Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 Tatiana Xavier de Almeida.pdf: 937180 bytes, checksum: e0cda53e5c0c9829836d98da482b3737 (MD5) Previous issue date: 2010-02-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Fungal and mycotoxin contamination in food is one of the greatest problems around the world. Some natural products, such as essential oils, have been shown to be effective inhibitors of fungal growth and production of their toxins. In this study, a total of 40 vegetal extracts were tested for inhibition on Aspergillus section Flavi growth. These extracts were obtained from ten herbs cultivated in Brazil: basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove (Eugenia caryophyllata), cumin (Cuminum cyminum), marjoram (Origanum majorana), nutmeg (Myristica fragrans), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis), spearmint (Mentha piperita) and sweet fennel (Pimpinella anisum). Preliminary in vitro tests using the agar diffusion method showed that essential oils were more effective in inhibiting the fungal growth when compared with ethanol and aqueous extracts. Essential oils obtained from oregano and rosemary, clove and cumin ethanolic extracts and the essential hexanic oil of oregano and cinnamon were selected for advanced tests. Differents concentrations of plant material (0, 50, 100, 150, 300 and 600 μg/g) were added to culture medium. A suspension of 103 conidia per inoculation were used. Plates were incubated for ten days while daily measurements of colony diameters were made to obtain growth rate (mm/day) and lag phase (h) of toxigenic strains. The comparison of selected essential oils effect on A. flavus and A. parasiticus showed better inhibition by incorporating oregano essential oil in the culture medium at 600 μg/g concentration. Nevertheless, the hexanic essential oil of oregano showed no statistical difference with the control group, a change occurred in the plant product due to the extraction method. Among the evaluated ethanol extracts, clove had the best result. Aspergillus flavus showed a slight reduction of growth (4.5 to 3.5 mm/day) when compared to control group, but a large increase in the latency period (37 h at a 600 μg/g concentration). Aspergillus parasiticus showed gradual decreases as increased concentrations were assayed, except for the 150 μg/g concentration, and a significant increase on lag phase at the highest concentrations tested (45 and 48 h, respectively). The data presented here are very important for the continued efforts of microbiological control in food, especially when it comes to control fungi. / A contamina??o de alimentos por fungos e micotoxinas constitui um grande problema mundial. Alguns produtos naturais, como por exemplo, ?leos essenciais, s?o demonstrados como potenciais inibidores do crescimento f?ngico, bem como da produ??o de suas toxinas. Neste estudo, um total de 40 extratos de plantas foi avaliado como inibidores do desenvolvimento f?ngico de cepas pertencentes ao g?nero Aspergillus se??o Flavi. Tais extratos foram obtidos a partir de dez ervas, utilizadas especialmente como condimentos e cultivadas no Brasil: alecrim (Rosmarinus officinalis), canela (Cinnamomum zeylanicum), cominho (Cuminum cyminum), cravo-da-?ndia (Eugenia caryophyllata), erva-doce (Pimpinella anisum), hortel? (Mentha piperita), manjeric?o (Ocimum basilicum), manjerona (Origanum majorana), noz moscada (Myristica fragrans) e or?gano (Origanum vulgare). Ensaios preliminares in vitro utilizando o m?todo de difus?o em agar demonstraram que os ?leos essenciais foram os mais efetivos na inibi??o do crescimento f?ngico quando comparados com extratos etan?licos e aquosos. Foram selecionados os ?leos essenciais de or?gano e alecrim, os extratos etan?licos de cravo-da-?ndia e cominho e, os ?leos essenciais hex?nicos de or?gano e canela para ensaios mais aprofundados. Diferentes concentra??es de extratos de plantas arom?ticas (0, 50, 100, 150, 300 e 600 μg/g) foram adicionadas ao meio de cultivo. Utilizou-se uma suspens?o de 103 con?dios por inocula??o. As placas foram incubadas por dez dias enquanto medi??es di?rias do di?metro da col?nia eram realizadas a fim de obter velocidade de crescimento (mm/dia) e fase de lat?ncia (h) das cepas tox?genas. Comparando os ?leos essenciais selecionados, tanto A. flavus quanto A. parasiticus apresentaram uma melhor inibi??o atrav?s da incorpora??o do ?leo essencial de or?gano no meio de cultivo a uma concentra??o de 600 μg/g. Apesar disso, o ?leo essencial hex?nico de or?gano n?o demonstrou qualquer diferen?a estat?stica em rela??o ao grupo controle, havendo ent?o altera??o em algum componente do extrato devido a forma de extra??o. Entre os extratos etan?licos avaliados, o cravo-da-?ndia obteve o melhor resultado. Aspergillus flavus demonstrou uma discreta diminui??o de crescimento (4,5 a 3,5 mm/dia), quando comparado ao grupo controle, mas um grande aumento em sua fase de lat?ncia (37 h a 600 μg/g). Aspergillus parasiticus apresentou uma diminui??o gradativa conforme ocorria o aumento das concentra??es, exceto pela concentra??o de 150 μg/g, e um aumento significativo da fase lag nas maiores concentra??es testadas (45 e 48 h respectivamente). Os dados apresentados aqui s?o de extrema relev?ncia para os constantes esfor?os de controle microbiol?gico em alimentos, principalmente em se tratando de controle f?ngico.

Made available in DSpace on 2016-04-28T20:17:27Z (GMT). No. of bitstreams: 1 2008 - Ana Claudia da Silva Vila Nova.pdf: 3100692 bytes, checksum: fceb0ab719732f64ea3e29199427d13f (MD5) Previous issue date: 2008-04-16 / Many areas are involved in the search for new substances from plants. The new global trends of a concern for biodiversity and the ideas of sustainable development have brought new air to studies of Brazilian medicinal plants. New lines of research were established in Brazilian universities, some of them seeking solid basis for the validation of the use of medicinal plants, thus justifying this work, using aqueous extracts of plants place of occurrence, such as Cinnamomum spp., Mentha piperita and Passiflora foetida, inhibition of growth of some species of the genus Aspergillus tox?genas, previously studied in the literature and identified as antifungal and encouraging the use of plants to replace chemicals. Using the method of minimum inhibitory concentration in agar, with the technique of dilution plate (Pour-Plate), being held dilutions of different extracts, from a solution of 5% and resulting in the dilution of 2.5% and 1.25%. Batteries tests were incubated at the 36oC. It was held also test for sensitivity to antifungal commercial - Ketoconazole. It was observed that the aqueous extract of Cinnamomum spp. was able to inhibit the mycelial growth of A. flavus and A. carbonarius at concentrations of 2.5% and 5% in readings of 48 hours. The other aqueous extracts, used in this work, showed no inhibitory effect of mycelial growth of fungi tested. / Muitas ?reas est?o envolvidas na pesquisa de novas subst?ncias oriundas de plantas. As novas tend?ncias globais de uma preocupa??o com a biodiversidade e as id?ias de desenvolvimento sustent?vel trouxeram novos ares aos estudos das plantas medicinais brasileiras. Novas linhas de pesquisa foram estabelecidas em universidades brasileiras, algumas delas buscando bases s?lidas para a valida??o do uso de plantas medicinais, justificando, assim, a realiza??o deste trabalho, utilizando extratos aquosos de plantas de ocorr?ncia local, como a Cinnamomum spp., Mentha piperita e Passiflora foetida, na inibi??o do crescimento de algumas esp?cies tox?genas do g?nero Aspergillus, previamente pesquisadas na literatura e apontadas como antif?ngicas e favorecendo a utiliza??o de plantas em substitui??o de produtos qu?micos. Utilizando o m?todo da concentra??o m?nima inibit?ria em agar, com a t?cnica de dilui??o em placa (Pour-Plate), sendo realizadas dilui??es dos diferentes extratos, partindo de uma solu??o de 5% e obtendo-se as dilui??es de 2,5% e 1,25%. As baterias testes foram incubadas a 36o C. Foi realizado, tamb?m, teste de sensibilidade ao antif?ngico comercial Cetoconazol. Observou-se que o extrato aquoso de Cinnamomum spp. mostrou-se capaz de inibir o crescimento micelial de A. flavus e A. carbonarius nas concentra??es de 2,5% e 5%, nas leituras de 48 horas. Os demais extratos aquosos, utilizados neste trabalho, n?o apresentaram efeito inibit?rio do crescimento micelial dos fungos testados.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Alexander Santos.pdf: 2028526 bytes, checksum: 5876d3f1d1f305df3af598f0bef1531d (MD5) Previous issue date: 2008-04-15 / This work was developed in the Department of Microbiology and Immunology of the Institute of Veterinary Rural Federal University of Rio de Janeiro in Serop?dica-RJ. We evaluated the antifungal activity plant Cymbopogon citratus (lemon-grass), Peumus boldus (boldo) and Shinus terebinthifolia (aroeira), inhibition of growth of species of the genus Aspergillus (A. flavus, A. niger, A. ochraceus, A. Parasiticus and A. carbonarius. Using the method of minimum inhibitory concentration in agar, with the technique of dilution plate (Pour-Plate), were held dilutions of different extracts, resulting in the final concentrations of 5%, 2.5% and 1.25%. Testing of commercial sensitivity to antifungal - ketoconazole were made in a final concentration of 1933.18 ? g /mL, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The results obtained in the experiments have shown that aqueous extracts of C. citratus and S. terebinthifolia, after 24 hours of incubation, were able to inhibit the growth of A. flavus and A. carbonarius, respectively. The other extracts being studied and tested in the concentrations were not able to inhibit the growth of mycelial species of the genus Aspergillus. / Este trabalho foi desenvolvido no Departamento de Microbiologia e Imunologia do Instituto de Veterin?ria da Universidade Federal Rural do Rio de Janeiro, em Serop?dica-RJ. Foram avaliadas a atividade antif?ngica das plantas Cymbopogon citratus (capim-lim?o), Peumus boldus (boldo) e Shinus terebinthifolia (aroeira), na inibi??o do crescimento de esp?cies do g?nero Aspergillus (A. flavus, A. niger, A. ochraceus, A. parasiticus e A. carbonarius). Utilizando o m?todo da concentra??o inibit?ria m?nima em ?gar, com a t?cnica de dilui??o em placa (Pour-Plate), foram realizadas dilui??es dos diferentes extratos, obtendo-se as concentra??es finais de 5%, 2,5% e 1,25%. Testes de sensibilidade ao antif?ngico comercial - cetoconazol foram realizados numa concentra??o final de 1933,18 ?g/mL, conforme recomendada pelo National Committee for Clinical Laboratory Standards (NCCLS). Os resultados obtidos nos experimentos demonstraram que os extratos aquosos de C. citratus e S. terebinthifolia, ap?s 24 horas de incuba??o, foram capazes de inibir o crescimento de A. flavus e A. carbonarius, respectivamente. Os demais extratos em estudo e nas concentra??es testadas, n?o foram capazes de inibir o crescimento micelial das esp?cies do g?nero Aspergillus.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-09-13T12:31:48Z No. of bitstreams: 1 2017 - Daniel Paiva Barros de Abreu.pdf: 1307092 bytes, checksum: b384d4db2cb316d3f640b111bc8efba4 (MD5) / Made available in DSpace on 2017-09-13T12:31:48Z (GMT). No. of bitstreams: 1 2017 - Daniel Paiva Barros de Abreu.pdf: 1307092 bytes, checksum: b384d4db2cb316d3f640b111bc8efba4 (MD5) Previous issue date: 2017-02-20 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Dimorphic fungi belonging to Sporothrix schenckii complex are responsible for sporotrichosis, important fungal infection with worldwide distribution. The anthropozoonotic characteristic is of high relevance in the state of Rio de Janeiro, where an increasing in the number of cases in human patients was observed in the last decades, highlighting the role of domestic cat as a transmitter agent. The description of new species compounding de Sporothrix genus, based on phenotypic and genotypic evaluations, showed the involvement of other members of this group in the epidemic status installed in Rio de Janeiro. The verification of strains resistant to itraconazole, a widely used antifungal in human and animal medicine for the treatment of this mycosis, is an important factor that possibly results in relapse and therapeutic failure of this disease. The present study aimed to identify, by phenotypic and molecular approaches, 168 isolates obtained from the routine of Veterinary Clinical Microbiology Laboratory ? UFRRJ, and the determination of minimal inhibitory concentration (MIC) for amphotericin B (AMB), ketoconazole (KTC), itraconazole (ITC), terbinafine (TRB) and voriconazole (VRC). Based on morphophysiological characteristics it was possible to identify 159 (94.64%) isolates as S. brasiliensis and 9 (5.36%) as S. luriei. However, applying PCR-RFLP of calmodulin 168 (100%) samples were identified as S. brasilensis. The susceptibility test, based on M38-A2 document (CLSI), showed that TRB was the most effective antifungal tested, followed by ITC, KTC, AMB, and VRC, respectively. No ITC resistant isolates were detected in the present study. These results demonstrate that the identification reached only by phenotypic evaluation is not recommended for the characterization of Sporothrix schenckii complex components. It also proves the predominance of S. brasiliensis in other regions of RJ state. The better efficacy of TRB added to the absence of isolates resistant to ITC support the necessity of pharmacodynamics and pharmacokinetics studies for the optimization of the therapeutic protocols. More information about isolates from dogs and cats correlated with the species from the S. schenckii complex, as well as in vitro antifungal efficacy evaluation provide knowledge about therapeutic alternatives. In this way, the present study also provides relevant information about the endemic status in Rio de Janeiro and important data for the treatment of human and animal sporotrichosis. / Fungos dim?rficos pertencentes ao complexo Sporothrix schenckii s?o respons?veis pela esporotricose, importante infec??o f?ngica que apresenta distribui??o mundial. Sua conhecida caracter?stica antropozoon?tica apresenta grande relev?ncia no estado do Rio de Janeiro, onde se verifica aumento significativo no n?mero de pacientes humanos e animais acometidos pela doen?a nas ?ltimas d?cadas, destacando-se em tais casos o papel do felino como agente transmissor. A descri??o de novas esp?cies pertencentes ao g?nero Sporothrix, baseada em caracter?sticas fenot?picas e genot?picas, demonstrou o envolvimento de outros componentes deste g?nero na epidemia instalada no estado. A verifica??o de isolados resistentes a itraconazol, antif?ngico amplamente utilizado na medicina humana e veterin?ria para o tratamento da doen?a, ? fato preocupante e tem poss?vel associa??o a recidivas e falhas terap?uticas. O presente estudo objetiva a identifica??o fenot?pica e genot?pica de 168 exemplares oriundos de pacientes felinos e caninos, obtidos na rotina do Diagn?stico Microbiol?gico Veterin?rio - UFRRJ, com determina??o da Concentra??o Inibit?ria M?nima (CIM) frente ? anfotericina B (AMB), cetoconazol (KTC), itraconazol (ITC), terbinafina (TRB) e voriconazol (VRC). A partir de caracter?sticas morfofisiol?gicas foi poss?vel identificar 159 (94,64%) isolados como S. brasiliensis e 9 (5,36%) como S. luriei. Contudo, metodologias moleculares identificaram 168 (100%) S. brasiliensis, a partir de PCR-RFLP em gene respons?vel pela s?ntese de calmodulina. O teste de sensibilidade, realizado a partir do documento M38-A2 (CLSI) determinou maior efic?cia in vitro para TRB, seguido por ITC, KTC, AMB e VRC, respectivamente. Cepas resistentes a ITC n?o foram detectadas no presente estudo. Tais resultados demonstram que a identifica??o alcan?ada exclusivamente por m?todos fenot?picos n?o ? recomendada para caracteriza??o de componentes do complexo Sporothrix schenckii. Comprova-se ainda a predomin?ncia de S. brasiliensis em outras regi?es do estado do RJ. A maior efic?cia de TRB, somada a aus?ncia de exemplares resistentes a ITC, refor?a a necessidade de estudos farmacodin?micos e farmacocin?ticos para otimiza??o dos protocolos terap?uticos atualmente utilizados. Obten??o de maiores informa??es acerca dos isolados provenientes de amostras provenientes de c?es e gatos correlacionados a esp?cies dentro do complexo S. schenckii, bem como a avalia??o da efic?cia in vitro de antif?ngicos proporcionam conhecimento sobre alternativas terap?uticas. Tais informa??es auxiliam no entendimento do quadro instalado no estado do Rio de Janeiro e fornece dados de grande utilidade para o tratamento humano e veterin?rio

La nature extraordinaire du r??gime national-socialiste a longtemps paralys?? les tentatives d???en expliquer les causes, les m??canismes et les assises id??ologiques. Pourtant, depuis les ann??es 1960 et de mani??re toujours croissante par la suite, il s???av??re que ce qu???il y a de plus significatif sur le Troisi??me Reich semble ??tre r??v??l?? gr??ce ?? son inscription dans une trame sociohistorique qui le d??passe. En ce sens, la tendance misogyne du r??gime nazi ne peut ??tre comprise que par un exercice g??n??alogique. Caract??ristique fondamentale souvent n??glig??e, l???antif??minisme nazi s???inscrit dans une tendance paternaliste occidentale qui, bien qu???elle n???explique pas ?? elle seule les politiques et mesures nazies concernant les femmes, permet de saisir la nature id??ologique de leurs motivations. Plongeant au c??ur d???un XIXe si??cle post-Lumi??res aux contradictions nombreuses, aux revendications sociales certaines et aux maintes red??finitions ??pist??mologiques provoqu??es, entre autres, par le mouvement ?? anti-Lumi??res ??, notre m??moire propose d???observer les ??chos du discours allemand sur la diff??renciation des sexes et les femmes sous le Troisi??me Reich. Les id??es nazies sur les femmes ne naissent ainsi pas ex nihilo ; elles s???inscrivent dans une r??cup??ration id??ologique et une continuit?? philosophique. Tant les philosophes Arthur Schopenhauer, Friedrich Nietzsche et Otto Weininger que le m??decin Paul M??bius et le sociologue Georg Simmel s???expriment abondamment sur la ?? question f??minine ??. Repr??sentants du discours allemand de la seconde moiti?? du XIXe si??cle et du premier tiers du XXe si??cle, ils s???inscrivent ??videmment dans la tendance europ??enne, voire occidentale, d???une remise en question des acquis des Lumi??res et des revendications f??ministes. Se pronon??ant tant sur le corps f??minin que sur les capacit??s intellectuelles et morales des femmes, ces penseurs discr??ditent le mouvement d?????mancipation f??minine et mettent de l???avant des valeurs in??galitaires et d??terministes qui caract??riseront ??galement l???Allemagne d???Hitler.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-16T21:49:55Z No. of bitstreams: 1 LeandroViniciusFernandesDeMorais_DISSERT.pdf: 1776137 bytes, checksum: 5be63e56c13449063a3882ed83b373a2 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-19T23:34:37Z (GMT) No. of bitstreams: 1 LeandroViniciusFernandesDeMorais_DISSERT.pdf: 1776137 bytes, checksum: 5be63e56c13449063a3882ed83b373a2 (MD5) / Made available in DSpace on 2016-08-19T23:34:37Z (GMT). No. of bitstreams: 1 LeandroViniciusFernandesDeMorais_DISSERT.pdf: 1776137 bytes, checksum: 5be63e56c13449063a3882ed83b373a2 (MD5) Previous issue date: 2015-03-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Licania rigida Benth e a Turnera ulmifolia Linn. var. elegans s?o esp?cies de plantas regionais do semi?rido empregadas no tratamento de diversas doen?as. Objetivos: O intuito desse trabalho foi caracterizar quimicamente os extratos e fra??es e investigar o potencial antimicrobiano e antioxidante. Metodologia: Para a an?lise qu?mica foram realizadas a quantifica??o total de compostos fen?licos por espectrofotometria e a caracteriza??o cromatogr?fica dos extratos e fra??es. A avalia??o da atividade antioxidante foi realizada pela determina??o da capacidade de sequestro do radical DPPH?. A atividade antimicrobiana foi avaliada pelos ensaios de difus?o em ?gar, microdilui??o em caldo e cin?tica de morte. Resultados: Os extratos e fra??es de L. rigida e T. ulmifolia apresentaram elevado conte?do fen?lico, com a presen?a de flavonoides, dos quais foram determinados como marcadores qu?micos. Observou-se que os extratos de ambas as esp?cies agiram como agentes sequestradores no ensaio da atividade antioxidante in vitro. O extrato da L. rigida foi a ?nica ativa contra cepas de S. aureus, S. aureus meticilina resistente, S. epidermidis, e as leveduras, Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida rugosa, Candida krusei eTrichosporon asahii. Conclus?o: Com base nesses resultados ? poss?vel afirmar que atividade antioxidante e antimicrobiana possivelmente ? atribu?da a presen?a de polifen?licos e flavonoides evidenciados nos extratos e fra??es. / Introduction: Licania rigida Benth and Turnera ulmifolia Linn. var. elegans are species of semi-arid regional plants used in the treatment of various diseases. Objectives: The purpose of this study was chemically characterize the extracts and fractions and investigate the antimicrobial and antioxidant potential. Methods: For chemical analysis, were performed spectrophotometric quantification of the total phenolic and characterization of the extracts by chromatographic analysis. Evaluation of antioxidant activity was done by determining the radical scavenging capacity DPPH ?. Antimicrobial activity was evaluated by agar diffusion, broth microdilutionand time-kill assays. Results: The extracts and fractions L. rigid and T. ulmifolia showed a high phenolic content, the presence of flavonoids, which were determined as chemical markers. It was observed that the extracts of both species performed as sequestering agents in the trial of antioxidant activity in vitro. The L. rigida extract was the only active front strains of S. aureus 33591 (methicillin-resistant), S. aureus 29213, S. epidermidis 12228, and also against the yeast, Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida rugosa, Candida krusei eTrichosporon asahii. Conclusions: Based on these results it is possibly affirm the antioxidant and antimicrobial activity of L. rigida and attributed the presence of polyphenolic flavonoid like responsible.

Made available in DSpace on 2014-12-17T14:13:50Z (GMT). No. of bitstreams: 1 MaizaRA_TESE.pdf: 1687383 bytes, checksum: 6a89b25caa7db5fc536b4051629db0e3 (MD5) Previous issue date: 2013-12-06 / Candid?ase ? um problema de import?ncia crescente, devido o aumento do n?mero de indiv?duos imunocomprometidos e o surgimento de cepas resistentes aos antif?ngicos convencionais. ? de fundamental import?ncia a busca por novos agentes antif?ngicos mais eficazes, menos t?xicos, sendo os ?leos essenciais (OEs) excelentes alternativas para esse prop?sito. Esse estudo investigou a atividade biol?gica do OE de Mentha spicata L. sobre Candida guilliermondii de origem anal e vaginal. Para tanto foram determinadas a Concentra??o Inibit?ria M?nima (CIM), Concentra??o Fungicida M?nima (CFM), cin?tica do crescimento das leveduras (Time-Kill), altera??es micromorfol?gicas (t?cnica do microcultivo em c?mara ?mida) e investiga??o do mecanismo de a??o antif?ngico, utilizando o bioensaio do sorbitol. O OE de M. spicata foi obtido pelo processo de extra??o por destila??o a vapor. Na an?lise fitoqu?mica desse ?leo foi observada a presen?a de carvona com 84,32%, seguida pelo limoneno (13,70%) e tra?os de iso-dihidrocarvona (0,82%). Os resultados da an?lise da CIM variou entre 32 e 128 μg/mL. A CFM variou entre 64 e 1024 μg/mL. Na avalia??o da a??o de OE e da nistatina 100UI/mL, o antif?ngico padr?o apresentou o efeito fungicida a partir de 4 horas e para OE de M. spicata foi observado efeito fungist?tico na CIM, CIMX2 e CIMX4 frente ?s cepas avaliadas. O OE de M. spicata apresentou forte atividade antif?ngica contra as cepas de C. guilliermondii, promovendo altera??es micromorfol?gicas vis?veis por microscopia ?ptica, nas concentra??es testadas (CIM, CIMx2), resultado semelhante ao que se observou com a nistatina (100UI/mL). Na investiga??o do mecanismo de a??o antif?ngico foi constatado que houve altera??o da CIM na presen?a de sorbitol, com eleva??o dos valores quatro vezes maior que a concentra??o inicial, o que indica que os componentes desse OE apresentam a??o direta sobre a parede celular das leveduras. Conclui-se que o OE de Mentha spicata ? um potencial agente terap?utico no tratamento de candid?ase

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-25T22:57:38Z No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-27T22:44:59Z (GMT) No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) / Made available in DSpace on 2016-04-27T22:44:59Z (GMT). No. of bitstreams: 1 DianaLuziaZuzaAlves_DISSERT.pdf: 2035213 bytes, checksum: 1604df1e90593ddd103ab0202e591e9e (MD5) Previous issue date: 2015-03-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / V?rios estudos t?m sido desenvolvidos com rela??o aos riscos ? sa?de humana associados ao uso recreativo de praias contaminadas com esgotos dom?sticos. Esses res?duos cont?m v?rios micro-organismos, incluindo Candida tropicalis, agente etiol?gico tanto de infec??es superficiais quanto sist?micas, al?m de indicador de contamina??o fecal do meio ambiente. Nesse contexto, o presente trabalho teve como objetivo caracterizar isolados de C. tropicalis oriundos da areia da Praia de Ponta Negra, Natal, Rio Grande do Norte, Brasil, quanto a express?o de fatores de virul?ncia in vitro, a adapta??o ao estresse osm?tico e a susceptibilidade ?s f?rmacos antif?ngicos. Foram analisados 62 isolados ambientais de C. tropicalis, observando-se grande varia??o entre os mesmos para os diversos fatores de virul?ncia avaliados. Em geral, os isolados ambientais foram mais aderentes a c?lulas epiteliais bucais humanas (CEBH) do que a cepa de refer?ncia de C. tropicalis ATCC13803, al?m de serem altamente produtoras de biofilme. Em rela??o ? morfog?nese, a maioria dos isolados exibiu fen?tipo rugoso em meio Spider (34 isolados, 54,8%). Na avalia??o da atividade enzim?tica, a maioria dos isolados teve maior produ??o de proteinase do que a cepa de refer?ncia de C. tropicalis ATCC13803. Adicionalmente, 35 isolados (56,4%) tiveram alta atividade hem?litica (?ndice de hem?lise > 55). Com rela??o ? resist?ncia de C. tropicalis ao estresse osm?tico, 85,4% dos isolados foram resistentes em meio contendo 15% de cloreto de s?dio, o que corrobora com a alta capacidade de sobreviv?ncia descrita para essa levedura no ambiente mar?timo. Finalmente, no que diz respeito ? sensibilidade aos antif?ngicos foi observada elevada resist?ncia aos az?licos testados (fluconazol, voriconazol e itraconazol), com ocorr?ncia do fen?meno ?Low-high? e de efeito semelhante ao crescimento paradoxal que ocorre para as equinocandinas. As cepas resistentes aos tr?s az?licos testados foram 15 (24,2%). Para a anfotericina B tamb?m ocorreu resist?ncia (14 isolados, 22,6%), ao passo que para as equinocandinas todas as cepas foram sens?veis. Portanto, nossos resultados demonstram que isolados de C. tropicalis oriundos da areia de praia do nordeste brasileiro podem expressar plenamente atributos de virul?ncia e apresentam alta capacidade de persist?ncia no ambiente costeiro, al?m de serem significativamente resistentes aos antif?ngicos mais empregados na pr?tica cl?nica atual. Isso constitui potencial risco ? sa?de dos frequentadores desse ambiente, especialmente indiv?duos imunocomprometidos e em extremos et?rios. / Several studies have been developed regarding health risks associated with the recreational use of beaches contaminated with domestic sewage. These wastes contain various microorganisms, including Candida tropicalis, etiologic agent of both superficial infections such as systemic, as well as indicator of fecal contamination for the environment. In this context, the objective of this study was to characterize C. tropicalis isolates from the sandy beach of Ponta Negra, Natal, Rio Grande do Norte, Brazil, regarding the expression of in vitro virulence factors, adaptation to osmotic stress and susceptibility to antifungal drugs. We analyzed 62 environmental isolates of C. tropicalis and observed a great variation between them for the various virulence factors evaluated. In general, environmental isolates were more adherent to CEBH than C. tropicalis ATCC13803 reference strain, besides the fact they were also highly biofilm producers. In relation to morphogenesis, most isolates presented wrinkled phenotype in Spider medium (34 isolates, 54.8 %). When assessing enzyme activity, most isolates had higher proteinase production than C. tropicalis ATCC13803 reference strain. In addition, 35 isolates (56.4 %) had high hemolytic activity (hemolysis index > 55). With regard to C. tropicalis resistance to osmotic stress, 85.4% of the isolates were able to grow in a liquid medium containing 15% sodium chloride, corroborating to high survival capacity described for this yeast at marine environment. Finally, with regard to sensitivity to antifungal drugs, it was observed high resistance to the azoles tested, with the occurrence of the "Low-high" phenomenon and similar effect to the paradoxical growth which occurs to the echinocandins. For the three azoles tested we verified that 15 strains were resistant (24.2 %). Some strains were also resistant to amphotericin B (14 isolates, 22.6 %), while all of them were sensitive for the echinocandins tested. Therefore, our results demonstrate that C. tropicalis isolated from the sand of northeast of Brazil can fully express virulence attributes and showed a high persistence capacity on the coastal environment, in addition of being significantly resistant to most applied antifungals in current clinical practice. This constitutes a potential health risk to visitors of this environment, especially immunocompromised individuals and those with extreme age range.

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 Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:22:35Z No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:23:00Z (GMT) No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Made available in DSpace on 2017-09-04T20:23:00Z (GMT). No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) Previous issue date: 2016-04-14 / Invasive fungal infections are a major public health problem in the world, since they increase morbidity and patients??? hospitalization time. In Brazil, the highest mortality rate among the systemic mycoses is caused by an endemic disease named paracoccidioimycosis, which the etiologic agent is the dimorphic fungus Paracoccidioides spp. The worldwide increased resistance to the commercially available antifungal agents, their limited spectrum of activity against some fungal pathogens and concerns with their toxic side effects are reasonable evidence of the necessity of novel therapeutic strategies, especially the development of new antifungal agents. Thus, the essential gene rim8 was identified by comparative genomics as an orthologous sequence in the genome of human pathogenic fungi absent in the human genome. In filamentous fungi and yeasts, gene expression regulation by the ambient pH involves components of a signaling pathway that mediate proteolytic activation of the transcription factor PacC/Rim101 in response to alkaline environmental pH. The rim8 gene in yeasts, also called palF in filamentous fungi, performs an essential step in this signaling pathway. It is important for host-pathogen interaction, leading to increased virulence and pathogen survival. Thus, Rim8 is a very interesting and promising drug target. The aim of this work is to optimize heterologous expression and purification of Rim8 protein from P. luzii to further perform its structural and functional characterization and also, to use it as a molecular target for drugs development. The rim8 gene was chemically synthesized with a histidine tag and cloned into a pET-21a vector. Heterologous expression of the gene was made using two strains of E. coli, obtaining the best conditions using LB medium with 0.5% glucose at 30 ??C for 2 hours and 0.25 mM IPTG and adding protease inhibitor cocktail. The Rim8 heterologous protein was purified using a nickel affinity chromatography column. Expression and purification results were analyzed by SDS-PAGE and in some cases, confirmed by western blot. Immunization of BALB/c mice was performed with the purified protein to obtain anti- Rim8 antibodies. The antibody production was confirmed by ELISA test. The protein immunocitolocalization in P. lutzii cells showed a difuse protein-plasma membrane association at acidic pH. At neutral pH the fluorescence pattern is showed as localized foci in plasma membrane and later, with extracellular alcalinization, it migrates into the cytosol. Thus, it can be inferred that this work gives some contribution to the development of new antifungal drugs, but still must undergo further production steps, proteolysis reduction and protein purification to allow structural and functional characterization of Rim8. / Infec????es f??ngicas invasivas s??o um problema de sa??de p??blica no mundo, j?? que aumentam a morbidade e o tempo de interna????o de pacientes. A micose sist??mica com o maior ??ndice de mortalidade no Brasil, onde ?? considerada end??mica, ?? a paracoccidioidomicose, causada pelo fungo dim??rfico Paracoccidioides spp. A tend??ncia global de aumento da resist??ncia aos agentes antif??ngicos dispon??veis comercialmente, o espectro limitado de atividade contra alguns fungos patog??nicos e a preocupa????o com a toxicidade s??o evid??ncias da necessidade de novas estrat??gias terap??uticas, sobretudo do desenvolvimento de novas drogas antif??ngicas. Nesse sentido, o gene essencial rim8 foi identificado por gen??mica comparativa como uma sequ??ncia ort??loga no genoma de fungos patog??nicos humanos e ausente em humanos. Em fungos filamentosos e leveduras, a regula????o da express??o g??nica pelo pH envolve componentes de uma via de sinaliza????o que levam ?? ativa????o proteol??tica do fator de transcri????o PacC/Rim101 em resposta ?? alcaliniza????o do pH externo. O gene rim8 em leveduras ou palF em fungos filamentosos possui um papel essencial nessa cascata de sinaliza????o, sendo importante para a intera????o pat??geno-hospedeiro, aumento de virul??ncia e sobreviv??ncia do pat??geno. Por isso, Rim8 ?? um alvo molecular promissor e bastante interessante para o desenvolvimento de drogas. O objetivo deste trabalho ?? otimizar a express??o heter??loga e purifica????o da prote??na Rim8 de P. lutzii, visando realizar a caracteriza????o estrutural e funcional da prote??na para futuramente utiliz??-la como alvo molecular para o desenvolvimento de drogas. O gene rim8 foi sintetizado quimicamente com cauda de histidinas e foi clonado em vetor pET-21a. A express??o heter??loga do gene foi padronizada usando duas estirpes de E. coli, obtendo as melhores condi????es para purifica????o com o uso de meio LB contendo 0,5% de glicose a 30??C por 2 h e 0,25 mM de IPTG e coquetel de inibidores de proteases. Os resultados da express??o e purifica????o foram analisados por SDS-PAGE e/ou confirmados por western blot. Realizou-se a imuniza????o de camundongos BALB/c com a prote??na purificada para obten????o de anticorpos anti-Rim8. A produ????o dos anticorpos foi confirmada por ELISA. A imunocitolocaliza????o em c??lulas de P. lutzii mostrou que a prote??na se encontra associada ?? membrana plasm??tica de forma difusa em pH ??cido, em seguida apresenta focos localizados em resposta a um pH pr??ximo da neutralidade e, por fim, migra para o interior da c??lula em pH alcalino. Sendo assim, pode-se inferir que o trabalho apresenta contribui????es para o desenvolvimento de novas drogas antif??ngicas, al??m de auxiliar o entendimento do processo biol??gico no qual a prote??na RIM8 est?? envolvida.