A thesis submitted to the school of graduate studies in the partial fulfillment of the requirements for the degree doctor of philosophy. / Fungal populations occupy a vast number of ecological niches across many geographic areas around the planet. Fungi act as essential nutrient recyclers, playing key roles as saprophytes, mutualists, and pathogens. As humans, we use these broad properties of fungi in biochemical and pharmaceutical industries, creating a plethora of products ranging from antimicrobials to food products. However, certain fungal species have become a devastating burden on human public health. Of these fungal species, my PhD thesis has focused on the critically important mold Aspergillus fumigatus. This mold is an opportunistic human pathogen, being the leading etiological cause of the spectrum of diseases termed aspergillosis that yearly affects over 8,000,000 people worldwide. In addition, the rising number of antifungal resistant strains around the world, especially within environmental populations, is of critical concern. Given that almost all aspergillosis infections result from environmental strains, and that soil is a major ecological niche for A. fumigatus, my thesis focused on characterizing genetic and phenotypic aspects of soil isolates of A. fumigatus obtained from many geographic and climatic regions around the world. My analyses revealed extensive allelic and genotypic diversity within and among populations. These A. fumigatus populations were defined by both historical differentiations, high gene flow, non-random recombination, and high susceptibility to triazole antifungals. Additionally, I tested the sexual fecundity of a subset of these global strains and found that geographic and genetic distance between the pairs of parental strains had little effect on sexual fecundity. Lastly, my research found broad variations in growth of a global sample of A. fumigatus strains at different temperatures. Again, no relationship of either geographic or genetic distance on strain growth was observed. Overall, my research highlights the extraordinary nature of A. fumigatus populations to quickly spread and adapt across diverse and complex environments. / Thesis / Doctor of Philosophy (PhD) / Aspergillus fumigatus is a cosmopolitan mold that causes opportunistic infections in humans termed aspergillosis. To better understand the environmental reservoirs of aspergillosis infection, I investigated soil populations of this fungus, as soil is likely the largest reservoir of A. fumigatus. I isolated A. fumigatus strains from 11 countries across 6 continents and genetically compared these soil populations to each other and to clinical A. fumigatus populations. I found extensive genetic diversity within most local soil populations, along with different relationships among geographic populations. When a sample of these global strains were sexually crossed, I uncovered high variation in their sexual fecundity, which lowered at higher geographic distances. Lastly, strains exhibited high variations in growth at different temperatures regardless of climatic, genetic, and geographic factors from where they were isolated. My thesis highlights the extraordinary phenotypic variations and complex population structure of A. fumigatus populations isolated from soil across the globe.

No description available.

In unserer Studie sollte die Genauigkeit der PCR zur Bestimmung von Zytokinspiegeln ermittelt werden. Mittels Blutproben von mit A. Fumigatus infizierten Mäusen, sollte eine Aussage bezüglich der Immunantwort getroffen werden. Wir griffen TNFα, IL-12p40 und IL-10 heraus, um einschätzen zu können, ob die Immunantwort eher humoral oder zellvermittelt abläuft. Zur möglichen Bestimmung der Sensitivität und Genauigkeit, wurden die crossing points der Standardverdünnungsreihen jeweils einmal in einem Lauf dreifach, ausserdem jeweils in drei unabhängigen Läufen von einander einfach eingesetzt, und miteinander verglichen. Unsere Ergebnisse decken sich mit den Ergebnissen aktueller Literatur und Etablierungen anderer Zytokine. Die Etablierung des ELISAs sollte dem Vergleich zwischen mRNA-Ebene und Proteinebene dienen. Zur richtigen Einordnung unserer Arbeiten mit dem Immunoassay müssen die Limitierungen der Ergebnisse beachtet werden. Die Versuche zur Quantifizierung der mRNA murinen TNFαs aus den Versuchsserien misslang. Auch die erzielten Ergebnisse mit Protein-basierten Nachweisverfahren konnten letztendlich nicht suffizient beurteilt werden. Die großen Schwankungen in der Konzentration und die Widersprüchlichkeit im Vergleich der Ergebnisse aktueller Literatur, machen eine Verfälschung durch Kontamination mit Proteinen aus lysierten Zellen sehr wahrscheinlich. Die erzielten Ergebnisse der RT-PCR anhand der Inter- und Intra-Assay- Vergleiche jedoch können nachfolgenden Projekten dazu dienen, hauptsächlich das Instrument LightCycler in seiner Sensitivität und Genauigkeit einschätzen zu können, und so die ermittelten Daten besser verarbeiten zu können. / In this study we analysed the accuracy of PCR for identification of murine cytokine levels. Using blood samples of with A. Fumigatus infected mice, we wanted to reach a conclusion concerning the immune reaction. We picked TNFα, IL-12p40 and IL-10 to decide if the immune reaction is humoral or cell mediated. For determination of sensitivity and accuracy we compared the crossing points of the dilution standard series. We used the standard series once three times in one run and on the other hand in three independent runs. Our results correspond with the results in current literature. The establishment of the ELISA should have served for comparing the mRNA level with the protein level. But the tests failed. We couldn´t find mRNA of murine TNFα, and there was big variability in the concentration of protein. Probably the falsification happend because of the contamination with proteins from lysis of cells. But the conclusions from the RT-PCR inter- and intra-assay comparison could be helpful to assess the LightCycler instrument in its sensitivity and its accuracy and so facilitate the assessment of the results.

No description available.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-08-02T20:48:43Z No. of bitstreams: 2 Dissertacao_DimorfismoSexualTamanho.pdf: 143594477 bytes, checksum: 84a6bb6c410ca1c3fe5b6e69b41d397d (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-08-16T14:40:54Z (GMT) No. of bitstreams: 2 Dissertacao_DimorfismoSexualTamanho.pdf: 143594477 bytes, checksum: 84a6bb6c410ca1c3fe5b6e69b41d397d (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2013-08-16T14:40:54Z (GMT). No. of bitstreams: 2 Dissertacao_DimorfismoSexualTamanho.pdf: 143594477 bytes, checksum: 84a6bb6c410ca1c3fe5b6e69b41d397d (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 1997 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Três espécies de sabiás se substituem ecologicamente nas florestas primárias e secundárias na Amazônia Oriental: Turdus albicollis, T. fumigatus e T. leucomelas . Estas três espécies são monocromáticas, isto é, machos e fêmeas possuem plumagem semelhante. O que não se conhecia é se estas espécies são também monomórficas, isto é, se machos e fêmeas possuem tamanho igual. Estudos nas florestas mexicanas indicam que algumas aves monocromáticas Neotropicais são de fato cripticamente dimórficas, ou seja, machos e fêmeas diferem estatisticamente em tamanho quando técnicas estatísticas apropriadas são usadas. Este trabalho teve três objetivos principais: (a) avaliar o padrão de dimorfismo sexual quanto ao tamanho em T. albicollis phaeopygus, T. fumigatus fumigatus e T. leucomelas albiventer; (b) contribuir para o estudo do dimorfismo sexual quanto ao tamanho em aves monocromáticas Neotropicais e (c) fornecer subsídios para o estudo ecológico-evolutivo do gênero Turdus , em particular, e da família Turdidae, em geral. A hipótese de trabalho era que as três espécies de Turdus analisadas seriam cripticamente dimórficas, tais como os outros passeriformes florestais estudados nas florestas mexicanas. Concluiu-se que das três espécies estudadas, duas são monomórficas ( T. f. fumigatus e T. a. phaeopygus ) e uma é cripticamente dimórfica ( T. l. albiventer ). Na única espécie cripticamente dimórfica, machos diferem significativamente das fêmeas quanto ao comprimento da asa, cauda, tarso e unha do quarto dedo. Mesmo assim, a função linear discriminante gerada, não permite uma sexagem segura dos espécimes. A razão de as três espécies de Turdus mostrarem-se monomórficas ou cripticamente dimórficas talvez esteja associada ao seu comportamento pré-reprodutivo. Durante o período de acasalamento, a vocalização seria um instrumento mais importante de atração de fêmeas e determinação do território do que a plumagem ou o tamanho. Assim, existiria forte pressão seletiva sobre a vocalização dos machos é fraca ou inexistente pressão seletiva sobre o tamanho do corpo. Sugere-se a realização de mais estudos de dimorfismo sexual em outras espécies de Turdus e de análise filogenética deste gênero, para se esclarecer a evolução dos padrões de dimorfismo sexual em sabiás. / Three species of Brazilian thrushes replace one another ecologlcally along the primary and secondary forests of the Eastern Amazonian Region, Turdus T fumigatus and T. leucomelas. These three species are monochromatic, i. e., me and female have similar plumages. Whether these species are monomorphic (i.e., if males and females are of similar size) or not has not been previously investigated. Studies in Mexican forests indicated that some monochromatic birds from the Neotropical Region are in fact cryptically dimorphic, i. e., males and females differ statistically in size when suitable statistic techniques are appiled. This work has three main objectives: (a) to evaluate the pattern of sexual dimorphism in size in T. albicollis phaeopygus, T. fumigatus fumigatus and T. ieucomelas albiventer, (b) to contribute to the study of the sexual dimorphism in size of Neotropical monochromatic birds, and (c) to provide subsidies for evolutionary and ecological studies on the genus Turdus, and also on the family Turdidae as a whole. The working hypothesis here was the three species of Turdus studied would be cryptically dimorphic in a pattern similar to the passeriform forest birds previously studied in the Mexican forests. Of the three species studied, two were found to be monomorphic (T. f fumigatus and T. a. phaeopygus) and one cryptically dimorphic (T. 1. albiventer). In the only cryptically dimorphic species, males differ significantly from females in the length of the wing, tad, tarsus and fourth toe claw. However, a reliable sexual identification cannot be performed from the discriminant linear function obtained. The reason the three species of Turdus are monomorphic or cryptically dimorphic may be associated with their pre-reproductive behavior. During the mating season. vocalization seems to be more important to attract females and for territorial defense than plumage or size. Thus, there is a strong selective pressure for vocalization of males and weak or non-existent pressure for body size. It is suggested that more research for the evaluation of sexual dimorphism in other species of Turdus and. a phylogenetic analysis of this large genus are indispensable in clarifying the evolution of patterns of sexual dimorphism in thrushes.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Apesar do desenvolvimento de novas drogas antifúngicas e da sua utilização como terapia profilática visando à prevenção de infecções fúngicas invasivas, estas ainda constituem-se num problema emergente, com elevadas taxas de mortalidade. Neste contexto, destaca-se a aspergilose invasiva, uma infecção fúngica oportunista que acomete pacientes com neutropenia profunda e prolongada, principalmente os pacientes com leucemia aguda ou submetidos a transplante de medula óssea. Aspergillus fumigatus, um fungo filamentoso, é o principal agente etiológico da aspergilose invasiva, sendo um patógeno angioinvasivo. As hifas deste fungo são capazes de causar injúria e ativação endotelial, induzindo o endotélio a um fenótipo pró-trombótico, que por sua vez, é mediado pela secreção de citocinas pró-inflamatórias, em especial, o TNF-α. O presente trabalho teve como objetivo estudar a capacidade de cepas mutantes de A. fumigatus em ativar células endoteliais, avaliando o perfil de secreção de citocinas em meio condicionado e a expressão de fator tecidual. Resumidamente, monocamadas confluentes de células endoteliais isoladas da veia umbilical humana foram incubadas com conídios e tubos germinativos de cepas selvagens (Af293 e Ku80) e mutantes (Δugm1, ΔcalA, ΔcrzA, ΔprtT) de A. fumigatus. A taxa de adesão e endocitose destas cepas às monocamadas de HUVEC foi avaliada a partir de um ensaio quantitativo de imunofluorescência diferencial. O perfil cinético de secreção de citocinas foi determinado em meio condicionado das HUVECs, por ensaio de multiplex para IL-6, IL-8 e TNF-α. A ativação endotelial, por sua vez, foi determinada pela expressão de fator tecidual por RT-PCR em tempo real. Os resultados obtidos demonstraram que a mutante para o gene ugm1, responsável por codificar a enzima UDP-galactopiranose mutase, que converte resíduos de galactopiranose a galactofuranose, apresentou um fenótipo hiperaderente às células endoteliais e um estímulo 10 vezes maior à secreção de TNF-α e 2,5 vezes maior a secreção de IL-6, quando comparada a ativação observada para as cepas selvagens. A galactofuranose é um componente importante de glicoconjugados da parede celular de A. fumigatus. Dessa forma, a ausência desse monossacarídeo na célula fúngica leva a um mecanismo compensatório caracterizado por um aumento na expressão de moléculas de galactosaminogalactana na parede celular. De maneira contrária, mutantes para os genes calA e crzA, apresentaram um fenótipo hipoaderente às HUVECs e uma perda na capacidade de induzir a secreção de citocinas e ativar o endotélio. Essas mutantes apresentam deleções que interferem na via de cálcio/calcineurina, responsável por regular a morfogênese e virulência de A. fumigatus, além de apresentarem alterações no conteúdo de beta-1-3 glucana. Já a cepa ΔprtT, mutante para o fator de transcrição prtT que regula a secreção de múltiplas proteases, apresentou um fenótipo de adesão, estímulo e ativação endotelial semelhante ao observado para as cepas selvagens. A comparação entre a capacidade de conídios e tubos germinativos em ativar células endoteliais, corroborou achados anteriores da literatura que reportam que só hifas são capazes de ativar células endoteliais, independentemente da sua viabilidade. Os dados deste estudo permitiram concluir que dentre os componentes de superfície celular de A. fumigatus, os polímeros de galactose, em especial a galactosaminogalactana, parecem ser responsáveis, pelo menos em parte, pelos mecanismos de interação e ativação endotelial. / Besides the emergency of more active and less toxic antifungal agents and the conventional use of antifungal prophylaxis, invasive mold infections have still high mortality rates, especially, invasive aspergillosis (IA). This life-threatening disease is a predominant fungal opportunistic infection for patients with long-term neutropenia, mostly HSCT recipients. Aspergillus fumigatus is the most important species causing IA and is already known as an angioinvasive fungal pathogen. Upon filamentation this fungus can damage and activate human vein endothelial cells (HUVEC) which in turn switch to a pro-thrombotic phenotype. HUVEC activation is known to be mediated by TNF-α once cell-cell contact occurs. The aim of this study was to investigate the endothelial activation ability of several mutants of A. fumigatus. Briefly, HUVECs were infected with germ tubes and conidia of A. fumigatus wild type (WT) Af293 and Ku80 and mutant (Δugm1, ΔcalA, ΔcrzA, ΔprtT) strains and a differential quantitative fluorescence assay performed to determine adhesion and internalization rates. Thus, a kinetic study of secreted pro-inflammatory cytokines and chemokines in HUVEC conditioned medium was achieved by a multiplex immuneassay. The cytokine production was assayed at three time points (4, 8 and 16 hours) using dead germ tubes, and at one time point (16 hours) using dead conidia, for an E:T ratio of 2:1. Additionally, to investigate endothelial activated phenotype, the expression of tissue factor was performed by a real time RT-PCR assay. The ugm1 mutant, which lacks the ugm1 gene encoding UDP-galactopyranose mutase, an enzyme responsible for the conversion of galactopyranose in galactofuranose, showed a hiperadherent phenotype and an increased capability to cause endothelial cell stimulation and activation. This mutant stimulated at least a 10-fold and 2.5-fold increase of TNF-alfa and IL-6 secretion, respectively and 2-fold increase of tissue factor expression by host cells, as compared to WT strains. Galactofuranose is an uncommon 5-membered ring form of galactose and a very important component of glycostructures of the A. fumigatus cell wall. The lack of this monosaccharide in the fungal cell wall leads to a compensatory mechanism characterized by an increment in the galactosaminogalactan expression. In contrast, the calA and crzA mutants, with upstream and downstream dysfunction in the calcium/calcineurin pathway, with alteration in the cell wall β-1,3-glucan content, showed a decrease capacity to adhere to HUVECs and did not induce both the secretion of pró-inflammatory cytokine and activation on endothelial cells. Furthermore, the mutant ΔprtT, witch lacks the transcriptional factor (prtT) that regulates the secretion of multiple proteases, showed the same adhesion and endothelial cell activation phenotype as observed for WT strains. As indicated in previous investigations, our data showed that conidia of A. fumigatus werent able to cause the same endothelial cell cytokine stimulation observed for germ tubes, and that endothelial cell activation is independent on fungal cell viability. Finally, its possible to conclude that the polymers of galactose in the cell wall of A. fumigatus, especially the galactosaminogalactan molecule, seem to be responsible for the endothelial cell interaction mechanisms and activation.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, uma infecção fúngica oportunista que acomete, principalmente, pacientes de Unidades Hematológicas, como aqueles com neutropenia profunda e prolongada. Após a filamentação este fungo angioinvasivo é capaz de ativar e causar danos em células endoteliais de veia umbilical humana (HUVEC) que passam a expressar um fenótipo pró-trombótico. A ativação destas células, dependente de contato célulacélula, é mediada por TNF-α e caracterizada pela expressão de moléculas próinflamatórias, como citocinas, quimiocinas e moléculas de adesão. Recentemente, nosso grupo comparou a ativação endotelial de HUVECs desafiadas com cepas selvagens e uma cepa mutante para o gene UGM1. Nestes experimentos a cepa mutante Δugm1, que apresenta um fenótipo de maior produção de galactosaminogalactana (GAG) na parede celular, mostrou um fenótipo hiperadesivo e uma capacidade maior de ativar células endoteliais. Entretanto, os receptores e as vias de sinalização envolvidos nesta ativação permanecem desconhecidos. Assim, o objetivo deste trabalho foi verificar as proteínas envolvidas nestes processos através do estudo das proteínas diferencialmente expressas nas HUVECs após a interação com A. fumigatus, usando a técnica proteômica 2D-DIGE. Brevemente, as HUVECs foram infectadas com tubos germinativos da cepa selvagem (AF293) e da cepa Δugm1 de A. fumigatus. Em seguida, as proteínas foram marcadas com diferentes fluorocromos e separadas por eletroforese bidimensional. A análise quantitativa foi realizada utilizando o software DeCyder. Foram identificadas por MS/MS cinco proteínas diferencialmente expressas, incluindo a galectina-1 e a anexina A2, ambas mais expressas após a interação, sendo a primeira ~25% mais expressa após a interação com a mutante Δugm1. Este trabalho propõe que a galectina-1 poderia ser o receptor endotelial para polímeros de galactose presentes na parede celular do A. fumigatus, e que a Anexina A2 poderia estar envolvida na sinalização intracelular em resposta a este patógeno. No entanto, experimentos complementares, em curso, são necessários para comprovar esta hipótese. / Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, the main opportunistic fungal infection of Hematologial Unitys patients, especially those with long-term neutropenia. Upon filamentation, this angioinvasive fungus can activate and damage the human umbilical vein endothelial cells (HUVEC), which in response switch to a pro-thrombotic phenotype. HUVEC activation is mediated by TNF-α once cell-cell contact occurs. This activation is characterized by the expression of pro-inflammatory molecules such cytokines, chemokines and adhesion molecules. Recently, our group performed the comparison of HUVEC activation upon interaction with a wild type and the UGM1 mutant strains of A. fumigatus. The Δugm1 strain, which presents an increased production of the cell wall galactosaminogalactan, showed a hyper adherent phenotype and an increased capability to cause endothelial cell stimulation and activation, when compared with the wild type strain. The receptors involved in the pathogen-host interaction or the signaling pathways after endothelial activation by A. fumigatus remain unknown. Thus, the aim of this study was to investigate the differentially expressed proteins in HUVECs upon interaction with A. fumigatus, using the 2D-DIGE proteomic approach. Briefly, HUVECs were challenged with germlings of A. fumigatus wild type Af293 and Δugm1 strains and then submitted to protein extraction. The total HUVEC protein extracts were labeled with different CyDyes and fractionated by 2D electrophoresis. Quantitative analysis to determine the differences in protein abundance amongst interacted cells vs. control endothelial cells was performed using the software DeCyder. Five differentially expressed proteins were identified by MS/MS including galectin-1 and annexin A2, both overexpressed after the interaction. These two proteins are described elsewhere to be associated with host-pathogen interaction. Besides, galectin-1 showed an ~25% increase after interaction with the Δugm1 strain and it is plausible that this particular protein could be a putative receptor for galactose-containing polymers of the A. fumigatus cell wall and annexin A2 could be involved in signalizing pathways upon interaction. However, other experimental evidences, under development, are necessary to confirm this hypothesis.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-16Bitstream added on 2014-06-13T19:14:40Z : No. of bitstreams: 1 silva_rr_me_sjrp.pdf: 961028 bytes, checksum: 0d7989443c42fd25745d208364a42a88 (MD5) / A produção de proteases de origem microbiana depende das condições de cultivo e da diversidade bioquímica de cada espécie. Estudos comparativos entre fermentação em estado sólido (FES) e fermentação submersa (FSm) usando farelo de trigo e meio sintético, respectivamente, foram realizados para a determinação dos parâmetros de produção de proteases pelo fungo Aspergillus fumigatus Fresenius. A melhor produção de protease foi em FES no período de 96 horas utilizando farelo de trigo, temperatura de 30 ºC e 1x106 esporos/5g de substrato com 1.517 U/mL. Em FSm o pico de produção foi em pH 6,0, a 30ºC, 5x105 esporos/mL de meio no período de 72 e 96 horas em meio contendo 0,5 e 0,25% de caseína, respectivamente, ambos com 40 U/mL. Conforme a produtividade dos processos fermentativos, o extrato enzimático da FES foi utilizado para estudos de purificação e caracterização bioquímica. Neste estudo, a protease purificada apresentou atividade ótima em pH 7,5 e 50ºC, sendo inibida por Fenil-metil-sulfonil-fluoreto (PMSF) e mais intensamente por antipaína (1,6 µM). Sobre efeito de íons, foi observado modulação da atividade proteolítica, principalmente com inibição por AlCl3, cuja atividade proteolítica residual foi de 18% após incubação com este íon. Na presença de Ditiotreitol (DTT) e uréia houve diminuição da atividade proteolítica, apresentando atividades residuais de 63% em 200 mM de DTT e 10% com 5 M de uréia. Comparativamente, na concentração de 0,1% de cada surfactante estudado, notou-se redução da atividade proteolítica, sendo 97% em presença de Brometo de cetil-trimetil amônio (CTAB), 79% para 4 - (1,1,3,3 - Tetrametilbutil) fenil- polietileno glicol (Triton X-100), 55% com Polyoxyethylenesorbitan monolaurato (Tween-20) e completa redução da atividade (0%) em... / The microbial protease production depends on growing conditions and the biochemical diversity of each species. Comparative studies between solid-state fermentation (SSF) and submerged fermentation (SmF) using wheat bran and synthetic medium, respectively, were performed to determine the optimum parameters for protease production by the fungus Aspergillus fumigatus Fresenius. The best protease production was in SSF within 96 hours using wheat bran, temperature 30°C and 1x106 spores/5g of substrate, with 1,517 U/mL. In SmF peak production was at pH 6.0 at 30°C, 5x105 spores/mL of media within 72 and 96 hours in medium containing 0.5 and 0.25% casein, respectively, with 40 U/mL. According to the productivity of the fermentative processes, enzymatic extract was used from SSF to study purification and biochemical characterization. In this study, purified protease showed optimum activity at pH 7.5 and 50°C, and inhibited by Phenylmethylsulfonyl fluoride (PMSF) and more intensely for antipain (1,6 µM). Concerning to the effect of ions, we observed modulation of the proteolytic activity, especially with inhibition by AlCl3, which residual activity was of 18 % after incubation with this ion. In the presence of Dithiothreitol (DTT) and ureia, we observed progressive decrease in proteolytic activity, presenting residual activities of 63% with 200 mM DTT, and 10% with 5 M ureia. Comparatively, in the concentration of 0.1% of each surfactant studied, there was a reduction in the proteolytic activity in 97% in presence of Cetyl trimethylammonium bromide (CTAB), 79% with 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), 55% with Polyoxyethylenesorbitan monolaurate (Tween-20) and a complete inactivation in the presence of Sodium dodecyl sulfate... (Complete abstract click electronic access below)

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Apesar do desenvolvimento de novas drogas antifúngicas e da sua utilização como terapia profilática visando à prevenção de infecções fúngicas invasivas, estas ainda constituem-se num problema emergente, com elevadas taxas de mortalidade. Neste contexto, destaca-se a aspergilose invasiva, uma infecção fúngica oportunista que acomete pacientes com neutropenia profunda e prolongada, principalmente os pacientes com leucemia aguda ou submetidos a transplante de medula óssea. Aspergillus fumigatus, um fungo filamentoso, é o principal agente etiológico da aspergilose invasiva, sendo um patógeno angioinvasivo. As hifas deste fungo são capazes de causar injúria e ativação endotelial, induzindo o endotélio a um fenótipo pró-trombótico, que por sua vez, é mediado pela secreção de citocinas pró-inflamatórias, em especial, o TNF-α. O presente trabalho teve como objetivo estudar a capacidade de cepas mutantes de A. fumigatus em ativar células endoteliais, avaliando o perfil de secreção de citocinas em meio condicionado e a expressão de fator tecidual. Resumidamente, monocamadas confluentes de células endoteliais isoladas da veia umbilical humana foram incubadas com conídios e tubos germinativos de cepas selvagens (Af293 e Ku80) e mutantes (Δugm1, ΔcalA, ΔcrzA, ΔprtT) de A. fumigatus. A taxa de adesão e endocitose destas cepas às monocamadas de HUVEC foi avaliada a partir de um ensaio quantitativo de imunofluorescência diferencial. O perfil cinético de secreção de citocinas foi determinado em meio condicionado das HUVECs, por ensaio de multiplex para IL-6, IL-8 e TNF-α. A ativação endotelial, por sua vez, foi determinada pela expressão de fator tecidual por RT-PCR em tempo real. Os resultados obtidos demonstraram que a mutante para o gene ugm1, responsável por codificar a enzima UDP-galactopiranose mutase, que converte resíduos de galactopiranose a galactofuranose, apresentou um fenótipo hiperaderente às células endoteliais e um estímulo 10 vezes maior à secreção de TNF-α e 2,5 vezes maior a secreção de IL-6, quando comparada a ativação observada para as cepas selvagens. A galactofuranose é um componente importante de glicoconjugados da parede celular de A. fumigatus. Dessa forma, a ausência desse monossacarídeo na célula fúngica leva a um mecanismo compensatório caracterizado por um aumento na expressão de moléculas de galactosaminogalactana na parede celular. De maneira contrária, mutantes para os genes calA e crzA, apresentaram um fenótipo hipoaderente às HUVECs e uma perda na capacidade de induzir a secreção de citocinas e ativar o endotélio. Essas mutantes apresentam deleções que interferem na via de cálcio/calcineurina, responsável por regular a morfogênese e virulência de A. fumigatus, além de apresentarem alterações no conteúdo de beta-1-3 glucana. Já a cepa ΔprtT, mutante para o fator de transcrição prtT que regula a secreção de múltiplas proteases, apresentou um fenótipo de adesão, estímulo e ativação endotelial semelhante ao observado para as cepas selvagens. A comparação entre a capacidade de conídios e tubos germinativos em ativar células endoteliais, corroborou achados anteriores da literatura que reportam que só hifas são capazes de ativar células endoteliais, independentemente da sua viabilidade. Os dados deste estudo permitiram concluir que dentre os componentes de superfície celular de A. fumigatus, os polímeros de galactose, em especial a galactosaminogalactana, parecem ser responsáveis, pelo menos em parte, pelos mecanismos de interação e ativação endotelial. / Besides the emergency of more active and less toxic antifungal agents and the conventional use of antifungal prophylaxis, invasive mold infections have still high mortality rates, especially, invasive aspergillosis (IA). This life-threatening disease is a predominant fungal opportunistic infection for patients with long-term neutropenia, mostly HSCT recipients. Aspergillus fumigatus is the most important species causing IA and is already known as an angioinvasive fungal pathogen. Upon filamentation this fungus can damage and activate human vein endothelial cells (HUVEC) which in turn switch to a pro-thrombotic phenotype. HUVEC activation is known to be mediated by TNF-α once cell-cell contact occurs. The aim of this study was to investigate the endothelial activation ability of several mutants of A. fumigatus. Briefly, HUVECs were infected with germ tubes and conidia of A. fumigatus wild type (WT) Af293 and Ku80 and mutant (Δugm1, ΔcalA, ΔcrzA, ΔprtT) strains and a differential quantitative fluorescence assay performed to determine adhesion and internalization rates. Thus, a kinetic study of secreted pro-inflammatory cytokines and chemokines in HUVEC conditioned medium was achieved by a multiplex immuneassay. The cytokine production was assayed at three time points (4, 8 and 16 hours) using dead germ tubes, and at one time point (16 hours) using dead conidia, for an E:T ratio of 2:1. Additionally, to investigate endothelial activated phenotype, the expression of tissue factor was performed by a real time RT-PCR assay. The ugm1 mutant, which lacks the ugm1 gene encoding UDP-galactopyranose mutase, an enzyme responsible for the conversion of galactopyranose in galactofuranose, showed a hiperadherent phenotype and an increased capability to cause endothelial cell stimulation and activation. This mutant stimulated at least a 10-fold and 2.5-fold increase of TNF-alfa and IL-6 secretion, respectively and 2-fold increase of tissue factor expression by host cells, as compared to WT strains. Galactofuranose is an uncommon 5-membered ring form of galactose and a very important component of glycostructures of the A. fumigatus cell wall. The lack of this monosaccharide in the fungal cell wall leads to a compensatory mechanism characterized by an increment in the galactosaminogalactan expression. In contrast, the calA and crzA mutants, with upstream and downstream dysfunction in the calcium/calcineurin pathway, with alteration in the cell wall β-1,3-glucan content, showed a decrease capacity to adhere to HUVECs and did not induce both the secretion of pró-inflammatory cytokine and activation on endothelial cells. Furthermore, the mutant ΔprtT, witch lacks the transcriptional factor (prtT) that regulates the secretion of multiple proteases, showed the same adhesion and endothelial cell activation phenotype as observed for WT strains. As indicated in previous investigations, our data showed that conidia of A. fumigatus werent able to cause the same endothelial cell cytokine stimulation observed for germ tubes, and that endothelial cell activation is independent on fungal cell viability. Finally, its possible to conclude that the polymers of galactose in the cell wall of A. fumigatus, especially the galactosaminogalactan molecule, seem to be responsible for the endothelial cell interaction mechanisms and activation.