Submitted by PPG Engenharia e Tecnologia de Materiais (engenharia.pg.materiais@pucrs.br) on 2018-05-17T18:27:28Z No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-05-23T14:59:14Z (GMT) No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) / Made available in DSpace on 2018-05-23T15:10:26Z (GMT). No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) Previous issue date: 2018-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The present study focuses on the development of nanoparticles with an iron oxide magnetic core, with different biocompatible coatings, and on a comparative study of their toxicities. Uncoated and dextran-, chitosan-, polyethylene glycol- and silica-coated nanoparticles were synthesized. The addition of optical markers of the benzo-thiazoles class was also accomplished. The physico-chemical properties of the nano-particles were characterized, including their magnetic, optical and contrast properties in nuclear magnetic resonance imaging (relaxivities). In the particular case of nanopar-ticles functionalized with 6-OH-BTA-1 molecules, the affinity to the beta-amyloid pep-tide was also investigated. A second step was to evaluate the toxicological effects of these nanoparticles in vitro (using in VERO cells), and in vivo with zebrafish as an animal model. The size of the nanoparticles with the coatings ranged from 13 to 30 nm. Their crystalline structure was consistent with the ferrite spinel. The nanoparticles, independent of the coating, did not present residual magnetization and hysteresis, in-dicating superparamagnetic behaviour. For most nanoparticles, the r2 transverse re-laxivity values ranged from 76-64 mM-1.s-1, exceptfor uncoated and chitosan-coated nanoparticles, which present higher values, possibly due to the aggregation. The val-ues of r1 were similar for all nanoparticles (12.6 to 18 mM-1.s-1), with the exception of silica-coated nanoparticles (r1=2.1 mM-1.s-1). The r2/r1 ratios were between 4 and 17, typical of commercially available negative contrast-agents. The nanoparticles function-alized with benzothiazoles showed fluorescence with a Stokes shift of the emission peak of ~ 197 nm. The interaction of the beta-amyloid peptide with the 6-OH-BTA-1 molecule analyzed by fluorescence suppression is characterized by a static mecha-nism and Stern-Volmer constants of 1.53x104 mM-1 for the monomeric form, 1.40x104 mM -1 for oligomers) and 1.33x104 mM -1 for amyloid plaques.The in vitro toxicity assays indicated acceptable values of cell viability for iron concentration up to 2 mmol.L-1. The nanoparticles with the carboxysilane and polyethylene glycol showed higher biocom-patibility and silica-coated nanoparticles had the highest cytotoxicity. The in vivo as-says did not show significant changes in survival and hatchability rates, except for doses greater than 2 mmol.L-1 in the case of the chitosan-coated nanoparticles. The percentages of animals with anatomical alterations were similar between the treated and control groups. In the locomotion and exploration tests, only chitosan- and silica-coated nanoparticles induced significant changes. / O presente trabalho aborda o desenvolvimento de nanopart?culas compostas por um n?cleo magn?tico de ?xido de ferro com diferentes revestimentos biocompat?-veis e estudo comparativo de suas toxicidades. Foram sintetizadas nanopart?culas de ?xido de ferro sem revestimento e com revestimentos de dextrana, quitosana, polieti-lenoglicol e s?lica, e com a adi??o de marcadores ?pticos da classe dos benzotiaz?is. As propriedades f?sico-qu?micas das nanopart?culas foram caracterizadas, incluindo as suas propriedades magn?ticas, ?pticas e de contraste em imagens por resson?ncia magn?tica nuclear (relaxividades), bem como a afinidade ao pept?deo beta-amiloide, no caso particular de funcionaliza??o com a mol?cula 6-OH-BTA-1. Em uma segunda etapa, foram avaliados os efeitos toxicol?gicos dessas nanopart?culas em ensaios bi-ol?gicos in vitro em c?lulas VERO, e in vivo tendo como animal modelo o peixe zebra. O tamanho das nanopart?culas com revestimentos variou entre 13 a 30 nm, e estrutura cristalina coerente com o espin?lio de ferrita. As nanopart?culas n?o apresentaram magnetiza??o residual e histerese, indicando superparamagnetismo, independente do revestimento. Para a maioria das nanopart?culas, os valores de relaxividade transver-sal r2 variaram de 76-64 mM-1.s-1, com exce??o das nanopart?culas sem revestimento e de quitosana, os quais foram mais elevados, possivelmente devido ao efeito de agrega??o. Os valores de r1 foram semelhantes para todas as nanopart?culas (12,6 a 18 mM-1.s-1), com exce??o das nanopart?culas de s?lica (r1 = 2,1 mM-1.s-1). As raz?es r2/r1 foram entre 4 e 17, valores t?picos de agentes de contraste negativos comercial-mente dispon?veis. As nanopart?culas funcionalizadas com os benzotiaz?is mantive-ram sua fluoresc?ncia com deslocamentos de Stokes na ordem de 197 nm para o pico de emiss?o. As an?lises da intera??o do pept?deo beta-amiloide com a mol?cula 6-OH-BTA-1, mostraram valores de constante de Stern-Volmer para supress?o de fluo-resc?ncia de 1,53x104 mM-1 (mon?mero), 1,40x104 mM-1 (olig?mero) e 1,33x104 mM-1 (placa), indicando quenching por um mecanismo est?tico. O pept?deo na forma mo-nom?rica demonstrou maior facilidade de acesso ?s mol?culas de 6-OH-BTA-1. Os resultados dos ensaios in vitro indicaram valores aceit?veis de viabilidade celular para concentra??o de ferro inferior a 2 mmol.L-1. As nanopart?culas com o carboxisilano e polietilenoglicol demostraram maior biocompatibilidade e as nanopart?culas de s?lica tiveram a maior citotoxicidade. Os resultados dos ensaios in vivo n?o mostraram alte-ra??es significativas na taxa de sobreviv?ncia e de eclos?o do corium, exceto para as doses maiores que 2 mmol.L-1 das nanopart?culas revestidas com quitosana. Os per-centuais de animais com altera??es anat?micas foram similares entre os grupos tra-tados e de controle. Nos ensaios de locomo??o e explora??o, apenas as nanopart?cu-las de quitosana e de s?lica induziram altera??es adversas significativas.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-11-01T22:37:28Z No. of bitstreams: 1 RaimundoNazarenoDaSilvaConceicaoFilho_DISSERT.pdf: 12242083 bytes, checksum: 01e4da6c38f0cf877f871a80f9e59811 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-11-14T00:09:04Z (GMT) No. of bitstreams: 1 RaimundoNazarenoDaSilvaConceicaoFilho_DISSERT.pdf: 12242083 bytes, checksum: 01e4da6c38f0cf877f871a80f9e59811 (MD5) / Made available in DSpace on 2017-11-14T00:09:04Z (GMT). No. of bitstreams: 1 RaimundoNazarenoDaSilvaConceicaoFilho_DISSERT.pdf: 12242083 bytes, checksum: 01e4da6c38f0cf877f871a80f9e59811 (MD5) Previous issue date: 2017-09-01 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Neste trabalho utilizamos o m?todo qu?mico poliol para a prepara??o de nanopart?culas de Sm-Co. As amostras sintetizadas foram nomeadas das seguintes formas: ARS-0%; ARS-5%;ARS-7% e ARS-9%, sendo as porcentagens referente a varia??o do ?cido Ac?tico utilizado na s?ntese. A caracteriza??o estrutural foi realizada via difra??o de raio-X e microscopia eletr?nica de transmiss?o (MET). Os par?metros estruturais foram determinados pelo m?todo de Rietvelt utilizando o software Maud. As seguintes fases foram identificadas: Sm2Co7; Sm2Co17 e Sm5Co19. Para o estudo das propriedades magn?ticas, medidas com o magnet?metro de amostra vibrante foram utilizadas para obten??o de curvas de magnetiza??es em fun??o do campo magn?tico aplicado ? temperatura ambiente. O PPMS foi utilizado para medir curvas de magnetiza??o em fun??o da temperatura. As temperaturas de Curie obtidas est?o de acordo com as fases obtidas pelos m?todos de caracteriza??o estrutural. Foram obtidas imagens das amostras atrav?s do MEV e MET para estudo morfol?gico e determina??o do tamanho das nanopart?culas das amostras. Nossos resultados mostram a viabilidade do m?todo poliol para obten??o de nanopart?culas de Sm-Co e a influencia do ?cido Ac?tico no processo de s?ntese. / In this work we used the chemical polyol method for the preparation of Sm-Co nanoparticles. The synthesized samples were named in the following forms: ARS-0%; ARS-5%, ARS-7% and ARS-9%, being the percentages referring to the variation of Acetic Acid used in the synthesis. The structural characterization was performed by X-ray diffraction and transmission electron microscopy (TEM). The structural parameters were determined by the Rietvelt method using the Maud software. The following phases were identified: Sm2CO7; Sm2Co17 and Sm5Co19. For the study of magnetic properties, measurements with the vibrating sample magnetometer were used to obtain magnetization curves as a function of the magnetic field applied at room temperature. The PPMS was used to measure magnetization curves as a function of temperature. The Curie temperatures obtained are in accordance with the phases obtained by the structural characterization methods. Images of the samples were obtained through the SEM and MET for morphological study and determination of the size of the nanoparticles of the samples. Our results show the feasibility of the polyol method to obtain Sm-Co nanoparticles and the influence of Acetic Acid in the synthesis process.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-01-24T12:09:54Z No. of bitstreams: 1 SilasSarmentoPedrosa_TESE.pdf: 13307553 bytes, checksum: 616db20e12e34747afe3ca24c303b31a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-01-26T12:09:55Z (GMT) No. of bitstreams: 1 SilasSarmentoPedrosa_TESE.pdf: 13307553 bytes, checksum: 616db20e12e34747afe3ca24c303b31a (MD5) / Made available in DSpace on 2018-01-26T12:09:55Z (GMT). No. of bitstreams: 1 SilasSarmentoPedrosa_TESE.pdf: 13307553 bytes, checksum: 616db20e12e34747afe3ca24c303b31a (MD5) Previous issue date: 2017-09-15 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / H? presentemente grande interesse de pesquisa em aglomerados de nanopart?culas superparamagn?ticas, devido em parte ? alta demanda para aplica??es biom?dicas, e ao mesmo tempo ao grande interesse, do ponto de vista fundamental, em novas fases magn?ticas. A suscetibilidade magn?tica inicial e o campo de fuga, s?o fatores essenciais para otimiza??o de sistemas para aplica??es biom?dicas. H?, ao mesmo tempo, grande interesse em confirmar a exist?ncia de ferromagnetismo dipolar, em sistemas onde a energia de troca n?o ? fator dominante. Desenvolvemos um estudo te?rico do impacto da intera??o dipolar sobre as fases magn?ticas de nanopart?culas superparamagn?ticas, confinadas em aglomerados esf?ricos e elipsoidais. Consideramos nanopart?culas de Fe3O4 com tamanhos no intervalo de 9 nm a 12 nm, arranjadas com densidade uniforme em aglomerados de tamanho da ordem de centenas de nan?metros. Mostramos que as fases magn?ticas, e a suscetibilidade inicial, s?o controladas pela intera??o dipolar, e que a topologia do arranjo de nanopart?culas, o tamanho das nanopart?culas e a densidade de empacotamento s?o fatores que controlam as propriedades magn?ticas. Mostramos que a intera??o dipolar pode estabilizar fases magn?ticas cl?ssicas, conhecidas apenas para sistemas com alto conte?do de energia de troca e de anisotropia. Al?m disso, as fases magn?ticas em reman?ncia t?m uma caracter?stica peculiar: a m?dia t?rmica do momento de cada nanopart?cula pode se aproximar do valor de satura??o, mantendo o aglomerado superparamagn?tico. Aglomerados elipsoidais de alta excentricidade s?o os sistemas de escolha para aplica??es biom?dicas porque podem exibir expressivo aumento de suscetibilidade magn?tica, mantendo um campo de fuga de baixa intensidade em reman?ncia. O modelo te?rico reproduz satisfatoriamente resultados experimentais de aglomerados esf?ricos de Fe3O4, e de sistemas de part?culas de Fe e Co de baixa dimensionalidade. / Superparamagnetic nanoparticles clusters are currently driving considerable research attention. The interest stems from chances of designing systems with promising potential for technological applications, and from the fundamental viewpoint, tailoring new magnetic phases. The initial magnetic susceptibility and the stray field, at remanence, are key features for the optimization of magnetic systems for biomedical applications. Also, the existence of dipolar ferromagnetism, in the absence of exchange energy, has been one of the focus of magnetism for decades. We report a theoretical discussion of the impact of the dipolar interactions on the magnetic phases of superparamagnetic nanoparticles confined in spherical and ellipsoidal clusters. We consider Fe3O4 nanoparticles, with size ranging from 9 nm to 12 nm, arranged with uniform density in hundreds nanometer size volumes. We show that the magnetic phases, and the initial susceptibility, are controlled by the dipolar interaction. Also, the topological nanoparticle arrangement, the nanoparticle size, and the packing density, are key features. We show that the dipolar interaction alone may stabilize classical magnetic phases, well known for systems with large content of exchange and anisotropy energies. In addition, we have found that at remanence the nanoparticles clusters magnetic phase have a unique property. The dipolar energy leads to thermal stabilization of the individual nanoparticles moments. Large nanoparticles densities may allow nearly full thermal value of the nanoparticles magnetic moments. Despite this, the nanoparticles cluster is superparamagnetic, with a rather small stray field at remanence, as required for biomedical safety. Nanoparticle clustering in large eccentricity ellipsoidal volumes are promising systems for both low field and large field biomedical applications. For low field applications, there is a large increase in the initial susceptibility, with enhancement in the efficacy of vector targeting and also for hyperthermia absorption rate. For high field applications, the enhancement of the stray is much stronger than that for spherical clusters. Our theoretical model reproduces typical properties of Fe3O4 nanoparticles spherical clusters, as well as intriguing results for Fe and Co quasi-one-dimensional systems.

Made available in DSpace on 2014-12-17T15:42:15Z (GMT). No. of bitstreams: 1 FabioPF_TESE.pdf: 3376603 bytes, checksum: 15dfaa7fe12ca918fd7e1b98c4378dd9 (MD5) Previous issue date: 2011-09-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 ?C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media / S?ntese enzim?tica de pept?deos usando proteases tem atra?do uma enorme aten??o nos ?ltimos anos. Um desafio chave na s?ntese de pept?deos ? encontrar suportes para imobiliza??o de proteases capazes de apresentar um alto desempenho em meio aquoso, produzindo oligopept?deos sol?veis em ?gua, j? que at? o presente momento, pouco tem sido descrito usando essa estrat?gia. Dessa forma, o objetivo dessa tese foi imobilizar proteases usando diferentes m?todos (imobiliza??o por liga??o covalente, aprisionamento em g?is polim?ricos de PVA e imobiliza??o em nanopart?culas magn?ticas de Glicidil) para a produ??o de oligopept?deos derivados da lisina. Tr?s proteases foram utilizadas: tripsina, α-quimotripsina e bromela?na. De acordo com as estrat?gias de imobiliza??o associadas ao tipo de protease empregada, foi provado que os sistemas tripsina-resinas mostraram os melhores desempenhos em termos de atividade hidrol?tica e s?ntese de oligopept?deos. A atividade hidrol?tica das enzimas livres e imobilizadas foi determinada por espectrofotometria com base na mudan?a de absorb?ncia em 660 nm ? temperatura de 25 ?C (Casein method). O rendimento de oligolisina e o c?lculo do grau de polimeriza??o m?dio foram monitorados por RMN H. A protease tripsina foi covalentemente imobilizada em quatro diferentes resinas (Amberzyme, Eupergit C, Eupergit CM and Grace 192). O m?ximo rendimento de prote?na imobilizada foi 92, 82, 60, e 71 mg/g de suporte para cada resina, respectivamente. A efici?ncia desses sistemas (Tripsina-resinas) foi avaliada pela hidr?lise do substrato case?na e a s?ntese de oligolisina em meio aquoso. A maioria dos sistemas foram capazes de catalisar a s?ntese de oligopept?deos, entretanto com diferentes efici?ncias, tais como: 40, 37 e 35% para os suportes Amberzyme, Eupergit C e Eupergit CM, respectivamente, em compara??o com a enzima livre. Esses sistemas produziram olig?meros em somente 1 hora com grau de polimeriza??o m?dio mais alto que a enzima livre. Dentre esses sistemas, Eupergit CTripsina mostrou ser mais eficiente que os outros sistemas em termos de atividade hidrol?tica e estabilidade t?rmica, ao passo que n?o exibiu a mesma efici?ncia como era esperado para a s?ntese de oligolisina. Tripsina-amberzyme provou ser mais eficiente para a s?ntese de oligopept?deos, al?m de exibir um excelente reuso, mantendo 90% de sua atividade hidrol?tica e sint?tica ap?s sete reusos. As intera??es hidrof?bicas da tripsina com o suporte Amberzyme s?o respons?veis por proteger a enzima contra as fortes mudan?as conformacionais no meio reacional. Al?m disso, a alta concentra??o de grupos oxiranos na superf?cie da resina promoveu liga??es covalentes multipontuais e, consequentemente, preveniu a enzima imobilizada do processo de desor??o (Leaching process). Os resultados acima mencionados sugerem que a tripsina imobilizada nesses suportes pode ser eficientemente usada para a s?ntese de oligopept?deos em meio aquoso