolamina (PE-Rodamina) e do conjugado ?cido Hialur?nico DOPE (HA-DOPE). Estes sistemas foram caracterizados da mesma forma que as nanoemuls?es e tamb?m foram avaliados estabilidade, influ?ncia do tempo, tamanho de material gen?tico e presen?a ou aus?ncia de endotoxinas na forma??o dos lipoplexos. Os resultados obtidos permitem afirmar que os sistemas s?o promissores para posterior utiliza??o na terapia g?nica e que esta ?rea promete ser uma ?rea f?rtil de pesquisa cient?fica e cl?nica por muitos anos, e provavelmente se tornar? uma pr?tica cl?nica importante neste s?culo. No entanto, da possibilidade ? pr?tica existe um longo caminho a percorrer

Made available in DSpace on 2014-12-17T14:16:32Z (GMT). No. of bitstreams: 1 AndreLS_DISSERT.pdf: 5066827 bytes, checksum: 73399e5a8ccc704bf54eb5cf1faba287 (MD5) Previous issue date: 2013-03-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355?, 1.2 % of Tween 80?, 0.8 % of Span 80?, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 &#956;S/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ?C. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/&#956;L); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier / A terapia g?nica consiste na transfer?ncia de material gen?tico ex?geno para c?lulas ou tecidos no intuito de corrigir, suplementar ou silenciar um determinado gene. Para que este objetivo seja alcan?ado, eficientes ve?culos carreadores devem ser desenvolvidos: virais ou n?o-virais. O objetivo deste trabalho foi produzir e avaliar um sistema nanoemulsionado como poss?vel ve?culo n?o-viral para terapia g?nica, capaz de veicular um modelo plasmidial (pIRES2-EGFP). A nanoemuls?o foi produzida pelo m?todo de sonica??o, ap?s ser escolhida em um digrama de fases pseudo-tern?rio. A formula??o ? constitu?da de 5 % de Captex 355?, 1,2 % de Tween 80?, 0,8 % de Span 80?, 0,16 % de estearilamina e ?gua (qsp 100 %). O sistema foi caracterizado quanto ao tamanho de got?cula, ?ndice de polidispers?o (PI), potencial zeta, pH e condutividade. Os resultados mostraram got?culas menores que 200 nm (PI < 0,2) e potencial zeta > 30 mV em uma formula??o de pH pr?ximo a 7,0 e condutividade compat?vel a formula??es de ?leo em ?gua (70 90 &#956;S/s). A possibilidade de escalonamento, a estabilidade do sistema e a melhor forma de esteriliza??o tamb?m foram avaliadas onde observou-se que o sistema pode ser escalonado adequando o volume produzido ao tempo de sonica??o e a forma mais eficiente de esteriliza??o foi a filtra??o em membrana. O sistema apresentou estabilidade maior que 180 dias quando armazenado a 4 ?C. Uma vez desenvolvido, o sistema foi testado quanto ? capacidade de compactar o modelo plasmidial (pDNA). Eletroforese em gel de agarose foi a metodologia empregada para este fim, onde verificou-se que o poder de compacta??o aumenta (de 46 para 115 ng/&#956;L) quando a estearilamina ? incorporada na fase aquosa, respeitando um per?odo de contato (nanoemuls?o/pDNA) de pelo menos 2 horas, em banho de gelo. A nanoemuls?o mostrou-se pouco t?xica nas concentra??es usuais para ensaios de transfec??o em c?lulas do tipo MRC-5, apresentando 81,49 % de viabilidade celular. Portanto, o processo de produ??o de um sistema que complexa eficientemente o modelo plasmidial foi obtido. Contudo, estudos de efici?ncia de transfec??o devem ser conduzidos para confirmar o sistema como um carreador n?o-viral de material gen?tico

Made available in DSpace on 2014-12-17T15:18:13Z (GMT). No. of bitstreams: 1 LourenaMV.pdf: 713655 bytes, checksum: d2c61219ecefc4633620bb6eebd45325 (MD5) Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Broadly speaking, the concept of gene therapy involves the transfer of a genetic material into a cell, tissue, or organ in order to cure a disease or at least improve the clinical status of a patient. Making it simple, gene therapy consists in the insertion of functional genes into cells containing defective genes by substituting, complementing or inhibiting them. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, it is a key issue to create systems able to transfer and protect the DNA until it reaches the target, the vectors. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easily produced, present controllable stability and enable transfection. The aim of this work was to develop an emulsion for gene therapy and evaluate its ability to compact nucleic acids by the development of a complex with the plasmid pIRES2-EGFP. The first step was to determine the Hydrophilic Lipophilic Balance (HLB) of the Captex? 355 (oily internal phase of the emulsion) through long and short term stability assays. Based on the results, emulsions composed of Captex? 355, Tween 20? and Span 60? with 10.7 HLB were produced by three different methods: phase inversion, spontaneous emulsification and sonication. The results showed that the lowest diameter and best stability of the emulsions were achieved by the sonication method. The cationic emulsions were made by adding DOTAP to the basic emulsion. Its association with pIRES2-EGFP was evaluated by electrophoresis. Several rates of emulsion and DNA were evaluated and the results showed that 100% of the complex was formed when the rate DOTAP/DNA(nmol/?g) was 130. In conclusion, the overall results show the ability of the proposed emulsion to compact pIRES2-EGFP, which is a requirement to a successful transfection. Therefore, such formulation may be considered a promising candidate for gene therapy / Terapia g?nica, em uma ampla defini??o, ? o tratamento de doen?as baseado na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local. Assim, ? necess?rio criar ve?culos, os vetores, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de emuls?es cati?nicas como vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi desenvolver um sistema emulsionado para terapia g?nica e avaliar sua capacidade de compacta??o de ?cidos nucl?icos atrav?s da sua associa??o com o plasm?deo pIRES2-EGFP. Primeiramente o EHLc do TCM utilizado, o Captex? 355, foi determinado atrav?s de ensaios de estabilidade acelerada e a longo termo. Com base nos resultados obtidos, emuls?es de EHL 10,7 compostas de Captex? 355, Tween 20? e Span 60? foram preparadas pelos m?todos de invers?o de fases, emulsifica??o espont?nea e sonica??o e elegeu-se o melhor m?todo para o preparo das emuls?es cati?nicas. As emuls?es de menor granulometria e maior estabilidade foram obtidas atrav?s do m?todo de sonica??o. As emuls?es cati?nicas foram preparadas acrescendo-se ? emuls?o base o DOTAP e a sua associa??o com o pIRES2-EGFP foi avaliada atrav?s da t?cnica de eletroforese em gel de agarose. V?rias propor??es de emuls?o e DNA foram testadas e os resultados demonstraram que houve forma??o de 100% dos complexos quando a propor??o DOTAP/DNA(nmol/?g) foi igual a 130. Em conclus?o, o conjunto dos resultados obtidos demonstra a capacidade da emuls?o proposta neste trabalho de compactar o DNA, requisito necess?rio para uma boa transfec??o, tornando a formula??o uma forte candidata ? utiliza??o em terapia g?nica