Cytokines play an important regulatory role during immune responses against pathogens. The outcome of an induced cytokine pattern is determined by many factors. It strongly depends on the nature of the pathogen and the host’s ability to control the quality and strength of cytokine signals. In pulmonary infection with Cryptococcus neoformans T helper (Th) 1 and Th17 cell subsets and their associated cytokines confer protection, whereas a Th2-biased response with production of interleukin (IL) -4 confers susceptibility. Since inappropriate Th responses often lead to death in immunosuppressed human patients, especially HIV-1 infected patients, this work aimed to elucidate mechanisms of Th2 induction and regulation by assessing the Th2 hallmark cytokine IL-4 in an experimental model of cryptococcosis. Therefore, a kinetic study of IL-4 expression during 70 days after intranasal infection was performed in susceptible mice. The analyses included characterization of pulmonary leukocytes and Th cell cytokine profiling. IL-4 profiling revealed Cryptococcus-specific IL-4 production not before six weeks after infection. This unexpected finding was further validated by equal results observed in a kinetic study done in IL-4 reporter mice. These mice express a green fluorescent protein simultaneously to IL-4 expression in the same cell and this protein can be detected by flow cytometry. Two cellular sources of IL-4 were identified: Th2 cells were found as expected, but also, as shown for the first time, eosinophilic granulocytes could be demonstrated to secrete IL-4. Next, the influence of eosinophils on pulmonary inflammation and disease development was investigated using ΔdblGATA-1 mice constitutively devoid of eosinophilic granulocytes. Experiments with infected ΔdblGATA-1 mice revealed novel regulatory functions of eosinophils in cryptococcosis. In the absence of eosinophils pulmonary Th cell recruitment was significantly diminished. In addition, Th2 polarization was reduced in ΔdblGATA-1 mice as shown by reduced numbers of Th2 cells expressing the Th2-related surface marker T1/ST2 and reduced albeit not absent IL-4 production by Th cells. In addition to reduced IL-4 production, in the absence of eosinophils Th cells with enhanced interferon-γ and IL-17 production were observed. However, control of pulmonary fungal growth was only slightly enhanced in the absence of eosinophils and dissemination of cryptococci to the brain was unaltered. This may be related to the shared IL-4 production by not only eosinophils but also Th2 cells. Blocking more than one cellular source of IL-4 could be required to prevent immunopathology. To test the hypothesis of gradual IL-4-dependent immunopathology, experiments were conducted using mice expressing only one allele of the IL-4receptor (R) alpha (α) chain (+/-) instead of two (+/+). Indeed, mono-allelic expression of the IL-4Rα resulted in an intermediate expression of the IL-4R on the surface of myeloid and lymphoid cells indicating a gene-dosage effect for IL-4R expression. Infected IL-4Rα+/- mice displayed reduced susceptibility as compared with IL-4Rα+/+ mice, and IL-4Rα-/- mice completely lacking IL-4R expression were found to be protected with survival for the complete time period of the experiment (i.e. up to 275 days). Reduced susceptibility found in infected IL-4Rα+/- mice was associated with decreased serum levels of immunoglobulin E, reduced mucus production by airway epithelia, attenuation of airway hyper-reactivity, and reduced formation of alternatively activated macrophages in lung parenchyma – pathophysiological features, which are typically found in experimental models of asthma but also in asthma of humans and animals. Since no up-regulation of IL-4R by the infection with Cryptococcus neoformans was found, the experimental pulmonary infection model used appears to be a very sensitive low-level IL-4 system. This work highlights the outstanding role of IL-4 and its different cellular sources as well as its receptor in cryptococcosis and provides novel insights into pathogenesis. Moreover, a cellular (i.e. eosinophils) and a molecular (i.e. IL-4R) target for treatment of this mycosis and possibly of asthma is provided.

<p>Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH. </p><p>Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms. </p><p>The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi. </p><p> To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.</p> / Dissertation

Submitted by DEBORA DE OLIVEIRA LOSNAK null (deboralosnak@hotmail.com) on 2017-03-08T18:48:25Z No. of bitstreams: 1 Dissertação Débora Losnak.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-13T18:04:26Z (GMT) No. of bitstreams: 1 losnak_do_me_bot.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) / Made available in DSpace on 2017-03-13T18:04:26Z (GMT). No. of bitstreams: 1 losnak_do_me_bot.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A emergência e reemergência de doenças infecciosas é impulsionada por vários fatores e a busca de patógenos em amostras animais podem oferecer oportunidades para estudos eco-epidemiológicos e também dados sobre a evolução dos patógenos. O objetivo deste estudo foi avaliar a ocorrência de fungos patogênicos importantes em saúde pública, em exemplares de animais silvestres mortos por atropelamento no estado de Santa Catarina e identificar e mapear áreas de risco para a infecção humana. Grande parte destes fungos apresenta em comum dimorfismo, distribuição geográfica restrita e produção de conídios infectantes que são aspirados pelo hospedeiro por meio das vias respiratórias. Cães e tatus são apontados como transmissores de Paracoccidioides brasiliensis, os morcegos ao Histoplasma spp., assim como as fezes de pombos ao Cryptococcus spp.. No presente trabalho foram analisadas 1063 amostras de pulmão, fígado, baço, pele e coração de 297 animais silvestres, para detecção de Paracoccidioides brasiliensis, Histoplasma capsulatum e Cryptococcus spp. pela técnica de Reação em Cadeia de Polimerase (PCR). Utilizou-se primers universais para detecção de fungos em geral e obteve-se positividade em 102 amostras de 59 animais. Para a análise de P. brasiliensis, utilizou-se os primers específicos, obtendo oito amostras positivas em cinco animais (quatro Oxymycterus spp. e um Euryoryzomys russatus). Não houve a detecção molecular para Histoplasma spp.. Foi possível a identificação de três amostras para Cryptococcus spp.. O sequenciamento foi realizado, porém em 89 amostras de 49 animais foi possível somente a identificação em Fungal sp. (GenBank KT923226.1), duas amostras para Cryptococcus neoformans (GenBank KY107218.1) obtidas de Oxymycterus spp. e Akodon spp. e três amostras de Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1) obtidas de Gracilinanus spp., Oxymycterus spp. e Philander spp. Importante salientar que houve coinfecção de P. brasiliensis e Cryptococcus neoformans em amostra de um Oxymycterus spp. Esta pesquisa mostra a importância dos animais silvestres na transmissão de doenças e auxilia no mapeamento dos locais de ocorrência de determinados patógenos e doenças em uma região ainda não avaliada. / The emergence and reemergence of infectious diseases is propelled by many diverse factors and the search for pathogens in animal samples may offer opportunity for eco-epidemiologic studies as well as data on the evolution of pathogens. The objective of this study was to evaluate in samples of road-killed wild animals the occurrence of pathogenic fungi of importance for public health. A great part of these fungi presented, in common, dimorphism, restricted geographic distribution and production of conidia infecting, which are aspirated by the host by means of their respiratory tract. Dogs and armadillos are normally related to the transmission of Paracoccidioides brasiliensis, bats to Histoplasma spp., as well as pigeons feces to Cryptococcus spp.. In this study we analyzed 1063 samples of organs of 297 wild animals for the detection of Paracoccidioides brasiliensis, Histoplasma capsulatum and Cryptococcus spp. by the technique of Polymerase Chain Reaction (PCR). Universal primers were employed for the detection of fungi in general and positivity was obtained in 102 samples from 59 animals. For the P. brasiliensis analysis was used specific primers, resulting in eight positive samples from five animals (four Oxymycterus spp. and one Euryoryzomys russatus). There was no molecular detection to Histoplasma spp.. Was possible the identification of three samples to Cryptococcus spp.. The sequencing was performed, however in 89 samples from 49 animals was possible to identify Fungal sp. (GenBank KT923226.1), two samples for Cryptococcus neoformans (GenBank KY107218.1) obtained from Oxymycterus spp. and Akodon spp. and three samples from Gracilinanus spp., Oxymycterus spp. and Philander spp. were positive for Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1). Is important emphasize the coinfection with P. brasiliensis and Cryptococcus neoformans in a sample from Oxymycterus spp.. This research shows the importance of the wild animals in transmissions of diseases and assists in the mapping of pathogen and disease sites in a region that has not yet been evaluated. / FAPESP: 2015/17519-4

Orientador : Ana Marisa Fusco Almeida / Coorientador: Maria José Soares Mendes Giannini / Banca: Juliana Campos Junqueira / Banca: Fernando Rogério Pavan / Banca: Patricia Albuquerque de Andrade Nicola / Banca: Dulce Helena Siqueira Silva / Resumo: 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... / Abstract: 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 ... / Doutor

Orientador: Ana Marisa Fusco Almeida / Coorientador: Jean Leandro dos Santos / Coorientador: Maria José Soares Mendes Giannini / Banca: Daniel Assis dos Santos / Banca: Luciana Trilles / Banca: Valéria Valente / Banca: Reginaldo dos Santos Pedroso / Resumo: Criptococose é uma importante micose sistêmica que acomete principalmente pacientes imunocomprometidos e é classificada como a infecção fúngica com maior mortalidade entre indivíduos portadores de HIV. Cryptococcus neoformans é uma levedura capsulada e o principal agente etiológico da criptococose; encontra-se dispersa no meio ambiente na forma de basidiósporos, os quais são responsáveis pela infecção em humanos e animais. As principais manifestações clínicas estão associadas à infecção pulmonar e meningite. A terapia se dá basicamente com a administração de anfotericina B (AMB) na terapia de ataque associada ou não a 5-fluocitosina e como manutenção é indicado ao longo do tratamento o fluconazol (FCZ). Apesar de esta terapêutica ser eficiente, é constatado elevado número de casos de reincidência e desenvolvimento de resistência aos azóis, além de problemas de toxicidade. Diante desta problemática, este estudo propõe o desenvolvimento de novas alternativas terapêuticas para o tratamento da criptococose, visando duas abordagens distintas. A primeira, aplicando novo composto antifúngico e a segunda, o estudo de microRNAs (miRNAs) envolvidos na interação da levedura e células de glioblastoma humano (U87-MG), com a finalidade de usá-los como reguladores da infecção, sendo esta uma estratégia recentemente divulgada em uma variedade de doenças. Para tanto, inicialmente moléculas análogas de resveratrol foram avaliadas quanto a atividade antifúngica e o derivado orto,orto-HDZ (HDZ) mostrou forte atividade fungicida contra isolados de C. neoformans, com valores de concentração fungicida mínima (CFM) variando entre 0,97 a 7,81 µg/mL. A associação entre HDZ e FCZ mostrou sinergia com potencialização do efeito do FCZ em até 64 vezes para um isolado clínico resistente. Baixa toxicidade foi... / Abstract: Cryptococcosis is an important systemic mycosis that mainly affects immunocompromised patients and is classified as the fungal infection with higher mortality among individuals with HIV. Cryptococcus neoformans is an encapsulated yeast and the main etiologic agent of cryptococcosis. This yeast is dispersed into the environment in the form of basidiospores, which are responsible for infection in humans and animals. The main clinical manifestations are associated with pulmonary infection and meningitis. The treatment is basically the administration of amphotericin B (AMB) as therapy consolidation with or without the use of 5-flucytosine and, for the maintenance therapy, fluconazole (FCZ) is indicated. Although this therapy is effective, it is observed high number of cases of relapses, development of resistance to azoles and toxicity problems. In front of these problems, this study proposes the development of new therapies for the treatment of cryptococcosis, targeting two distinct approaches. The first aims a new antifungal compound and the second the study microRNAs (miRNAs) involved in the interaction between yeast and human glioblastoma cells (U87-MG), in order to use them as regulators of infection, since this is a recently disclosed strategy in a variety of diseases. For this purpose, initially, analogs of resveratrol molecule were evaluated for antifungal activity and the derived ortho,ortho-HDZ (HDZ) showed a strong fungicidal activity against isolates of C. neoformans with values of minimum fungicidal concentration (MFC) ranging between 0.97 to 7.81 µg/mL. The association between HDZ and FCZ showed synergy with potentiation of the effect of FCZ in up to 64 times for a resistant clinical isolate to FCZ. Low toxicity was observed in in vitro and in vivo assays (Galleria mellonella and mice), despite showing changes in ... / Doutor

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-09T11:45:00Z No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-20T10:55:13Z (GMT) No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-20T10:55:13Z (GMT). No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-05-14 / Fungal infections caused by yeasts of Cryptococcus neoformans complex have increased considerably among immunosuppressed especially in patients infected with human immunodeficiency virus (HIV). Antifungal drugs available to treat these infections have a wide spectrum of action, but the high costs, side effects, besides the acquisition of resistance of fungi make their limited effectiveness. In these circumstances, the search for new drugs is needed. In the present study, we evaluated the antifungal activity of tamoxifen, bisabolol, indinavir, didanosine, UFMG, Labiocon 241, solasodine, Labiocon 237, Clonazepam, semicarbonado Benzaldehyde, Cardanol, AB 36 and three natural extracts of Psychotria spp, Sclerolobium paniculatum, Hymenaea courbaril and cell viability and mechanism of action of the compound showed antifungal activity of 20 isolates of Cryptococcus neoformans. To evaluate the antifungal activity , we used the in vitro susceptibility to yeast broth microdilution protocol M27 - A3 ( CLSI 2008) and to verify the mechanism of action, flow cytometry was performed with the cellular marker Propidium Iodide ( PI ) and cell viability assay with MTT salt. Among the compounds analyzed, only tamoxifen and olanzapine showed inhibitory action on fungal samples at concentrations 128-256 μg/mL. Analysis by flow cytometry was performed with tamoxifen and showed that the compound did not alter the cell membrane of Cryptococcus neoformans ATCC 28957 , however, the cell viability test showed that tamoxifen was able to inhibit the metabolism of the fungus in different concentrations ranging 4-1024 μg/mL. / As infecções fúngicas causadas por leveduras do complexo Cryptococcus neoformans tem sido reduzida entre os imunossuprimidos principalmente nos pacientes infectados pelo vírus da imunodeficiência humana (HIV). Os antifúngicos disponíveis para o tratamento destas infecções apresentam um amplo espectro de ação, porém os altos custos, efeitos colaterais, além da aquisição de resistência dos fungos tornam sua eficácia limitada. Nestas circunstâncias, a busca por novos fármacos é necessária. No presente trabalho, foi avaliada a atividade antifúngica de tamoxifeno, bisabolol, indinavir, didanosina, Olanzapina, UFMG, Labiocon 241, Solasodina, Labiocon 237, Clonazepam, Benzaldeído semicarbonado, Cardanol, AB 36 e três extratos naturais obtidos de Psychotria spp, Sclerolobium paniculatum, Hymenaea courbaril. A viabilidade celular e o mecanismo de ação do composto que apresentou ação antifúngica sobre 20 isolados de Cryptococcus neoformans formam determinados. A atividade antifúngica foi realizada utilizando-se o método microdiluição em caldo para leveduras. Para verificar do mecanismo de ação, foi realizada a citometria de fluxo com o marcador celular Iodeto de Propídio (PI), enquanto que o teste da viabilidade celular foi obtido usando-se o sal MTT. Dentre os compostos analisados, apenas o tamoxifeno e olanzapina apresentaram ação inibitória sobre as amostras fúngicas, em concentrações de 128 a 256 μg/mL. Pela análise por citometria de fluxo com o tamoxifeno não se verificou alteração da membrana celular de Cryptococcus neoformans ATCC 28957. Entretanto, o tamoxifeno inibiu o metabolismo do fungo em diferentes concentrações, variando de 4 a 1024 μg/mL verificado pelo teste da viabilidade celular.

Made available in DSpace on 2016-04-28T20:17:26Z (GMT). No. of bitstreams: 1 2006-Felipe Lopes Campos.pdf: 553352 bytes, checksum: e4d71e5d911a1305af8111fa2c150966 (MD5) Previous issue date: 2006-03-16 / The aim of this survey was the isolation of the Cryptococcus neoformans yeast from cats and dogs central nervous system in Rio de Janeiro City. Periodic collections were made from brain samples from April 2004 to July 2005 at the rabies diagnosis laboratory in the Jorge Vaitsman Veterinary Medicine Municipal Institute in Mangueira, RJ. According to the results, of the 201 clinical samples, we found four positives samples; one of these was positive for C. laurentii, three were positive for Cryptococcus neoformans; of which two were neoformans variety and one was gattii variety. Secondarily we compared with strains from the ICB-USP, previously isolated and identified as for urease and fenol-oxidase production in qualitative character, in the same way that the potential of virulence demonstrated to fosfolipase and protease production. Killer biotyping used in all isolates showed a predominance of biotype II to neoformans variety and biotype VIII to gattii variety. All isolates showed protease and fosfolipase production in varied degrees. lipase / Objetivando o isolamento da levedura Cryptococcus neoformans do sistema nervoso central de c?es e gatos no Munic?pio do Rio de Janeiro, procedeu-se ?s coletas peri?dicas, no per?odo de abril de 2004 a julho de 2005, de amostras de c?rebro, no Laborat?rio de Diagn?stico de Raiva do Instituto Municipal de Medicina Veterin?ria Jorge Vaitsman no bairro da Mangueira, Rio de Janeiro. Das 201 amostras obteve-se um total de quatro positivas para o g?nero; sendo uma de Cryptococcus laurentii e tr?s de C. neoformans, das quais duas revelaram-se pertencentes ? variedade neoformans e uma ? variedade gattii. Secundariamente, confrontamos estas com amostras obtidas junto ao ICB-USP, previamente isoladas e identificadas, quanto ao potencial de virul?ncia espelhado pela produ??o de fosfolipase e de protease. A biotipagem Killer empregada em todos os isolados demonstrou uma predomin?ncia do biotipo II para a variedade neoformans e biotipo VIII para a variedade gattii. Todos os isolados apresentaram-se produtores de protease e de fosfolipase, em diferentes graus.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Pablo Waldeck Goncalves de Souza.pdf: 2011389 bytes, checksum: 2d903c70e0f497dfa879d5f386e128c6 (MD5) Previous issue date: 2008-03-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The objective of this paper was determined the presence of Cryptococcus neoformans in cockroach (Periplaneta americana) collected in public places. The motivation for this research was based on the fact that this species of cockroach is synanthropic and vehicle of several pathogenic agents for man and animals. The insect were collected already dead and also collected alive using different traps, at all, were 150 samples. The research of the presence of the agents in the insects was made in Laboratory of Pathogenic and Environmental Yeasts, Department of Microbiology and Immunology Veterinary, Institute of Veterinary, UFRRJ, by the process of the exoskeleton and the internal content of then, sowing in cultivation medium containing dopamine. The confirmations of isolation were made by macromorfology and micromorfology test, realization of auxanographic method and complementally proofs. Was not obtained isolation of Cryptococcus neoformans of exoskeleton. Were obtained 5 isolated (3%), starting by the content of the insects that, after the serogrouping in CGB medium, show them self as belonging to AD serum group. Test show that 80% of the stumps are big producing of phospholipase and 100% big producing of protease. We considerate that the cockroaches of the species Periplaneta americana are,potentially, a vector for the studied yeast. / O objetivo deste trabalho foi determinar a presen?a de Cryptococcus neoformans em baratas (Periplaneta americana) recolhidas de ambientes p?blicos. A motiva??o para esta pesquisa deve-se ao fato desta esp?cie de barata ser sinantr?pica e veiculadora de v?rios agentes patog?nicos para o homem e animais. Os insetos foram recolhidos mortos e tamb?m capturados vivos por meio de duas diferentes armadilhas, totalizando 150 amostras. A pesquisa da presen?a do agente nos insetos foi feita no Laborat?rio de Leveduras Patog?nicas e Ambientais, Depto de Microbiologia e Imunologia Veterin?ria, Instituto de Veterin?ria, UFRRJ, por processamento do exoesqueleto e do conte?do interno dos mesmos, com semeadura em meio de cultivo contendo dopamina. As confirma??es de isolamento foram realizadas por meio de testes de macromorfologia, micromorfologia, realiza??o de auxanograma e provas complementares. N?o foi obtido isolamento de Cryptococcus neoformans de exoesqueleto. Foram obtidos 5 isolamentos (3%) a partir do conte?do interno dos insetos que, ap?s sorogrupagem em meio CGB revelaram-se como pertencentes ao sorogrupo AD. Testes evidenciaram que 80% das cepas s?o forte produtoras de fosfolipase e 100% forte produtoras de protease. Consideramos que as baratas da esp?cie Periplaneta americana s?o um potencial vetor para a levedura estudada.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-29T15:27:32Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoFenotipicaIsolados.pdf: 1039438 bytes, checksum: 3c6c87cbf938c2196c47c8af4a7fe355 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-02T16:49:44Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoFenotipicaIsolados.pdf: 1039438 bytes, checksum: 3c6c87cbf938c2196c47c8af4a7fe355 (MD5) / Made available in DSpace on 2017-10-02T16:49:44Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoFenotipicaIsolados.pdf: 1039438 bytes, checksum: 3c6c87cbf938c2196c47c8af4a7fe355 (MD5) Previous issue date: 2005-02-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cryptococcus neoformans foi descrito como patógeno humano em 1894. Desde então, estudos abrangendo aspectos da biologia, taxonomia, ecologia e epidemiologia do fungo , vêm se desenvolvendo com significante importância para a medicina e para o meio ambiente. O fungo foi recentemente classificado em duas espécies distintas, C. neoformans (sorotipo A, D e AD) e C. gattii (sorotipos B e C), cada uma com características morfológicas e genéticas bem definidas, assim como seus aspectos ecológicos e epidemiológicos. C. neoformans acomete principalmente indivíduos imunocomprometidos, especialmente aqueles infectados pelo HIV, enquanto C. gattii infecta principalmente indivíduos imunocompetentes, atingindo todas as faixas etárias. Os fatores principais que determinam a patogênese da criptococose são relacionados com a situação de defesa do hospedeiro, o tamanho do inóculo e a virulência da cepa, sendo este último determinado por quatro características principais: a cápsula polissacarídica, a produção de melanina, a diferença de tipos sexuados (MAT e MATa) e a termotolerância. O fungo possui ciclo de vida transitório entre formas leveduriformes haplóides e formas filamentosas dicarióticas. A reprodução sexuada envolve mistura de material genético de parentais que formam progênies contendo características genéticas de ambos. A forma sexuada pode ocorrer através de um processo conhecido como frutificação haplóide. As espécies são heterotálicas, com dois tipos sexuados MAT e MATa. Estudos de caracterização fenotípica, incluindo a identificação da espécie, sorotipo e tipo sexuado tem grande relevância para a condução de estudos mais aprofundados devido ao estreito relacionamento entre seus aspectos ecológicos variáveis e seu ciclo de desenvolvimento e multiplicação, especialmente para o Norte e Nordeste do Brasil, onde o sorotipo B destaca-se como principal agente de meningite fúngica em hospedeiros normais. Este trabalho utilizou a técnica do CGB para a análise metabólica (identificação da espécie), onde 28 cepas apresentaram resultado positivo, caracterizando C. gattii e duas apresentaram resultado negativo, caracterizando C. neoformans . Os resultados de sorotipagem, realizados através de teste de aglutinação revelaram 28 cepas do sorotipo B e duas do sorotipo A. A determinação do tipo sexuado foi realizada através de técnica de PCR e todas as amostras eram MAT. / Cryptococcus neoformans was described as human pathogen in 1894. Since then, studies about fungal aspects as biology, taxonomy, ecology and epidemiology have been developed with great importance to medicine and the environment. The fungus was recently classified in two separated species, C. neoformans (serotypes A, D and AD) and C. gattii (serotypes B and C) each one with well defined genetic and morphological features as well as ecology and epidemiology aspects. C. neoformans occurs mainly in immunocompromised hosts, especially those infected by HIV. In the other hand, C. gattii infects mainly immunocompetents individuals to all lifetimes. The main factors determining the cryptococcosys pathogenesis have relations with the host defenses state, the size of particle and strain virulence, this last for four main features: the polyssacharide capsule, the melanin production, the mating types (MAT and MATa) and thermotolerance. The fungus has a transitory life cycle between haploid yeast form and dykariotic filamentous forms. The sexual cycles includes a mixture of genetic parental genes that form progenies containing the genetic characteristics of both. The asexual cycle occurs through a process know as haploid fruiting. The species are heterothallic with two mating types MAT and MATa. Phenotypic characterization studies, including the species identification, serotype and mating type have a great importance for conductions of profound studies because of the close relation between the ecological variants features and its development cycle and multiplication, especially in Brazilian regions North and Northeast where serotype B is the most prevalent cause of meningitis in ordinary hosts. This work used the CGB techniques for methabolic analysis (species identification), where 28 strains were positive, characterizing C. gattii and two were negative, characterizing C. neoformans. The serotyping results, make among agglutination test reveals 28 strains of serotype B and two of serotype A. The mating type was realized by PCR technique and all strains were MAT.

Publicación a texto completo no autorizada por el autor / Determina los genotipos y la sensibilidad a los antifúngicos de cepas de Cryptococcus neoformans, provenientes de aislamientos clínicos y ambientales efectuados en la ciudad de Lima. El estudio es descriptivo. La población de microorganismos está constituida por cepas de Cryptococcus neoformans provenientes de aislamientos clínicos y ambientales. Para determinar el genotipo se utiliza la técnica del análisis del polimorfismo en longitud de fragmentos amplificados (AFLP), según instrucciones del fabricante. Para determinar la sensibilidad se utiliza un panel de 3 compuestos antifúngicos: anfotericina B, fluconazol y voriconazol. Mediante la técnica del AFLP, se determina que el 50% de las cepas son del genotipo AFLP1 (16) y el 40,6% del genotipo AFLP1B (13), ambos correspondieron a C. neoformans var. grubii; asimismo, el 6,3% resulta ser del genotipo AFLP2 (2) (C. neoformans var. neoformans) y el 3,1% es del genotipo AFLP3 (1), es decir, un híbrido entre las dos variedades de C. neoformans. Los perfiles de sensibilidad antifúngica para la anfotericina B, fluconazol y voriconazol indicaron que las 32 cepas de C. neoformans son sensibles a los 3 compuestos antifúngicos. Se observa que C. neoformans var. grubii (AFLP1 y AFLP1B) y C. neoformans var. neoformans (AFLP2) son las únicas variedades de Cryptococcus implicadas en infecciones en humanos. Asimismo, se demuestra que todas las cepas son sensibles a los antifúngicos probados, de acuerdo a lo reportado en la literatura internacional. / Tesis