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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.