Sporothrix shenckii, um fungo dimórfico, termo-dependente, é encontrado na natureza associado com matéria orgânica em decomposição, como, espinhos, folhas secas, madeira e também na água. Este fungo é o agente causador da esporotricose, que ocorre com maior freqüência em pessoas que trabalham com o solo e vegetais contaminados com os esporos do fungo, sendo considerada uma doença ocupacional. A contaminação acontece com a inoculação traumática do material contaminado e em alguns casos por inalação dos esporos. Quando infecta o organismo humano, bem como outros mamíferos ou quando cultivado a 37°C, passa para a forma de levedura. É comum em áreas de clima tropical e subtropical, com alta umidade. Apresenta distribuição mundial, sendo que, atualmente no Brasil, são relatados casos em São Paulo, Rio de Janeiro e no Rio Grande do Sul. No Rio de Janeiro, a esporotricose tem sido relatada como doença endêmica, onde o agente etiológico é transmitido de gatos para homens. A esporotricose apresenta caráter crônico, polimórfico e com formação granulamatosa. As formas clínicas e a patogênese dependem do sítio de inoculação do fungo e da resposta imunológica do hospedeiro. O mecanismo de defesa e suscetibilidade do organismo, bem como a disseminação da infecção por este fungo ainda não são totalmente conhecidos. O estado de saúde do paciente contribui diretamente para o estabelecimento da doença, sendo que, alcoolismo, diabete, imunidade comprometida e uso extensivo de drogas imunosupressoras são fatores que podem predispor à infecções severas. Neste trabalho estudamos in vitro a interação do S. schenckii com macrófagos murinos. Por meio de técnicas de fagocitose, ELISA, dosagem de óxido nítrico, viabilidade de leveduras, citometria de fluxo e RT-PCR foram realizados ensaios da atividade de macrófagos. Desta forma foram gerados alguns dados para o melhor entendimento da resposta de defesa nesta doença. Nos estudos realizados, concluímos que os anticorpos gerados nos camundongos pela infecção por S. schenckii foram capazes de induzir a fagocitose do fungo. Também, após a fagocitose das leveduras pelos macrófagos, incubados com laminarina ou manana, houve inibição significativa da produção de TNF-α, indicando que os receptores dectina-l e manose, respectivamente, estão relacionados à produção desta citocina. O soro, contendo os anticorpos do animal infectado, também induziu forte produção desta citocina, mostrando que a entrada do fungo via receptor Fc pode também induzir a produção dessa citocina. Verificamos ainda que a fagocitose de leveduras, pelos macrófagos, na presença de soro de camundongo infectado, induziu alta produção de IL-10. Quando analisamos a expressão de genes presentes nos macrófagos, observamos forte expressão de TLR-2 e MyD-88, na presença das leveduras. Já a expressão de iNOS pelos macrófagos, observamos que foi maior na presença do fungo somente quando as leveduras foram incubadas com o soro, após o período de 72 horas. Em seguida, ao dosar a produção de Óxido Nítrico (NO), verificamos no período de 3 dias de incubação, que houve redução significativa na concentração de NO produzida pelos macrófagos, quando estes foram incubados com o fungo. Porém, quando as leveduras foram opsonizadas pelo soro, houve aumento da produção de NO após 72 horas de incubação. Outra verificação importante foi que os macrófagos foram capazes de destruir as leveduras fagocitadas na presença de soro. / Sporothrix schenckii is a dimorfic fungus, term-dependent, find in nature associated with organic material at decomposition, like thorns, wheat leaves, wood and water. This is the fungi agent that causes sporothricosis, which happens with more frequency with people that work with land and green, contaminated with mold of fungus. That is why it is considered a occupational disease. The contamination happens with traumatic inoculation of material and some cases with inhalation of mold. When a human is infected, as well as other mammals or when cultivate at 37°C, it tums into the yeast form. Is common at tropical and moisture weather areas. Is found all around the world, and nowadays at Brazil, with cases related São Paulo, Rio de Janeiro and Rio Grande do Sul. At Rio de Janeiro, sporothricosis are related like endemic disease, where the etiologic agent is transmitted by cats to men. Sporothricosis is a chronic, polimorfic disease, and with form granulamatosa. The clinical type and the pathogenesis depend on the way of inoculation and the response of immunological system of the host. The defense and suscebility of the organism, as dissemination of infection with this fungi is not yet totally known. The patient\'s health state contributes directly to the disease, as much as alcoholism, diabetes and immunity deficiency and the extensive use of immunosuppressive drugs are factors that can bring to harder infection. Here, we studied in vitro the interaction of S. schenckii with murine macrophages. Using phagocytosis assay, ELISA, dosage of oxide nitric, CFU assay, flow citometry and RT-PCR were analysed activity of macrophages. We produced some results in order to better understand the response of defense with this disease. In the present study, we conclude that antibodies generated in mice by infection with S. schenckii were capable to promote phagocytosis of fungus. Also, when macrophages and yeast were incubated with laminarin or mannan, we had significant decrease in production of TNF-α, showing that receptors dectin-l and manose, respectively, are related with production this cytokine. The antibodies of mice infected with S. schenckii, also produced hard level this cytokine showing that entrance of fungus by receptor Fc can produce TNF- α. Besides, we verify that phagocitosys of yeast, with sera of infected mice, induct hard production of IL-10. When we analysed the expression of genes of macrophages, we observed hard expression of TLR-2 and MyD-88, when yeast were present. However, the expression of iNOS by macrophages, was more hard with yeast only when sera was present, before 72 hours. Similarly, when yeast were opsonized by sera, we had increase of production of NO after 72 hours of incubation. After, macrophage were capable to destruct yeast phagocyted when sera was present.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-09-26T17:06:38Z No. of bitstreams: 1 2015 - Erika Caitano da Silva.pdf: 1958258 bytes, checksum: 61fcc63494dc0f8b0966ba187b5280a0 (MD5) / Made available in DSpace on 2017-09-26T17:06:38Z (GMT). No. of bitstreams: 1 2015 - Erika Caitano da Silva.pdf: 1958258 bytes, checksum: 61fcc63494dc0f8b0966ba187b5280a0 (MD5) Previous issue date: 2015-07-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The objective of this study was to evaluate the efficiency of a fungicide of the strobilurin group in reducing soil N2O production, a potent greenhouse gas, and a possible positive effect on the N use efficiency by sugarcane. Laboratory studies were conducted to evaluate the effect of fungicide in soil fertilized with N on N2O emissions, as well as greenhouse and field experiments to study the fungicide as controller of urea and ammonium nitrate induction of N2O emissions and as an enhancer of fertilizer N use efficiency by sugarcane. In addition to the monitoring of soil N2O fluxes with static chambers sugarcane leaves were collected for analysis of soluble forms of N and nitrate reductase activity together with plant parameters to estimate yield and the efficiency of fertilizer N use. Laboratory tests revealed the fungicide was capable of reducing soil N2O emissions. Regardless of N source, the fungicide did not affect the analyses of enzymes and plant N fractions in sugarcane, but there was a downward trend of N2O fluxes in the soil treated with urea in the greenhouse experiment. In the field experiment, N2O fluxes were 6 to 89 ?g N m-2h-1, 24 to 795 ?g N m-2h-1 and 27 to 508 ?g N m-2h-1 respectively to the control (no added N), urea and urea + fungicide treatments, indicating that N2O emissions were reduced in approximately 30% by the fungicide. The treatment with ammonium nitrate did not induce N2O emissions from soil. There was no significant difference between treatments in all analyses of soluble fractions and enzyme activity. Based on the dimensions of stalks, sugarcane yield was estimated at 190 Mg ha-1for the control treatment (without added N) and at 238 Mg ha-1 for urea treatment. The application of fungicide had no effect on yield. The use of fungicide reduced soil N2O emissions, but does not induce greater N use efficiency by sugarcane / O objetivo deste trabalho foi avaliar a efici?ncia de fungicida do grupo das estrobilurinas na redu??o da produ??o de N2O no solo, um potente g?s de efeito estufa, al?m de poss?veis efeitos positivos na efici?ncia de uso de N pela cana-de-a??car. Foram conduzidos estudos em laborat?rio para avaliar o efeito do fungicida em solo fertilizado com N sobre as emiss?es de N2O, assim como um ensaio em casa de vegeta??o e outro em campo, onde se estudaram a ureia e o nitrato de am?nio, tratados ou n?o com fungicida, sobre as emiss?es de N2O e indicadores de efici?ncia de uso de N pela cana-de-a??car. Al?m do monitoramento dos fluxos de N2O com c?maras est?ticas, foram tamb?m coletadas folhas da cultura para an?lise das fra??es sol?veis de N e an?lise da enzima nitrato redutase, incluindo-se tamb?m a estimativa da produtividade e efici?ncia do uso de N. Os ensaios de laborat?rio mostraram que o fungicida aplicado ao solo reduz emiss?es de N2O. No ensaio em casa-de-vegeta??o, a an?lise de enzimas e fra??es de N na cana-de-a??car n?os e alteraram com o uso do fungicida, independente da fonte de N, mas houve tend?ncia de redu??o dos fluxos de N2O no solo tratado com ureia. No ensaio de campo, os fluxos de N2O foram de 6 a 89 ?g N m-2h-1, 24 a 795 N m-2 h-1 e 27 a 508 N m-2 h-1respectivamente para os tratamentos controle (sem adi??o de N), ureia e ureia +fungicida, indicando queda nas emiss?es de N2O de aproximadamente 30% no tratamento com fungicida.O tratamento com nitrato de am?nio n?o induziu emiss?es de N2O do solo.N?o houve diferen?a significativa entre os tratamentos em todas as an?lises das fra??es sol?veis e tamb?m na an?lise enzim?tica. A produtividade de colmos (TCH), estimada a partir das dimens?es dos colmos, variou significativamente de 190 Mg ha-1, no tratamento controle (sem adi??o de N), para 238 Mg ha-1, no tratamento com ureia, por?m a aplica??o do produto n?o influenciou na produ??o da cultura. O uso de fungicida reduz emiss?es de N2O, por?m n?o induz maior efici?ncia de uso de N pela cana-de-a??car

O aumento da complexidade dos resíduos produzidos nos processos de mineração, que acompanha a crescente expansão deste setor no Brasil, tem revelado a necessidade de novas técnicas para o tratamento ecologicamente correto dos rejeitos de mineração. Desta forma, a biorremediação, uma técnica de baixo custo que utiliza microrganismos extremófilos na recuperação de metais tóxicos, se apresenta como um método economicamente viável no tratamento dos rejeitos contendo íons metálicos. Assim, mediante uma análise proteômica dos fungo isolados do ambiente de mineração, quando submetidos a estresse com ions de cobre, podemos compreender seu comportamento fisiológico no processo de biorremediação. Para isso, Aspergillus niger VC e Rhizopus microsporus VC, isolados do ambiente de mineração foram incubados junto a íons de cobre e mediante analise proteômica foram identificados biomarcadores de estresse oxidativo nos fungos. O proteoma realizado utilizando a técnica de NanoLC-ESI-Q-TOF identificou a expressão de proteínas que mudaram sob a influência do cobre em comparação a seus respectivos controles. Os resultados mostraram que as duas cepas obtidas do ambiente de mineração possuem diferentes mecanismos de resistência. Sendo assim, A.niger VC apresentou uma redução na expressão proteica, das quais 132 foram diferencialmente expressas na presença de íons Cu2+. Por sua vez, a cepa R.microsporus VC exibiu uma superexpressão proteica, com 389 proteínas expressas na presença do metal. Nestes cultivos foram identificadas proteínas relacionadas ao choque térmico e à adsorção de metais, conhecidas como proteínas de choque térmico (HSPs) e metaloproteínas, produzidas em resposta ao estresse imposto pela presença do agente indutor de estresse. No entanto, as enzimas envolvidas na defesa contra o estresse oxidativo, identificadas na ausência de metal, são um indicativo de adaptação metabólica em resposta ao ambiente de mineração. Assim concluímos que tanto A.niger VC como R.microsporus VC, são fungos que apresentam importantes características que permitem sua utilização como agentes de biorremediação dos rejeitos de mineração. / The increase in the complexity of the waste produced in the mining process, with the growing expansion of this sector in Brazil, has revealed the need for new techniques for the ecologically correct treatment of this toxic waste. Therefore, bioremediation, a low cost technique that uses endophilic microorganisms in the recovery of toxic metals, presented as an economically viable method in the treatment of metal ion-containing wastes. In this way, through proteomic analysis of the isolated fungus from the mining environment, when submitted to stress with copper ions, we can understand their physiological behavior in the bioremediation process. In this regard, Aspergillus niger VC and Rhizopus microsporus VC, isolated from the mining environment were incubated with copper ions and in result of proteomic analysis, biomarkers of oxidative stress were identified in the fungus. The proteome performed using the NanoLC-ESI-Q-TOF technique identified the expression of proteins that changed under the copper influence compared to their respective controls. The results showed that the two strains obtained from the mining environment have different resistance mechanisms. Thus, A.niger VC presented a reduction in protein expression, of which 132 were differentially expressed in the presence of Cu2+ ions. In turn, the strain R.microsporus VC exhibited a protein overexpression, with 389 proteins expressed in the metal presence. In these cultivations, proteins related to thermal shock and the adsorption of metals, known as HSPs and metalloproteins, produced in response to the stress imposed by the presence of the stress-inducing agent. However, the enzymes involved in defense against oxidative stress, identified in absence of metal, are indicative of metabolic adaptation in response to the mining environment. Therefore we conclude that both A.niger VC and R.microsporus VC are fungus that present important characteristics that allow their to be used as bioremediation agents for mining waste.

This thesis focuses on the edible fungus Lepista (Pied Bleu or Wood Blewit). Factors affecting its potential commercial cultivation were explored and a contribution to knowledge of the morphology and cultivation of Australian species of Lepista has been made. Australian collections of Lepista were made within a 200 km zone of Sydney. A study of the morphology and taxonomic species of these collections was undertaken. Intra- and inter-fertility crosses were completed with French L. nuda and L. sordida to determine genetic relationships and biological species. Suitable substrates for agar medium, spawn production and cultivation were explored. The response to temperature of French and Australian Lepista in vitro, and Australian Lepista under cultivation, using cold shock, was observed. The effect of modified atmosphere exchanges per hour, CO2 levels, and cold shock during the cultivation cycle and sporophore production were investigated. A genebank of Australian Lepista was established. Three species of Lepista were found in Australia : L. nuda, L. sordida and L. saeva. Two other groups of Lepista were identified. The use of A. bisporus compost appeared to be optimal for experimental and commercial applications. Australian isolates of Lepista tolerate higher temperatures than French isolates, and grew at double the rate of the French at all temperatures except 5 degrees centigrade. The length of the spawn run was reduced from 43-58 days to 12-16 days with introduced CO2 of 9,000-11,000 ppm, but an erratic cyclic pattern of net CO2 production occurred which could only be stabilised by increasing ventilation. This initial cyclic pattern appeared to inhibit subsequent sporophore formation. / Doctor of Philosophy (PhD)

Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium, the lignocellulolytic activity of P. carnosa has not been investigated. An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium, it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels.

Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium, the lignocellulolytic activity of P. carnosa has not been investigated. An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium, it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2002. / Thesis documentation sheet inserted. Appendix reprints three manuscripts and one published paper, three of which are co-authored with others. Includes bibliographical references. Also issued electronically via World Wide Web in PDF format; PDF version lacks abstract, ack., and appendix. One ill. in PDF version is in col.

Thesis (Ph. D.)--University of California, Davis and San Diego State University, 2003. / Includes bibliographical references (leaves 142-144). Also available via the World Wide Web. (Restricted to UC campuses).

The general prevalence of sexual reproduction over asexual reproduction among metazoans testifies to the evolutionary, long-term benefits of genetic recombination. Despite the benefits of genetic recombination under sexual reproduction, asexual organisms sporadically occur throughout the tree of life, and a few asexual lineages persisted over significant evolutionary time without apparent recombination. The study of asexual organisms therefore may provide clues to answer why almost all eukaryotes reproduce via meiosis and syngamy and why asexual eukaryotes are almost always evolutionarily short-lived. Towards understanding the evolution of asexual lineages in the Hymenoptera, I first review the diversity of reproductive systems in the Hymenoptera, introduce the study organism, the fungus-gardening ant Mycocepurus smithii, and discuss my research objectives. Second, I integrate information from reproductive physiology, reproductive morphology, natural history and behavior, to document that that queens of M. smithii are capable of thelytokous parthenogenesis, workers are sterile, and males are absent from the surveyed population. These results suggest that M. smithii might be obligately asexual. To place the origin and maintenance of asexual reproduction in M. smithii in an evolutionary context, I use molecular phylogenetic and population-genetic methods to (i) test if M. smithii reproduces asexually throughout its distribution range; (ii) infer if asexuality evolved once or multiple times; (iii) date the origin of asexual reproduction in M. smithii; and (iv) elucidate the cytogenetic mechanism of thelytokous parthenogenesis. During field collecting for these studies throughout the Neotropics, I discovered a new species of obligate social parasite in the genus Mycocepurus. Social parasites are of great interest to evolutionary biology in order to elucidate mechanisms demonstrating how parasites gained reproductive isolation from their host species in sympatry. I describe this new parasite species, characterize its morphological and behavioral adaptations to the parasitic lifestyle, and discuss the parasite’s life history evolution in the context of social parasitism in fungus-growing ants. The dissertation research integrates population-genetic, phylogenetic, physiological and morphological approaches to advance our understanding of the evolution of reproductive systems and diversity of life-history traits in animals. / text

Revised 02/2015; Originally published: 2000. / The most important disease of woody dicotyledonous plants in Arizona is Phymatotrichopsis root rot (Cotton or Texas root rot) caused by a unique and widely distributed soil-borne fungus, Phymatotrichopsis omnivora. The fungus is indigenous to the alkaline, low-organic matter soils of the southwestern United States and central and northern Mexico.