Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-02-21T00:14:47Z No. of bitstreams: 1 YagoQueirozDosSantos_DISSERT.pdf: 1419350 bytes, checksum: 3ae7b369d8ed52af2d85454e8aa5fdbb (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-03T21:49:39Z (GMT) No. of bitstreams: 1 YagoQueirozDosSantos_DISSERT.pdf: 1419350 bytes, checksum: 3ae7b369d8ed52af2d85454e8aa5fdbb (MD5) / Made available in DSpace on 2017-03-03T21:49:40Z (GMT). No. of bitstreams: 1 YagoQueirozDosSantos_DISSERT.pdf: 1419350 bytes, checksum: 3ae7b369d8ed52af2d85454e8aa5fdbb (MD5) Previous issue date: 2016-06-16 / A atual demanda por fontes de energia renov?veis tem impulsionado a busca de alternativas capazes de substituir o uso de combust?veis f?sseis. Uma das inova??es mais promissoras para impactar positivamente o cen?rio mundial de energia ? a produ??o de bioetanol de segunda gera??o (B2G), derivado de monossacar?deos obtidos a partir da degrada??o enzim?tica de material lignocelul?sico que normalmente ? descartado nos atuais processos agroindustriais. No presente trabalho, uma celulase halotolerante secretada por uma linhagem marinha Bacillus sp. SR22 com grande resist?ncia ?s mudan?as de temperatura e pH foi isolada e caracterizada. A endoglucanase de massa aproximada de 37.35 kDa nomeada como Bc22Cel foi purificada por precipita??o de sulfato de am?nio, cromatografia de gel filtra??o e extra??o do gel ap?s eletroforese em gel de poliacrilamida em condi??es nativas. O valor ideal de pH e temperatura de Bc22Cel foram 6,5 e 60 ? C, respectivamente. A enzima purificada demonstrou consider?vel propriedade halof?lica ao manter mais de 70% de atividade residual mesmo quando pr?-incubadas com 1,5 M de NaCl durante 1 hora. Ap?s a an?lise cin?tica de enzima purificada os valores de Km e Vmax foram estabelecidos em 0,3953 nmol.ml-1 e 0,0167 ?mol.ml-1.min-1, respectivamente. Avaliados conjuntamente, os presentes dados apontam a Bc22Cel como uma candidata potencial para aplica??es industriais de degrada??o de celulose. / The current demand for environment-friendly renewable energy sources has driven the search for alternatives capable of replacing the use of fossil fuels. One of the most promising innovations to positively impact the world energy scenario is the second-generation bioethanol production (B2G) from reducing sugars derived from enzymatic degradation of lignocellulosic material that is ordinarily discarded at agroindustrial processes. In this work, a salt-tolerant cellulase secreted by a marine Bacillus sp. SR22 strain with wide resistance to temperature and pH was isolated and characterized. The 37.35 kDa endoglucanase named as Bc22Cel was purified by ammonium sulphate precipitation, gel filtration chromatography and extraction from the gel after non-reducing sodium dodecylsufate-polyacrylamide gel electrophoresis. The optimal pH value and temperature of Bc22Cel were 6.5 and 60 ?C, respectively. The purified Bc22Cel showed a considerable halophilic property being able to maintain more than 70% of residual activity even when pre-incubated with 1.5 M NaCl for 1 hour. Kinetic analysis of purified enzyme showed the Km and Vmax to be 0,3953 nmol ml-1 and 0,0167 ?mol ml-1 min-1, respectively. Taking together, the present data indicates the Bc22Cel as a potential and useful candidate for industrial applications.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-09T20:08:05Z No. of bitstreams: 1 PabloDeCastroSantos_TESE.pdf: 3735284 bytes, checksum: 5bc846ee3890e15ca33dc1e32e7349d8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-13T20:05:04Z (GMT) No. of bitstreams: 1 PabloDeCastroSantos_TESE.pdf: 3735284 bytes, checksum: 5bc846ee3890e15ca33dc1e32e7349d8 (MD5) / Made available in DSpace on 2017-03-13T20:05:04Z (GMT). No. of bitstreams: 1 PabloDeCastroSantos_TESE.pdf: 3735284 bytes, checksum: 5bc846ee3890e15ca33dc1e32e7349d8 (MD5) Previous issue date: 2016-08-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A urolit?ase afeta aproximadamente 10% da popula??o mundial e est? associada fortemente a presen?a de cristais de oxalato de c?lcio (OxCa). Atualmente n?o existe nenhum composto eficiente que pode ser utilizado para prevenir esta doen?a. No entanto, alguns polissacar?deos sulfatados (PS) de algas marinhas marrons demonstraram capacidade de inibir a forma??o de cristais de OxCa e alterar a morfologia destes in vitro. Os PS da alga vermelha Gracilaria birdiae t?m atividades biol?gicas importantes, mas at? o presente momento n?o foi avaliada como inibidora da forma??o de cristais de OxCa. O presente estudo teve como objetivo obter PS e verificar seu efeito na forma??o do OxCa. Para tal, extratos ricos em polissacar?deos foram obtidos utilizando extra??o alcalina, sonica??o, digest?o proteol?tica, seguida por precipita??o com etanol. Esses extratos ricos em PS (EB) foram separados em 5 fra??es (F-0.25; F-0.5; F-0.75; F-1.0 e F-1.25) atrav?s de cromatografia de troca i?nica DEAE-celulose. A eletroforese em gel de agarose, infra vermelho e an?lises qu?micas mostraram que essas fra??es cont?m o mesmo pool de polissacar?deos sulfatados ricos em galactose. F-0.25; F-0.5; F-0.75; F-1.0 foram capazes de inibir etapas importantes da forma??o dos cristais de OxCa, como a nuclea??o e agrega??o, no entanto a F-0.25 promoveu a maior inibi??o da nuclea??o em 76,92% e da agrega??o em 68,57%. As fra??es F-0.25 e F-0.5 foram capazes de inibir em 6 e 7 vezes, respectivamente, o n?mero de cristais OxCa monohidratados (COM) formados in vitro, enquanto que as fra??es F-0.75 e F-1.0, reduziram apenas em 1,5 vezes. A morfologia dos cristais de OxCa foi afetada principalmente pelas amostras, EB, F-0.25; F-0.5 e F-0.75, pois promoveram as varia??es mais destoantes em rela??o ao controle. A an?lise do potencial zeta (?) dos cristais formados na presen?a das amostras, constatou um aumento de cargas negativas em suas superf?cies. Atrav?s das imagens obtidas por microscopia de fluoresc?ncia, foi constatado que os PS est?o distribu?dos de forma homog?nea nos cristais dihidratados (COD) e de forma perif?rica nos COM. Os dados obtidos atrav?s da microscopia eletr?nica de varredura(MEV) e espectroscopia de energia dispersiva (EDS) revelaram grandes varia??es na distribui??o de ?tomos de oxig?nio e c?lcio na superf?cie dos cristais na presen?a das F-0.25 e F-0.5. A c?lulas renais HEK-293, MDCK e 786-0 tiveram baixa toxicidade na presen?a do extrato bruto e das fra??es F-0.25; F-0.5; F-0.75. Estas amostras protegeram c?lulas renais MDCK e verificou-se um efeito reparador contra danos causados pelo H2O2 e OxCa. Por fim, as c?lulas MDCK submetidas ao tratamento pr?vio e simult?neo com a F-0.25 e a F-0.5 na presen?a de H2O2 e OxCa, reduziram a atividade das enzimas super?xido dismutase (SOD) e catalase (CAT). Com isto, verifica-se que PS da G. birdiae podem ser potenciais alvos farmacol?gicos do ponto de vista preventivo, terap?utico e reparador de danos causados pela urolit?ase, por?m ? importante que outros estudos sejam realizados in vivo, bem como, sejam realizados experimentos para elucidar os mecanismos precisos de a??o, pelos quais estes PS protegem as c?lulas contra danos por H2O2. / The urolithiasis disease affects approximately 10% of the world's population and is strongly associated calcium oxalate crystals (CaOx). Until now, there is not an efficient compound that can be used to prevent this disease. However, some sulfated polysaccharides (SP) from brown seaweeds exhibited an ability to inhibit the formation of CaOx crystals and change the morphology in vitro. SP from the red seaweed Gracilaria birdiae has important biological activities, but they have not been evaluated as inhibitor of CaOx crystals formation. This study aimed to obtain SP from this seaweed and evaluate their effect on CaOx crystals formation. Thus, sulfated polysaccharide-rich extract (EC) was obtained using alkaline extraction, sonication, proteolytic digestion followed by ethanol precipitation. This extract was fractionated into five fractions (F-0.25; F-0.5; F-0.75; F-1.0; F-1.25) by DEAE-cellulose ion-exchange chromatography. Agarose gel electrophoresis, infrared and chemical analysis showed that these fractions contain the same pool of sulfated galactose-rich polysaccharides. F-0.25; F-0.5; F, 0.75; F-1.0 were able to inhibit important formation steps of the CaOx crystals, as nucleation and aggregation, we highlight F-0.25 that promoted the highest inhibition of nucleation in 76.92% and aggregation in 68.57%. The F-0.25 and F-0.5 fractions were able to reduce approximately 6 and 7 times, respectively, the number of these CaOx monohydrated crystals (COM) formed in vitro, whereas F-0.75 and F-1.0 fractions reduced this number only 1.5 times. The morphology of CaOX crystals was mainly affected by the samples EC, F-0.25; F-0.5 and F-0.75, they promoted the most discordant variations in the control. The analysis of the zeta potential (?) of the crystals formed in the presence of the samples was found an increase of negative charges on their surfaces. Through the images obtained by fluorescence microscopy revealed that the PS are distributed homogeneously in the dehydrated crystals (COD) and peripherally in COM. The data obtained by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) revealed wide variations in the distribution of oxygen and calcium atoms on the surface of the crystals in the presence of F-0.25 and F-0.5. The kidney cells HEK-293, MDCK and 786-0 showed low toxicity in the presence of EC and fractions F-0.25; F-0.5; F, 0.75. These samples protected kidney MDCK cells against damage caused by H2O2 and CaOx. Finally, the MDCK cells treated beforehand and concomitant with both F-0.25 and F-0.5 in the presence of H2O2 and CaOx, reduced the activity of superoxide dismutase and catalase enzymes. Overall, the data showed that PS G. birdiae may be potential pharmacological targets for preventive, therapeutic and repairing damage caused by urolithiasis. However, it is important that to make set in vivo experiments, as well as, experiments to elucidate the precise mechanism of action by which these PS protect the H2O2 damage on cells.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-09T20:08:05Z No. of bitstreams: 1 PaulaIvaniMedeirosDosSantos_TESE.pdf: 2576570 bytes, checksum: ad723940125128ab00057ee1698f195a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-13T20:16:47Z (GMT) No. of bitstreams: 1 PaulaIvaniMedeirosDosSantos_TESE.pdf: 2576570 bytes, checksum: ad723940125128ab00057ee1698f195a (MD5) / Made available in DSpace on 2017-03-13T20:16:47Z (GMT). No. of bitstreams: 1 PaulaIvaniMedeirosDosSantos_TESE.pdf: 2576570 bytes, checksum: ad723940125128ab00057ee1698f195a (MD5) Previous issue date: 2015-07-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / As esponjas marinhas se apresentam como uma rica fonte de novos compostos bioativos de grande interesse biotecnol?gico, vimos, neste trabalho, relatar a purifica??o e caracteriza??o de uma lectina da esp?cie Aplysina fulva, denominada AFL com atividade imunomoduladora, bem como avaliar se a mesma possue efeito citot?xico sobre c?lulas em cultura (normais, tumorais e leishmanias). As prote?nas totais foram extra?das da esponja com tamp?o Tris-HCl 20 mM, pH 7,5, e fracionadas por precipita??o com acetona. A fra??o obtida com 1 volume de acetona possu?a maior atividade hemaglutinante e foi submetida a uma cromatografia em matriz de goma guar com tamp?o tetraborato de s?dio 20 mM, pH 7,5. Em seguida foi aplicada em uma coluna de Superdex 75 (FPLC-AKTA) contendo tamp?o Tris- HCl 20 mM, pH 7,5. A pureza da lectina foi atestada em uma coluna HILIC (HPLC). A sequ?ncia de 15 res?duos de amino?cidos do N-terminal da lectina foi determinada por degrada??o de Edman e n?o apresentou similaridade com nenhuma outra prote?na dos bancos de dados mais conhecidos. A massa foi estabelecida em 62 kDa, por gel filtra??o em Superdex 75. An?lise ap?s tratamento com agentes redutores permitiu deduzir que AFL ? uma prote?na homotetram?rica. Sua atividade hemaglutinante foi reduzida quando a lectina foi exposta em meio ?cido (pH < 4) ou quando submetida a temperaturas acima de 60 ?C. AFL apresentou especificidade para lactose e parcial para D-glicose e D-galactose. A lectina purificada AFL n?o apresentou efeito citot?xico para as linhagens de c?lulas cancer?genas (PANC, HeLa, HT-29, Bf16F10, A549) e de fibroblastos (MRC5). AFL foi capaz de aglutinar formas promastigotas vivas de Leishmania amazonensis e Leishmania. braziliensis,e teve sua atividade revertida pela adi??o de lactose ao ensaio. AFL n?o diminuiu de forma significante a viabilidade de Leishmania amazonensis. A lectina AFL apresentou atividade mitog?nica em concentra??es a partir de 40 ?g/mL para c?lula mononucleares do sangue perif?rico (mon?citos e linf?citos). A lectina AFL foi capaz de induzir na linhagem de macr?fagos (RAW264.7) de murinos a libera??o de TNF- ?, mas n?o de IL-6, na concentra??o de 10,0 ?g/mL. Quando AFL foi incubada com c?lulas espl?nicas de camundongos BALB/c, foi capaz de estimular a produ??o de IFN-? e promover a diferencia??o de c?lulas T para uma resposta imune celular do tipo Th1. A capacidade de modular a resposta imune do tipo Th1 jamais foi relatada em lectinas de esponjas marinhas. Todos os resultados corroboram com o alto potencial que a lectina AFL apresenta como uma nova mol?cula capaz de modular o sistema imune, podendo ser utilizada como adjuvante de vacinas contra microrganismos, dentre eles, os do g?nero leishmania. / Marine sponges are presented as a rich source of new bioactive compounds of great biotechnological interest. Here in this research we report the purification and characterization of a lectin from Aplysina fulva species, called AFL, that presents immunomodulatory activity, and it was also evaluated whether it possesses cytotoxic effects on cultured cells (normal, tumor and Leishmania). Total proteins were extracted from the sponge with Tris-HCl buffer 20 mM, pH 7.5, and fractionated by precipitation with acetone. The fraction obtained with 1 volume of acetone had greater hemagglutinating activity and was subjected to a column chromatography on guar gum matrix with 20 mM sodium tetraborate buffer, pH 7.5. It was then applied to a Superdex 75 column (AKTA-FPLC) containing 20 mM Tris-HCl buffer, pH 7.5. The purity of the lectin was attested in a HILIC (HPLC) column. The sequence of 15 residues of N-terminal amino acid of the lectin was determined by Edman degradation and showed no similarity to any other protein of the known databases. The molecular mass was set at 62 kDa by gel filtration on Superdex 75. Analysis after treatment with reducing agents enabled to deduce that the AFL is a homotetrameryc protein. Their hemagglutinating activity was reduced when the lectin was exposed in an acidic medium (pH <4) or when subjected to temperatures above 60 ? C. AFL showed specificity for partial lactose and D-glucose and D-galactose. The purified lectin LFA showed no cytotoxic effect on cancer cell lines (PANC, HeLa, HT-29, Bf16F10, A549) and fibroblast (MRC5). AFL was able to agglutinate living promastigotes of Leishmania amazonensis and Leishmania braziliensis, and had their activity reversed by the addition of lactose to the test. AFL did not decrease significantly the viability of Leishmania amazonensis. The AFL lectin showed mitogenic activity at concentrations from 40 ug / ml for mononuclear peripheral blood cells (lymphocytes and monocytes). The AFL lectin was able of induce the macrophage murine lineage (RAW264.7) release of TNF-?, but not IL-6 at a concentration of 10.0 mg / mL. When LPA was incubated with spleen cells from BALB / c mice was able to stimulate the production of IFN-? and to promote the differentiation of T cells to a cellular Th1 immune response. The ability to modulate the immune response of the Th1-type lectins has never been reported in marine sponges. All results confirm the high potential that the lectin AFL, that appears as a new molecule capable of modulating the immune system and can be used as an adjuvant for vaccines against microorganisms, including the gender Leishmania.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-20T22:52:54Z No. of bitstreams: 1 AntonioMoreiraMarquesNeto_DISSERT.pdf: 1777931 bytes, checksum: f46a9d1741f7574f2fd804826fb17637 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-24T19:13:59Z (GMT) No. of bitstreams: 1 AntonioMoreiraMarquesNeto_DISSERT.pdf: 1777931 bytes, checksum: f46a9d1741f7574f2fd804826fb17637 (MD5) / Made available in DSpace on 2017-03-24T19:13:59Z (GMT). No. of bitstreams: 1 AntonioMoreiraMarquesNeto_DISSERT.pdf: 1777931 bytes, checksum: f46a9d1741f7574f2fd804826fb17637 (MD5) Previous issue date: 2015-02-02 / Uma lectina com elevada atividade aglutinante sobre hem?cias humanas dos v?rios tipos do sistema ABO foi purificada da esponja marinha Aplysina sp. por extra??o hidroalco?lica e uma sequ?ncia de etapas de purifica??o envolvendo cromatografias de gel filtra??o em Superdex 75 10/300 GL e de troca-i?nica Resource Q (FPLC-AKTA Purifier). Ap?s purificada, a lectina denominada AplyL-1, apresentou uma ?nica cadeia pept?dica com uma massa molecular de 40 kDa e com especificidade de liga??o para D-galactose, D-galactosamina e lactose. A atividade hemaglutinante de AplyL-1 foi independente da presen?a de ?ons bivalentes e n?o foi alterada em condi??es b?sicas (acima de pH 7,0), mas bastante reduzida quando submetida a condi??es ?cidas (abaixo de pH 6,0). Testes de termoestabilidade mostraram que AplyL-1 perde gradativamente sua atividade hemaglutinante a partir de 40?C e n?o mais exibe atividade em 100?C. Aply-L1 (10 ?g/mL) foi testada frente a diversas linhagens tumorais, onde apresentou atividade citot?xica significativa para a linhagem de adenocarcinoma cervical humano (HeLa). Para a linhagem de c?lula normal 3T3 n?o foi vista atividade citot?xica. Em testes realizados com Leishmania braziliensis e Leishmania amazonensis, Aply-L1 exibiu a capacidade de aglutinar a esp?cie L. amazonensis (concentra??o de 77,5 ?g/mL). Os resultados mostram que esta nova lectina ligante de derivados de galactose pode ser importante no desenvolvimento de produtos com import?ncia biotecnol?gica e filogen?tica. / A lectin with high binding activity under human erythrocytes of different types of ABO system was isolated from the marine sponge Aplysina sp. by hydroalcoholic extraction and a sequence of purification steps involving gel filtration chromatography on Superdex 75 10/300 GL and ion exchange chromatography on Resource Q (FPLC-AKTA Purifier). Once purified, the lectin, named AplyL-1, showed a single peptide chain with a molecular of weight 40 kDa and binding specificity for D-galactose, D-galactosamine and lactose. The AplyL1 hemagglutinating activity was independent of bivalent ions and was not changed in basic conditions (pH > 7.0), but significantly reduced when submitted into acid conditions (pH <7.0). Thermal stability tests showed that AplyL-1 gradually loses its hemagglutinating activity at 40 ?C and no longer displays any activity at 100 ?C. AplyL-1 has been tested against several tumour cell lines, and howed significantly cytotoxic activity (up to 10 ?g/mL) only for human cervical adenocarcinoma cell line (HeLa). For the 3T3 normal cell line no cytotoxic activity was seen. In tests performed with Leishmania braziliensis and Leishmania amazonensis, AplyL-1 exhibited the ability to agglutinate only the species L. amazonensis (at a concentration 77.5 ?g/mL). The results show that this new binding galactose derivatives lectin, could be important to development of new products with biotechnological and phylogenetic significance.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-20T22:52:55Z No. of bitstreams: 1 MilenaSimoesPeixoto_DISSERT.pdf: 3229732 bytes, checksum: 280f7905e8a552967b42f0794a5a52f9 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-24T19:20:37Z (GMT) No. of bitstreams: 1 MilenaSimoesPeixoto_DISSERT.pdf: 3229732 bytes, checksum: 280f7905e8a552967b42f0794a5a52f9 (MD5) / Made available in DSpace on 2017-03-24T19:20:37Z (GMT). No. of bitstreams: 1 MilenaSimoesPeixoto_DISSERT.pdf: 3229732 bytes, checksum: 280f7905e8a552967b42f0794a5a52f9 (MD5) Previous issue date: 2016-09-19 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A polui??o atmosf?rica ? um fator de risco ambiental, e consequentemente, um problema a sa?de. Diversas subst?ncias s?o lan?adas diariamente por meio de atividades antropog?nicas ou naturais, tornando o ar impr?prio e nocivo ao bem-estar de seres humanos e prejudicial ? fauna e ? flora. Na regi?o amaz?nica, os desmatamentos e as queimadas t?m causado preju?zos para a popula??o exposta. Estudos demonstram que essas part?culas presentes no ar causam s?rios problemas respirat?rios e cardiovasculares, incluindo danos ao DNA. Diante disso, o objetivo desse estudo foi avaliar o estresse oxidativo, a integridade mitocondrial e morte celular desencadeados por compostos org?nicos do material particulado menor que 10 ?m (MP10) oriundos da queima de biomassa da floresta Amaz?nica, assim como os efeitos do reteno, marcador de queima de biomassa, em c?lulas epiteliais do pulm?o humano (A549). Para tal, foi avaliado a forma??o de esp?cies reativas de oxig?nio (ERO) com os corantes DCF e MitoSOX e o processo de autofagia por express?o e distribui??o das isoformas da prote?na LC3, marcadora de autofagossomo maduro, em c?lulas A549 expostas a 200 ?g/mL e 400 ?g/mL de MP10 nos tempos de 24 h e 72 h. Da mesma forma, foi analisado os efeitos do reteno sobre estresse oxidativo nas concentra??es de 3,3 ng/mL, 10 ng/mL e 30 ng/mL. Tamb?m foi observado a fun??o mitocondrial com TMRM e Mitotracker e o processo de morte celular via marca??o por anexina e iodeto de prop?deo. Com rela??o ? fra??o org?nica do material particulado, esta induziu um aumento da produ??o de ERO intracelulares e de super?xido mitoncondrial. Al?m disso, a exposi??o ao MP10 desencadeou a forma??o de autofagossomos, sugerindo o aumento da autofagia. Nas an?lises biol?gicas com o reteno, os dados mostraram que este composto levou ao aumento de ERO e de super?xido mitocondrial, a hiperpolariza??o da membrana mitocondrial, assim como o aumento do conte?do mitocondrial em todos os tempos testodos. Por?m, o reteno s? foi capaz de induzir a morte celular na maior concentra??o utilizada e no per?odo de 72 h. Com esses resultados, ? importante enfatizar a necessidade de redu??o da emiss?o de poluentes por queima de biomassa, buscando pol?ticas de controle. Al?m disso, a toxicidade apresentada pelo reteno levanta um alerta em rela??o a inclus?o desse composto, marcador de queima de biomassa, na avalia??o de risco dos hidrocarbonetos polic?clicos arom?ticos (HPA). / In recent discussions on environmental issues, air pollution has been considered an important environmental risk factor, and, consequently, a burden to human health. Several poluents are released daily by natural or human activities, causing the air to be improper and harmful to the welfare of humans and ecosystems. In the Amazon region, deforestation and forest fires have been causing damage to the exposed population. Studies already demonstrated that airborne particles can lead to serious cardiorespiratory effects, including DNA damage. Therefore, the aim of this study was to evaluate oxidative stress, mitochondrial integrity and cell death caused by organic chemical compounds from particulate matter smaller than 10 ?m (PM10) originated from biomass burning of the Amazon forest, as well as the effects of retene, a biomass burning marker, in human lung epithelial cells (A549). It was evaluated reactive oxygen species (ROS) generation (DCF and MitoSOX) and autophagy process by expression and distribution of LC3 protein, autophagosome marker, in A549 cells exposed to 200 ?g/mL and 400 ?g/mL for 24 h and 72 h. Likewise, it was examined the effects of retene on oxidative stress on the concentrations of 3,3 ng/mL, 10 ng/mL and 30 ng/mL. Also, mitochondrial function and cell death was observed with TMRM and Mitotracker dyes and annexin and propidium iodide markers, respectifully. Regarding the extracted organic particulate matter, this led to the increased production of reactive oxygen species and intracellular mitochondrial superoxide. Additionally, PM10 exposure triggered the formation of autophagosomes, suggesting increased autophagy. In the biological analysis with retene, the data showed that this compound led to an increase in reactive oxygen species and mitochondrial superoxide, hyperpolarization of the mitochondrial membrane, as well as increased mitochondrial content at all tested times. However, the retene was only able to induce cell death in the greatest concentration used and over a 72-hour period. From these results, it is important to emphasize the reduction of emissions by biomass burning, searching for new control policies. In addition, the toxicity of the retene, a biomass burning marker, raises an alert about the inclusion of this compound in the risk assessment of polycyclic aromatic hydrocarbons (PAHs).

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-06-02T22:15:26Z No. of bitstreams: 1 FelipeEmmanuelDoEspiritoSantoGomes_DISSERT.pdf: 3727906 bytes, checksum: 5ccaabce900c234137ecb8f5d64843dd (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-06-08T18:54:49Z (GMT) No. of bitstreams: 1 FelipeEmmanuelDoEspiritoSantoGomes_DISSERT.pdf: 3727906 bytes, checksum: 5ccaabce900c234137ecb8f5d64843dd (MD5) / Made available in DSpace on 2017-06-08T18:54:49Z (GMT). No. of bitstreams: 1 FelipeEmmanuelDoEspiritoSantoGomes_DISSERT.pdf: 3727906 bytes, checksum: 5ccaabce900c234137ecb8f5d64843dd (MD5) Previous issue date: 2017-03-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A criptococose, causada pelas esp?cies f?ngicas Cryptococcus neoformans e Cryptococcus gattii, ? uma das micoses oportun?sticas e/ou sist?micas mais importantes no mundo. Cada esp?cie possui quatro gen?tipos, usualmente definidos pelo PCR-RFLP do gene URA5, os quais apresentam diferen?as em sua ecologia, epidemiologia, distribui??o geogr?fica e susceptibilidade a antif?ngicos. Marcadores moleculares de acesso mais direto s?o atrativos para um r?pido reconhecimento de gen?tipos ou de carater?sticas relevantes como virul?ncia e susceptibilidade antif?ngica. Neste sentido, introns autocatal?ticos do grupo I, no rRNA LSU mitocondrial foram aqui avaliados como potencial marcador molecular para os gen?tipos de C. neoformans e C. gattii, bem como quanto a sua rela??o com a susceptibilidade a antif?ngicos. Foram utilizados 77 isolados brasileiros, sendo a maioria do gen?tipo VNI (39 cepas), seguido de 20 VGII, 5 VNIV, 4 VNII, 3 VNIII, 2 VGI, 2 VGIII e 2 VGIV. Os introns Cne.mL2449 e Cne.mL2504 foram amplificados em um s? PCR com primers complementares a regi?o do gene rRNA LSU flanqueadora dos introns. Os produtos de PCR mostraram um polimorfismo de comprimento significativo entre gen?tipos de C. neoformans e C. gattii. O sequenciamento destes produtos indicou que algumas cepas apresentaram nenhum, um, dois, tr?s ou quatro introns em s?rie. Estes dois novos introns, n?o descritos anteriormente, foram nomeados de Cne.mL2439 e Cne.mL2584 em C. neoformans e Cga.mL2439 e Cga.mL2584 em C. gattii. Os introns Cne.mL2439/Cga.mL2439 foram classificados como pertences a subclasse IB2 ao passo que Cne.mL2584/Cga.mL2584, pertencentes a subclasse IA1. Curiosamente, os gen?tipos com isolados sem introns, VNI, VGII, VGI e VNIV, s?o aqueles conhecidos como mais virulentos e menos suscept?veis a agentes antif?ngicos. De fato, tais isolados apresentaram MICs significativamente superiores para 5-flucitosina. Estes achados sugerem que estes elementos podem ser utilizados como potenciais marcadores moleculares para a resist?ncia deste antif?ngico. Por fim, an?lises filogen?ticas sugeriram alta similaridade de sequ?ncia entre os introns Cne.mL2449, Cne.mL2504, Cne.mL2439/Cga.mL2439 e Cne.mL2584/Cga.mL2584 com outros introns mitocondriais presentes nos genes COX1, COX2, COX3, NAD5, ATP9, COB, LSU de fungos distintos, sustentando a hip?tese de origem antiga dos introns (hip?tese ?introns early?), al?m da dispers?o destes elementos em s?tios heter?logos, via splicing reverso. / Cryptococcosis, caused by the fungal species Cryptococcus neoformans or Cryptococcus gattii, is one of the most important systemic and/or opportunistic diseases in the world. Each species has four genotypes, usually accessed by PCR-RFLP of the URA5 gene, which present differences in their ecology, epidemiology, geographical distribution and antifungal susceptibility. Easier accessible molecular markers are attractive for rapid recognition of genotypes or relevant characteristics such as virulence and antifungal susceptibility. In this way, group I autocatalytic introns in the mitochondrial LSU rRNA were evaluated as potential molecular marker for the genotypes of C. neoformans and C. gatti, as well as their relationship to antifungal susceptibility. Seventy-seven Brazilian isolates were used, most of the genotype VNI (39 strains) followed by 20 VGII, 5 VNIV, 4 VNII, 3 VNIII, 2 VGI, 2 VGIII and 2 VGIV. The introns Cne.mL2449 and Cne.mL2504 were amplified in a single PCR with complementary primers to the flanking region of the introns LSU rRNA gene. PCR products showed a significant polymorphism between C. neoformans and C. gattii genotypes. Sequencing of the PCR products indicated that some strains had none, one, two, three or four introns followed. This new two introns, not previously described in the mitochondrial genome of Cryptococcus, were named Cne.mL2439 and Cne.mL2584 in C. neoformans and Cga.mL2439 and Cga.mL2584 in C. gattii. Cne.mL2439/Cga.mL2439 introns were classified as belonging to IB2, whereas Cne.mL2584/Cga.mL2584, as belonging IA1 subclass. Interestingly, genotypes with some intronless strains, VNI, VGII, VGI and VNIV, are those known to be more virulent and less susceptible to antifungal agents. Here, we observed that those intronless isolates had significant higher MICs values for 5-flucytosine. The findings suggest that these elements can be used as potential molecular markers for antifungal resistance. Finally, phylogenetic analyzes suggested high sequence similarity between the introns Cne.mL2449, Cne.mL2504, Cne.mL2439/Cga.mL2439 and Cne.mL2584/Cga.mL2584 with other mitochondrial introns present in the genes COX1, COX2, COX3, NAD5, ATP9, COB, LSU of fungi supporting the ?introns early? hypothesis, as well as its dispersion to heterologous sites by reverse splicing.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-06-02T22:15:27Z No. of bitstreams: 1 LarisseAraujoDantas_DISSERT.pdf: 2039205 bytes, checksum: c6567bc4fed86a3072ac9d28162203b1 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-06-08T19:00:32Z (GMT) No. of bitstreams: 1 LarisseAraujoDantas_DISSERT.pdf: 2039205 bytes, checksum: c6567bc4fed86a3072ac9d28162203b1 (MD5) / Made available in DSpace on 2017-06-08T19:00:32Z (GMT). No. of bitstreams: 1 LarisseAraujoDantas_DISSERT.pdf: 2039205 bytes, checksum: c6567bc4fed86a3072ac9d28162203b1 (MD5) Previous issue date: 2017-04-17 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Nanopart?culas de prata (AgNPs) possuem diversas aplica??es biom?dicas, dentre elas destaca-se o potencial antiproliferativo. In?meros tipos de s?ntese de nanopart?culas de prata encontram-se descritos na literatura, no entanto, um tipo de s?ntese menos agressiva e mais economicamente vi?vel, a s?ntese verde, tem ganhado import?ncia dentre as demais. Neste processo de s?ntese ? necess?rio o uso de um agente redutor e estabilizante que n?o sejam t?xicos para as c?lulas animais e para o meio ambiente. Nesse contexto, a associa??o de biomol?culas de origem marinha com atividades biol?gicas j? descritas, como os polissacar?deos ?cidos de algas, ?s AgNPs ganham import?ncia. Muitos polissacar?deos ?cidos (PA) extra?dos de algas marinhas destacam-se por exibir diversas atividades, como exemplo as fucanas da alga Spatoglossum schr?ederi. Estas fucanas apresentam atividade antioxidante, antiangiog?nica, antitumoral, dentre outras. Devido aos poucos relatos de s?ntese de AgNPs envolvendo polissacar?deos de algas, este trabalho teve por objetivo sintetizar AgNPs com uma fra??o rica em ?cido alg?nico e fucana A da alga S. schr?ederi por um processo de s?ntese verde, e testar as AgNPs obtidas frente ? linhagem de melanoma B16F10. Os PA da alga S. schr?ederi foram extra?dos por um processo de digest?o proteol?tica e fracionados com acetona. A fra??o de maior rendimento e rica em ?cido alg?nico e fucana A, a F0.5 v, foi empregada para a s?ntese das AgNPs. A caracteriza??o das AgNPs obtidas contemplou a an?lise de absor??o de luz UV, composi??o qu?mica, di?metro m?dio, polidispers?o, potencial zeta, espectroscopia de infravermelho (FTIR) e a espectroscopia de energia dispersiva (EDS), microscopia eletr?nica de varredura (MEV) e microscopia de for?a at?mica (MFA). As an?lises por espectroscopia de UV-vis?vel na faixa entre 400 e 440 nm confirmaram a forma??o das AgNPs, estas apresentaram di?metro de 196 ? 13 nm, valores de polidispers?o abaixo de 0,4 e potencial zeta negativo. As dosagens qu?micas, o FTIR e o EDS revelaram que a maior parte das AgNPs ? composta por polissacar?deos ?cidos. As imagens obtidas por MEV e MFA indicaram um formato esf?rico para as AgNPs. A atividade antiproliferativa foi avaliada pelo m?todo do MTT frente ?s linhagens celulares B16F10 e fibroblastos 3T3, e por citometria de fluxo foi investigado sua a??o na morte e no ciclo celular. As AgNPs afetaram a capacidade de redu??o do MTT das linhagens 3T3 e B16F10, sendo esta mais evidenciada para a B16F10, pois as AgNPs (0,5 mg/mL) conseguiram reduz?-la em torno de 50%, enquanto a prata i?nica e a F0.5 v em nenhuma das concentra??es influenciaram na redu??o do MTT. As an?lises por citometria de fluxo apontaram uma elevada taxa de marca??o para anexina V e iodeto de prop?dio para as c?lulas B16F10 expostas ?s AgNPs, al?m de um aumento na porcentagem das c?lulas em Sub-G1 em detrimento ?s outras fases. Estudos adicionais s?o necess?rios para elucidar completamente o mecanismo de a??o antiproliferativo das AgNPs e descobrir outras propriedades da fra??o que s?o intensificadas pela s?ntese de nanopart?culas. Entretanto, pode-se concluir que ? poss?vel sintetizar AgNPs com polissacar?deos ?cidos de S. schr?ederi usando um m?todo verde e que essas AgNPs apresentaram atividade antiproliferativa mais pronunciada que seus percussores. / Silver nanoparticles (AgNPs) have several biomedical applications, among them the antiproliferative potential. Numerous types of synthesis of silver nanoparticles are found in the literature, however, a type of synthesis less aggressive and more economically viable, a green synthesisl, has gained value among the others. In this synthesis process, it is necessary to use a reducing agent and stabilizer which are non-toxic to animal cells and to the environment. In this context, an association of biomolecules of marine origin with biological activities already described, such as the polysaccharide acids of algae, to the AgNPs gain importance. Many acidic polysaccharides (PA) extracted from marine algae are distinguished by their different activities, such as the algae Spatoglossum schr?ederi. These fucans have antioxidant, antiangiogenic, antitumor activity, among others. Due to the few reports of AgNPs synthesis involving algae polysaccharides, the objective of this work was to synthesize AgNPs with a rich fraction of alginic acid and fucan A from the S. schr?ederi algae by a green synthesis process and to test the AgNPs obtained on the lineage of melanoma B16F10. The PA of the S. schr?ederi algae was extracted by a proteolytic digestion process and fractionated with acetone. The higher yield fraction rich in alginic acid and fucan A, F0.5 v, was used for synthesis of the AgNPs. The characterization of the AgNPs obtained contemplated an analysis of UV light absorption, chemical composition, average diameter, polydispersity, zeta potential, infrared spectroscopy (FTIR), dispersive energy spectroscopy (EDS), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The analyzes by UV-visible spectroscopy in the range between 400 and 440 nm confirmed the formation of AgNPs, they presented 196 ? 13 nm diameter, polydispersion values below 0.4 and negative zeta potential. The chemical dosages, FTIR and EDS revealed that most AgNPs are composed of acidic polysaccharides. The images obtained by SEM and AFM indicated a spherical format for AgNPs. The antiproliferative activity was evaluated by the MTT method against B16F10 cell lines and 3T3 fibroblasts, and by flow cytometry, its action on death and cell cycle was investigated. The AgNPs affected the ability to reduce MTT from the 3T3 and B16F10 lineages, but these were evidenced for a B16F10, the AgNPs (0.5 mg/mL) were able to reduce by around 50%, while a silver ion and F0.5 v at any of the concentrations influenced the reduction of MTT. Flow cytometry analyzes indicated a high labeling rate for annexin V and propidium iodide for the B16F10 cells exposed to AgNPs, in addition to an increase in the percentage of cells in SubG1 in detriment to the other phases. Additional studies are needed to fully elucidate the mechanism of antiproliferative action of AgNPs and to discover other properties of the fraction that are enhanced by the synthesis of nanoparticles. However, it can be concluded that it is possible to synthesize AgNPs with acidic polysaccharides from S. schr?ederi using a green method and that these AgNPs showed more pronounced antiproliferative activity than their precursors.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-06-13T19:42:47Z No. of bitstreams: 1 FrancianneMedeirosAmorim_TESE.pdf: 4988511 bytes, checksum: 0e073467f779cb0d1dfda10542efe955 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-06-16T22:18:03Z (GMT) No. of bitstreams: 1 FrancianneMedeirosAmorim_TESE.pdf: 4988511 bytes, checksum: 0e073467f779cb0d1dfda10542efe955 (MD5) / Made available in DSpace on 2017-06-16T22:18:03Z (GMT). No. of bitstreams: 1 FrancianneMedeirosAmorim_TESE.pdf: 4988511 bytes, checksum: 0e073467f779cb0d1dfda10542efe955 (MD5) Previous issue date: 2017-04-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A hansen?ase ? uma doen?a de evolu??o espectral causada pelo Mycobacterium leprae. Pacientes podem apresentar desde les?es ?nicas, com pequena carga bacilar (paucibacilares - PB) a les?es disseminadas e alta carga bacteriana (multibacilares - MB). Os primeiros apresentam uma forte resposta imune celular e os ?ltimos uma resposta predominantemente humoral. O Brasil ? o segundo pa?s em n?mero de casos, com ?reas hiperend?micas em diversos estados, incluindo o Rio Grande do Norte. A alta morbidade da doen?a est?, em parte, relacionada a ocorr?ncia de rea??es hans?nicas: a rea??o reversa (RR) e o eritema nodoso hans?nico (ENH). Essas ocorrem predominantemente em pacientes MB. Nosso objetivo foi determinar o papel de c?lulas B e de anticorpos na patog?nese da hansen?ase e das rea??es hans?nicas. Para isto, o trabalho foi subdividido em dois estudos: 1. Determina??o do perfil de anticorpos espec?ficos utilizando os ant?genos recombinantes LID-1 e LIDNDO ao longo do espectro cl?nico da hansen?ase e em comunicantes; 2. An?lise de altera??es envolvidas na regula??o da produ??o de anticorpos em c?lulas B de pessoas com diferentes formas cl?nicas. Neste ?ltimo foram avaliadas: a frequ?ncia de diferentes subpopula??es de c?lulas B, a express?o de CD32 e CD21 nestas c?lulas, subclasses de imunoglobulinas presentes no sangue, complexos imunes (IC) e prote?nas envolvidas na via cl?ssica de ativa??o do sistema complemento. No estudo 1, observou-se um aumento na quantidade de anticorpos espec?ficos ao longo do espectro cl?nico da doen?a e este foi correlacionado com o ?ndice bacilosc?pico. Al?m disso, foi verificado que mais de 82% dos comunicantes haviam sido expostos ? infec??o pelo M. leprae. Esse achado mostra que a quantifica??o de anticorpos espec?ficos pode ser utilizada para estimar o risco para o desenvolvimento de hansen?ase. No estudo 2, observou-se que a exacerbada resposta imune humoral de e pessoas com MB est? associada a altera??es num?ricas e funcionais em c?lulas B, com aumento na frequ?ncia de plasmoblastos e reduzida express?o de CD32 nestes. Pessoas com hansen?ase MB apresentaram maior concentra??o de IgG1 e imunocomplexos (IC) no sangue perif?rico quando comparados a PB. Pessoas com hansen?ase MB que desenvolveram ENH (durante ou ap?s a poliquimioterapia) j? apresentavam, ao diagn?stico n?veis mais elevados de IgM, IgG1, anticorpos espec?ficos e IC quando comparados ?queles que n?o desenvolveram rea??o. Durante o ENH h? uma expans?o na popula??o de plasmoblastos, contudo h? diminui??o na concentra??o destas imunoglobulinas no sangue. Indiv?duos com n?veis elevados de anticorpo anti-LID-NDO, ao diagn?stico, apresentaram um risco at? 20 vezes maior de desenvolverem rea??o. Nossos resultados mostram que o uso de ant?genos recombinantes em testes sorol?gicos pode contribuir para um diagn?stico mais r?pido da doen?a, levando a diminui??o da transmiss?o de M. leprae. Al?m disso, verificamos que a exacerbada resposta imune humoral de pacientes MB pode ser, em parte, explicada por altera??es em c?lulas B. A quantifica??o de anticorpos anti-M. leprae e das subclasses IgM e IgG1 ao diagn?stico da hansen?ase pode contribuir para identifcar indiv?duos em risco de desenvolverem rea??o. Do ponto de vista cl?nico, esse ? um dado importante pois pode direcionar interven??es terap?uticas futuras. / Leprosy is a spectral disease caused by Mycobacterium leprae infection. Subjects can present single lesions with a reduced number of bacilli (paucibacillary - PB), but also disseminated lesions and a high bacterial load (multibacillary - MB). The former present a strong cellular immune response and the latter have a predominantly humoral response. Brazil is the second country in number of cases, with hyperendemic areas in several states, including Rio Grande do Norte. Disease?s high morbidity is directly associated with the occurrence of leprosy reactions: reversal reaction (RR) and erythema nodosum leprosum (ENL). These reactions occur predominantly in MB patients. Our aim was to determine the role of B cells and antibodies in the pathogenesis of leprosy and its immune reactions. For this, the work was subdivided in two studies: 1. Determination of the profile of specific antibodies to the recombinant antigens LID-1 and LID-NDO according to the clinical spectrum of the disease and in household contacts. 2. Analysis of changes involved in the regulation of antibody production in B cells of patients with different clinical forms of leprosy. For this latter, the frequency of different B cell subpopulations, the expression of CD32 and CD21 in these cells, subclasses of immunoglobulins present in the blood, immune complexes (IC) and proteins involved in the classical pathway of complement activation were evaluated. In the study 1, it was observed a gradual increase in the level of specific antibodies along with the clinical spectrum of the disease, and this increase was correlated with the bacterial index. More than 82% of the household contacts recruited in the study had been previously exposed to M. leprae infection, with a potential risk of developing leprosy. This finding shows that the quantification of specific antibodies in the blood can be used to define groups at risk for leprosy development. MB subjects presented an exacerbated humoral immune response that was associated with numerical and functional alterations in B cells, with increased frequency of plasmoblast and reduced expression of CD32 in these cells. MB patients presented higher concentrations of IgG1 and IC in the blood when compared to PB. MB patients that developed ENL (during or after multidrug therapy) presented increased levels of IgM, IgG1, specific antibodies, and IC in the blood, when compared to those that did not develop reactions. During ENL, there is an expansion in the plasmoblast population, however a decrease in the concentration of these immunoglobulins in the blood. Patients with elevated levels of anti-LID-NDO antibody at diagnosis presented a 20- fold increased risk of developing reactions. Our results show that the use of recombinant antigens in serological tests can contribute to an early diagnosis, decreasing the risk of M. leprae transmission. The exacerbated humoral immune response of MB patients may be explained, at least in part, by changes in B cells. The quantification of anti-M. leprae antibodies and IgM and IgG1 subclasses at leprosy diagnosis may contribute to identify individuals at risk of developing reaction. Clinically, this is an important data since it can direct future therapeutic interventions.

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.

Submitted by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-03T14:45:12Z No. of bitstreams: 1 Tese_AdrianoTort_2005.pdf: 1861214 bytes, checksum: 93eb092dffbce7414f26253895b0ad28 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-03T14:46:05Z (GMT) No. of bitstreams: 1 Tese_AdrianoTort_2005.pdf: 1861214 bytes, checksum: 93eb092dffbce7414f26253895b0ad28 (MD5) / Made available in DSpace on 2017-11-06T11:41:34Z (GMT). No. of bitstreams: 1 Tese_AdrianoTort_2005.pdf: 1861214 bytes, checksum: 93eb092dffbce7414f26253895b0ad28 (MD5) Previous issue date: 2005-12 / Os objetivos da presente tese de doutorado foram os de buscar novos antipsic?ticos at?picos de baixo pre?o comercial e tamb?m procurar entender o mecanismo de a??o que leva a um perfil antipsic?tico at?pico. Os resultados da tese s?o divididos em duas partes, de acordo com sua natureza, em experimentais (primeira parte) e te?ricos (segunda parte). Para o desenvolvimento da primeira parte, foi necess?ria primeiramente a programa??o de um software para medir locomo??o em roedores ap?s filmagem com webcam. A seguir, foram investigados os efeitos da guanosina, flunarizina e cinarizina em modelos animais de psicose, bem como em outros paradigmas comportamentais. A guanosina foi escolhida para estudo uma vez que tem se mostrado que ela interage com o sistema glutamat?rgico ? que sabidamente est? envolvido na fisiopatologia da esquizofrenia ? promovendo a capta??o astrocit?ria de glutamato. J? a flunarizina e a cinarizina, dois bloqueadores de canal de c?lcio empregados para tratar enxaqueca e vertigem foram escolhidas pelo fato delas produzirem sinais e sintomas extrapiramidais em pacientes idosos, o que posteriormente foi relacionado ?s suas propriedades como antagonistas moderados dos receptores dopamin?rgicos do tipo D2. A guanosina diminuiu o aumento de locomo??o induzido por um antagonista NMDA (MK-801), enquanto que n?o apresentou efeito sobre o aumento de locomo??o induzido por anfetamina, de forma que sua utilidade como potencial antipsic?tico deve ser ainda melhor estudada. Tanto a flunarizina quanto a cinarizina foram capazes de diminuir o aumento de locomo??o induzido por MK-801 e por anfetamina em doses que n?o causam efeitos catal?pticos importantes. Portanto, foi conclu?do que estes dois compostos apresentam um potencial perfil de antipsic?tico at?pico, com as vantagens de j? estarem dispon?veis para uso comercial, boa tolerabilidade e baixo custo quando comparados com os antipsic?ticos at?picos dispon?veis comercialmente nos dias de hoje. A segunda parte da tese apresenta alguns resultados te?ricos matem?ticos que podem ser derivados da teoria da lei de a??o das massas aplicada ao binding de receptores, utilizando tamb?m resultados experimentais j? conhecidos de PET. Estes resultados apresentam insights ao entendimento das diferen?as entre os perfis antipsic?ticos at?picos e t?picos em rela??o ? gera??o de sinais extrapiramidais. ? discutido que fatores culturais e comerciais relacionados ? posologia atual empregada no tratamento com antipsic?ticos t?picos podem ser os respons?veis pelas diferen?as de perfis, uma vez que alguns deles s?o prescritos em doses proporcionalmente maiores em rela??o ? sua afinidade, atingindo assim maiores n?veis de bloqueio dopamin?rgico no estriado. Uma curta meia-vida plasm?tica tamb?m ? apontada como um poss?vel par?metro importante na gera??o de um perfil at?pico. ? mostrado ainda alguns erros de concep??o relacionados ao curso temporal da ocupa??o dopamin?rgica que tem sido atualmente cometidos na literatura cient?fica, como o conceito de meia-vida de ocupa??o de receptores. Como um ?ltimo resultado te?rico, ? proposto um algoritmo para a redu??o de dose em pacientes tratados com antipsic?ticos apresentando sinais e sintomas extrapiramidais. / The aims of this work were the search for new atypical antipsychotics presenting low cost and the understanding of the mechanism of action leading to atypical antipsychotic profile. The results obtained are presented in two distinct parts based on their nature, namely, experimental (first part) or theoretical (second part). For the development of the first part, a webcam based software to measure locomotion of rodents was programmed. After that, it was investigated the effect of guanosine, flunarizine and cinnarizine on animal models of psychosis, as well as in other behavioral tasks. Guanosine was chosen because it has been shown to interact with the glutamatergic system ? which is known to be involved in the pathophysiology of schizophrenia ? by promoting astrocytic glutamate reuptake. Flunarizine and cinnarizine, two calcium channel blockers commonly used in many countries to treat vertigo and migraine, were chosen because they were shown to induce extrapyramidal signs in elder patients, which was later related to moderate antagonist properties at dopamine D2 receptors. Guanosine was able to reduce a NMDA antagonist (MK-801) induced hyperlocomotion, whereas it had no effect on the hyperlocomotion induced by amphetamine, and it is discussed that its utility as antipsychotic drug should be further evaluated. Both cinnarizine and flunarizine were able to reduce the hyperlocomotion induced by MK-801 and amphetamine at doses that presented no significant cataleptic behavior. It was therefore concluded that these compounds have a potential atypical antipsychotic profile, with the advantage of already approved for commercial use, presenting well tolerability and very low cost when compared to current commercially available atypical antipsychotics. The second part of this thesis presents some theoretical mathematical results that can be derived from the law of mass action theory applied to receptor binding linked with known PET experimental data. These results present insights to the understanding of the differences between typical and atypical profile of antipsychotics regarding the generation of extrapyramidal syndrome. It is argued that cultural and commercial aspects related to the nowadays employed posology of typical antipsychotics can be responsible for the difference seen in profile, once some typical antipsychotics are prescribed in proportionally higher doses in relation to their affinities, leading therefore to higher dopaminergic blockade. A short plasmatic half-life is also pointed as a possible important factor leading to an atipical profile. Moreover, the second part of this thesis also points to some misconception currently being used in the scientific literature regarding the time-course of dopaminergic occupation, such as the concept of receptor occupation half-life. As a last theoretical based result, it is proposed an algorithm for antipsychotic dose reduction in patients presenting extrapyramidal signs and symptoms.