Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ceilândia, Programa de Pós-Graduação em Ciências e Tecnologias em Saúde, 2014. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2014-10-14T21:29:48Z No. of bitstreams: 1 2014_AmandaLiraNogueiraBarros.pdf: 3370997 bytes, checksum: 85117556d8ae68f1292b6073cbb5fc91 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2014-10-15T13:24:45Z (GMT) No. of bitstreams: 1 2014_AmandaLiraNogueiraBarros.pdf: 3370997 bytes, checksum: 85117556d8ae68f1292b6073cbb5fc91 (MD5) / Made available in DSpace on 2014-10-15T13:24:45Z (GMT). No. of bitstreams: 1 2014_AmandaLiraNogueiraBarros.pdf: 3370997 bytes, checksum: 85117556d8ae68f1292b6073cbb5fc91 (MD5) / As proteínas Velvet compreendem quatro membros altamente conservados entre ascomicetos e basidiomicetos, os quais compartilham o domínio Velvet. Eles regulam diferentes vias de sinalização em resposta a estímulos ambientais e também coordenam o metabolismo secundário e diferenciação assexual e sexual em diferentes espécies de fungos. Recentemente, as proteínas Velvet foram implicadas na virulência de alguns agentes patogênicos de plantas. Buscou-se avaliar o papel das proteínas Velvet no fungo patogênico humano Cryptococcus neoformans, causador da criptococose, importante micose sistêmica e a terceira doença mais prevalente em indivíduos portadores do vírus HIV. Entre as micoses sistêmicas, ela é classificada como tendo a maior incidência em pacientes imunocomprometidos. A infecção por C.neoformans ocorre pela inalação de células infecciosas e é considerada uma infecção pulmonar primária, o que pode levar a uma infecção disseminada e meningoencefalite. O objetivo geral do trabalho foi avaliar o papel do gene VELB na patogenidade e morfogênese de C. neoformans. O primeiro passo realizado no estudo foi a deleção do gene VELB em Cryptococcus neoformans. O cassete foi transformado em células leveduriformes haploides de C. neoformans diretamente através de biobalística. Confirmou-se a deleção por PCR e Southern Blot. Após confirmação, os transformantes foram submetidos a diferentes condições de crescimento para determinação de seus fenótipos e virulência. Foram realizados testes de estresse osmótico (NaCl), estresse na parede celular (CongoRed, SDS, cafeína) e estresse oxidativo com peróxido de hidrogênio. Foram avaliados os fatores de virulência de C. neoformans como fosfolipase, urease, melanina e cápsula. Os testes fenotípicos não apresentaram alterações entre a linhagem e mutante indicando que o gene VELB parece não ter nenhum envolvimento em virulência e resposta a agentes estressores. Para avaliação da capacidade de acasalamento foram realizados co-cultivos no escuro em diferentes meios de cultura (Filament, SLAD e MS), além disto foi testada a capacidade de fusão dos mutantes. Estes testes demonstraram que a deleção de VELB bloqueia a produção de hifas no acasalamento entre mutantes e que estes apresentam também defeito no processo de fusão celular. Estes resultados gerados neste trabalho sobre o papel de VELB corroboram os dados anteriormente obtidos pelo nosso grupo de pesquisa de que os membros da família velvet estão diretamente envolvidos na regulação do ciclo sexual de C. neoformans. _________________________________________________________________________________ ABSTRACT / The Velvet proteins consist of four highly conserved members in ascomycetes and basidiomycetes, sharing the Velvet domain. They regulate different signaling ways in response to environmental stimulation and also coordinate secondary metabolism and asexual to sexual differentiation in different species of fungi. Recently, Velvet proteins have been implicated in the virulence of some pathogenic agents of plants. It was evaluated the role of Velvet proteins in the human pathogenic fungus Cryptococcus neoformans that causes cryptococcosis, an important systemic mycosis and the third most prevalent disease in HIV positive individuals. Compared to other systemic mycoses, it is classified as having the highest incidence in immunocompromised patients. C.neoformans infection is by inhalation of infectious cells and is considered a primary pulmonary infection, which can lead to widespread infection and meningoencephalitis. The overall objective of this study was to evaluate the role of the VELB gene in morphogenesis and pathogenicity of C. neoformans. The first step was performed at study deletion of the gene VELB in Cryptococcus neoformans. The cassette was transformed into haploid yeast cells of C. neoformans directly through biolistic. The deletion was confirmed by PCR and Southern blot. After confirmation, the transformants were submitted to different growth conditions to determine their phenotypes and virulence. Tests of osmotic stress (KCl, NaCl, sorbitol), stress in the cell wall (Congo Red, Calcofluor, SDS, caffeine) and oxidative stress with hydrogen peroxide were performed. Virulence factors of C. neoformans as phospholipase, urease, melanin and capsule were evaluated. Phenotypic tests showed no change between the strain and mutant indicating that the VELB gene seems to have no involvement in virulence and response to stressors. To evaluate the ability of mating co-cultures were performed in the dark in different culture media (Filament, SLAD and MS), in addition it was tested the ability of fusion of mutants. These tests showed that the deletion of VELB blocks the production of hyphae in mating and that these mutants also exhibit defective cell fusion process. These results generated in this paper about the VELB function corroborate data previously 13 obtained by our research group that the members of the velvet family are directly involved in the regulation of the sexual cycle of C. neoformans.

Dissertação (mestrado)—Universidade de Brasília, Pós-Graduação em Patologia Molecular, 2014. / Submitted by Raquel Viana (raquelviana@bce.unb.br) on 2014-10-17T20:16:57Z No. of bitstreams: 1 2014_ThaísAmandadePinhoSilva.pdf: 4485973 bytes, checksum: 3c18aced7d776760ef9a8f71cdac3533 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2014-10-17T20:20:32Z (GMT) No. of bitstreams: 1 2014_ThaísAmandadePinhoSilva.pdf: 4485973 bytes, checksum: 3c18aced7d776760ef9a8f71cdac3533 (MD5) / Made available in DSpace on 2014-10-17T20:20:32Z (GMT). No. of bitstreams: 1 2014_ThaísAmandadePinhoSilva.pdf: 4485973 bytes, checksum: 3c18aced7d776760ef9a8f71cdac3533 (MD5) / Granulócitos contribuem de forma significativa na resposta imunológica contra infecções. Dentre eles, os mais presentes na circulação sanguínea são os neutrófilos e eosinófilos. Cryptococcus neoformans é uma levedura patogênica adquirida pela inalação e pode causar desde pneumonia até meningoencefalite em indivíduos imunocomprometidos. Alguns fatores de virulência do fungo tornam possível a infecção, dentre eles o principal é a presença de uma cápsula polissacarídica. Durante a infecção, ocorre um recrutamento de neutrófilos, eosinófilos, macrófagos entre outras células. Esses fagócitos podem matar ou inibir o crescimento do fungo ao ativarem uma resposta inflamatória. Um dos marcadores de ativação celular durante a resposta inflamatória são os corpúsculos lipídicos (CLs). Estas organelas multifuncionais, são locais de síntese de importantes mediadores inflamatórios. Durante a resposta inflamatória, ocorre uma importante produção de eicosanóides, como os leucotrienos e as prostaglandinas. Dentre as prostaglandinas, a prostaglandina D2 é um dos principais mediadores lipídicos que medeia a resposta inflamatória de granulócitos. assim, o objetivo desse trabalho foi investigar os mecanismos inflamatórios envolvidos na ativação do metabolismo lipídico de granulócitos por C. neoformans, analisando o papel da cápsula nesse processo. Demonstramos que C. neoformans acapsular Cap67 induz o aumento do tamanho e granulosidade celular em granulócitos. Essa modificação não foi causada pelos componentes da cápsula ou lipídeos totais do fungo. A infecção por Cap67 também induziu o aumento de CLs, porém os lipídeos do fungo contribuíram para este processo. Esse aumento de CLs foi dependente da enzima COX (mediadora de prostaglandinas) e independente de 5-LO e 15-LO (mediadoras de leucotrienos). A infecção pelo sorotipo D B3501 induziu um aumento da expressão de PGD2 em granulócitos o qual foi revertido pelo mutante acapsular Cap67. A expressão do receptor nuclear PPARgamma foi aumentada e o fator de transcrição NFkB não foi translocado durante a infecção por Cap67. Os receptores de PGD2, DP1 e CRTH2, não interferem na biogênese de CLs, mas modulam a capacidade fagocítica e de eliminação dos fungos pelos granulócitos. Todos esses resultados mostram a importância da via das PG na biogênese de CL, na fagocitose e na eliminação de C. neoformans por granulócitos. Nesse trabalho foi proposta uma nova via de escape do patógeno C. neoformans. __________________________________________________________________________ ABSTRACT / Granulocytes significantly contribute to immune responses against infections. Among ganulocytes, the most common cell type in bloodstream is neutrophil and eosinophil. Cryptococcus neoformans is a pathogenic yeast acquired by inhalation and may cause pneumonia and even meningoencephalitis in immunocompromised individuals. Some fungal virulence factors, such as presence of polysaccharide capsule, make infection more feasible to occur. During infection, occurs a recruitment of neutrophils, eosinophils, and macrophages, among other cells. These phagocytes may kill or inhibit the fungal growth, activating an inflammatory response. One of the markers of cellular activation during the inflammatory response is an organelle called lipid body (LB). These multifunctional organelles are sites of important inflammatory mediators synthesis. During an inflammatory response, there is substantial production of eicosanoids such as leukotrienes and prostaglandins. Among the prostaglandins, Prostaglandin D2 is the key lipid mediator that mediates inflammatory response of granulocytes. Thus, the aim of this study was to investigate the inflammatory mechanisms involved in activation of lipid metabolism of granulocytes caused by C. neoformans infection, analyzing the role of the fungal capsule in this process. Here we demonstrate that C. neoformans acapsular Cap67 induced the increase of the cell size and granulosity in granulocytes. The fungal lipid extract or capsule components did not cause this modification. The infection by Cap67 also induced the increase of LBs, however the fungal total lipids may contribute to this process. This increase of LBs was dependent on COX (prostaglandin mediator) and independent on 5-LO and 15-LO (leukotriene mediator). The infection with serotype D B3501 induced an increased expression of PGD2 in granulocytes, which was reversed by the acapsular mutante Cap67. The expression of the nuclear receptor PPARgamma increased and the transcription factor NFkB did not translocated during the infection by Cap67. The PGD2 receptors, DP1 and CRTH2, did not interfere in the biogenesis of LBs, but modulated the phagocytic capacity and killing of fungi by granulocytes. All these results show the importance of the PG signaling pathway in the biogenesis of LBs, the phagocytosis and the killing of C. neoformans by granulocytes. In this work we have proposed a new escape pathway for the pathogen C. neoformans.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Cryptococcus neoformans species Complex comprises the main agents of cryptococcosis, an important systemic mycosis of nature and opportunistic. These species are capable of expressing multiple virulence factors that contribute to the development of resistance to antifungal therapeutic use. Research seeking new effective therapy measures are necessary. Heat shock proteins (Hsp) have been shown to be a promising molecular target for medical treatment of various diseases such as various forms of cancer and atherosclerosis. Studies have demonstrated that the expression of these chaperonins is also required for the maintenance of cellular homeostasis in different fungal pathogens. From this perspective, this study aimed to investigate the effect of blocking the protein Hsp90 front of sensitivity to antifungal agents in vitro planktonic cells and sessile, production of virulence factors in vitro and in vivo virulence in Cryptococcus neoformans species Complex. Therefore, the action of yeast Hsp90 protein was blocked with radicicol drug - an antibiotic capable of inhibiting the ATPase activity of Hsp90 - and then the cultures were evaluated for in vitro susceptibility to antifungal agents for therapeutic use in planktonic cells and associated in biofilms. We also assessed the effect of inactivation of Hsp90 on the production of virulence factors capsule, melanin and proteases. In addition, the in vivo virulence was investigated in a model of experimental infection using the nematode Caenorhabditis elegans. The results revealed that radicicol inhibited the growth of planktonic cells Cryptococcus spp. (n=12) with values of minimum inhibitory concentration (MIC) ranging from 0.5 to 2 &#956;g/mL. We observed synergy between pharmacological and radicicol azoles. Furthermore, blockade of Hsp90 potentiated the effect of in vitro antifungal agents, particularly for the fluconazole and itraconazole, which MIC values were reduced by up to 64 times the face of isolated cells (n=12). Although the blockade of Hsp90 by isolated radicicol was not able to inhibit biofilm formation and biofilm formed of Cryptococcus spp. (n=12) (p>0.05), the inactivation of Hsp90 potentiated the effect of amphotericin B, azoles, especially fluconazole, when tested in combination with radicicol (p<0.05). Moreover, blockade of Hsp90 significantly reduced the volume of capsular Cryptococcus spp. (n=4) (p<0.05). However, no difference was observed in production of melanin by radicicol treated cells (n=4) (p>0.05) as well as the production of protease by strains of Cryptococcus spp. (n=4) (p>0.05). By contrast, inactivation of Hsp90 decreased virulence Cryptococcus spp. in vitro and in vivo in C. elegans (p<0.0001) as well as maximize the effect of fluconazole in vitro and in vivo. Therefore, Hsp90 is an important target for the development of antifungal strategies. However, further studies are needed to better understand the role of Hsp90 in fungal pathogens of the genus Cryptococcus spp. / O Complexo Cryptococcus neoformans/C. gattii compreende os principais agentes da criptococose, uma importante micose de natureza sistÃmica e carÃter oportunista. Essas espÃcies sÃo capazes de expressar mÃltiplos fatores de virulÃncia que contribuem para o surgimento de resistÃncia aos antifÃngicos de uso terapÃutico. Pesquisas que buscam novas medidas eficazes de terapia sÃo necessÃrias. ProteÃnas do choque tÃrmico (Hsp) tÃm se demonstrado um alvo molecular promissor em Ãrea mÃdica para o tratamento de diversas doenÃas, tais como diferentes tipos de cÃncer e aterosclerose. Estudos demonstram que a expressÃo dessas chaperoninas tambÃm à necessÃria para a manutenÃÃo da homeostase celular em diferentes patÃgenos fÃngicos. Nessa perspectiva, o presente estudo teve como objetivo investigar o efeito do bloqueio das proteÃnas Hsp90 frente à sensibilidade a antifÃngicos in vitro em cÃlulas planctÃnicas e sÃsseis, produÃÃo de fatores de virulÃncia in vitro e virulÃncia in vivo no Complexo Cryptococcus neoformans/C. gattii. Para tanto, a aÃÃo das proteÃnas Hsp90 fÃngicas foi bloqueada com o fÃrmaco radicicol â um antibiÃtico capaz de inibir a atividade ATPÃsica das Hsp90 â e, em seguida, as culturas foram avaliadas quanto à sensibilidade in vitro a antifÃngicos de uso terapÃutico em cÃlulas planctÃnicas e associadas em biofilmes. TambÃm foi avaliado o efeito da inativaÃÃo das Hsp90 sobre a produÃÃo dos fatores de virulÃncia cÃpsula, melanina e proteases. AlÃm disso, a virulÃncia in vivo foi investigada em modelo de infecÃÃo experimental utilizando o nematÃdeo Caenorhabditis elegans. Os resultados revelaram que o radicicol inibiu o crescimento das cÃlulas planctÃnicas de Cryptococcus spp. (n=12), com valores de concentraÃÃo inibitÃria mÃnima (CIM) que variaram de 0,5 â 2 &#956;g/mL. Observou-se sinergismo farmacolÃgico entre o radicicol e os azÃlicos. Ademais, o bloqueio das Hsp90 potencializou o efeito dos antifÃngicos in vitro, sobretudo para o fluconazol e o itraconazol, os quais tiveram os valores de CIM reduzidos em atà 64 vezes frente Ãs cÃlulas isoladas (n=12). Embora o bloqueio das Hsp90 pelo radicicol isolado nÃo tenha sido capaz de inibir a formaÃÃo de biofilme e biofilme formado de Cryptococcus spp. (n=12) (p>0,05), a inativaÃÃo das Hsp90 potencializou o efeito da anfotericina B e derivados azÃlicos, sobretudo o fluconazol, quando testados em combinaÃÃo com o radicicol (p<0,05). AlÃm disso, o bloqueio das Hsp90 reduziu significativamente o volume capsular de Cryptococcus spp. (n=4) (p<0,05). PorÃm, nÃo observou-se diferenÃa na produÃÃo de melanina por cÃlulas tratadas com o radicicol (n=4) (p>0,05) assim como na produÃÃo de proteases pelas cepas de Cryptococcus spp. (n=4) (p>0,05). Em contrapartida, a inativaÃÃo das Hsp90 diminuiu a virulÃncia de Cryptococcus spp. in vitro e in vivo em C. elegans (p<0,0001) alÃm de potencializar o efeito do fluconazol in vitro e in vivo. Portanto, as Hsp90 sÃo um importante alvo para o desenvolvimento de estratÃgias antifÃngicas. Todavia, estudos mais aprofundados sÃo necessÃrios para melhor compreender o papel das Hsp90 nos patÃgenos fÃngicos do gÃnero Cryptococcus spp.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Tendo em vista que algumas propriedades terapÃuticas da curcumina jà sÃo bem descritas, dessa forma o presente estudo aponta para um efeito antifÃngico da curcumina frente a cepas de leveduras patogÃnicas resistentes ao fluconazol, cepas de Candida spp. e Cryptococcus neoformans. Todas as cepas estudadas foram inibidas pela curcumina, com diferentes graus de inibiÃÃo entre as espÃcies com CIMs variando entre 8-64 &#956;g/mL. ApÃs a exposiÃÃo das cepas de C. albicans a curcumina, foi observado uma diminuiÃÃo no nÃmero de cÃlulas viÃveis, indicando assim danos Ãs membranas celulares, com possÃvel comprometimento de suas funÃÃes. Os nossos resultados revelaram que apÃs o tratamento com a curcumina, constatamos uma intensificaÃÃo na marcaÃÃo com PI (Iodeto de propÃdio), indicando alteraÃÃes nas membranas celulares. No presente trabalho, a funÃÃo mitocondrial das cÃlulas de C. albicans parece ter sido afetada apÃs exposiÃÃo à curcumina. O colapso no &#916;&#968;m (Potencial Transmembrana Mitocondrial) pode conduzir a aberturas de poros transientes nas membranas mitocondriais. Em relaÃÃo aos diversos mecanismos de aÃÃo descritos para a curcumina, existem evidÃncias de que o DNA à um dos alvos celulares desta molÃcula. Nossos dados sugerem que apÃs a exposiÃÃo à curcumina, as cÃlulas de C. albicans, apresentaram quebras totais nas cadeias de DNA. Em conclusÃo, o composto curcumina apresenta atividade antifÃngica in vitro contra cepas de Candida spp. e Cryptococcus neo-formans resistentes ao fluconazol. AlÃm de promover danos ao DNA e externalizaÃÃo da fosfatidilserina, o respectivo composto parecem atuar em sÃtios especÃficos prÃximos a mitocÃndria das cÃlulas de C. albicans, levando à morte por apoptose. / Considering that some therapeutic properties of curcumin are well described, so the present study points to an antifungal effect of curcumin against strains of pathogenic yeasts resistant to fluconazole, strains of Candida spp. and Cryptococcus neoformans. All strains studied were inhibited by curcumin with different degrees of inhibition among species with MICs ranging from 8-64 mg / ml. After exposure strains of C. albicans, curcumin a decrease in the number of viable cells was observed, thus indicating damage to cell membranes, with possible compromise of their functions. Our results showed that after treatment with curcumin, found in more intense staining with PI (Propidium iodide), indicating changes in cell membranes. In this study, the mitochondrial function of C. albicans cells appears to have been affected after exposure to curcumin. The collapse in &#916;&#968;m (Mitochondrial Transmembrane Potential) can lead to openings of transient pores in the mitochondrial membranes. In relation to the different mechanisms of action described for curcumin, there is evidence that DNA is one of the celular targets of this molecule. Our data suggest that after exposure to curcumin, cells of C. albicans showed total breaks in DNA strands. In conclusion, the compound curcumin has antifungal activity in vitro against strains of Candida spp. and Cryptococcus neoformans resistant to fluconazole. In addition to promoting DNA damage and externalization of phosphatidylserine, the respective compound seem to act at specific sites near mitochondria of C. albicans cells, leading to death by apoptosis.

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cryptococcus neoformans is one yeast opportunist that it unchains meningitis in imunocompetentes patients in such a way as in patients This work had as objective to study the varieties (neoformans and gattii) of isolated C.neoformans in the State of Sergipe from the liquor liquid of patients with infectious miningitis. Also had as objective to determine the sensitivity of C. neoformans variety neoformans and variety gattii, isolated of the líquor of patients with infectious meningite in the State of Sergipe, through the microdilution method (NCCLS) and to determine the sexual type - mating type of isolated physicians of Cryptococcus neoformans through the Reaction in Chain of DNA Polimerase (PCR) in Sergipe. Samples of the LCR had been used that had given entered in the IPH/LACEN/SE in the period of 2001 the 2004. Direct microscopy through the coloration with Nanquim Ink was carried through. An aliquot one of the líquor (0,1mL) was transferred to pipes contends Agar Sabouraud with clorafenicol and incubado in ambient temperature 25ºC and 37ºC. Tests for presumptive, fenotípica identification had been carried through in half Níger and of the varieties in half CGB. In the analyzed period, only one sample presented fenotípicas characteristics in half Níger differentiated of the others. Of the total of 15 samples analyzed in half CGB, 02 samples (13,33%) if had presented positive, or either, had been identified as variety gattii, being one of them isolated one of soronegativo patient for HIV (patient not aidético). The others 13 samples (86,67%) had presented negative CGB, or either, they had been identified as variety neoformans. The majority of the isolated samples of Cryptococcus neoformans of the líquor of patients with infectious meningite is of the variety neoformans in Sergipe. For the accomplishment of the sensitivity test variety had been used two samples of C. neoformans var. gattii and 11 of C. neoformans listed variety neoformans above. For control of the tests standard ATCC 32608 was used. After that suspension of fúngicas cells. The following antifungal had been used: Anphotericin B, Fluconazol, Cetoconazol, Miconazol, Itraconazol, 5- Fluorocitosina and Terbinafrina. The fungal suspension was adjusted in a final concentration of 0,5x10³ for 2,5x10³ UFC in the half RPMI. The experiment was carried through in microtitulação plates. After the inoculation, the microdilution plates had been incubadas 37ºC and deal in 24, 48 and 72 hours. The end point of the CIM was determined in the readings of 48 and 72 hours. The isolated physicians of C. neoformans had presented high values of CIM for the antifungal agents tested, what he mainly symbolizes one high tax of resistance Anfotericina B and to the Fluconazol, given that he can be correlated with the clinical data. For analysis of mating type sexual of C. neoformans from the líquor of patients with infectious meningite in Sergipe during the period of 2001 and 2004 had been used 13 samples. The related laboratoriais procedures to molecular biology (extration of DNA and accomplishment of the PCR) had followed as Chatuverdi reference et al. (2000). All the samples of C.neoformans tested, as much that pertaining to the variety neoformans as gattii are matting type, considered of bigger virulence. / Cryptococcus neoformans é um fungo oportunista que desencadeia meningite tanto em pacientes imunocompetentes como em pacientes hígidos Este trabalho teve como objetivo estudar as variedades (neoformans e gattii) de Cryptococcus neoformans isolados no Estado de Sergipe a partir do LCR de pacientes com meningite infecciosa. Também teve como objetivo determinar a sensibilidade de C. neoformans variedade neoformans e variedade gattii, isolados do líquor de pacientes com meningite infecciosa no Estado de Sergipe, através do método de microdiluição (NCCLS) e determinar o mating type de isolados clínicos de Cryptococcus neoformans através da Reação em Cadeia da DNA Polimerase em Sergipe. Foram utilizadas amostras do LCR que deram entrada no IPH/LACEN/SE no período de 2001 a 2004. Foi realizada microscopia direta através da coloração com Tinta Nanquim. Uma alíquota do líquor foi transferida para tubos contendo Agar Sabouraud com clorafenicol e incubado em temperatura ambiente 25ºC e a 37ºC.Foram realizadas provas para identificação presuntiva, fenotípica em meio Níger e das variedades em meio CGB. No período analisado, apenas uma amostra apresentou características fenotípicas em meio Níger diferenciada das outras. Do total de 15 amostras analisadas em meio CGB, 02 amostras (13,33%) se apresentaram positiva, ou seja, foram identificadas como variedade gattii, sendo uma delas isolada de paciente soronegativo para HIV (paciente não aidético). As outras 13 amostras (86,67%) apresentaram CGB negativo, ou seja, foram identificadas como variedade neoformans. A maioria das amostras de Cryptococcus neoformans isolados do líquor de pacientes com meningite infecciosa são da variedade neoformans em Sergipe. Para a realização do teste de sensibilidade foram utilizadas duas amostras de C. neoformans variedade gattii e 11 de C. neoformans variedade neoformans listadas acima. Para controle dos testes foi utilizada a cepa padrão ATCC 32608. Em seguida foi realizada a preparação do inóculo. Foram utilizados os seguintes antifúngicos: Anfotericina B, Fluconazol, Cetoconazol, Miconazol, Itraconazol, 5- Fluorocitosina e Terbinafrina. A suspensão fúngica foi ajustada numa concentração final de 0,5x10³ para 2,5x10³ UFC no meio RPMI. O experimento foi realizado em placas de microtitulação. Após a inoculação, as placas de microdiluição foram incubadas a 37ºC e lida em 24, 48 e 72 horas. O ponto final da CIM foi determinado nas leituras de 48 e 72 horas. Os isolados clínicos de C. neoformans apresentaram valores altos de CIM para os agentes antifúngicos testados, o que simboliza uma alta taxa de resistência principalmente a Anfotericina B e ao Fluconazol, dado que pode ser correlacionado com os dados clínicos. Para análise do mating type sexual foram utilizadas 13 amostras de C. neoformans a partir do líquor de pacientes com meningite infecciosa em Sergipe durante o período de 2001 e 2004. Os procedimentos laboratoriais relacionados à biologia molecular seguiram como referência Chatuverdi et al. (2000). Todas as amostras de C. neoformans tanto aquelas pertencentes à variedade neoformans como gattii são matting type a, considerado de maior virulência.

Orientador: Márcia Marinho / Banca: Luzia Helena de Queiroz / Banca: Simone Baldini Lucheis / Resumo: Foram acompanhados 310 parturições de receptoras, com embriões fertilizados in vivo (FV) e in vitro (FIV) da raça Nelore, para determinar os índices de partos distócicos em receptoras meio sangue das raças Braford/Nelore, Hereford/Nelore, Simental/Nelore, Red Angus/Nelore, Nelore e anelorados, com idade entre três e seis anos, assim como avaliar o estado de saúde dos bezerros obtidos por intermédio de transferência de embriões, resultantes das técnicas de fertilização in vivo (FV) ou fertilização in vitro (FIV). Observou-se a ocorrência de 38 partos distócicos (38/310) e oito abortos (8/310), dos quais cinco fetos eram oriundos da técnica de fertilização in vivo (FV) e três da técnica de fertilização in vitro (FIV). A taxa de mortalidade em animais neonatos foi de 3,4% (10/290). Os bezerros nascidos de partos distócicos mais duradouros, entre quatro e seis horas, apresentaram os maiores valores médios de freqüência respiratória. A temperatura retal foi mais elevada em animais nascidos de partos laboriosos do que naqueles nascidos de partos normais. / Abstract: Cryptococcosis is an opportunistic fungal infection caused by Cryptococcus yeasts, especially C. neoformans and Cryptococcus gattii, which may be associated with immunosuppression or not. The fungus is found in substrates of animal and vegetable origin, and infection occurs through inhalation and seedlings present in the environment. The present study aimed to investigate the existence of microfocus Cryptococcus sp. from the environmental samples Araçatuba city, São Paulo, featuring new niches, by decoupling the direct relationship between fungus and host. in order to minimize the risk of contamination of man and animals, understanding the ecoepidemiology of Cryptococcus. Samples 50 originated from hollow logs and trees (Cassia sp., Ficus sp. and Caesalpinea peltophorides) of ten point the urban perimeter points. The samples were immediately sent to the Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine Araçatuba - Unesp where they were processed and plated on Petri dishes containing agar seed Niger and Sabouraud dextrose agar with chloramphenicol, incubated at 30ºC for a period not less than 5 days. Colonies yeast growth were analyzed considering the macro and micro morphological compatible gender Cryptococcus, and later peaked in Niger agar, grown in time and above temperature and subjected to biochemical tests: urease production, thermotolerance at 37°C and quimiotipagem in CGB agar (L- Canavanine, Glinica bromothymol blue). The results showed that 17 (34%) cultures were positive for Cryptococcus, and 9 (18% ) were Cryptococcus gattii and 8 (16%) for Cryptococcus neoformans. Other yeast correlated as Rhodotorula sp. and Candida sp. were isolated. For these reasons, we conclude that the infectious propagules of Cryptococcus are dispersed in nature constitute environmental microfocus, not necessarily being bound to a single host / Mestre

Orientador: Liliana Scorzoni / Banca: Luciane Dias de Oliveira / Banca: Haroldo Cesar de Oliveira / Resumo: Cryptococcus neoformans são leveduras que acometem principalmente indivíduos imunocomprometidos, podendo causar a meningoencefalite dependendo do estado imunológico do hospedeiro. Os tratamentos convencionais têm enfrentado grandes desafios. Dessa forma, o objetivo desse estudo foi avaliar os efeitos antifúngicos dos fármacos cloridrato de verapamil (CV), cloridrato de fluoxetina (CF) e cloridrato de paroxetina (CP) isolados e combinados com anfotericina B (AmB) contra C. neoformans. Foram determinados os valores de Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM) de acordo com a técnica de microdiluição em caldo proposta pelo Comitê Europeu de Teste de Susceptibilidade Antimicrobiana (EUCAST). Posteriormente, foi avaliada a atividade sinérgica dos fármacos combinados com AmB (EUCAST). Os efeitos das CIMs e das concentrações sinérgicas selecionadas foram avaliados em biofilmes, quantificando a biomassa por cristal violeta e sua viabilidade por contagem de colônias (UFC/mL). Além disso, foram analisados os efeitos dos mesmos na cápsula induzida desta levedura. Os ensaios in vivo foram realizados em Galleria mellonella avaliando a toxicidade dos compostos e a eficácia por análise de curva de sobrevivência e também o efeito em concentração de hemócitos. Os fármacos CV, CF e CP apresentaram valores de CIM de 113, 9,6 e 41µg/mL, respectivamente, e quando combinados com AmB resultaram em vinte concentrações sinérgicas, e uma concentração de cada combinação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cryptococcus neoformans are yeasts that mainly affect immunocompromised individuals, and may cause meningoencephalitis depending on the immunological state of the host. Conventional treatments have faced major challenges. Verapamil hydrochloride (CV), fluoxetine hydrochloride (CF) and paroxetine hydrochloride (CP) were isolated and combined with amphotericin B (AmB) against C. neoformans. Minimum Inhibitory Concentration (MIC) and minimum Fungicidal Concentration (CFM) values were determined according to the broth microdilution technique proposed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST). Subsequently, the synergistic activity of drugs combined with AmB (EUCAST) was evaluated. The effects of MICs and selected synergistic concentrations were evaluated in biofilms, quantifying the biomass by violet crystal and its viability by colony-forming unit analysis (CFU / mL). In addition, the effects of the same in the induced capsule of this yeast were evaluated. In vivo assays were performed in Galleria mellonella evaluating the toxicity of the compounds and the efficacy by analysis of survival curve and also the effect on hemocyte concentration. The CV, CF and CP drugs had MIC values of 113, 9.6 and 41 μg / mL, respectively, and when combined with AmB resulted in twenty synergistic concentrations, and a concentration of each combination was chosen for the following trials. CV reduced biofilm biomass to 38%, while CF and CP reduced biomass to 22-30% w... (Complete abstract click electronic access below) / Mestre

Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“