<p>Fungi have a genetically controlled sex determination system, which is governed by a small, sex-specific region in the genome called the mating-type locus (MAT). In the basidiomycetous yeast Cryptococcus neoformans, the pathogen that causes cryptococcal meningitis and cryptococcosis, sex has been associated with virulence. To further understand how sex is genetically regulated in C. neoformans, we focused our studies on the evolution of the MAT locus and molecular dissection of the pheromone signaling pathway that controls sexual development. Two MAT-linked meiotic recombination hotspots that likely drove the assembly and rearrangement of MAT were identified. Fine mapping through the integration of genetic markers established that two hotspots, one on each side of the MAT locus, are located in an ~10 kb and ~5 kb region. Plotting the G + C content along MAT and the flanking regions revealed a strong association between the location of these two hotspots and a high G + C content. By deletion and insertion of the G + C rich region, we demonstrated that the high G + C rich region is required but not sufficient to induce recombination. On the other hand, to provide direct experimental evidence to support the previously proposed model for the evolution of MAT, we sought to recapitulate the ancestral tetrapolar, and the intermediate tripolar mating systems of C. neoformans by manipulating the MAT structure to model a tetrapolar system. In the two modified "a" and "α" strains, the sex-determining genes SXI1α or SXI2a residing at the MAT locus were disrupted and the wild-type allele of these two genes was then reintroduced at another genomic location (URA5) that is unlinked to MAT. Our results show that C. neoformans can complete the sexual cycle with a tetrapolar mating configuration and the transitional tripolar state might be under strong negative selection pressure, which could have facilitated the transition from a tripolar state to the final bipolar mating system. </p><p>The MAT locus is the major determinant of the sexual identity of a cell, but several signaling pathways, including the pheromone signaling pathway, are required to regulate mating and sexual development. Many components of the pheromone signaling pathway have been identified; however, it is less clear what lies upstream of the MAPK cascade. To address this question, we studied the role of two Gα subunits (Gpa2, Gpa3) in mating and concluded that they share both redundant and divergent roles in mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. In their GTP-bound form, they signal in opposition: Gpa2 promotes mating whereas Gpa3 inhibits. Furthermore, we also studied the functions of a novel upstream component Cpr2, a pheromone receptor-like gene, in pheromone signaling and sexual development. All lines of evidence suggest that Cpr2 is a constitutive ligand-independent receptor that, when expressed, engages the same G-proteins and activates the same pheromone signaling pathway as the canonical ligand-activated pheromone receptors. Expression of Cpr2 is induced post cell fusion during mating, and likely introduces a positive feedback loop to allow a self-perpetuating signaling state to enable efficient mating. Cells lacking this receptor are fertile, but produce abnormal filamentous structures. Overexpression of CPR2 in a or α cells strongly enhances fruiting, an alternative same-sex mating process in C. neoformans. Therefore, Cpr2 establishes a new paradigm for a naturally occurring constitutively active GPCR that governs cell fate in fungi. </p><p>Finally, we described a sex-induced silencing (SIS) phenomenon in C. neoformans. Using genetic approaches, we showed that SIS is triggered by a tandem insertion of a transgene during the sexual cycle. Interestingly, only a proportion of progeny carrying the transgene are silenced. Gene deletion, RIP, or DNA methylation do not contribute to SIS but the RNAi machinery is required. In conclusion, these studies provide further understanding of sex in C. neoformans from different perspectives, which invites comparisons to other fungal and even more broadly, eukaryotic pathogens to address the role of sex in evolution.</p> / Dissertation

The increasing infection of Cryptococcus species and Tricosporon asahii emerged in clinical patients who were immunocompromised. They usually induce lung, skin, brain and systemic infection. Morbidity and mortality of immunocompromised patients are higher than normal healthy people. Cryptococcus neoformans var. grubii ¡]serotype A¡^ infections were reported in clinical cases predominantly and they were isolated from birds¡¦ droppings in large amount. Cryptococcus neoformans var. gattii ¡]serotype B, C¡^ had a natural life in plants, especially Eucalypticus trees. Isolations from other trees were reported increasingly in the tropical and subtropical areas. Comparing to Cryptococcus species, Tricosporon asahii is the normal mycoses of soil. In this study, we performed an environmental investigation concerning Cryptococcus species and Tricosporon asahii in Southern Taiwan. 120 droppings of racing pigeons and 114 samples from Eucalypticus trees were obtained. The results revealed that 30 Cryptococcus neoformans were isolated from racing pigeons¡¦ droppings ¡]25%¡^, as well as 4 Cryptococcus laurentii ¡]3.3%¡^ and 2 Cryptococcus albidus ¡]1.7%¡^. In addition, 25 Tricosporon asahii ( 20.8% ) were isolated from droppings of racing pigeons. But, none of Cryptococcus species or Tricosporon asahii is isolated from Eucalypticus trees ¡]0%¡^. All of Cryptococcus neoformans isolated from pigeons¡¦ droppings were var. grubii ¡]serotype A¡^ and their drug susceptibility tests showed sensitive to Amphotericin B ¡]minimal inhibitory concentration ¡Ø0.25£gg/ml¡^ and Fluconazole ¡]minimal inhibitory concentration 2£gg/ml¡^ and Flucytosine ¡]minimal inhibitory concentration ¡Ø1£gg/ml¡^. To sum up, both Cryptococcus species and Tricosporon asahii were isolated from droppings of racing pigeons in our study, especially Tricosporon asahii in large amount. Opportunistic infection caused by these species should be given more attention to racing pigeons which have close contact with human . Intensive investigation and surveillance should be carried out in the future to provide an information for the control and prevention of diseases.

Thesis (doctoral)--Universität Göttingen, 1999. / Includes bibliographical references (p. 249-277).

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Jianping Xu). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (D.P.H.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-27Bitstream added on 2014-06-13T20:24:16Z : No. of bitstreams: 1 diniz_jnm_dr_arafcf.pdf: 718250 bytes, checksum: ad07abef21dc44356500905c93cbf5e5 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cryptococcus neoformans é levedura capsulada saprobiota que acomete principalmente indivíduos com o sistema imunológico comprometido. Seu habitat está relacionado com algumas espécies de árvores, madeira em decomposição e fezes de pássaros, em especial de pombos do gênero Columba livia. Nas grandes cidades estas aves vêm causando diversos problemas devido a sua multiplicação intensa. Desta maneira, medidas saneantes serão de grande auxílio para controlar a população desta levedura. Assim, este estudo teve como objetivo reidentificar 67 isolados de C. neoformans de fontes ambientais e caracterizá-los quanto ao seu genótipo e perfil de sensibilidade aos antifúngicos anfotericina B, cetoconazol, itraconazol e fluconazol, utilizando o método da difusão em ágar seguindo o documento M44A e determinando a concentação inibitória mínima frente a fluconazol pelo método M27-A2, ambos preconizados pelo CLSI (Clinical Laboratory Standard Institute). Por outro lado, verificar o efeito antifúngico de extratos de plantas e sua atividade saneante em fezes de pombos comprovadamente contaminadas com isolado ATCC 90012 de C. neoformans. Todos isolados foram Mating Type α e tipo VNI. Pelo método de microdiluição, 21% (14/67) dos isolados ambientais apresentaram sensibilidade diminuída ao fluconazol com CIM de 16 μg/mL e mais de 50% (34/67) apresentaram CIM de 8 μg/mL.O extrato de Alibertia macrophylla apresentou efeito saneante com índice de eficiência de mais de 80% após 24 horas, na grande maioria dos isolados de C. neoformans. Este extrato foi estável... / Cryptococcus neoformans is a saprobiote capsuled yeast that proliferates mainly in individuals who have the immunologic system compromised. It natural habitat is related to some species of trees, decaying wood and bird excrement, in special Columba livia doves. In big cities, these birds have caused many problems due to its intense proliferation and the cleaning measures will be of great help in controlling this yeast. The aim of this study was to re-identify 67 C. neoformans isolates and characterize them according to their genotype and susceptibility to antifungal amphotericin B, cetoconazole, itraconazole e fluconazole, using the method of agar diffusion following the M44A document and determining the minimum inhibitory concentration to fluconazole by method M27-A2, both recommended by the CLSI (Clinical Laboratory Standard Institute). On the other hand, the verification of the effect of plant extracts e and their cleaning activity on dove excrement previously contaminated with isolate ATCC 90012 of C. neoformans. All the isolates were Mating Type α and VNI. For the microdilution method, 21% (14/67) of the environmental isolates showed decreased susceptibility to fluconazole with MIC of 16 mg/mL and more than 50% (34/67) had MICs of 8 mg/mL. The extract of Alibertia macrophylla presented clean effect with index of efficiency of 80% after 24 hours, in the great majority of C. neoformans isolates. This extract was stable to the light incidence, to the heat and variation of pH. Our results are of great use for the development and utilization of active natural compounds in the fight against C. neoformans from environmental and the study of other variables are necessary for the viability of its use as a domestic cleaner.

A Criptococose é uma enfermidade micótica sistêmica, subaguda ou crônica, que acomete a cavidade nasal, tecidos paranasais e pulmões do homem, animais domésticos e silvestres, podendo disseminar-se para o sistema nervoso central, olhos, pele e outros órgãos. Considerando as dificuldades terapêuticas no tratamento da micose em pequenos animais, incluindo toxicidade, desenvolvimento de resistência aos antifúngicos tradicionalmente utilizados e longos períodos de tratamento da enfermidade, o estudo objetivou avaliar a eficácia do imunomodulador ß (1-3) glucana isoladamente e em associação ao fluconazol no tratamento da criptococose experimental. Foram utilizados 100 camundongos (Mus musculus), cepa UFPel, albinos, os quais foram divididos em cinco grupos de 20 animais. O tratamento dos animais com criptococose experimental foi iniciado sete dias após a inoculação. O grupo Controle (G1) foi tratado com 0,1 ml de água destilada estéril, o grupo Fluconazol (G2) com 5 mg/kg de fluconazol, o grupo Fluconazol associado a ß (1-3) glucana (G3) com 5 mg/kg de fluconazol associado a 0,5 mg de ß (1-3) glucana, o grupo Glucana dose I (G4) com 0,5 mg de ß (1-3) glucana e o grupo Glucana dose II (G5) recebeu 0,25 mg de ß (1-3) glucana. Após acompanhamento clínico durante as seis semanas de tratamento os animais foram eutanasiados e necropsiados para avaliação anatomopatológica dos órgãos, quantificação das unidades formadoras de colônias fúngicas (UFCs), retroisolamento e avaliação histopatológica. Nas avaliações clínicas o G3 foi sempre superior ao G1 e G2, sendo que na última avaliação clínica individual, os animais pertencentes ao G3 não apresentavam nenhum sintoma clínico. O G2 teve um menor número de órgãos afetados e de alterações macroscópicas, seguido pelo G3, sendo que no primeiro não foram observadas lesões em órgãos-alvo, como pulmão e cérebro. O grupo com maior número de lesões e órgãos afetados foi o G1. Nos parâmetros retroisolamento e UFCs, o G3 foi superior aos outros tratamentos, seguido pelo G2, sendo que o pior grupo, ou seja, aquele que teve um maior número de isolamentos e maior quantificação de UFCs foi o G1. Conforme os resultados obtidos os tratamentos G2 e G3 foram os mais eficazes para a remissão da criptococose experimental, no entanto, G3 apresentou uma superioridade na maioria dos parâmetros avaliados. Portanto, de acordo com os resultados obtidos, a associação de fluconazol ao imunomodulador ß (1-3) glucana pode ser uma alternativa benéfica em considerável quantidade de pacientes com criptococose. / Cryptococcosis is a systematic sub acute or chronic mycotic condition that affects the nasal cavity, paranasal tissues and the lungs of humans, as well as domestic and wild animals, which can spread to the central nervous system, skin, and other organs. Considering the therapeutic difficulties in treating this mycosis in small animals, which include toxicity, resistance towards traditionally used antifungals, and the extended treatment of this condition, this study aimed to evaluate the ß (1-3) glucan immunomodulator efficacy when used both alone and in association with fluconazole in the treatment of experimental cryptococcosis. One Hundred albino UFPel strain mice (Mus musculus), divided into five groups of 20 animals each, were used. The treatment of these animals with experimental cryptococcosis was started seven days following inoculation. The control group (G1) was treated with 0,1 ml sterile distilled water. The fluconazole group (G2) was treated with 5 mg/kg fluconazole; the fluconazole associated with ß glucan (1-3) group (G3) was treated with 5 mg/kg fluconazole associated with 0,5 mg ß (1-3) glucan ; the group glucan dose I (G4) was treated with 0,5 mg ß (1-3) glucan, and the group glucan dose II (G5) was administered 0,25 mg ß (1-3) glucan. After the six-week treatment clinical follow-up, the aimals were euthanized and necropsied for anatomopathological evaluation of organs, quantification of fungal colony-forming units (FCUs), retro isolation and histopathological evaluation. In clinical evaluations, G3 was always superior to G1 and G2, and in the last individual clinical evaluation, G3 animals did not show any clinical symptoms. G2 had a smaller number of affected organs and microscopic alterations, followed by G3. In G2, target organ lesions, such as those in the lungs and brain, were not found. Considering retro isolation and FCU parameters, G3 was superior to other treatments, followed by G2; the worst group, that is, the one with the highest isolation occurrence and greatest FCU quantification, was G1. According to the results obtained, treatments G2 and G3 were the most efficient in experimental cryptococcosis remission; however, G3 was superior in most parameters evaluated. Therefore, according to the results obtained, the association of fluconazole with the ß (1-3) glucan immunomodulator can be a beneficial alternative to a considerable number of cryptococcosis-bearing patients.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2014-07-07T20:06:26Z No. of bitstreams: 1 Dissertação_ICS_ João Antônio Miranda Souza.pdf: 2240651 bytes, checksum: 74599c9ddde7f9d862894d9a27be25e0 (MD5) / Made available in DSpace on 2014-07-07T20:06:26Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ João Antônio Miranda Souza.pdf: 2240651 bytes, checksum: 74599c9ddde7f9d862894d9a27be25e0 (MD5) / CAPES / Cryptococcus é uma levedura zoonótica, frequentemente associada a excretas de pombos e seu isolamento já é relatado em todo o mundo. Trata-se de uma levedura encapsulada, de forma globosa ou ovalada, que apresenta acentuado tropismo pelo sistema nervoso central. O principal problema é que o fungo permanece viável em excretas secas dessas aves por um longo período, tornando-se um reservatório de partículas infectantes passíveis de serem inaladas. O trabalho teve como objetivo caracterizar a variedade fenotípica e genética de Cryptococcus spp. ambientais da cidade de Salvador, Bahia. Para a execução desse estudo, 200 amostras de excre-mentos secos de pombos foram coletadas no solo entre novembro 2011 e fevereiro de 2012, em praças públicas, centro de distribuição de alimentos e no Porto da cida-de Salvador, Bahia. Foram adicionados 4mL de solução salina esterilizada, acresci-da de cloranfenicol, para cada 1g de excreta. A mistura foi submetida à agitação em vortex por 3 minutos, e mantida em repouso por 60 minutos e, em seguida, diluições foram feitas. Foram aspirados 0,1 mL do sobrenadante e semeados com alça de Drigalski, de cada diluição, em placas de ASD contendo cloranfenicol. As placas fo-ram incubadas em estufa de demanda de oxigênio a 28oC por até 21 dias. Colônias sugestivas foram repicadas em caldo uréia e analisadas com tinta da China para pesquisa de cápsula e micromorfológica com coloração de Gram. Após a identifica-ção do gênero, as leveduras foram identificadas bioquimicamente por auxanograma, zimograma, crescimento a 37ºC e repicadas em ágar Níger (Guizotia abyssinica), e CGB. Foram isoladas 37 leveduras, das quais 22 são da espécie C. neoformans, 4 de C. gattii, 5 de C. albidus, 3 de C. uniguttulatus e 3 da espécie C. laurentii. A de-terminação dos genótipos foi realizada utilizando a técnica de PCR-RFLP do gene URA5 e foram verificados os perfis moleculares VNI (53,8%), VNIII (11,5%), VNIV (19,2%), VGII (7,7%), VGIII (3,8%) e VGIV (3,8%). A positividade para Cryptococcus nas amostras encontradas nos locais de estudo, associado ao grande número de pombos, apontam para a existência de riscos ambiental que devem servir de alerta aos órgãos de vigilância sanitária em Salvador. A identificação de espécies como C. gattii, C. albidus, C. uniguttulatus e C. laurentii em excretas de pombos evidencia a importância de novos estudos epidemiológicos para traçar os perfis de espécies de Cryptococcus que podem ser detectados de fontes ambientais em nossa cidade.

Made available in DSpace on 2015-12-10T14:24:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-28. Added 1 bitstream(s) on 2015-12-10T14:30:40Z : No. of bitstreams: 1 000851921.pdf: 11886045 bytes, checksum: 1cf15c01de888d44bc38aeeab19c7d88 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O aumento de micoses invasivas e o desenvolvimento de mecanismos de resistência de algumas espécies de fungos, frente aos fármacos utilizados na terapia, têm sido preocupante. O tratamento antifúngico, geralmente é agressivo e o surgimento da resistência antifúngica acrescenta dificuldades adicionais no tratamento dessas infecções. Cryptococcus neoformans e Cryptococcus gattii são os principais agentes etiológicos da criptococose, e afetam principalmente pacientes imunodeprimidos. Nesse trabalho foi realizada a caracterização morfológica e da virulência de três isolados clínicos de C. neoformans e dois isolados ambientais de C. gattii em Galleria mellonella, além da análise do nível de expressão dos genes MDR1 e AFR1 envolvidos no funcionamento de bomba de efluxo, através da quantificação relativa por PCR (polimerase chain reaction) em tempo real. Uma vez esses genes quantificados, esses isolados foram usados na prospecção de moléculas sintéticas para verificação da atividade inibitória dos genes selecionados, após o contato com as mesmas. Os isolados foram classificados como sensível (26S), sensibilidade intermediária (27I) e resistente (30R) da espécie neoformans e da espécie C. gattii, um foi resistente (118R) e outro sensível (CL), in vitro, a fluconazol. As moléculas selecionadas para os testes foram o ácido gálico e seus derivados sintéticos, e os melhores resultados observados foram obtidos a partir dos galatos de alquila G11, G12, G14, G15, G16 e G17, apresentando ótima atividade antifúngica contra os isolados testados, em especial os galatos G14, G16 e G17, obtendo ótima atividade antifúngica. O galato de n-dodecila (G16), foi selecionado para os demais testes apresentando atividade aditiva no teste de sinergismo, quando utilizado em associação com fluconazol, reduzindo os valores de CIM para o mesmo, o que mostra a possível... / The increase of invasive mycoses and the development of antifungal resistance by some fungi species have been worrying. The antifungal treatment is usually aggressive and inefficient, and the emergence of antifungal resistance increases the difficulties in treating these fungi infections.Cryptococcus neoformans e Cryptococcus gattii are the main etiologic agents of cryptococcosis andespecially affect immunocompromised patients. In this work the Galleria mellonella, was used to perform the morphologic characterization as well as determine the virulence of three sequential clinical isolates of C. neoformansand twoisolates environmental of C. gattii. In addition, was held the analysis of genes expressions (MDR1, ERG11 and AFR1), that are involved in efflux pumps and ergosterol synthesis, by real time PCR (polimerase chain reaction) assay. After these characterizations the isolates were used in the prospecting of synthetic molecules to verify the inhibitory activity of selected genes. After incubation in vitro with fluconazole the C.neoformans isolates were classified as sensitive (26S), intermediate sensitivity (27I) and resistant (30R), and the C. gattiiisolates were resistant (118R) and the other sensitive (LC). The galic acid molecules and their synthetic derivatives were tests against C.neoformans and C. gattii isolates.The best results were obtained from alkyl gallates G11, G12, G14, G15, G16 and G17 which shows a great antifungal activity, especially G14, G16 e G17. The gallate n-dodecyl (G16), was select to others tests once presented additive activity in synergism test with fluconazole, decreasing the MIC values. The gene expression assay shows that both isolates expressed MDR1 and AFR1 (encoding efflux pumps), and the G16 was capable to inhibit these genes which makes this molecule promising for inhibiting efflux. With virulence assays using the G. mellonella was possible ...