[Typescript] Includes bibliography.

Thirty-seven samples were taken from the water, step access and air/water interface surfaces of 10 commercial whirlpools in Delaware and Madison Counties, Indiana between September and March of 1986-1988. Dermatophytes were isolated from 19 of the samples, representing 51%. At least three species of the yeast genus Candid. were isolated from 13 (35%) of these samples. Fifty-eight percent of all dermatophytes isolated were found in male-only whirlpools, as were nearly 62% of all Candida species. Isolated dermatophytes included Trichophyton rubrum, T. menta,grophytes, T. tonsurans, T. verrucosum, Epidermophyton floccosum, Microsporum nanum, and one Trichophvton species. Whirlpools were grouped based on similarity of fungi isolated from each facility. Male whirlpools sorted into one group, except for one that clustered with female facilities. Coliform bacteria were found in 43% of all samples, and blue-green fluorescent pseudomonads were found in 54% of all samples. Most dermatophytes were isolated from the step access surface of the facilities tested, and more Candida species were isolated from the water than from either surface tested. / Department of Biology

To improve fungal identification using a single DNA sequence, I introduce the Barcode Similarity Group (BSG) defined as a cluster of sequences that share greater than or equal to a threshold amount of genetic similarity with each other. As a test case, I created 393 BSGs from 2463 Cortinarius ITS sequences using a 94% similarity cut-off value in DOTUR. Some BSGs may contain multiple species. The BSG database was used to label environmental sequences, find misidentified or mislabeled sequences, and find potential cryptic species and novel species. Expert taxonomists will be needed to perform detailed morphological and phylogenetic studies to identify the individual species within each BSG. The main advantage of using BSGs is that it clusters together sequences using total genetic relatedness and does not rely on any taxonomy for identification. A website was created where the RDP Classifier is used to classify a query sequence into a BSG.

To improve fungal identification using a single DNA sequence, I introduce the Barcode Similarity Group (BSG) defined as a cluster of sequences that share greater than or equal to a threshold amount of genetic similarity with each other. As a test case, I created 393 BSGs from 2463 Cortinarius ITS sequences using a 94% similarity cut-off value in DOTUR. Some BSGs may contain multiple species. The BSG database was used to label environmental sequences, find misidentified or mislabeled sequences, and find potential cryptic species and novel species. Expert taxonomists will be needed to perform detailed morphological and phylogenetic studies to identify the individual species within each BSG. The main advantage of using BSGs is that it clusters together sequences using total genetic relatedness and does not rely on any taxonomy for identification. A website was created where the RDP Classifier is used to classify a query sequence into a BSG.

Thesis (Ph. D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 253-281).

- / -

A grande diversidade de organismos que hoje encontramos em nosso planeta se deve à adaptação às diferentes condições ambientais de cada nicho ecológico existente e à resposta adaptativa originária das mudanças dessas condições. Pode-se considerar que a etapa inicial do processo de adaptação seja a reprogramação da expressão gênica dum organismo como resposta imediata a uma nova condição ambiental. De fato parte do genoma de todos os organismos é dedicada à codificação de proteínas relacionadas ao controle dos efeitos nocivos criados por diferentes tipos de estresse como choque térmico, ou osmótico, estresse oxidativo, ou aqueles resultantes de altas concentrações de íons de metais pesados. De forma semelhante, a ausência, ou exaustão, de fontes de macronutrientes, como carbono, nitrogênio, fósforo ou enxofre, exige uma reorganização do padrão de expressão gênica para adequação às novas condições nutricionais, também sendo considerada um estresse ambiental. Visto que a maioria dos estudos de análise da expressão gênica em resposta a estresses ambientais realizados em fungos se refere às leveduras unicelulares Saccharomyces cerevisiae e Schizossacharomyces pombe, nos propusemos a estudar tal resposta no fungo filamentoso multicelular Trichoderma reesei. Dessa forma analisamos por meio da técnica de \"microarrays\" de cDNAs a expressão gênica de aproximadamente 2.000 transcritos desse organismo em resposta a choque térmico, à alta concentração de íons de cádmio II e à ausência de fonte de carbono, ou nitrogênio, por período de 2 horas. Em geral, as respostas aos estresses se compuseram da regulação negativa da transcrição de genes envolvidos em processos com alta demanda de energia como a síntese protéica, evidenciada pela repressão da expressão de genes de proteínas ribossomais e do anabolismo. Em contrapartida, genes codificando proteínas relacionadas à defesa celular, como chaperonas, tiveram sua expressão induzida. As respostas ao choque térmico e ao tratamento com cádmio II se mostraram bastantes semelhantes, enquanto a ausência de fonte de nitrogênio também induziu a expressão de genes relacionados à degradação de proteínas e nucleotídeos. Genes relacionados à utilização de reservas lipídicas foram induzidos tanto na ausência de fonte de carbono quanto de nitrogênio. Foram identificados reguladores transcricionais e componentes de vias de sinalização celular com expressão diferenciada frente a esses diferentes estresses ambientais. A maior parte dos genes cuja expressão se alterou em função dos diversos estresses ambientais estudados ainda não tem função celular conhecida, sendo essa observação, portanto, uma contribuição importante para sua anotação funcional. Uma vez que o fungo filamentoso Trichoderma reesei vem se tornando um organismo de valor biotecnológico por sua característica de alto poder de síntese e secreção de proteínas, esperamos que os dados apresentados forneçam um maior entendimento dos processos celulares desse organismo e possam subsidiar futuros projetos visando uma melhor adaptação do mesmo a ambientes industriais. / The diversity of organisms found today in our planet is due to their adaptation to different environmental conditions present in each ecological niche, and to the adaptative response originated from changes in those conditions. The first step in the adaptation process is considered to be the reprogramming of gene expression as an immediate response to a new environmental condition. A fraction of the genome from all living organisms is dedicated to encoding proteins related to the control of deleterious effects created by different types of stresses like heat or osmotic shock, oxidative stress, or by the presence of high concentrations of heavy metal ions. Similarly, the absence or exhaustion of macronutrients as carbon, nitrogen, phosphorous or sulphur sources demand new patterns of gene expression in order to the organisms survive in a limited nutritional condition, which is also considered an environmental stress. Once the gene expression analyses in fungi as a response to environmental stresses have been widely studied in the yeasts Saccharomyces cerevisiae and Schizossacharomyces pombe, we proposed to study such response in the multicellular filamentous fungus Trichoderma reesei. To this purpose, we have utilized the cDNA microarray technique to analyze the gene expression of approximately 2,000 T. reesei transcripts in response to heat shock, to high concentration of cadmium II ions and to a 2-hour absence of carbon or nitrogen source. As a general response to the four studied stresses, we observed on one hand a negative transcriptional regulation of genes involved in processes that demand great amounts of energy, i.e. a negative regulation of protein synthesis, indicated by strong repression of ribosomal protein genes transcription, as well as a negative regulation of anabolism. On the other hand, genes that encode proteins associated with cellular defense, like chaperones, had their expression induced. The responses to heat shock and to cadmium poisoning were quite similar while nitrogen source absence also induced the expression of genes related to protein and nucleotide degradation. Genes implicated in the consumption of lipid reserves were induced in the absence of both carbon and nitrogen sources. We identified some transcription regulators as well as components of signal transduction pathways that have differential patterns of gene expression caused by these different environmental stresses. Most of the genes that had their expression altered in response to the studied environmental stresses has no known function yet. Their expression patterns towards such stresses are therefore an important contribution to their functional annotation. Since the filamentous fungus Trichoderma reesei has become a microorganism of biotechnological value for its high capacity of synthesis and secretion of proteins, we expect that the data presented on this work can provide a better understanding of its cellular processes and may support future projects for a better adaptation of this organism to industrial conditions.

Orientador: Liana Maria Cardoso Verinaud / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T19:49:25Z (GMT). No. of bitstreams: 1 Souto_PaulaCristinadeSouza_D.pdf: 7669317 bytes, checksum: eeec29689d95e50ddec45928c2c76343 (MD5) Previous issue date: 2006 / Resumo: O Paracoccidioides brasiliensis é um fungo dimórfico que cresce na forma miceliar a temperatura ambiente e na forma de levedura a 35-37ºC. Recentemente, no laboratório de Imunopatologia do Departamento de Microbiologia e Imunologia do Instituto de Biologia da Unicamp, foi observado que uma amostra da cepa virulenta Pb18 crescia de forma diferente das outras. A análise preliminar desta cultura mostrou que o fungo se apresentava, exclusivamente, em formas intermediárias entre levedura e micélio. Assim, este trabalho teve como objetivo a caracterização parcial deste isolado atípico através da comparação com a cepa selvagem. Para tanto, foram analisados: a morfologia do isolado a 25 e 37ºC; a composição de proteínas de parede celular; o desenvolvimento da doença e a resposta imunológica adaptativa em camundongos BALB/c. Os resultados mostraram que este isolado não alterou a sua forma de crescimento, independente da temperatura de cultivo ou da passagem por animal. Através da análise das proteínas de parede celular observou-se ausência de expressão de algumas proteínas e baixa concentração da glicoproteína 43 (gp43) no fungo atípico quando comparado com o selvagem. A infecção causada por este isolado atípico foi mais branda do que aquela observada com a cepa virulenta Pb18, com completa resolução das lesões de disseminação. Entretanto, não foi possível determinar diferenças na avaliação da resposta imune adaptativa dos animais infectados com os diferentes isolados. Apesar da necessidade de mais estudos, estes resultados indicam que o isolado atípico pode vir a ser uma importante ferramenta para o estudo da biologia do fungo e sua relação com o hospedeiro / Abstract: P. brasiliensis is a dimorphic fungus that grows in mycelial phase at room temperature and in yeast form at 35-37°C. Recently, in the Immunopathology Laboratory of the Department of Microbiology and Immunology (Biology Institute/Unicamp), it was observed that a sample of the virulent strain Pb18 grew in a different way from other ones. Preliminary analysis of this culture showed that the isolated exhibited, exclusively, an intermediate form of pseudohyphae. That way, the objective of this work was the partial characterization of the atypical isolated (Pb18PH) through the comparison with the wild strain Pb18. For this, there were analyzed: the morphology of the isolated to 25 and 37ºC; the composition of cell wall proteins; the disease development and the adaptative immune response in BALB/c mice. The results showed that Pb18PH didn't alter their growth form, independently of the cultivation temperature or the passage through animal. By the analysis of the cell wall proteins, it was observed that Pb18PH didn't express some proteins and presented low concentration of the glicoprotein 43 (gp43) when compared with the wild one. The infection caused by this atypical isolated was softer than that observed with the virulent strain Pb18. Besides, it was noted the complete resolution of disseminated lesions. However, it was not possible to determine differences in the adaptative immune response evaluation of the animais infected with the different isolated. To despite the need of more studies about this atypical isolated, our results indicate that Pb18PH would come to be an important tool for the study of the fungus biology and their relationship with the host / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / With developing of forensic observations, certain species of insects and microorganisms were described as indicators of periods of the degradation of the body. However, the literature is still scarce in the field of Forensic Mycology. It was investigated the microbiological characteristics of fungi presence in the post mortem change, as well as, it esteemed the value of the mycology exams in the study. 400 collections was accomplished in 60 human bodies (34 of the bloated stage, 06 of the putrefaction stage and 20 of the skeletonization stage) at the Fortaleza city morgue and public cemeteries in the state of CearÃ, The picked material was analyzed through macro/microcharacteristics and specific biochemical tests. Ally to such analyses, was also accomplished the test of the perforation of the hair in vitro in hair of corpses in the bloated stage and in hair of healthy adults. The material gathered was analyzed at the Specialized Medical Mycology Center of the Federal University of CearÃ. With the anthropological results, it was observed that male, in the interval of the 31 to the 40 years, was more commonly attacked, being the capital (Fortaleza) the more involved in the number of deaths. In the bloated stage, among the identified filamentous fungi, the presence of four orders was observed: Order Eurotiales (63 isolated Aspergillus spp and 21 isolated Penicillium spp), Order Mucorales (4 Mucor spp), Order Hypocreales (2 Acremonium spp, 1 Trichoderma spp and 1 Fusarium spp) and Order Saccharomycetales (2 Geotrichum spp). In the yeast distribution, it was observed the orders:Saccharomycetales (44 Candida spp) and Tremellales (5 Trichosporon spp). In the putrefaction stage, it was isolated the following orders: Eurotiales (Penicillium spp 2 and Aspergillus 2) and Hypocreales 1 (Acremonium spp). Regarding the yeasts, it was just isolated the Order Saccharomycetales (Candida spp 3). In the eskeletonization stage, the following orders were observed: Order Eurotiales (Aspergillus spp 22 and Penicillium spp 18), Order Mucorales (Mucor spp 10) and Order Hypocreales (Acremonium spp 2 and Trichoderma spp 1). Regarding the yeasts, two orders were found: Tremellales (Trichosporon spp 1) and Saccharomycetales (Candida spp 1). In the hair perforation test in vitro, it was positive in the bloated stage, differing of healthy adults,where the test was negative. For conclusion, Forensic Micology is still a rich field in data and fungi can come to be a tool in the aid of human post mortem diagnosis. / Com o evoluir das observaÃÃes mÃdico-legais, determinadas espÃcies de insetos e microrganismos foram descritos como indicadores de perÃodos da degradaÃÃo do corpo. Entretanto, a literatura cientÃfica ainda à escassa no campo da Micologia Forense. Assim, investigaram-se as caracterÃsticas microbiolÃgicas dos fungos partÃcipes na mudanÃa de flora post mortem em humanos, bem como estimou-se o valor da realizaÃÃo de exames micolÃgicos no estudo cronotanatolÃgico. Foram realizadas um total de 400 coletas em 60 corpos humanos (34 do perÃodo gasoso, 06 do coliquativo e 20 do esqueletizado) examinados no Instituto MÃdico-Legal de Fortaleza e em cemitÃrios do Estado do CearÃ. Foram realizados estudos macro/micromorfÃlogicos e testes bioquÃmicos especÃficos para cada grupamento fÃngico isolado. Aliado a tais anÃlises, foi tambÃm realizado o teste da perfuraÃÃo do pÃlo in vitro em pÃlos de cadÃveres no perÃodo gasoso e em pÃlos de adultos hÃgidos. Estas identificaÃÃes foram realizadas no Centro Especializado em Micologia MÃdica da Universidade Federal do CearÃ. Com os resultados antropolÃgicos, foi observado que o sexo masculino, dos 31 aos 40 anos, foi o mais comumente acometido, sendo a capital (Fortaleza) a mais envolvida no nÃmero de mortes. No perÃodo gasoso, dentre os fungos filamentosos identificados, foi observada a presenÃa de quatro ordens: Eurotiales (63 isolados de Aspergillus spp e 21 de Penicillium spp), Mucorales (4 Mucor spp) e Hypocreales (2 Acremonium spp, 1 Trichoderma spp e 1 Fusarium spp) e Saccharomycetales (2 Geotrichum spp). No referente Ãs leveduras, verificaram-se as ordens: Saccharomycetales (Candida spp 44) e Tremellales (Trichosporon spp 5). No perÃodo coliquativo, registraram-se as ordens: Eurotiales (Penicillium spp 2 e Aspergillus 2) e Hypocreales 1 (Acremonium spp). No referente Ãs leveduras, isolou-se apenas a Ordem Saccharomycetales (Candida spp 3). No perÃodo de esqueletizaÃÃo, verificaram-se as seguintes ordens: Eurotiales (Aspergillus spp 22 e Penicillium spp 18), Mucorales (Mucor spp 10) e Hypocreales (Acremonium spp 2 e Trichoderma spp 1). No referente Ãs leveduras, registraram-se duas ordens: Tremellales (Trichosporon spp 1) e Saccharomycetales (Candida spp 1). Quanto ao teste de perfuraÃÃo do pÃlo in vitro, a positividade foi observada no perÃodo gasoso, diferindo de adultos hÃgidos, em que foi negativa. Conclui-se que o estudo da Micologia Forense ainda à um campo rico em dados e que os fungos poderÃo vir a ser uma ferramenta complementar no estudo do tempo de morte em Medicina Legal.

O objetivo do presente trabalho foi estudar a ação de diversos antifúngicos isolados e associados a anfotericina B contra amostras sequenciais ou não recuperadas de pacientes com cromoblastomicose e submetidas ou não a tratamento. A partir de 18 pacientes foram isoladas 39 cepas, submetidas a testes de diluição em ágar contendo anfotericina B, cetoconazol, itraconazol, terbinafina, 5-fluorocitosina, fluconazol ou griseofulvina. Com base nas CIMs e nas concentrações séricas desses antifúngicos verificou-se que as amostras foram, de modo geral, sensíveis ao cetoconazol, a terbinafina e ao itraconazol. Somente 13 amostras foram sensíveis a anfotericina, 7 a 5-FC, 2 ao fluconazol e nenhuma à griseofulvina.Com relação as CFMs, 2 cepas foram sensíveis a AnB, 11 ao itraconazol, 20 ao cetoconazol e 3 a 5-FC. Seis não responderam a terbinafina e 38 ao fluconazol. As combinações de antifúngicos resultaram em interações principalmente aditivas ou indiferentes. O estudo de isolados seqüenciais evidenciou um ponto de corte para sensibilidade desses agentes ao itraconazol, correspondente a ClMs ≤0,06µg/ml desse azol. / The aim of the present work was to study the action of several antifungals isolated and combined with amphotericin B against sequential or non-sequential samples in patients infected with chromoblastomycosis and treated or not treated. Thirty nine strains from 18 patients were isolated and submitted to agar dilution testing containing amphotericin B, cetoconazole, itraconazole, terbinafine, 5-flucytosin, fluconazole or griseofulvin. Based on the CIMs and on the serum levels of these antifungals, the isolates were susceptible to ketoconazole, terbinafine and itraconazole. Only 13 of the isolates were susceptible to amphotericin; seven to 5-FC, two to fluconazole and none to a griseofulvin. Regarding the CFMs two strains were susceptible to AnB, 11 for itraconazole, 20 for ketoconazole and three for 5-FC. Six didn\'t respond to terbinafine and 38 for fluconazole. The combination of antifungals resulted in mainly additive or indifferent interaction. The study of the sequential isolated sample showed a breaking point for the susceptibility of these agents to itraconazole, corresponding CIMs ≤0,06µg/ml of this azole.