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.