Der Schimmelpilz Aspergillus (A.) fumigatus stellt den häufigsten Erreger der invasiven Aspergillose (IA) dar, die vor allem bei immunsupprimierten Patienten auftritt. Unter den unspezifischen klinischen Symptomen dieser Erkrankung ist Fieber das häufigste. Dennoch wurden physiologische Aspekte wie eine erhöhte Körpertemperatur in Arbei-ten zur Interaktion menschlicher Immunzellen mit A. fumigatus bisher nicht berück-sichtigt. Zahlreiche Studien konnten den Einfluss einer erhöhten Temperatur auf den Verlauf von Infektionserkrankungen in vivo sowie auf die Funktionen verschiedener Immunzellen – einschließlich dendritischer Zellen (DCs) – in vitro zeigen. DCs spielen eine wichtige Rolle in der Immunabwehr gegenüber A. fumigatus, ihre besondere Be-deutung liegt in der Verknüpfung der angeborenen mit der erworben Immunantwort. Ziel dieser Arbeit war die in vitro Analyse des Einflusses einer erhöhten Temperatur auf die Immunantwort humaner DCs gegenüber A. fumigatus. Dazu wurden DCs mit A. fumigatus oder Zymosan, einem ß-1,3-Glucan, bei Normo- (37 °C) und Hyperthermie (40 °C) für bis zu 24 h inkubiert und spezifische DC-Funktionen charakterisiert. Hierbei tolerierten DCs die Inkubation und Stimulation unter Hyperthermie ohne signifikanten Viabilitätsverlust. Die Zytokinexpression und -sekretion durch A. fumigatus-Stimulation wurde durch Hyperthermie nicht signifikant verändert. Die Fähigkeit zur Aufnahme von A. fumigatus-Konidien wurde durch eine kurzzeitige (1 h) Hyperthermie nicht beein-flusst, längerfristige (24 h) Hyperthermie reduzierte diese Fähigkeit jedoch signifikant. Ebenso bestand unter Hyperthermie eine verstärkte Expression von CD86 und HLA-DR auf unstimulierten DCs sowie von CD80, CD86 und HLA-DR auf stimulierten DCs. Die reduzierte Aufnahmekapazität für A. fumigatus-Konidien und die verstärkte Expression der kostimulatorischen Moleküle unter Hyperthermie zeigten, dass Hyper-thermie in vitro einen reiferen Phänotyp unstimulierter DCs bewirkt sowie die DC-Reifung durch A. fumigatus-Stimulation verstärken kann. Diese reiferen DCs könnten zu einer verbesserten T-Zell-Aktivierung und Abwehr von A. fumigatus und zu einem verbesserten Outcome der IA beitragen. Außerdem könnte Hyperthermie als Adjuvans zur in vitro Generierung A. fumigatus-spezifischer DCs eingesetzt werden. / In immunocompromised patients, invasive aspergillosis (IA) is the most frequent disease caused by the pathogenic mould Aspergillus (A.) fumigatus. Fever is one of the most common yet nonspecific clinical symptoms of IA. However, physiological aspects such as febrile body temperature have never been taken into account in studies investigating the interaction between human immune cells and A. fumigatus in vitro. It has been shown that elevated body temperatures can influence the course and out-come of infectious diseases in vivo as well as the functions of different immune cells in vitro, including dendritic cells (DCs). DCs play an important role in the immune response to A. fumigatus, as they act as a bridge between the innate and adaptive immune system. In order to determine the influence of elevated body temperature during IA, I investigated the effect of hyperthermia on human DCs confronted with A. fumigatus in vitro. DCs were stimulated with germ tubes of A. fumigatus or the fungal cell wall component zymosan, a ß-1,3-glucan, at 37 °C or 40 °C for up to 24 h, followed by characterization of specific DC functions. My results demonstrate that DC viability was maintained under hyperthermia. Expression and secretion of cytokines by DCs following A. fumigatus-stimulation were not influenced by hyperthermia. Short-time hyperthermia did not affect antigen uptake, however, long-time hyperthermia reduced uptake of A. fumigatus-conidia by DCs significantly. Hyperthermia enhanced CD86 and HLA-DR expression on unstimulated DCs as well as CD80, CD86 and HLA-DR expression on A. fumigatus-stimulated DCs. As the expression of costimulatory molecules on DCs was enhanced and uptake of A. fumigatus-conidia by DCs was reduced at 40°C, hyperthermia might cause a more mature phenotype in unstimulated DCs and enhance DC-maturation caused by A. fumi-gatus-stimulation. This could contribute to an increased T-cell-activation and to an improved A. fumigatus-defense and might influence the course and outcome of IA. Moreover, hyperthermia might even be a useful adjuvant in the in vitro generation of A. fumigatus-specific DCs for immunotherapy.

Pilzinfektionen zählen zu den häufigsten Infektionen beim Menschen. Sie verlaufen in den meisten Fällen unkompliziert und stellen keine vitale Bedrohung für den Betroffenen dar. Invasive Mykosen hingegen verlaufen oft tödlich und sind eine große Herausforderung für die moderne Medizin, da eine frühe Diagnose schwierig ist und die therapeutischen Möglichkeiten limitiert sind. Die Invasive Aspergillose (IA) zählt mit geschätzt über 200.000 Infektionen pro Jahr weltweit zu einer der häufigsten Invasiven Mykosen. Die bekanntesten Risikofaktoren für die Entstehung einer IA sind die Neutropenie, Organtransplantationen, hämatopoetische Stammzelltransplantationen und Erkrankungen, die mit einer Kompromittierung des Immunsystems einhergehen. Erreger der Invasiven Aspergillose ist in nahezu 90 Prozent Aspergillus fumigatus (A. fumigatus), ein ubiquitär vorkommender Schimmelpilz. Seine Verbreitung erfolgt aerogen durch Sporen, sogenannte Konidien, die aufgrund ihres geringen Durchmessers problemlos über die Atemwege in die Lunge gelangen können. Dendritische Zellen spielen als professionelle Antigen präsentierende Zellen eine wichtige Rolle in der Immunabwehr gegen A. fumigatus. Sie sind ein wichtiges Bindeglied zwischen angeborenem und adaptivem Immunsystem und sind mit einer Vielzahl von Rezeptoren (engl. pattern recognition receptor, PRR) zur Pathogen Erkennung ausgestattet. In der vorliegenden Arbeit wurde die Interaktion ausgewählter C Typ Lektin (CLEC) Rezeptoren auf Subtypen dendritischer Zellen (DCs) mit verschiedenen A. fumigtaus Morphologien untersucht. Es wurde mit in vitro generierten Monozyten abgeleiteten (moDCs) und in vivo vorkommenden myeloiden dendritischen Zellen (mDCs) gearbeitet und die Expression von CLEC4A, CLEC6A, CLEC7A, CLEC12A und CLEC4E und eine mögliche Regulation der Rezeptoren nach Stimulation mit Konidien, geschwollenen Konidien oder Keimschläuchen untersucht. Hierbei wurde bei beiden Subtypen eine Herabregulation von CLEC4A, CLEC7A und CLEC12A beobachtet. Dies ist vereinbar mit der Tatsache, dass C Typ Lektin Rezeptoren nicht nur eine Rolle bei der Pathogen Erkennung spielen, sondern auch als Phagozytose Rezeptoren fungieren. Auf molekularbiologischer Ebene wurde in Analysen von moDCs ebenfalls eine Reduktion der relativen mRNA Expression von CLEC4A, CLEC6A, CLEC7A und CLEC12A beobachtet. Weiterhin wurden die Auswirkungen einer Rezeptorblockade von CLEC7A mittels blockierender Antikörper auf das Maturierungsverhalten und Zytokinprofil beider Subtypen analysiert. Hier konnte durch die Zugabe eines CLEC7A blockierenden Antikörpers vor Stimulation mit A. fumigatus Konidien oder depletiertem Zymosan die Maturierung effektiv inhibiert werden. Die Sekretion der pro inflammatorischen Zytokine Tumornekrosefaktor α, Interleukin 8 und 1β, als auch des anti inflammatorischen Zytokins Interleukin 10 war durch die Rezeptorblockade ebenfalls signifikant vermindert. Diese Erkenntnisse stützen die bislang relativ gut untersuchte Rolle von CLEC7A auf Monozyten abgeleiteten dendritischen Zellen als spezifischen Rezeptor für A. fumigatus. Darüber hinaus konnte im Rahmen dieser Arbeit gezeigt werden, dass CLEC7A ebenfalls auf mDCs an der Erkennung von A. fumigatus und Initiierung einer Immunantwort beteiligt ist. Diese Tatsache ist von Bedeutung, da die beiden Subtypen nicht ohne weiteres miteinander verglichen werden können und die Relevanz von in vivo vorkommen myeloiden dendritischen Zellen an einer Immunantwort gegen A. fumigatus bislang noch viele Fragen offen lässt. Es bedarf weiterer Untersuchungen, insbesondere funktionaler Analysen von intrazellulären Signalwegen um ein besseres Verständnis zu erlangen. Die Übertragung in ein Tiermodell und die gezielte Ausschaltung von C Typ Lektin Rezeptor Genen könnte ein Ausblick auf zukünftige Forschungsprojekte sein. / Fungal infections are among the most common infections in humans. Most of them are minor superficial infections whereas invasive fungal infections are associated with high mortality rates due to difficulties in diagnosis and limited therpeutic options. Invasive aspergillosis (IA) is one of the most significant invasive fungal infection with 200.000 estimated life-threatening infections per year worldwide. Known risk factors are neutropenia, solid organ transplantation, haematopoetic stem cell transplantation and other immunosuppressive conditions. IA is caused primarily by Aspergillus fumigatus (A. fumigatus), an opportunistic pathogenic mould with a worldwide distribution. Hundreds of airborne spores are inhaled daily into the human lung. Dendritic cells are potent antigen-presenting cells and have an important role in the immune response against A. fumigatus. They are the sentinels of the immune system by bridging innate and adaptive immune responses against pathogens and express a large number of different pattern recognition receptors. This study aimed to analyze the interaction between selected C-type lectin (CLEC) receptors on human dendritic cell subsets and different A. fumigatus morphotypes. Therefore we analyzed the expression and regulation of CLEC4A, CLEC6A, CLEC7A, CLEC12A and CLEC4E on monocyte-derived dendritic cells (moDCs), which were generated in vitro, and myeloid dendritic cells (mDCs), which were directly isolated from peripheral blood. We show that CLEC4A, CLEC7A and CLEC12A are down-regulated in the presence of A. fumigatus on both substes, consistent with their role as pathogen-recognition receptors and phagocytic receptors. mRNA expression of CLEC4A, CLEC6A, CLEC7A and CLEC12A was also down-regulated after confrontation with A. fumigatus. In addition we investigated the effect of blocking CLEC7A, using a specific antibody, on cell maturation and cytokine profiles. Blocking of CLEC7A diminished the expression of maturation markers on both dendritic cell subsets and inhibited cytokine release of pro-inflammatory cytokines TNF-α, IL-8, IL-1β as well as of the anti-inflammatory cytokine IL-10. These findings support the role of CLEC7A on moDCs as specific receptor for A. fumigatus and furthermore confirme that CLEC7A is involved in the recognition of A. fumigatus and inducing immune responses on primary human mDCs. This is of special relevance as both subsets do not necessarily show the same biological behavior, stimulatory capability and pattern recognition receptors and the role of mDCs for initiating immune responses against A. fumigatus leaves many questions unanswered so far. Further investigations, in particular functional analyses of intracellular pathways, are necessary to acquire a deeper knowledge. Transfer to an animal model or targeted gene knock-out of C-type lectin receptors could be next steps in future research.

In this study, novel polyketide biosynthesis gene cluster of Aspergillus fumigatus was characterized and functionally analyzed. Analysis of the newly sequenced A. fumigatus genome for laccases, which are involved in melanin biosynthesis and detoxification in fungi, resulted in several putative laccase and multicopper oxidase gene sequences, one of which, Afu4g14490 (tpnJ), was selected for further characterization. The predicted amino acid sequence TpnJp showed 63% identity with the dihydrogeodin oxidase of Aspergillus terreus, which is involved in the biosynthesis of the antifungal geodin. When the genome region of tpnJ was investigated, the presence of a polyketide biosynthesis gene cluster containing 13 genes, hypothesized to be responsible for the production of trypacidin and monomethylsulochrin, was realized. By a comparative genomics approach, a putative geodin biosynthesis gene cluster containing 13 genes, including dihydrogeodin oxidase, in A. terreus and a putative trypacidin biosynthesis gene cluster containing 13 genes in N. fischeri were established. Targeted deletions of the polyketide synthase (tpnC) and multicopper oxidase (tpnJ) genes confirmed the hypothesis that TpnCp, a three-domain minimal polyketide synthase, is involved in trypacidin and monomethylsulochrin biosynthesis in A. fumigatus. TpnCp is the first fungal minimal polyketide synthase whose functional role was experimentally identified. Moreover, the fact that LC-MS analysis of DtpnJ strain showed the absence of trypacidin and the presence of a higher amount of monomethylsulochrin in DtpnJ strain, confirmed the hypothesis that TpnJp is involved in the oxidation of monomethylsulochrin into trypacidin. This novel multicopper oxidase having high substrate specificity is given the name monomethylsulochrin oxidase.

The aim of this study was to analyze the effect of hydrogen peroxide and selected phenolic compounds on growth and catalase production of Aspergillus fumigatus. As a result of growing A. fumigatus at different temperatures it was observed that, growth and catalase production of this species were highest at 37 &deg / C. Catalase production was highest in the presence of 1 mM H2O2, yielding a significant 3 fold increase with respect to the control. Biomass was also increased by 1,44 fold with respect to the control sample. H2O2 increased catalase production possibly by inducing oxidative stress as biomass production significantly increased after the depletion of H2O2. Both gallic acid and trans-resveratrol significantly enhanced biomass generation of A. fumigatus (1,17 fold increase at 10 mM gallic acid and 1,45 fold increase at 3 mM resveratrol with respect to controls) and decreased extracellular catalase production (4,33 fold at 25 mM gallic acid and 16,7 fold decrease at 3 mM resveratrol with respect to controls) especially in the first 5 or 6 days of the cultivation where the anti-oxidant activity of the compounds were possibly at their maximum. A sudden and significant rise was observed in extracellular catalase activity between 5th and 7th days of the cultivation in phenolic compound applied samples, possibly owing to the depletion of the antioxidant activity of gallic acid and resveratrol followed by fungal cells&rsquo / response to a sudden increase of oxidative stress by boosting catalase production.

This thesis describes the cultivation and extraction of filamentous fungi isolated from extreme environments in the search for new antibiotic compounds. Filamentous fungi are a rich source of medicines including antibiotics, and it is believed that many currently unknown fungal species and bioactive fungal metabolites remain to be discovered. Aspergillus fumigatus and Penicillium nalgiovense strains were isolated from an antibiotic-contaminated riverbed near Hyderabad, India, and soil taken from a penguin’s nest on Paulete Island, Antarctica, respectively. It was anticipated that the extreme conditions within these environments would exert unusual selective pressures on their filamentous fungi, possibly causing the secretion of new bioactive compounds. The cultivation, extraction and analysis of metabolites from the A. fumigatus strain resulted in the isolation of the antimicrobial substance gliotoxin. Subsequent investigations revealed that this strain’s secretion of gliotoxin was increased by as much as 65 % when it was cultivated in the presence of pathogen-associated molecular patterns. These results indicate the existence of a fungal receptor/signaling system for detecting nearby bacteria. The scope for using gliotoxin and the related metabolite bis(methyl)gliotoxin as biomarker metabolites for diagnosing the lethal pulmonary condition invasive aspergillosis was also investigated. Bronchoalveolar lavage fluid from 42 patients with and without possible invasive aspergillosis was extracted and analyzed. The results obtained suggest that gliotoxin and bis(methyl)gliotoxin are not suitable markers for diagnosing invasive aspergillosis. Studies on the P. nalgiovense strain from Antarctica resulted in the isolation of the antifungal agent amphotericin B. The secretion of this compound increased when P. nalgiovense was cultured on a potato-dextrose agar enriched with coconut flakes rather than liquid RPMI 1640 medium. This was the first time amphotericin B was isolated from any organism other than the bacterium Streptomyces nodosus. The results presented in this thesis will be useful in the continuing search for novel bioactive compounds, the diagnosis of fungal infections, and as a source of insight into the interactions between microorganisms. Moreover, they show that even extensively studied fungal genera such as Aspergillus and Penicillium are not completely understood and may produce unexpected or previously unknown bioactive metabolites under appropriate conditions.

Aspergillus fumigatus is a foodborne and airborne human pathogen which produces mycotoxins such as gliotoxin, helvolic acid, fumigallin, and fumigaclavin. The most common disease caused by A. fumigatus is aspergillosis, which is often fatal, especially among AIDS, cancer, and organ-transplant patients. In this study, random cDNA cloning was performed from a cDNA library of A. fumigatus (IMI 385708) constructed on &amp / #955 / ZAP Express. Some of these clones were selected according to their insert sizes and were further subjected to sequence analysis. The sequences were then analyzed by a BLAST search (NCBI Genome Database) to determine the possible functions of these genes. Two of the clones were identified as the primase and 60S ribosomal protein L1-b genes. These genes and a third gene corresponding to the antigenic cell wall galactomannoprotein gene of A. fumigatus were used for the design of three distinct primer pairs. For the primer design, a software was written to differentiate nonconserved regions by multiple sequence alignment. Designed primers were employed in PCR experiments against genomic DNAs of different Aspergillus species. Unique bands were obtained only against A. fumigatus genomic DNA. It was concluded that this PCR-based detection method can be further developed for the rapid detection of A. fumigatus spores from air and food samples.

In this study, molecular cloning studies were performed on the &amp / #945 / -galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable &amp / #945 / -galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results were obtained from these screenings. However, the DNA probes used were shown to be homologous to the &amp / #945 / -galactosidase gene (agl1) of Trichoderma reesei. After the completion of the genome project of A. fumigatus, regions with homology to &amp / #945 / -galactosidase genes were searched on the genome of A. fumigatus. PCR-based cloning studies were performed by designing specific primers for these regions. Two &amp / #945 / -galactosidase genes, namely aglA and aglB were amplified with these specific primers, sequenced, and ligated to vector pUC19. The recombinant plasmid was then used to transform E. coli XL1 Blue MRF&rsquo / cells. aglB gene consists of an open reading frame of 1684 bp containing six introns. The gene encodes a protein of 447 amino acids with a signal sequence of 22 amino acids and four N-glycosylation sites. aglA gene has an open reading frame of 1599 bp without introns. The gene encodes a protein of 532 amino acids with a putative signal sequence of 21 amino acids and four putative N-glycosylation sites. Cloning of &amp / #945 / -galactosidase genes represents a first step towards the high level expression of these enzymes in a GRAS host.

Aspergilose é uma das principais causas de mortalidade em aves imunocompetentes e imunodeprimidas. A manifestação clínica aguda da doença é geralmente observada em aves jovens, com episódios de surtos em aviários, enquanto a forma crônica é mais frequentemente observada em aves adultas. A inspeção das carcaças é fundamental para a detecção e monitoramento da prevalência de doenças. Os objetivos do trabalho foram avaliar a ocorrência de aspergilose causada por Aspergillus fumigatus em aves comerciais através do diagnóstico micológico e histopatológico e verificar a possibilidade de associação causal entre os critérios de diagnóstico de aspergilose e condenação por aerossaculite em frangos de corte através de um estudo de casocontrole. O estudo foi realizado com 380 amostras pulmonares. Foram coletados pulmões de frangos condenados (95) por aerossaculite e não condenados (285), diretamente na linha de abate de um frigorífico. Quarenta e seis (12%) amostras de pulmão foram positivas na cultura micológica. Do total de amostras, 177 (46,6%) apresentaram alterações histopatológicas, sendo as mais frequentes pneumonia fibrinoheterofílica necrótica, pneumonia heterofílica e hiperplasia linfóide. Do total de 380 pulmões analisados, 30 (65,2%) apresentaram concomitantemente alterações histopatológicas e isolamento fúngico. A relação entre a presença de lesões histopatológicas e isolamento de A. fumigatus testada por McNemar indicou que houve associação significativa entre a presença de alterações histopatológicas e o isolamento de A. fumigatus. A identificação molecular foi realizada em 44 isolados, sendo todos confirmados como sendo A. fumigatus através dos genes b-tub e rodA. O cultivo micológico e o exame histopatológico aumentam as chances de se detectar alterações pulmonares em aves condenadas pelo Sistema de Inspeção Final do que nas aves normais, sugerindo que tais critérios de diagnóstico são eficazes para aprimorar a avaliação e condenação de aves por aerossaculite. O perfil genético entre os isolados foi variado, mostrando que isolados de aves normais podem ser potencialmente causadores de aspergilose em aves suceptíveis. / Aspergillosis is one of the main causes of mortality in both immunocompetent and immunodepressed birds. The clinical manifestation of acute aspergillosis is usually observed in young birds, often with episodes of outbreaks in poultry farms, whereas chronic aspergillosis is more frequently observed in adult birds. The inspection of carcasses is fundamental for the detection of diseases and for monitoring their prevalence. The objectives of this study were to evaluate the occurrence of aspergillosis caused by Aspergillus fumigatus in poultry through mycological and histopathological diagnosis and to verify the possibility of a causal association between the criteria for aspergillosis diagnosis and carcass condemnation due to airsacculitis in broilers through a case-control study. The study was made with 380 lung samples. Lungs were collected from condemned (95) and not condemned (285) broilers due to airsacculitis, directly from the slaughter line of a slaughterhouse. Forty-six (12%) lung samples were positives in mycological culture. From the total samples, 177 (46.6%) showed histopathological alterations, the most frequent being necrotic, fibrinous, heterophilic pneumonia, heterophilic pneumonia and lymphoid hyperplasia. Of the 380 lungs analyzed, 30 (65.2%) showed histopathological alterations and isolation of fungi. The relation between the presence of histopathological lesions and the isolation of A. fumigatus, as observed with the McNemar test, indicated the significant association between the presence of histopathological alterations and the isolation of A. fumigatus.The molecular identification was made in 44 isolates, and all of them were confirmed to be A. fumigatus through analysis of the b-tub and rodA. The mycological cultivation and the histopathological test increase the chances of detecting pulmonary alterations in birds condemned by the Final Inspection System than in normal birds, suggesting that such diagnostic criteria are efficient to improve the assessment and condemnation of birds affected by airsacculitis. There were different genetic profiles among the isolates, which shows that isolates obtained from normal birds can potentially cause aspergillosis in those susceptible.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2007. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-25T18:56:16Z No. of bitstreams: 1 produo de biossurfactante e lipase por aspergillus fumigatus cultivado em estado slido e avaliao da biorremediao em derrames de leos e derivados.pdf: 999408 bytes, checksum: b510002262da45fc59e138e475cca986 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-19T17:01:49Z (GMT) No. of bitstreams: 1 produo de biossurfactante e lipase por aspergillus fumigatus cultivado em estado slido e avaliao da biorremediao em derrames de leos e derivados.pdf: 999408 bytes, checksum: b510002262da45fc59e138e475cca986 (MD5) / Made available in DSpace on 2013-06-19T17:01:49Z (GMT). No. of bitstreams: 1 produo de biossurfactante e lipase por aspergillus fumigatus cultivado em estado slido e avaliao da biorremediao em derrames de leos e derivados.pdf: 999408 bytes, checksum: b510002262da45fc59e138e475cca986 (MD5) Previous issue date: 2007 / Os biossurfactantes são compostos de superfície ativa sintetizados por diversos microrganismos e que têm recebido crescente interesse pelas vantagens que possuem sobre os surfactantes químicos, tais como biodegradabilidade, baixa toxicidade e produção a partir de fontes renováveis. Estes compostos apresentam propriedades emulsificante, solubilizante e dispersante, podendo ser aplicados em diversos processos, em especial, na biorremediação. O presente trabalho teve por objetivo estudar a produção de biossurfactante e lipase a partir do fungo filamentoso Aspergillus fumigatus através de fermentação em estado sólido, avaliar a estabilidade do biossurfactante em diferentes condições físico-químicas e estudar a biorremediação em derrames de compostos de origem animal (óleo de pescado) e mineral (tolueno) ocasionados em solos utilizando o biosurfactante produzido. Para o estudo da produção de biossurfactante e lipase foram testados dois meios fermentativos (farelo de arroz e farelo de trigo), dois níveis de aeração (60 e 100 mL.g-1.h-1) e três fontes adicionais de carbono (óleo de soja degomado, óleo de pescado bruto e tolueno). Os experimentos foram realizados em biorreatores de coluna com leito fixo durante 144 h. Foram avaliados a cada 24 h, os teores de umidade, pH, atividade emulsificante água em óleo (AEw/o) e óleo em água (AEo/w), atividade lipolítica, tensão superficial e interfacial. Para o estudo da estabilidade, foi utilizado um planejamento experimental fatorial 23 com três pontos centrais, em que foram testados pH 5,0, 7,0 e 9,0, salinidades 3,0, 16,5 e 30,0, e temperaturas de 10, 20 e 30°C. Os experimentos de biorremediação foram realizados durante 90 dias, sendo avaliado a influência da adição de biossurfactante ou dispersante químico e adição de fertilizantes (uréia e superfosfato triplo) na proporção C:N:P de 100:15:3 nos solos contaminados. Durante o processo foram realizadas análises de concentração do contaminante, teor de fósforo e nitrogênio total nos solos e contagem microbiológica. Os resultados mostraram que a máxima atividade emulsificante água em óleo (25,49 UE.g-1) e atividade emulsificante óleo em água (58,51 UE.g-1) foram obtidas quando se utilizou tolueno, farelo de trigo e aeração de 60 mL.g-1.h-1. A maior redução na tensão superficial (36,2 mN.m-1) e interfacial foi obtida quando se utilizou óleo de soja degomado, farelo de trigo e aeração de 60 mL.g-1.h-1. O surfactante produzido mostrou ser estável em diferentes níveis de temperatura, pH e salinidade, apresentando maior estabilidade quando mantido em pH 7,0, salinidade 16,5 e 20°C. A máxima atividade lipolítica (112,46 U.g-1) foi obtida quando se utilizou farelo de trigo, aeração de 100 mL.g-1.h-1 e tolueno como fonte adicional de carbono. Os ensaios de biorremediação em solos mostraram que a maior taxa de remoção do contaminante óleo de pescado foi 59,47% obtido no experimento contendo biossurfactante e bioestimulante em 90 dias de processo. A maior taxa de remoção do contaminante tolueno (100%) foi obtido em 14 dias para os experimentos contendo biossurfactante, dispersante químico e biossurfactante com bioestimulação. Os resultados demonstraram a possibilidade de produzir biossurfactantes e enzimas a partir de Aspergillus fumigatus utilizando resíduos agroindustriais e menores taxas de aeração, contribuindo para a minimização de custos de processos e impactos ambientais. O biosurfactante foi eficaz no processo de descontaminação de solos, favorecendo a remoção do poluente quando utilizado juntamente com bioestimulante. A utilização de fertilizantes mostrou favorecer o crescimento microbiano e o emprego de biossurfactante mostrou aumentar a biodisponibilidade do contaminante, acelerando o processo de degradação dos compostos. / Biosurfactants are compounds of active surface synthesized by several microorganisms which have been increasingly receiving attention due to the advantages they show over chemical surfactants, such as biodegradability, low toxicity, and yield from renewable sources. Such compounds show emulsifying, solubilizing, and spreading features, being able to be applied to several processes, especially in bioremediation. This paper aimed at studying the production of biosurfactant and lipase from the filamentous fungus Aspergillus fumigatus through in the solid state fermentation, evaluating biosurfactant stability under different physical-chemical conditions, and studying the bioremediation in animal (fish oil) and mineral (toluene) origin compounds shedding that occur in soils using the biosurfactant produced. For studying the production of both biosurfactant and lipase two fermentation media have been tested (rice and wheat bran), two aeration levels (60 and 100 mL.g-1.h-1), and three additional carbon sources (degummed soybeans oil, raw fish oil, and toluene). The experiments have been performed in column, fix bed bioreactors for 144h. Moisture contents, pH, emulsifying activity water-in-oil (AEw/o) and oil-in-water (AEo/w), lipolytic activity, superficial and interfacial tension have been evaluated at 24h intervals. For studying stability a 2³ factorial experimental desing has been used with three central points in which pH 5.0, 7.0, and 9.0, salt contents 3.0, 16.5, and 30.0, and temperatures at 10, 20, and 30°C. Bioremediation experiments have been performed for 90 days, being evaluated the influence of adding biosurfactant or chemical spreading and adding fertilizers (urea and triple super phosphate) in the ratio C:N:P 100:15:3 on contaminated soils. During the process analyses have been performed on contaminant content, phosphorus content, and total nitrogen in the soils and microbiological count. The results have shown that the maximum emulsifying activity water-in-oil (25.49 UE.g-1) and emulsifying activity oil-in-water (58.51 UE.g-1) have been achieved when toluene, wheat bran and aeration 60 mL.g-1.h-1 have been used. The highest superficial and interfacial tension reduction (36.2 mN.m-1) has been achieved when degummed soybeans oil, wheat bran and aeration 60 mL.g-1.h-1 have been used. The surfactant produced has shown to be stable at different temperature, pH, and salt levels, presenting higher stability when maintained under pH 7.0, salt content 16.5, and at 20°C. The maximum lipolytic activity (112.46 U.g-1) has been obtained when wheat bran, aeration 100 mL.g-1.h-1 and toluene as carbon additional source were used. The bioremediation assays in soils have shown that the highest rate of fish oil contaminant removal was 59.47% obtained in the experiments containing biosurfactant and biostimulate in 90 days of process. The highest rate of toluene contaminant removal (100%) has been achieved in 14 days for experiments containing biosurfactant, chemical spreading, and biosurfactant with biostimulation. The results have shown the possibility of producing biosurfactants and enzymes from Aspergillus fumigatus by using agroindustrial residues and lower aeration rates, contributing to minimizing processes costs and environmental impacts. The biosurfactant has shown efficacy in the process of decontaminating soils, favoring the polluting removal when used along with a biostimulate. The use of fertilizers has shown to favor microbial growth and the use of biosurfactant has shown to increase contaminant bioavailability, accelering the process of degrading compounds.

Les hydrophobines sont des protéines fongiques caractérisées par leurs propriétés amphipathiques et un motif de quatre ponts disulfures. Leur forme soluble s’auto-assemble aux interfaces hydrophobe/hydrophile pour former une couche amphipatique. Ces protéines sont utilisées par les champignons pour franchir la barrière air/eau, former des hyphes aériennes ou recouvrir les spores les rendant hydrophobes, ce qui facilite leur dispersion dans l’air. L’hydrophobine RodA du pathogène opportuniste Aspergillus fumigatus forme une couche de fibres amyloïdes avec une morphologie en bâtonnets qui recouvre la surface des spores ce qui les rend inertes vis-à-vis du système immunitaire. Nous aspirons à décrire l’auto-association de RodA en bâtonnets, caractériser la structure des fibres et établir les potentiels liens entre structure et inertie immunologique. La protéine recombinante RodA exprimée chez E. coli peut être correctement repliée in vitro et s’auto-associe sous forme de fibres amyloïdes. Comme première étape, la structure et la dynamique de RodA ont été étudiées par RMN en solution. Par rapport aux autres hydrophobines, RodA présente de nouveaux éléments structuraux ainsi que d’autres conservés. Grâce à une étude de mutagénèse, des régions importantes dans la formation des fibres ont été identifiées, certaines impliquées dans le cœur des fibres et d’autres dans les interactions latérales des bâtonnets. Les relations entre la structure et les propriétés immunologiques ont également été établies. L’étude d’autres hydrophobines d’A. fumigatus, probablement impliquées dans la formation du biofilm ou importantes pour la conidiation et la survie des spores, a été initiée.dC), a été initiée. / Hydrophobins are fungal proteins characterised by their amphipatic properties and a pattern of four disulfide bridges. Their soluble form self-assembles at hydrophobic/hydrophilic interfaces to form an amphipatic layer. These proteins are used by fungi to breach the air/water barrier, to form aerial hyphae, or to cover spores rendering them hydrophobic, thus facilitating spore dispersal. The RodA hydrophobin of the opportunistic pathogen Aspergillus fumigatus forms an amyloid monolayer with a rodlet morphology that covers the surface of spores rendering them inert relative to the immune system. We aim at describing the self-association of RodA into rodlets, characterising the structure of the amyloid rodlets and shedding light on the possible relationships between structure and immunological inertness. Recombinant RodA expressed in Escherichia coli can be successfully refolded in vitro and it can auto-associate into amyloid rodlets. As a first step, we have studied the structure and dynamics of RodA by solution NMR and shown that the protein displays new as well as conserved structural features relative to other hydrophobins. A mutational analysis has highlighted important residues for rodlet formation that may be involved on the one hand in the spine of the amyloid fibres and on the other hand on the lateral association of the rodlets to form a monolayer. We have also established the relationship between structure and immunological inertness. We have initiated the study of other hydrophobins from A. fumigatus, that are most likely involved in biofilm formation or in conidiation and spore survival.