Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-08T21:45:26Z No. of bitstreams: 1 PhilippeDeCastroMesquita_DISSERT.pdf: 4902837 bytes, checksum: f6da7808fb637146d14c266f835d467b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-13T21:11:08Z (GMT) No. of bitstreams: 1 PhilippeDeCastroMesquita_DISSERT.pdf: 4902837 bytes, checksum: f6da7808fb637146d14c266f835d467b (MD5) / Made available in DSpace on 2016-07-13T21:11:08Z (GMT). No. of bitstreams: 1 PhilippeDeCastroMesquita_DISSERT.pdf: 4902837 bytes, checksum: f6da7808fb637146d14c266f835d467b (MD5) Previous issue date: 2015-07-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Acidentes notificados envolvendo o envenenamento por escorpi?es ainda s?o frequentes no Brasil, ocasionados principalmente pela esp?cie Tityus serrulatus, conhecido como escorpi?o amarelo. Embora os soros antiescorpi?nicos sejam produzidos rotineiramente por diversos laborat?rios oficiais, a efic?cia de sua utiliza??o depende da rapidez com que se inicia o tratamento e da efici?ncia na produ??o de anticorpos pelos animais imunizados. No presente trabalho, o desenvolvimento de nanopart?culas polim?ricas cati?nicas de poli(?cido l?tico) teve como objetivo a busca de um sistema de libera??o modificada para os pept?deos e prote?nas presentes na pe?onha do escorpi?o T. serrulatus, capaz de potencializar a produ??o de anticorpos em soro anti-veneno. As nanopart?culas cati?nicas foram obtidas por nanoprecipita??o, ap?s o estudo do efeito dos par?metros da t?cnica sobre as propriedades f?sico-qu?micas das part?culas, com a otimiza??o de um m?todo de baixa energia. A funcionaliza??o da superf?cie das nanopart?culas com a polietilenoimina hiper-ramificada foi comprovada pela an?lise de potencial zeta e possibilitou a adsor??o por intera??o eletrost?ticade diferentes tipos de prote?nas. A efici?ncia de incorpora??o de 40-80 % de albumina de sorobovino (BSA) e 100 % de pept?deos da pe?onha do escorpi?o avaliadas por espectrofotometria e eletroforese em gel de poliacrilamida, confirmou o sucesso na escolha dos par?metros do m?todo de obten??o das nanopart?culas, produzidas com tamanho entre 100 a 250 nm. A an?lise de microscopia de for?a at?mica e estudos in vitro de libera??o, mostraram que as nanopart?culas esf?ricas fornecem um perfil sustentado de libera??o das prote?nas pelo mecanismo de difus?o, que potencializou a produ??o de anticorpos antiveneno em animais imunizados com as nanopart?culas contendo a pe?onha do escorpi?o, comprovando o potencial para aplica??o in vivo das nanopart?culas. / Reported accidents involving the poisoning scorpions are still frequent in Brazil, mainly caused by Tityus serrulatus, known as yellow scorpion. Although antivenom sera are produced routinely by various government laboratories, the effectiveness of its use depends on how quickly treatment is initiated and efficiency in the production of antibodies by the immunized animals. In this study, the development of cationic polymeric nanoparticles of poly(lactic acid) aimed to create a modified delivery system for peptides and proteins of T. serrulatus venom, able to enhance the production of serum antibodies against the scorpion toxins. The cationic nanoparticles were obtained by a low energy nanoprecipitation, after study of the parameters? variations effects over the physicochemical properties of the particles. The surface functionalization of the nanoparticles with the hyperbranched polyethyleneimine was proved by zeta potential analysis and enabled the adsorption by electrostatic interaction of different types of proteins. The protein loading efficiency of 40-80 % to bovine serum albumin (BSA) and 100 % to scorpion venom peptides evaluated by spectrophotometry and polyacrylamide gel electrophoresis confirmed the success of the selected parameters established for obtainment of nanoparticles, produced with size between 100 to 250 nm. The atomic force microscopy analysis and in vitro release showed that the spherical nanoparticles provided a sustained release profile of proteins by diffusion mechanism, demonstrating the potential for application of the nanoparticles in vivo.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:03Z No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:33Z (GMT) No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) / Made available in DSpace on 2018-06-06T14:01:33Z (GMT). No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) Previous issue date: 2018-04-03 / Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study. / Os problemas ambientais gerados por pl??sticos ?? base de tereftalato de polietileno (PET) se devem ao extenso tempo de decomposi????o desses materiais no meio ambiente e a sua fonte de origem que ?? dependente de petr??leo. Diante disso, biopl??sticos t??m sido uma alternativa promissora devido ao fato de serem biologicamente degrad??veis, al??m de terem como origem mat??rias-primas renov??veis, o que os tornam sustent??veis. Um dos pol??meros mais relevantes desse cen??rio ?? o poli (??cido l??tico) (PLA) formado a partir de mon??meros de ??cido l??tico. As principais caracter??sticas do PLA s??o baixa toxicidade aos humanos devido ?? alta biocompatibilidade, como por exemplo em mat??rias biom??dicos, e biodegradabilidade, o que reduz seu tempo em aterros devido ao processo mais r??pido de decomposi????o. Essas propriedades proporcionam uma ampla aplicabilidade deste pol??mero em diversas ??reas como embalagens, ??reas t??xteis e materiais biom??dicos. Comumente, o processo qu??mico de polimeriza????o do PLA pode ser realizado por meio de duas formas, (1) abertura do anel para posterior polimeriza????o ou (2) por condensa????o dos ??cidos l??ticos. Nos dois casos, ?? necess??ria a presen??a de catalisadores met??licos como zinco, alum??nio e magn??sio. Estes, al??m de serem t??xicos atrapalham na utiliza????o do pol??mero, por exemplo, na ??rea biom??dica, por gerar res??duos met??licos. Uma alternativa a esses catalisadores ?? a utiliza????o de biocatalisadores, como a polihidroxialcanoato sintase (phaC), j?? foi previamente utilizada para polimeriza????o de ??cido l??tico produzido em cepas recombinantes de Escherichia coli. Assim, dentro da plataforma de produ????o de ??cido l??tico, em cepas de Komagataella phaffii recombinantes, o objetivo deste trabalho ?? referente ?? produ????o da enzima phaC com muta????es pontuais nos s??tios S325N e Q481I, pois essas altera????es proporcionam uma maior especificidade do complexo enzima-substrato, para que atue como biocatalisador na polimeriza????o de ??cido l??tico em Komagataella phaffi. Neste estudo, foram realizadas tr??s estrat??gias de clonagem entre o inserto phaCPs e vetor pGAPZ??B. At?? o presente, n??o houve transformantes em nenhuma das estrat??gias. Entretanto, a Estrat??gia C ainda n??o foi executada completamente, o que resulta ainda na possibilidade de clonagem entre inserto phaCPs e vetor de express??o pGAPZ??B com a sequ??ncia correta. A expectativa deste estudo ?? a conclus??o da clonagem, verifica????o da sequ??ncia correta do DNA recombinante atrav??s do resultado do sequenciamento e inser????o do plasm??deo ao genoma da levedura Komagataella phaffii.

Made available in DSpace on 2014-12-17T14:16:27Z (GMT). No. of bitstreams: 1 AliceRO_DISSERT.pdf: 2104659 bytes, checksum: 208850f5293dd4764037ec4c490d3636 (MD5) Previous issue date: 2011-12-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Methotrexate (MTX) is a drug used in the chemotherapy of some kind of cancers, autoimmune diseases and non inflammatory resistant to corticosteroids uveits. However, the rapid plasmatic elimination limits its therapeutic success, which leads to administration of high doses to maintain the therapeutic levels in the target tissues, occurring potential side effects. The aim of this study was to obtain spray dried biodegradable poly-lactic acid co-glycolic acid (PLGA) microparticles containing MTX. Thus, suitable amounts of MTX and PLGA were dissolved in appropriate solvent system to obtain solutions at different ratios drug/polymer (10, 20, 30 and 50% m/m). The physicochemical characterizing included the quantitative analysis of the drug using a validate UV-VIS spectrophotometry method, scanning electron microscopy (SEM), infrared spectrophotometry (IR), thermal analyses and X-ray diffraction analysis. The in vitro release studies were carried out in a thermostatized phosphate buffer pH 7.4 (0.05 M KH2PO4) medium at 37?C ? 0.2 ?C. The in vitro release date was subjected to different kinetics release models. The MTX-loaded PLGA microparticles showed a spherical shape with smooth surface and high level of entrapped drug. The encapsulation efficiency was greater then 80%. IR spectroscopy showed that there was no chemical bond between the compounds, suggesting just the possible occurrence of hydrogen bound interactions. The thermal analyses and X-ray diffraction analysis shown that MTX is homogeneously dispersed inside polymeric matrix, with a prevalent amorphous state or in a stable molecular dispersion. The in vitro release studies confirmed the sustained release for distinct MTX-loaded PLGA microparticles. The involved drug release mechanism was non Fickian diffusion, which was confirmed by Kornmeyer-Peppas kinetic model. The experimental results demonstrated that the MTX-loaded PLGA microparticles were successfully obtained by spray drying and its potential as prolonged drug release system. / O metotrexato (MTX) ? um f?rmaco utilizado na quimioterapia de alguns tipos de c?ncer, doen?as autoimunes e uve?tes n?o inflamat?rias resistentes aos corticoster?ides. No entanto, sua r?pida elimina??o plasm?tica limita o sucesso terap?utico, levando ? necessidade de altas doses para manuten??o da concentra??o efetiva no tecido alvo, ocasionando o potencial surgimento de rea??es adversas. O objetivo principal desse estudo foi obter um sistema microparticulado biodegrad?vel ? base de ?cido poli (?cido l?tico-co-?cido glic?lico) (PLGA) por spray drying para libera??o prolongada do MTX. Para isso, quantidades distintas de MTX e PLGA foram dissolvidas em sistema solvente adequado para obter solu??es com diferentes propor??es de f?rmaco em rela??o ao pol?mero (10, 20, 30 e 50% m/m). A caracteriza??o f?sicoqu?mica incluiu an?lise quantitativa do f?rmaco incorporado na matriz polim?rica por espectrofotometria UV-VIS em 303nm previamente validada, microscopia eletr?nica de varredura (MEV), espectrofotometria de infravermelho (IV), an?lises t?rmicas e difra??o de raios-X (DRX). O perfil de libera??o in vitro do f?rmaco nas micropart?culas foi realizado em tamp?o fosfato (0.05 M KH2PO4) em banho termostatizado 37 ?C ? 0.2 ?C. Os dados obtidos do estudo de libera??o in vitro foram submetidos a diferentes modelos cin?ticos de libera??o. As micropart?culas de PLGA contendo o MTX apresentaram a forma esf?rica, uniforme, com superf?cie aparentemente lisa. O n?vel de efici?ncia de encapsula??o foi superior a 80%. A espectroscopia na regi?o do infravermelho demonstrou que n?o ocorreu liga??o qu?mica entre os componentes dos sistemas, no entanto foi observado forte intera??o entre o MTX e PLGA indicando prov?vel ocorr?ncia de pontes de hidrog?nio. An?lise XII t?rmica e DRX demonstraram que o MTX est? distribu?do na matriz polim?rica com a preval?ncia do estado amorfo ou em dispers?o molecular. O estudo de libera??o in vitro confirmou o perfil de libera??o prolongada para as diferentes micropart?culas. O mecanismo de libera??o envolvido foi por difus?o n?o Fickiana, ao qual foi determinado a partir do modelo cin?tico de Kornmeyer- Peppas. Os resultados experimentais demonstraram o sucesso na obten??o das micropart?culas de PLGA contendo o MTX por spray drying e seu potencial como sistema de libera??o prolongada do f?rmaco.