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.

Diagnostic medical mycology has been slow to advance due to a lack of species specific antigens in organisms which cause serious diseases in man. Toward this end, an HPLC analysis was done of the following fungal antigens: histoplasmins HKC-43 and H-42, blastomycin KCB-26, an alkali-soluble, water soluble antigen from Blastomyces dermatitidis (b-ASWS), a coccidioidin prepared from a toluene lysate of the mycelial-arthroconidia phase of Coccidioides immitis, and an alkali-soluble, water-soluble antigen from Coccidioides immitis (c-ASWS). The HPLC survey included size-exclusion chromatography (SEC), ion exchange chromatography (HPIEC), and reversephase chromatography (RP). Resolution was poor with both SEC and HPIEC but was excellent with RP chromatography. The use of RP will allow sufficient separation for further antigenic and structural analysis.

O ciclo de vida de Dictyostelium discoideum é composto de duas fases independentes. Durante o crescimento vegetativo, as amebas crescem isoladas até que a fonte de nutrientes seja esgotada. A carência nutricional induz sua entrada num processo de desenvolvimento, que inclui a parada do crescimento, a agregação das células e a formação de um organismo multicelular onde as células se diferenciam em esporos que sobrevivem as condições desfavoráveis. A proteína quinase YakA é requerida para a transição entre o crescimento e o desenvolvimento. YakA regula os níveis da PKA. Mutantes yakA- apresentam o crescimento acelerado, são deficientes no processo de agregação e são hiper-sensíveis a estresse oxidativo e nitrosoativo. Uma mutação em um segundo sítio em keaA, suprime a morte induzida por SNP (um gerador de óxido nítrico) no mutante yakA-. O papel de keaA foi determinado em resposta a estresse oxidativo, nitrosoativo e carênica nutricional. O gene keaA é necessário para o crescimento e desenvolvimento. Uma mutação em keaA confere resistência a estresse nitrosoativoloxidativo confirmando que uma mutação em keaA confere resistência a estresse. Um segundo supressor da morte induzida por SNP no mutante yakA- foi isolado pela mesma técnica de REMI e identificado como pkaC um regulador da resposta a estresse. YakA e PKA integradam a resposta a vários estresse em Dictyostelium. Os resultados indicam que a yakA regula a parada do ciclo celular em resposta a estresses através da modulação de keaA. keaA regula, por sua vez, a expressão da pkaC, um regulador chave da produção de cAMP e do processo de desenvolvimento. A interação gênica entre estes elementos é complexa e deve ser ajustada para permitir que as células sobrevivam a mudanças ambientais encontradas durante o seu ciclo de vida. / Dictyostelium discoideum\'s life cycle is composed of two phases. During the vegetative phase, amoebae grow as single cells until the nutrients are depleted. Starvation induces a developmental program where isolated amoebae adopt a multicellular mode of living and differentiate into spores to survive the harsh conditions. The protein Kinase YakA is required for the growth to development transition. YakA regulates the leveis of PKA. yakA null cells have a faster cell cycle, are aggregation deficient and are hypersensitive to nitrosoative/oxidative stress. A second-site mutation in keaA, supresses the death induced by SNP, a generator of nitric oxide. The role for keaA has been determined in response to oxidative, nitrosoative and nutrient starvation stresses. keaA is required for proper growth and development. The keaA- cells are more resistant to nitrosoative and oxidative stress confirming that a mutation in keaA confers stress resistance. A second supressor of the death induced by SNP in the mutant yakA- was isolated with REMI and identified pkaC as a regulator of the nitrosoative stress response. YakA and PKA may integrate the responses to several stresses in Dictyostelium. The results indicate that yakA regulates the cell cycle arrest in response to stress through the modulation of keaA. KeaA regulates the expression of pkaC, a key regulator of cAMP prodution and development. Genetic interactions among these elements is complex and must be finely tuned in the many environmental changes cells endure during the life cycle.

Criptococose é uma doença causada pelo fungo C. neoformans que têm grande importância atualmente, devido ao aumento da população imunocomprometida,. Além disso, existem poucas opções terapêuticas contra micoses profundas. Neste trabalho foi avaliado se a via de biossíntese do triptofano seria um bom alvo para o desenvolvimento de novos antifúngicos. Com o uso da tecnologia de RNA de interferência, concluiu-se que esta via de síntese é essencial para a sobrevivência desta levedura, sendo, portanto, um ótimo alvo. Ainda neste estudo, demonstrou-se que a letalidade decorre da baixa captação de triptofano pelas permeases de aminoácidos, as quais sofrem repressão catabólica pela fonte de nitrogênio e efeito negativo da temperatura. Foram testados dois inibidores específicos que atuam sobre a antranilato sintase e a triptofano sintase, duas enzimas cruciais para a conversão do corismato em triptofano. Ambos compostos causaram inibição do crescimento de C. neoformans e C. gattii. / Cryptococcosis is a disease caused by C. neoformans, currently of great importance due to the increase in immunocompromised population. Furthermore, there are few therapeutic options for treating this disease. This study evaluated the tryptophan biosynthetic pathway as a possible target for the antifungal development. By using RNA interference technology we concluded that this metabolic pathway is essential for the survival of this yeast, and, therefore, it is a good target. In the same study, it was demonstrated that lethality results from the low uptake of the tryptophan amino acid by permeases, which undergo nitrogen catabolite repression and negative effect of temperature. Two specific inhibitors acting on the anthranilate synthase and tryptophan synthase, two key enzymes for the conversion of chorismate into tryptophan were tested. Both compounds caused growth inhibition of C. neoformans and C. gattii.

How do we hold each other through a change of phase? This is the seminal question asked in this thesis as it explores networked systems populated by urban metabolisms and waste streams. Using an embodied feminist framework, this paper attempts to highlight short comings in the current treatment of road snow sludge in the metropolitain areas of Stockholm, Sweden and proposes the use of a systemic, intergrated mycological strategy to filter and detoxify snow-melt-water before it re-enters natural bodies of water.

Investigating functional traits is an important tool for understanding fungal communities, yet ecological functions of fungal pigmentation remain poorly investigated. Recent studies support the thermal-melanism hypothesis for fungal color lightness in relation to macroclimate, but it remains unclear if microclimate also affects fungal pigmentation. In this study I use fruit bodies of wood-living polypores to further investigate this subject. While previous studies have used database-derived color lightness from photos often taken under unknown and non-standardized conditions, here I extract all color values from standardized photos taken in situ. In contrast to previous studies that have only examined fungal color lightness, I also studied the other components of the CIE L*a*b* color space, which may be related to e.g. protection, thermoregulation or signaling. I investigated community-level fungal pigmentation of pore surfaces and caps of polypores in fragmented forest patches in northern Sweden, in relation to microclimate proxies, deadwood availability and surrounding landscape structure. Two types of multiple linear regression models were used, one for community weighted means (CWM) and one for the coefficient of variance (CV) of patch level pigmentation. While no support was found for the thermal-melanism hypothesis in this study, saturation in the yellow spectrum was positively related to canopy cover and forest patch size. Variance in lightness of pore surfaces and in saturation in the red spectrum of caps was also positively related to diversity of decay stages and forest patch size respectively. These results indicate previously unknown relationships between fungal pigmentation and environment which warrant further research.

<p dir="ltr">Rapid ʻōhiʻa death (ROD) is a disease complex caused by two <i>Ceratocystis fungi</i>, <i>C. lukuohia</i> and <i>C. huliohia</i>, that is devastating the keystone tree of the Hawaiian Islands, ʻōhiʻa lehua (<i>Metrosideros polymoropha</i>). The causal agents of ROD were identified in 2015 and I began researching entomological aspects of the complex in 2016. Much like other <i>Ceratocystis</i> diseases, my colleagues and I suspected that beetles and frass might be involved in the system. Together, we identified four species of invasive ambrosia beetles (Coleoptera: Curculionidae) that contribute to the spread of ROD: <i>Xyleborinus saxesenii, Xyleborus affinis, Xyleborus ferrugineus</i>, and <i>Xyleborus perforans</i>. Both ROD-<i>Ceratocystis</i> fungi and the ambrosia beetles inhabit the xylem of ʻōhiʻa. When these beetles create their home galleries, they produce frass particles infested with resting chlamydospores that can be transported in the environment through the soil, wind, and water. Secondly, the beetles are capable of vectoring the fungi directly to stressed trees via viable propagules attached to their exoskeleton. The natural progression of this research was to investigate the chemical ecology of the system, therefore building the foundations for management strategies to reduce the spread of ROD. In addition, I satisfied my curiosity to explore the fungal mutualisms of these beetles through the use of phylogenetics.</p><p dir="ltr">In Chapter 1, I review the literature describing ROD and the four ROD-associated ambrosia beetle species. I report all of the research to date regarding ROD, including current monitoring and management strategies. Then, I introduce ambrosia beetles and the Xyleborini tribe, focusing on the life history of the ROD-associated beetles and current literature describing the use of semiochemicals to control them.</p><p dir="ltr">In Chapter 2, I determine the volatile organic compounds associated with the ROD <i>Ceratocystis</i> – ʻōhiʻa pathosystem and the response of the associated beetles to those compounds. I investigated the volatiles produced by <i>C. lukuohia</i> and <i>C. hulihia</i> in culture in addition to when inoculated into ʻōhiʻa seedlings. Then, I describe olfactometer assays to determine if the ROD-associated beetles are attracted to the volatiles emitted from ROD-<i>Ceratocysti</i>s in culture.</p><p dir="ltr">In Chapter 3, I investigate semiochemicals for attracting and repelling ambrosia beetles in ʻōhiʻa forests. I describe separate trapping experiments, first, testing the attraction of beetles to 100% ethanol and 1:1 methanol ethanol. Second, we investigate the use of two beetle repellent products, one with verbenone and the other with verbenone + methyl salicylate active ingredients.</p><p dir="ltr">In Chapter 4, I describe the testing of the repellent, verbenone, in the SPLAT^® Verb formulation, to deter ambrosia beetle attack from both healthy ʻōhiʻa trees and trees infested with ROD-<i>Ceratocystis</i>. Over two field seasons, we monitored ambrosia beetle attacks on trees treated with verbenone and measured the abundance of verbenone released from the repellents over time during the first season.</p><p dir="ltr">In Chapter 5, I investigate the ambrosia fungi of the ROD-associated beetles and native Hawaiian ambrosia beetles on the Island of Hawaiʻi. We isolated a dozen fungal symbionts from the mycetangia of ambrosia beetles, most of which are first reports in Hawaiʻi, and use phylogenetics to investigate putative new species of <i>Raffaelea</i> and <i>Ambrosiozyma.</i></p><p dir="ltr">Finally, in Chapter 6, I synthesize the results and future directions of the aforementioned chapters. Together, these dissertation chapters provide insights into ambrosia beetle monitoring and management strategies in Hawaiʻi and beyond. I describe the groundwork for understanding the pathosystem from a chemical ecology perspective and touch on the understudied world of Hawaiʻi fungi and potential pathogens.</p>

Shiitake growers in Virginia are experiencing considerable diminution of the fruiting life of oak logs, due primarily to Ascomycetous, competing, wood-rotting, contaminating "weed fungi" that either invade the logs after trees are felled or are present in tree tissues before felling. We surveyed several shiitake farms, and, although the fungal flora differed among them, the predominant colonizing fungi were identified as Graphostroma platystoma (also identified as Diatrype stygma), Eutypa spinosa and Slereum aff. complicatum. Other less frequently observed fungi were Stereum ostrea, Schizophyllum commune, Hymenochaete sp., Poria sp., Hypoxylon atropunctatum and Hypoxylon punctulatum. Site and means of invasion (colonization) of these "weed fungi" were studied by monitoring endophytic fungal populations in stressed and non-stressed oak trees in the Jefferson National Forest Montgomery County, Virginia. Twenty-six samples from Poverty Creek (non-stressed) and 23 from Brush Mountain (stressed) were studied. Fifty percent of the samples from the non-stressed were sterile, 23% yielded Paecilomyces variota., and 27% were colonized by other fungi. On the other hand, 100% of the samples from stressed trees (Brush Mountain) were colonized by fungi; 74% yielded P. variotii and 26% by other fungi. Thirty samples from a pin oak (Quercus paluslris) plot at Virginia Tech (non-stressed) were 74% sterile, 16% colonized by an unidentified yeast, and 10% colonized by other unknown fungi. / Master of Science

Nesse trabalho avaliamos a proteção que o anticorpo monoclonal (AcM) contra CMH e contra Hsp60 fornecem aos animais infectados com as leveduras de Paracoccidioides brasiliensis e P. lutzii em modelo profilático. O tratamento com os AcMs de isotipos IgG2a e IgG2b foram protetores, induzindo a secreção das citocinas IL-12p70, IFN-<font face=\"Symbol\">g e TNF-<font face=\"Symbol\">a, padrão de resposta imune Th1. Nós mostramos por imunomarcação que moléculas de CMH e Hsp60 estão acessíveis na célula. E o anticorpo monoespecífico contra CMH e os anticorpos policlonais contra melanina, gerados nesse trabalho foram eficazes nos ensaios in vitro de proteção. Foram gerados anticorpos monoclonais contra glicoproteina extraída de Pb18, o AcM reconheceu, por imunomarcação, estruturas internas e a parede celular da levedura. Avaliamos um novo modelo de hospedeiro, com o uso da larva Galleria mellonella, o que pode servir de triagem e reduzir desta forma o uso excessivo de camundongos, as leveduras P. lutzii e H. capsulatum são letais para as larvas e evocam resposta celular que se organizam semelhante a um granuloma. / In this work we evaluated the protection that the monoclonal antibody (MAb) against Hsp60 and against CMH provides the animals infected with the yeast Paracoccidioides brasiliensis and P. lutzii in prophylactic model. The treatment with the MAbs of IgG2a and IgG2b isotypes were protective, inducing the secretion of IL-12p70, IFN-<font face=\"Symbol\">g and TNF-<font face=\"Symbol\">a, Th1 pattern of immune response. We show by immunogold that Hsp60 and MHC molecules are accessible on the cell. And the monospecific antibody against HCM and polyclonal antibodies against melanin, generated in this study were effective in in vitro assays of protection. Monoclonal antibodies were generated glycoprotein extracted from Pb18, Mab recognized by immunogold, internal structures and cell wall material. Evaluated a new type of host, using the larvae Galleria mellonella, which can serve as a screening and reduce thus the overuse of mice, yeast P. lutzii and H. capsulatum are lethal to larvae and evoke cellular responses that are organized like a granuloma.

Os objetivos deste estudo foram: re-identificar 70 isolados de Candida em micoteca por método tradicional e API 20C; identificar a presença de C. dubliniensis; determinar e comparar as concentrações inibitórias mínimas (CIMs) das amostras, frente a sete antifúngicos (E-test); caracterizar molecularmente as amostras seqüenciais de Candida pela técnica de PFGE; estudar e comparar o perfil genotípico de isolados de C. albicans e C. parapsilosis, através da análise de marcadores microsatélites e cariotipagem. O nível de concordância entre o método tradicional e o API 20C foi de 93%. Nenhum isolado de C. dubliniensis foi identificado no estudo. Em relação à sensibilidade antifúngica, a maioria das amostras apresentou alta porcentagem de sensibilidade às sete drogas testadas. A análise molecular pela técnica de PFGE, mostrou seis perfis de cariótipos diferentes em 24 amostras seqüenciais. Em relação à comparação das técnicas de PFGE e microssatélites, observamos que a última mostrou maior poder discriminatório para as amostras de estudadas. / This study was aimed to: identify the 70 isolates of Candida in a culture collection by the traditional method and by API 20C; identify the presence of C. dubliniensis; determine and compare the minimum inhibitory concentrations (MICs) of these samples in regard to the seven drugs (E-test); molecularly characterize sequential samples from the same patient by the PFGE technique; study the genotypic profile of all the isolates of C. albicans and C. parapsilosis by means of the microsatellite technique and compare the results with those obtained by the PFGE technique. The level of agreement between the traditional method and the API 20C was 93%. No isolate of C. dubliniensis was identified in the study. In relation to antifungal susceptibility, most of the samples presented a high percentage of susceptibility to the seven drugs tested. The molecular analysis by the PFGE technique revealed six different karyotype profiles among 24 sequential samples. In the comparison between the PFGE and microsatellite, the latter showed a greater discriminatory power for samples.