Tesis: "Aspergillus fumigatus"

1

Weaver, Sean. "Heterokaryon incompatibility in Aspergillus fumigatus". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/heterokaryon-incompatibility-in-aspergillus-fumigatus(c0db26be-8326-4a93-8bcb-2648069e256c).html.

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Invasive aspergillosis (IA) is associated with high mortality rates and can be difficult and expensive to treat with current drugs. The drugs used to treat IA are also associated with undesirable, and often severe, side-effects of the patient. The main causative agent of this disease is the opportunistic pathogen Aspergillus fumigatus. This study identifies genes which play a role in a fungal-specific type of programmed cell death (PCD) in A. fumigatus, known as heterokaryon incompatibility. The development of drugs specifically targeting the products of these genes could lead to fewer side-effects than those arising from currently available anti-fungal drugs. The drug amphotericin B is currently used to treat IA and has been shown to induce an apoptotic-like phenotype in A. fumigatus; however, the sterols targeted are present in both fungal and mammalian cell membranes. HI is a fungal-specific self/non-self recognition system that results in rapid compartmentalisation and cell death of hyphal fusion sites if the two fusing fungi are not genetically compatible. The HI system could be exploited as a novel drug target against invasive fungal pathogens through targeting a component of the molecular pathway to induce cell death. In contrast to current drugs, novel drugs could target HI components to induce PCD without affecting non-desirable targets that cause side-effects. The non-self recognition systems used by Neurospora crassa, Aspergillus Nidulans and Podospora anserina are the well characterised, and they each differ significantly in their modes of action. BLAST searches found 30 homologues of HI genes from other the systems of characterised species in A. fumigatus, with 8 containing the fungal-specific het domain. The first assay to determine whether disruption of het genes could affect HI was to observe the barrage phenotype between incompatible A. fumigatus individuals. However, there was no barrage visible as the leading edge of colonies stopped growing when in close proximity to another colony. Instead, nitrate non-utilising (Nit) A. fumigatus mutant strains were generated using chlorate and pair-wise crosses of 46 environmentally and clinically isolates on nitrate-containing media resulted in the formation of 16 viable heterokaryons. All of the heterokaryons fell into exclusive compatibility groups where no intergroup crossing was possible. Homologous recombination was used to disrupt five of the identified het domain genes with gene replacement cassettes, generated through fusion-PCR, in an akuB(KU80Delta) A. fumigatus strain. The mutant strains displayed both detrimental growth on standard agar growth media and reduced ability to recognise non-self strains. Full and partial heterokaryons were formed during intergroup pair-wise compatibility crosses using the mutants and strains that the akuB(KU80Delta) parent strain was previously incompatible with. This was followed with a non-bias approach of gene disruption using the Fusarium oxysporum impala160 transposable element in a Nit A. fumigatus mutant. Inducing transposon mutagenesis through exposure to low temperature generated a mutant library of spores in which the transposon had disrupted different open reading frames at different locations across the A. fumigatus genome. The mutant spore library was also screened for the ability to form viable intergroup heterokaryons with strains belonging to different compatibility groups. PCR recovery and DNA sequencing was able to identify the locus of impala160 in three isolates able to form viable heterokaryons. The sequences revealed the transposable element had disrupted the same gene, AFUA_2G05070, in each of the three isolates. This gene encodes an uncharacterised conserved hypothetical protein which may be a critical component for non-self recognition in A. fumigatus HI, and a potential target for novel anti-fungal drugs to induce PCD.

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2

Robertson, Maura Diane. "Host defences against Aspergillus fumigatus". Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/26892.

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The potential of the filamentous fungus Aspergillus fumigatus to act as an opportunistic pathogen may be related to its ability to resist the host defence network. Whilst phagocytic cells are clearly important in host defences against invading microorganisms their precise role in the killing of A. fumigatus remains undefined. The purpose of this study was to examine the basic interactions between phagocytic cells, from humans and rodents, with spores of A. fumigatus. In particular the mechanisms whereby phagocytic cells bind and kill spores of A. fumigatus, when compared with the relatively non-pathogenic fungus Penicillium ochrochloron were investigated. In order to investigate why people with asthma may develop some hypersensitivity reactions to A. fumigatus, in particular, rather than to the many other fungi in the atmosphere, the possibility that there may be a defect in the handling of the fungus by such patients has been tested. A comparison of the fungal handling by phagocytes from asthmatic patients, both sensitised and unsensitised to A. fumigatus with phagocytes from non-asthmatic subjects has been made. The principal findings from this study are that spores of A. fumigatus bind to the surface of the phagocytic cell yet are relatively resistant to phagocytosis. The spores also fail to trigger the phagocytic cells into releasing the potentially microbicidal reactive oxygen intermediates. These results may be related to a further finding which is that spores of A. fumigatus release a low molecular weight substance (diffusate) which interferes with various aspects of phagocytic cell activation. Spore diffusates were shown to inhibit the phagocytosis of radiolabelled antibody-coated sheep red blood cells and to suppress the spontaneous release of reactive oxygen intermediates by Corynebacterium parvum stimulated mouse peritoneal exudate cells. In addition spores diffusates inhibited the ability of phagocytic cells to spread on glass and reduce the number of phagocytic cells migrating towards a known chemoattractant. Studies on spore killing showed that spores of A. fumigatus opsonised in autologous serum were more resistant to killing by phagocytic cells from humans and rodents than similarly opsonised spores of P. ochrochloron. However, the ability of the phagocytic cells to kill spores of A. fumigatus was substantially increased when the spores were opsonised in sera which had been heat-treated for 30 minutes at 56?C. No increased killing was found with P. ochrochloron. People with asthma sensitised to A. fumigatus showed significant differences in their handling of A. fumigatus in vitro when compared with the control group. Monocytes from these sensitised patients killed significantly fewer spores of A. fumigatus (opsonised in auto? logous sera) whilst their polymorphonuclear leucocytes killed significantly more. No such differences were found for P. ochrochloron. The work reported in this Thesis has given us a clearer understanding of why Aspergillus fumigatus is an important cause of disease in man, and how the defence mechanisms that it has evolved in its natural environment the soil, enable it to act as a saprophyte or parasite in the lungs of humans and animals. The results also suggest a mechanism whereby heat-labile serum components may be an advantage to the survival of the fungus, thus perhaps explaining why it may be a particular problem in the airways of asthmatic patients.

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3

Philippe, Bruno. "Aspergillose pulmonaire invasive : interactions entre Aspergillus fumigatus et macrophage alvéolaire". Paris 12, 2004. https://athena.u-pec.fr/primo-explore/search?query=any,exact,990003948260204611&vid=upec.

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L'aspergillose pulmonaire invasive (AI) due à Aspergillus fumigatus est une infection grave du malade immunodéprimé. Un modèle murin d'AI a montré que le macrophage alvéolaire (MA) représente la première ligne des défenses pulmonaires innées contre A. Fumigatus. Le killing est lent et fait intervenir les réactifs oxydants. Les corticostéroi͏̈des diminuent les capacités de killing en inhibant la production de réactifs oxydants par les MA. L'analyse du killing de A. Fumigatus par des MA de malades transplantés pulmonaires a montré des résultats similaires aux MA murins. La dose de corticostéroi͏̈des prise quotidiennenement > 0,25 mg/kg/j, la dose totale > 1,5 mg/kg/j et la période précoce < 6 mois après la transplantation ont été retrouvés comme facteurs diminuant le niveau de killing des conidies par les MA. Ces résultats démontrent le rôle essentiel du MA dans la résistance de l'homme à A. Fumigatus
Pulmonary invasive aspergillosis (lA) due to Aspergllus fumigatus is a severe infection in immunocompromised patients. A murine model of invasive aspergillosis showed that alveolar macrophages (AM) are the first une of pulmonary innate defence against A. Fumigatus. The killing is slow and involves reactive oxidants intermediates. Corticosteroids decrease killing capacity of the AM du to an inhibition of reactive oxidants intermediates production. Killing study of lung transplant recipients showed similar resuits as murin AM. Several factors that influence the killing were identified daily dose of corticosteroids > 0,25 g/kg/d total dose> 1,5 mg/kg/d and early period post transplantation <6 months were found to decrease significatively the killing rate. These data demonstrated unequivocally that the alveolar macrophage is the first une of defence against A. Fumigatus

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4

Philippe, Bruno Latgé Jean-Paul. "Aspergillose pulmonaire invasive interactions entre Aspergillus fumigatus et macrophage alvéolaire /". Créteil : Université de Paris-Val-de-Marne, 2007. http://doxa.scd.univ-paris12.fr:8080/theses-npd/th0394826.htm.

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Thèse de doctorat : Sciences de la vie et de la santé : Paris 12 : 2004.
Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 305 réf.

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5

Leleu, Christopher. "Evaluation du risque lié à l'exposition aérienne à Aspergillus fumigatus". Paris 6, 2012. http://www.theses.fr/2012PA066413.

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Aspergillus fumigatus est un champignon filamenteux pathogène responsable de différentes formes d’infections pulmonaires allergiques sur les sujets immunocompétents et d’infections invasives chez les patients neutropéniques. L’inhalation de spores est le mode habituel de contamination suggérant un rôle majeur de l’environnement dans l’épidémiologie de l’aspergillose. Cependant, la relation entre les concentrations d’Aspergillus dans l’air et la probabilité d’infections ne sont pas connues. Dans cette étude, trois approches complémentaires ont été proposées pour analyser cette relation. In vitro, nous avons utilisé un dispositif de culture en interface air-liquide pour analyser les conséquences de l’exposition de cellules pulmonaires A549 à différentes concentrations de spores d’Aspergillus fumigatus. Aucun effet significatif sur la production de cytokines pro-inflammatoires n'a été retrouvé suite à cette exposition, même lorsque cette exposition aspergillaire était combinée avec une exposition au formaldéhyde. In vivo, la relation entre l’exposition à des spores d’Aspergillus et la survenue d’une infection a été étudiée dans un modèle murin d’aspergillose invasive en utilisant la souche de référence Af293 d’Aspergillus fumigatus. Dans une approche bayésienne la relation dose-infection entre probabilité d’infection et exposition aux spores a été estimée en utilisant le modèle exponentiel et le modèle plus flexible bêta-Poisson. Ceci a permis d’estimer la dose infectieuse 50 à 1,8-1,9. 104 spores inhalées viables. Secondairement, ce modèle a été utilisé pour mettre au point un nouveau modèle de réactivation d’aspergillose et étudier l’efficacité de l’amphotéricine B liposomale dans la prophylaxie de l’aspergillose invasive. Chez l’homme, nous avons tenté d’estimer la relation entre l’exposition environnementale aux spores fongiques et l’incidence de la colonisation ou de l’infection aspergillaire chez 44 transplantés pulmonaires étudiée de façon consécutive. A l'aide d'un modèle de régression par GEE, nous avons trouvé une relation significative entre la contamination des surfaces par Aspergillus et l’incidence de la colonisation. De plus, nous avons montré des identités génotypiques entre les isolats cliniques et environnementaux d’Aspergillus, ce qui confirme les risques d’acquisition d’Aspergillus dans le cadre hospitalier. Globalement, ces résultats apportent des données nouvelles sur la relation entre la contamination environnementale et la probabilité d’aspergillose chez les patients immunodéprimés
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for various respiratory diseases in normal hosts and severe invasive infections in neutropenic patients. Spore inhalation is the usual route of Aspergillus infection, suggesting a determining role of environmental contamination in the epidemiology of aspergillosis. However the relationship between Aspergillus concentration in the air and probability of infection is not quantitatively known. In this study, three different approaches were proposed to analyse this relationship. In vitro we used an air-liquid interface module to expose pulmonary A549 cells to high concentrations of A. Fumigatus spores, but found not effect of exposure on the production of pro-inflammatory cytokines, even when exposure was combined with exposure to formaldehyde. In vivo, the relationship between spore exposure and infection was examined in a murine model of invasive aspergillosis, using the reference Af293 strain of A. Fumigatus. In a bayesian approach, the dose-response relationship between the probability of infection and spore exposure was approximated using the exponential model and the more flexible beta-Poisson model. It allowed estimating the median infective dose at 1. 8-1. 9x104 inhaled viable spores. Further, this model was used to develop a unique model of reactivating aspergillosis and then to examine the efficacy of liposomal amphotericin B on prophylaxis of aspergillosis. In human, we attempted to estimate the relationship between environmental exposure to fungal spores and the incidence of Aspergillus colonization or infection in 44 consecutive lung transplant recipients. In a GEE multivariate analysis, we found a significant relationship between surface contamination by Aspergillus and the incidence of colonization. Furthermore, we found genotypic similarities between clinical and environmental isolates of Aspergillus, which confirm the risk of acquisition of Aspergillus in the hospital setting. Altogether, this result provides new insights into the relationship between airborne exposure and probability of aspergillosis in immunocompromised hosts

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6

Bertout, Sébastien. "Polymophisme génétique de souche d'Aspergillus fumigatus isolées d'aspergilloses pulmonaires au cours d'une étude multicentrique". Montpellier 1, 2000. http://www.theses.fr/2000MON13512.

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7

Bernard-Cardona, Muriel. "Protéines et paroi chez Aspergillus fumigatus". Phd thesis, INAPG (AgroParisTech), 2003. http://tel.archives-ouvertes.fr/tel-00005702.

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La paroi du champignon filamenteux A. fumigalus conditionne la croissance et est responsable du maintien de l'intégrité cellulaire lors de l'infection. Les protéines de la paroi de ce champignon filamenteux n'avaient pas été étudiées jusqu'à présent alors qu'elles semblent jouer un rôle essentiel dans la structuration de la paroi de la levure modèle S. cereviciae. Une approche essentiellement biochimique a permis de caractériser les protéines associées à la paroi de A..fumigatus. La majorité des protéines pariétales chez A. fumigatus sont des protéines solubles. Une seule protéine est relarguée à partir de la paroi par un traitement f3- (1 .3) glucanase : c'est une phosphatase acide qui possède une ancre GPl et dont l'expression est réprimée en présence de phosphate inorganique. Par ailleurs, une étude des protéines GPl chez A. fumigatus par génomique comparative a montré que les protéines GPI décrites comme associées de façon covalente à la paroi chez la levure n'ont pas d'homologue chez A. fumigatus. Ainsi. l'organisation des protéines au sein de la paroi de A..fumigatus est différente de celle de la levure : les protéines pariétales ne semblent pas avoir un rôle essentiel dans l'élaboration de la paroi. Ensuite, une nouvelle famille de glycoprotéines portant un N-glycane lié à un galactofuranose en position terminale a été décrite. Cette famille comprend une phospholipase C, une phosphatase alcaline et une phytase. Enfin, une analyse morphologique de deux mutants chitine synthase et gluconosyltransférase a permis d'associer la réduction de la croissance à un hyper-branchement du mycélium et à une diminution de la taille de la cellule apicale sans que l'organisation globale des polysaccharides pariétaux et le taux de croissance spécifique soient affectés.

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8

Melloul, Elise. "Aspergillose aviaire : développement d’un modèle d’aspergillose chez la dinde (Meleagris gallopavo) et évaluation de l’efficacité de l’énilconazole". Thesis, Paris Est, 2015. http://www.theses.fr/2015PEST1183/document.

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Aspergillus fumigatus est un agent pathogène respiratoire majeur chez les oiseaux d’ornement comme de production. L’aspergillose qui peut être responsable de mortalités importantes et de chutes de performances est difficile à traiter. Nous avons développé un modèle d’aspergillose aiguë chez le dindonneau en inoculant différents lots d’oiseaux âgés de moins d’une semaine via une aérosolisation intratrachéale de doses croissantes de conidies (105 à 108/animal) en utilisant un MicroSprayer®. Le développement de la masse fongique a été évalué par qPCR, dosage du galactomannane (GM), culture fongique et évaluation histopathologique dans le but de comparer les résultats obtenus en fonction du nombre de conidies inoculées. Une mortalité significative a été observée dans les 4 jours suivant l’inoculation uniquement pour l’inoculum le plus concentré. Les résultats des différents marqueurs du développement du champignon (culture, qPCR et GM), sont très bien corrélés avec la dose de l’inoculum administrée. Les moyennes d’équivalents conidies/g de poumon obtenues par qPCR étaient 1,3 log10 plus importantes que les numérations obtenues par culture sur gélose. Ce nouveau modèle incluant une combinaison inédite de biomarqueurs chez la dinde a été utilisé pour évaluer l’efficacité de l’énilconazole, seule molécule utilisée en élevage avicole pour lutter contre l’aspergillose
Aspergillus fumigatus remains a major respiratory pathogen in both ornamental and poultry. Aspergillosis can be responsible for high mortality rates and induces significant economic losses, particularly in turkey production, and it is still difficult to treat. We developed a new model of acute aspergillosis in young turkeys by inoculating few-days-old turkeys via intratracheal aerosolization with increasing concentrations (105 up to 108) of conidia using a MicroSprayer® device. The fungal burden was assessed and compared by real-time PCR, galactomannan (GM) dosage, fungal colony (CFU) counting and by histopathology. Early death occurred in the first 96 h post-inoculation only at the highest inoculum dose. We observed a correlation between inoculum size and results obtained by real-time PCR, GM dosage and CFU counting. The mean fungal burden detected by qPCR was 1.3 log10 units higher than the mean values obtained by CFU measurement. Furthermore, this new model, with its unique combination of markers, has been used to evaluate the efficacy of enilconazole

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9

Brown, Jeremy Stuart. "Signature tagged-mutagenesis of aspergillus fumigatus". Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322287.

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10

Kong, Yun-cheung y 江潤祥. "Multilocus sequence typing of aspergillus fumigatus". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B4593972X.

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Marsh, Rachael. "Cytochrome P450 studies in Aspergillus fumigatus". Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364267.

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12

Rosa, Carla Maria de Gouveia. "Detecção de Aspergillus fumigatus em Hemoculturas". Master's thesis, Faculdade de Farmácia da Universidade do Porto, 2007. http://hdl.handle.net/10216/9491.

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SPENTCHIAN, MARC. "Interaction aspergillus fumigatus - laminine : etude preliminaire". Nantes, 1990. http://www.theses.fr/1990NANT016M.

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Rosa, Carla Maria de Gouveia. "Detecção de Aspergillus fumigatus em Hemoculturas". Dissertação, Faculdade de Farmácia da Universidade do Porto, 2007. http://hdl.handle.net/10216/9491.

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15

Khoufache, Khaled. "Aspects toxicologiques d'Aspergillus fumigatus sur l'épithélium repiratoire in vitro". Paris 12, 2006. https://athena.u-pec.fr/primo-explore/search?query=any,exact,990002459590204611&vid=upec.

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Le rôle des mycotoxines d'Aspergillus fumigatus sur l'épithélium respiratoire a été peu étudié. Pour étudier ces interactions, nous avons utilisé deux modèles de culture primaire de cellules épithéliales en interface air liquide l'un humain (CENH ou cellules épithéliales nasales humaines), l'autre porcin (CETP ou cellules épithéliales de trachée de porc). Afin d'identifier les mycotoxines d'A. Fumigatus impliquées dans ces interactions, nous avons travaillé à la fois sur la phase organique et sur la phase aqueuse d'un filtrat de culture d'A. Fumigatus. La phase organique des filtrat de culture mimait les effets des filtrats de culture totaux déjà décrits sur les CENH. Parmi les molécules candidates de cette phase organique, seul le verruculogène reproduisait les mêmes effets sur les paramètres électrophysiologiques. Le verruculogène est produit par toutes les souches d'A. Fumigatus et associé aux conidies. De la même manière que pour la phase organique, l'activité de la phase aqueuse a été étudiée. Les résultats semblent montrer un effet électrophysiologique marqué sur les CENH, mais aucune molécule candidate n'a, pour l'instant, été identifiée comme responsable de ces effets. Dans la 2ème partie de notre travail, une mise au point d'un modèle de CETP était effectuée. Ce modèle, de façon identique au modèle CENH, était bien différencié et composé uniquement de cellules épithéliales. L'étude des interactions antre CETP - filtrat de culture d'A. Fumigatus, verruculogène et conidies d'A. Fumigatus a montré des résultats similaires à ceux obtenus avec le modèle humain. Une immortalisation des CETP était obtenue à partir du 22ème passage. Les cellules conservaient leur aspect épithélial, mais perdaient leur différenciation mucociliaire. Le verruculogène, métabolite secondaire d'A. Fumigatus, a été pour la 1ère fois mis en évidence dans la modification des paramètres électrophysiologiques des CENH. L'activité du verruculogène pourrait être immédiate sur l'épithélium respiratoire, intervenir dans la modification de l'activité anti-microbienne du fluide de la surface apicale des cellules. Cette activité pourrait s'exercer via les canaux K+ et Na+ des CENH. Par ailleurs, le développement d'un modèle de CETP permet de dispose d'un grand potentiel de cellules afin de travailler sur de nombreux domaines impliquant A. Fumigatus et cellules des voies aériennes supérieures (phagocytose du champignon, étude de la réponse inflammatoire face à l'agression fongique, étude d'autres mycotoxines,. . . )
The role of Aspergillus fumigatus mycotoxins on the respiratory epithelium has been poorly studied. To study these interactions, we have used two models of primary culture of epithelial cells in interface air liquid : one model of human nasal epithelial cells (CENH), and one model of pig tracheal epithelial cells pig (CETP). To identify the Aspergillus fumigatus mycotoxins implicated in these interactions, we worked on the organic phase and the aqueous phase of A. Fumigatus culture filtrate. The organic phase of the culture filtrates showed similar effects than totals filtrates of culture, already described on the CENH. Among the molecules candidates of this organic phase, only the verruculogen reproduced the same effects on the electrophysiological parameters. The verruculogen is produced by all the strains of A. Fumigatus and is associated to the conidia. Same manner as for the organic phase, the activity of the aqueous phase was studied. The results seem to show an electrophysiological effect marked on the CENH, but no molecule candidate, for the moment, has been identified. In the 2nd part of our work, a development of a model of CETP was carried out. This model, in a way identical to model CENH, well was differentiated and composed only of epithelial cells. The study of the interactions between CETP - culture filtrates of A. Fumigatus, verruculogen and A. Fumigatus conidia’s showed similar results with those obtained with the human model. An immortalization of the CETP has been obtained starting from the 22nd passage. The cells preserved their epithelial aspect, but lost their mucociliary differentiation. The verruculogen, secondary metabolite of A. Fumigatus, was for the 1st time highlighted in the modification of the electrophysiological parameters of the CENH. The activity of verruculogen could be immediate on the respiratory epithelium, to interfere in the modification of the antimicrobial activity of apical surface fluid of the cells. This activity could be exerted via the K+ and Na+ channels of the CENH. In addition, the development of a model of CETP makes it possible to have a great potential of cells in order to work on many fields implying A. Fumigatus and cells of the higher air routes (phagocytosis of fungi, study of the inflammatory response against the fungus aggression, study of other mycotoxins. . . )

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Khoufache, Khaled Bretagne Stéphane. "Aspects toxicologiques d'Aspergillus fumigatus sur l'épithélium repiratoire in vitro". Créteil : Université de Paris-Val-de-Marne, 2006. http://doxa.scd.univ-paris12.fr:80/theses/th0245959.pdf.

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Haddad, Ziad. "Monozytäre Zellantwort gegen Aspergillus fumigatus Untersuchung der Phagozytose, Genexpression und Peptidpräsentation = Monocytic cell responses to Aspergillus fumigatus /". [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980508169.

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Frandon, Isabelle. "Actions physiologique et moléculaire de glucocorticoïdes sur Aspergillus fumigatus". Université Joseph Fourier (Grenoble), 1998. http://www.theses.fr/1998GRE18006.

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19

Urb, Mirjam. "Mechanisms of «Aspergillus fumigatus» chronic airway disease". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110500.

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Colonization of airways by Aspergillus fumigatus hyphae in immunocompetent patients with chronic lung disease leads to progressive decline in pulmonary function. While a minority of these patients develop a severe allergic response to the fungus, the majority of patients show a decline in lung function and airway hyperreactivity despite the absence of elevated IgE levels, eosinophilia or other evidence of hypersensitivity. Treatment of these non-allergic patients with antifungal drugs improves the symptoms, suggesting that pulmonary complications might be directly caused by A. fumigatus. The mechanisms underlying the acquisition of A. fumigatus colonization and the pathogenesis of pulmonary inflammation have not been studied. Our central hypothesis is that A. fumigatus interacts directly with elements of the immune system to facilitate its own colonization and induces ineffective inflammatory responses that damage host airways. To begin to investigate this hypothesis we used two complementary approaches to examine the interactions of A. fumigatus with elements of the pulmonary immune system. First, we studied the in vitro interactions of A. fumigatus with mast cells, key effector cells involved in orchestrating inflammatory responses and airway reactivity. We found that A. fumigatus triggers mast cell degranulation, while suppressing cytokine expression in an IgE-independent manner. While both degranulation and cytokine transcription required direct contact with mature hyphae, cytokine suppression could also be induced in part by A. fumigatus culture supernatant. Further studies revealed that A. fumigatus hyphae modulated mast cell function by downregulating protein tyrosine phosphorylation and in part by cleavage-dependent activation of protein tyrosine phosphatase 1B (PTP1B). The cleavage of PTP1B was caused by A. fumigatus serine proteases. In parallel, we developed a murine model of A. fumigatus colonization, where airways of healthy mice were colonized with live A. fumigatus by intratracheal injection of conidia embedded within agar beads. Unlike previously described models that induce airway hyperreactivity by repeated challenge with antigen or A. fumigatus spores, infection with this approach resulted in chronic colonization with fungi for up to 28 days. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammation and peribronchial infiltration of lymphocytes. During the first 2 weeks of infection, low level Th2 type responses were seen in the infection, including increased pulmonary IL-4 levels, elevated serum IgE, and a mild increase in airway responsiveness. In addition, significant levels of pro-inflammatory cytokines and chemokines including TNF-α and MIG were observed, suggesting a mixed inflammatory picture. Furthermore, elevated IL-17 levels and presence of RORγ-positive mononuclear cells during late phase of the infection indicated the development of a Th17 response in association with reduction in pulmonary fungal load. Collectively, these results provide insight into the pathogenesis of A. fumigatus-induced chronic airways disease in patients without allergic bronchopulmonary aspergillosis, and suggest a possible role for mast cells in the pathogenesis of this condition.
La colonisation des voies respiratoires par les hyphes d'Aspergillus fumigatus chez des patients immunocompétents, mais avec une maladie pulmonaire chronique, entraîne le déclin progressif de la fonction des poumons. Alors qu'une minorité de ces patients développe une réponse allergique aigue au champignon, la majorité montre un déclin dans la fonction des poumons et une hyperréactivité des voies respiratoires malgré l'absence d'une augmentation du niveau des IgE, des éosinophiles ou d'autres indications d'hyper-sensibilité. Le traitement antifongique chez ces patients améliore les symptômes suggérant que le champignon peut être la cause directe des complications observées. Les mécanismes qui sous-tendent la colonisation par A. fumigatus et la pathogenèse de l'inflammation des voies respiratoires restent largement indéterminés. Notre hypothèse centrale stipule que le champignon interagit directement avec des éléments du système immunitaire pour faciliter la colonisation et induire une réponse inflammatoire inefficace qui cause des dégâts aux voies respiratoires de l'hôte. Pour vérifier cette hypothèse, nous avons développé deux approches complémentaires visant à tester l'interaction d'A. fumigatus avec des éléments du système immunitaire pulmonaire. D'abord, nous avons étudié l'interaction in vitro entre A. fumigatus avec les mastocytes, une population clé de cellules impliquées dans la réponse inflammatoire. Nous avons montré que le champignon déclenche la dégranulation des ces cellules, tout en bloquant l'expression des cytokines indépendamment des IgE. Les processus de dégranulation et de transcription des cytokines nécessitent un contact direct avec les hyphes matures, alors que la suppression des cytokines quant à elle peut être induite en partie par le surnageant de culture d'A. fumigatus. Une étude plus approfondie nous a permis de montrer que les hyphes d'A. fumigatus peuvent moduler la fonction des mastocytes via une régulation négative de la phosphorylation d'une protéine tyrosine kinase et, en partie, via l'activation clivage-dépendante de la protéine tyrosine phosphatase 1B (PTP1B). Ce clivage de PTP1B est causé par la serine protéase du champignon. Dans une seconde approche, nous avons développé un modèle murin pour la colonisation par A. fumigatus, où nous avons fait coloniser les voies respiratoires de souris saines avec le champignon par injection intra-trachéale de conidies encapsulées dans des billes en agar. Ce modèle, au contraire des précédents, qui induisent l'hyperréactivité des voies respiratoires par exposition répétée à un antigène ou des spores de A. fumigatus, permet une colonisation chronique par le champignon allant jusqu'à 28 jours. Après ce traitement, les lésions des voies respiratoires, causées par le champignon, se retrouvent entourées par une inflammation neutrophilique robuste ainsi qu'une infiltration péri-bronchiale des lymphocytes. Durant les deux premières semaines qui suivent l'infection, nous avons détecté des niveaux bas d'une réponse type Th2, y compris une augmentation des niveaux des IL-4 pulmonaires, élévation des IgE dans le sérum, et une augmentation légère de la réponse des voies respiratoires. En plus, une augmentation significative des cytokines et des chémokines pro-inflammatoires, y compris les TNF-α et MIG, a été observée suggérant une réponse inflammatoire mixte. Les niveaux élevés des IL-7 et la présence des cellules mononucléaires RORγ-positives durant la phase tardive de l'infection indiquent le développement d'une réponse de type Th-17 associée à une réduction de la charge fongique pulmonaire. Collectivement, ces résultats nous permettent de mieux comprendre le processus de pathogenèse lié aux maladies chroniques des voies respiratoires induites par A. fumigatus chez les patients sans aspergillose bronchopulmonaire allergique, et suggèrent que les mastocytes peuvent jouer un rôle dans cette pathogenèse.

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20

Leslie, Carolyn Elizabeth. "Studies on clinical isolates of Aspergillus Fumigatus". Thesis, Heriot-Watt University, 1985. http://hdl.handle.net/10399/1653.

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21

Baillie, George Scott. "Characterisation of cytochromes P450 from Aspergillus fumigatus". Thesis, University of Kent, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334500.

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Taib, Mariam. "The complex chitinolytic system of 'Aspergillus fumigatus'". Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416841.

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23

Rajendran, Ranjith. "Adaptive resistance mechanisms of Aspergillus fumigatus biofilms". Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4333/.

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Biofilm formation is one of several significant virulence factors associated with life threatening pulmonary infections in immunocompromised individuals caused by Aspergillus fumigatus. Previous studies have demonstrated phase dependant antifungal activity against A. fumigatus biofilms. Antifungal resistance associated with fungal biofilms is a complex multifactorial phenomenon, and it remains unclear specifically how this manifests itself in A. fumigatus. This study therefore aimed to investigate adaptive resistance mechanisms in A. fumigatus biofilms. Different phases of A. fumigatus biofilms were grown for 8, 12, 24 and 48h in polystyrene plates in RPMI media. Functional efflux pump activity was subsequently assessed using an Ala-Nap fluorescent uptake assay. Extracellular material was extracted from each phase and the level of extracellular DNA (eDNA) was quantifiedusing a microplate fluorescence assay. The minimum inhibitory concentrations (MIC) of different classes of antifungals were assessed in the presence and absence of different inhibitors using a checkerboard assay, or with a fixed concentration, by the broth microdilution method to assess synergism, antagonism, or otherwise. The presence of eDNA and phenotypic changes in biofilm caused by antifungal agents and inhibitors were assessed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) techniques. The resultant biofilm biomass for different experiments was evaluated using a crystal violet assay. SYBR green qRT-PCR was used to assess the expression of different genes implicated in biofilm resistance (AfuMDR 1-4, ChiA-E, HSP90 and Fks1) over the period of multicellular development, using a diffusion chamber in a murine model and a Galleria mellonella infection model. The results from this study demonstrated phase dependant expression of efflux pumps in A. fumigatus biofilm populations, which actively contributes to azole resistance. Moreover, voriconazole treatment induced efflux pump expression in both in vitro and in vivo models.These data suggest that A. fumigatus efflux pump proteins, which evolved to become integral to their natural physiological function, have inadvertently induced resistance to azole drugs, albeit in the early phases of biofilm development. Assessment of A. fumigatus biofilm extracellular matrix (ECM), associated with maturing biofilms, showed that eDNA is an important architectural component of the biofilm, helping to maintain its stability. The antifungal sensitivity of different phases of A. fumigatus growth decreased significantly in the presence of DNase, indicating that decreased susceptibility to antifungals in the A. fumigatus is mediated in part by eDNA.Its release was shown to correlate withchitinase activity, a marker of autolysis, suggestive that autolysis was associated with eDNA release. It was hypothesised that heat shock protein 90 (HSP90) was involved in this autolytic pathway. Therefore, when HSP90 was pharmacologically inhibited this led to a decrease in matrix eDNA level, providing a compelling mechanism through which HSP90 might regulate biofilm antifungal resistance. To test whether these mechanisms of adaptive resistance had any bearing clinically, a G. mellonella model was developed. It was shown that each of the key genes were expressed during infection, both in control and antifungal treated larvae. This validates the potential use of this insect model for resistance and virulence studies. Overall, this study establishes several novel adaptive resistance mechanisms regulating biofilm drug resistance in A. fumigatus biofilms. Moreover, it highlights the potential to target these mechanisms as a therapeutic strategy for managing and improving clinical outcomes in these hard-to-treat infections.

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24

Pillé, Ariane. "Amyloïdes fonctionnelles du pathogène opportuniste Aspergillus fumigatus". Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066696/document.

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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

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25

Chauvin, David. "Nouvelles stratégies de traitement de l'aspergillose : ciblage d'Aspergillus fumigatus par des anticorps thérapeutiques et ciblage du microenvironnement fongique". Thesis, Tours, 2018. http://www.theses.fr/2018TOUR3310.

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Due au champignon Aspergillus fumigatus, l’aspergillose pulmonaire invasive représente une grave menace pour les individus souffrant d’immunodépression sévère. En parallèle d’un diagnostic manquant de spécificité, les traitements actuels présentent une forte toxicité. Ces travaux se sont dans un premier temps intéressés au développement d’anticorps thérapeutiques dirigés contre les protéines pariétales Chitin ring formation du champignon. Le ciblage de ces protéines impliquées dans la croissance fongique a permis la mise en évidence d’effets modérés in vitro, et ont induit, in vivo, un recrutement significatif de cellules immunitaires impliquées dans la défense anti-aspergillaire. Dans un second temps, ces travaux se sont intéressés au ciblage du microenvironnement et de la réponse de l’hôte au cours de l’aspergillose, afin de mieux comprendre les processus physiopathologiques induits au cours de la maladie, et de permettre l’identification de nouveaux biomarqueurs et cibles thérapeutiques. L’utilisation de la spectrométrie de masse iTRAQ®, chez des rats et des manchots, a permis la mise en évidence de plusieurs voies de signalisation surreprésentées. Ces travaux se sont également intéressés à la caractérisation immunologique d’un modèle rat d’API. En plus de la mise en évidence des effets du champignon sur le recrutement de certaines populations de cellules immunitaires, l’utilisation de l’iTRAQ® a permis la mise en évidence de la surexpression de l’interleukine-33 et de son récepteur ST2 au cours de la maladie. Ces travaux ouvrent d’intéressantes perspectives dans la mise en place de nouveaux traitements contre l’API
Caused by the fungus Aspergillus fumigatus, invasive pulmonary aspergillosis is a serious threat for individuals suffering from severe immunosuppression. In parallel of a diagnosis lacking specificity, current treatments present a high toxicity. This work first focused on the development of therapeutic antibodies directed against cell wall proteins Chitin ring formation of the fungus. Targeting of these proteins involved in fungal growth highlighted moderate effects in vitro, and induced, in vivo, a significant recruitment of immune cells involved in anti-aspergillary defense. In a second time, this work focused on targeting the microenvironment and the host response during aspergillosis, in order to better understand pathophysiological processes induced during the disease, and allow the identification of new biomarkers and therapeutic targets. Use of iTRAQ® mass spectrometry in rat and penguins allowed the identification of several overrepresented signaling pathways. This work also focused on the immune characterization of a rat model of IPA. In addition of highlighting the effects of the fungus in the recruitment of some immune cell populations, use of iTRAQ® exhibited an overexpression of interleukin-33 and its receptor ST2 during the disease. Overall, this work is bringing interesting insights in the establishment of new treatments against IPA

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26

Khatiri, Mariam. "Vergleich zweier PCR-Nachweisverfahren zur Früherkennung einer Aspergillusinfektion bei Hochrisikopatienten manuelle DNA-Extraktion im Vergleich zur automatischen MagNa Pure DNA-Extraktion; konventionelle PCR-ELISA im Vergleich zur LightCycler R -Methode /". [S.l. : s.n.], 2006.

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27

Bart-Delabesse, Emmanuelle. "Développement d'un système de typage d'Aspergillus fumigatus au moyen de microsatellites polymorphe : implications écologiques et épidémiologiques". Paris 12, 1999. http://www.theses.fr/1999PA120046.

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L'aspergillose invasive (ai) due a aspergillus fumigatus est devenue une cause majeure de deces chez les patients immunodeprimes. La pathogenese et l'epidemiologie de cette mycose opportuniste sont mal connues, ce qui rend sa prevention particulierement difficile. Devant le manque de fiabilite et/ou de rapidite des methodes de typage actuelles, un systeme de differenciation infraspecifique d'aspergillus fumigatus a ete developpe, au moyen de microsatellites. Le polymorphisme de 4 sequences riches en (ca), issues d'une banque genomique criblee avec un oligonucleotide (ca) 1 0, a ete evalue par pcr en determinant la taille des alleles sur un sequenceur automatique. L'analyse de 50 isolats environnementaux, 50 isolats de patients et de deux souches de reference a permis de detecter respectivement 12, 11, 10 et 23 alleles aux 4 loci (pouvoir discriminant = 0,994). Les resultats, hautement reproductibles, peuvent etre obtenus directement a partir de conidies. Aucun regroupement n'a ete observe selon l'origine clinique ou environnementale des isolats, suggerant que tout isolat est potentiellement pathogene. Afin d'etablir l'origine nosocomiale de l'ai, les isolats, recueillis sur 12 mois, de patients souffrant de maladies hematologiques et d'ai a l'hopital h-mondor (27 isolats, 12 patients) et de leur environnement (62 isolats), ont ete types. Une origine nosocomiale a ete trouvee pour 42% des patients. La grande biodiversite revelee au sein de la population environnementale (51 genotypes), la possibilite qu'un genotype persiste au cours du temps (8 genotypes), et l'absence de methodes exhaustives de prelevements constituent les facteurs limitants pour prouver la nosocomialite et reclame une meilleure connaissance des populations environnementales d'a. Fumigatus. Dans cette perspective, le typage par microsatellites apparait comme un systeme plus approprie que le typage par rapd, de par sa fiabilite, ou que le typage par rflp, de par sa rapidite et sa perfectibilite.

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28

Burc, Laurence. "Suivi épidémiologique des "Aspergillus" pathogènes dans les services d'hématologie et de réanimation pneumologique à l'hôpital Beaujon : typage moléculaire de souches d'"Aspergillus fumigatus" isolées de patients et de leur proche environnement". Paris 5, 1997. http://www.theses.fr/1997PA05P098.

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29

Luther, Kathrin. "Interaktionen des humanpathogenen Schimmelpilzes Aspergillus fumigatus mit Wirtszellen". Diss., kostenfrei, 2007. http://edoc.ub.uni-muenchen.de/7678/.

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30

Amaar, Yousef Grera. "Molecular characterizations of nitrate assimilation in Aspergillus fumigatus". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24287.pdf.

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31

Fraczek, Marcin Grzegorz. "The molecular analysis of allergens in Aspergillus fumigatus". Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508896.

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32

Albarrag, Ahmed. "Azole resistance in clinical isolates of Aspergillus fumigatus". Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487927.

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Aspergillus fil1nigatus is the most common aetiological agent of aspergillosis. Invasive aspergillosis is a major cause of death in leukaemic and organ transplant patients. The azoles are the largest and the most widely used class of antifungals. Antifungal drug resistance has been observed in many fungi. In many organisms, acquiring resistance to a drug can confer a biological fitness cost that is expressed for example as decreased growth rate or virulence. A total number of 21 A. fill11igatus clinical isolates were used in this project. Sixteen of these isolates were recovered serially from four patients at different times during the treatment period with azoles, including three cases of acquired azole resistance. The antifungal susceptibilities of isolates were determined. The genetic relatedness of the isolates was confirmed by microsatellite length polymorphism typing. Various measures of fitness were used to determine variation between susceptible and resistant isolates. Growth rate (colony radial growth rate and specific growth rate), germination time and conidial yields were determined on various media. Although some resistant isolates had a reduced growth rate compared to their susceptible match, there was no clear evidence of fitness cost of resistance has been found. The mechanisms of drug resistance in these isolates were investigated. Sequencing of the cyp5JA gene was carried out. Novel and previously described mutations in cyp5JA were identified. Three new mutations were detected in isolates recovered from patient D which were shown to have the same genotype. These mutations were G138C (in six isolates), Y431C (in one isolate) and G434C (in one isolate). These isolates have decreased susceptibility to itraconazole (>8.0 mg/l), voriconazole (4-8.0 mg/l), ravuconazole (4-8.0 mg/l), and posaconazole 0-4.0 mg/l). Azole cross-resistance observed in these isolates was confirmed to be caused by two of these mutations, G138C and Y431C, by expression of the mutated cyp5JA alleles in the yeast S. cerevisiae. The relative levels of expression of cyp5JA, cyp5JB and five efflux transporters, AfuMDRJ, AfilMDR2, AfiIMDR3, AfuMDR4 and atrF, genes were analysed using real-time RT-PCR. Gene expression analysis revealed that isolates from patient D had their cyp5JA gene up-regulated by 7.2- to 13.4-fold. Generally, susceptible and resistant isolates had the same level of expression of all five efflux transporters examined. Interestingly, a type II transposon insertion (1882 bp) in the region upstream of the start codon of cyp5JA, at position -317, was detected in one isolate from patient D, which exhibited the highest level of cyp5JA expression and so transposon insertion was associated with elevation of cyp5JA expression. In conclusion, this thesis describes novel mutations in the cyp5JA gene of clinical isolates that confer and azole cross-resistance. It also identified up-regulation of the cyp5JA gene as an additional azole resistance mechanism in some isolates. An active transposon was identified and found to be associated with resistance by elevating gene expression, an observation not previously made in A. fill11igatus for any phenotype. Up-regulation of 5 transporters and mutations in the cyp5JB gene were not related to resistance.

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33

Mowat, Eilidh. "In vitro modelling of chronic Aspergillus fumigatus infections". Thesis, Glasgow Caledonian University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501309.

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Recent studies have demonstrated the potential of Aspergillus fumigatus to form biofilm structures similar to those identified with Candida albicans. In contrast to the wide berth of knowledge relating to the biology of C. albicans biofilms, relatively little is known regarding the development and morphology of multicellular communities of A. fumigatus. This alternate lifestyle may have profound clinical implications with regard to the pathogenic potential of the organism and the likelihood of successful detection and treatment. A reproducible biofilm model of A. fumigatus was developed using several different substrates, which were used to macroscopically and microscopically investigate the phenotypic attributes and general characteristics of biofilm formation, and to investigate antifungal susceptibility profiles.

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Júnior, André Oliveira Mota. "Caracterização molecular do gene ncsA de Aspergillus fumigatus". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-05012009-123104/.

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O foco do trabalho está direcionado para a construção da linhagem knock-out do gene ncsA de A. fumigatus, bem como a caracterização do fenótipo da linhagem DncsA e a investigação de interações deste gene com vias de sinalização importantes para o desenvolvimento do fungo. A linhagem mutante ncsA foi construída através da transformação do WT (Dku80) com o gene marcador pyrG de A. fumigatus. Foi confirmada a deleção por Southern blot e as alterações fenotípicas da linhagem foram analisadas. O gene ncsA em A. fumigatus não é essencial e a linhagem DncsA apresentou-se sensível EGTA e SDS, alé, de tolerante a altas concentrações de íons cálcio. Na linhagem NcsA:mRFP foi localizado a proteína NcsA no citoplasma das células e sua localização não é alterada pela em resposta ao aumento da concentração de cálcio. A linhagem DncsA foi sensível a drogas que afetam a biossíntese e a manutenção da membrana plasmática, tais como voriconazole, anfotericina e itraconazole. O crescimento polarizado em presença de lovastatina nessa linhagem foi consideravelmente mais afetado que no tipo selvagem. O Spitzenkörper não foi visualizado no mutante DncsA, e existe uma significante diminuição de estruturas vacuolares e endossomos. Os resultados obtidos em ensaios de TRPCR em tempo real sugerem que NcsA modula a expressão dos genes pmcA e pmcB, que codificam proteínas ATPases transportadoras de íons cálcio. Os ensaios de virulência no modelo animal revelaram que a mutação do gene ncsA não causa perda de virulência no A. fumigatus.
Here, we characterize the A. fumigatus Neuronal Calcium Sensor, ncsA homologue. We showed that ncsA is not an essential gene and ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm that its cellular localization is not affected by the cellular response to calcium chloride. The ncsA mutant strain is more sensitive to voriconazole, itraconazole, and the ergosterol intercalating agent, amphotericin. Polar growth in the DncsA mutant strain was also considerably more affected by lovastatin than in the wild type mutant strain. The Spitzenkörper cannot be visualized in the DncsA mutant, and there is a significant decrease of the endosome/vacuole structures. NcsA supports pmcA and pmcB expression therefore reduced expression of these ion pumps, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

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35

Jamal, Atif. "Incidence and characterisation of mycoviruses from Aspergillus fumigatus". Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6092.

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This study investigated the incidence and characterisation of mycoviruses in a range of different fungi including Phytophthora spp. Phlebiopsis gigantea and Aspergillus fumigatus and their effects on their hosts. The investigation developed methodology for rapid molecular characterisation of dsRNA elements from different fungi which were applied in detail to a range of isolates of A. fumigatus. DsRNA elements or mycoviruses are present in almost all major classes of fungi where they lack an extracellular phase and are transmitted intracellulary via anastomosis or spores. Mycoviruses can confer a range of phenotypes on their fungal hosts ranging from symptomless to debilitating and hypovirulence to hypervirulence. In most cases most fungal mycovirus infections are symptomless and the effects of A. fumigatus mycoviruses on fitness and growth of infected isolates were also investigated. A screen of thirty nine clinical isolates of A. fumigatus, which is an opportunistic human pathogen and causes aspergillosis in immunocompromised patients, revealed the presence of dsRNA elements which were hitherto unknown. Two mycoviruses were identified including a chrysovirus (isolate A-56) and an unclassified, triripartite dsRNA-containing mycovirus (isolate A-54). All four dsRNA segments of the A-56 chrysovirus were sequenced in their entirety using the methodologies developed earlier in the investigation and the sequences analysed and compared to other members of the family Chrysoviridae and other characterised mycovirus families. Also the effects of the chrysovirus on the fitness of the host fungus were studied as was transfer of the purified chrysovirus to cured strains of A. fumigatus via protoplast fusion and direct transfection by different methods. It is intended to develop the use of dsRNA elements for gene silencing in A. fumigatus and towards this aim a full-length clone of the smallest A-56 dsRNA has been constructed and characterised and used to produce dsRNA transcripts for infection and amplification in the fungus.

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36

Griffiths, James. "CLRs and their role during Aspergillus fumigatus infection". Thesis, Cardiff University, 2018. http://orca.cf.ac.uk/114588/.

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CLRs are vital for orchestrating anti-fungal immunity. These receptors are expressed on myeloid immune cells and induce robust anti-A.f. responses including phagocytosis, cytokine and chemokine release, respiratory burst, and inflammasome activation. The role of Dectin-1 has been thoroughly investigated; however, the CLRs requirement during anti-A.f. response is controversial. The impact of Dectin-2, Mcl and Mincle during anti-A.f. immunity is not well understood. A.f. is complex and poses many challenges for the immune system. PRR collaboration is likely required for A.f. clearance. Collaboration between TLRs and CLRs has been identified. Recently, the first CLR:CLR collaboration was demonstrated between Dectin-1 and Dectin-2; however, this was not in response to A.f. The research in this thesis describes many novel interactions between CLRs and A.f. and seeks to further the understanding of the complex collaborative anti-A.f. immune response. Firstly, a novel role for NE inducing Dectin-1 Isoform A cleavage was described. This impaired A.f. recognition and the anti-A.f. response. CF patients possess high airway NE activity and experience a severe A.f. disease burden. My research suggests blocking the action of NE in CF patient’s airway may restore Dectin-1 expression and improve patient’s anti-A.f. immune response. Secondly, in vivo CLR KO and DKO models were used to elicit alveolar macrophages reliance on CLRs when generating anti-A.f. responses. My research suggests Dectin-1 might exclusively be required during early anti-A.f. responses. Unfortunately, discrepancies between the sex and microbiome of mice restricted the conclusions drawn from in vivo CLR KO and DKO A.f. infection experiments. Finally, I identified novel risk factors that can be used to stratify patients according to their susceptibility to A.f. infection. A.f. disease incidence and mortality rates are unacceptably high in immune-suppressed patients. Patients are often prophylactically treated with inadequate anti-fungal therapeutics. Stratifying patients according to their A.f. disease susceptibility would allow a personalised medicine approach and reduce unnecessary treatment.

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37

Fillaux, Judith Sophie. "Évaluation de la sensibilisation à Aspergillus fumigatus et du portage persistant comme facteurs de détérioration de la fonction respiratoire des patients atteints de mucoviscidose au CHU de Toulouse". Toulouse 3, 2013. http://thesesups.ups-tlse.fr/2323/.

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Aspergillus fumigatus (Af) est responsable de maladies respiratoires. La mucoviscidose est une maladie génétique fréquente, à transmission autosomique récessive au cours de laquelle les manifestations respiratoires déterminent le pronostic de la maladie. Les publications sur les infections fongiques au cours de la mucoviscidose sont rares. Au CHU de Toulouse, deux CRCM, pédiatrique et adulte, suivent les patients de la région Midi-Pyrénées. Les médecins implémentent à chaque consultation un dossier informatisé. A partir de ces données, une étude a été menée afin de déterminer si le fait de présenter des signes directs et/ou indirects de la présence d'Af, en l'absence d'ABPA, pouvait être responsable de l'altération de la fonction pulmonaire. Dans un deuxième temps, après avoir identifié deux nouvelles entités morbides, la sensibilisation à Af et le portage persistant de ce champignon, un algorithme de calcul de risque a été proposé
Aspergillus fumigatus (Af) is a ubiquitous fungus that causes a wide range of pulmonary diseases. Cystic fibrosis (CF) is one of the most common life-shortening autosomal recessive diseases in which chronic endobrochial infection contributes to progressive obstructive pulmonary disease. The literature provides scarce information about the impact of fungal infection on the pulmonary function of CF patients. At the Toulouse CF Resources and Competence Centre, details of patients with CF are entered into a database during each visit. From these data, a study was conducted to assess Af related-status modulating the forced expiratory volume in one second of CF patients. We have determined that Af may be of clinical relevance in some CF patients who do exhibit manifestations of sensitisation or persistent carriage. Secondly, we assess the putative predictive factors for CF patients to become either sensitised to or carriers of Af, and we proposed a tree diagram for risk calculation

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38

Durand-Perdriel, François. "Aspergillose broncho-pulmonaire allergique (ABPA) (ou maladie de Hinson-Pepys) : à propos de six nouvelles observations dans l'Ouest". Nantes, 1985. http://www.theses.fr/1985NANT3351.

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Hamet, Nicole. "Contribution à la prophylaxie de l'aspergillose des volailles". Université Joseph Fourier (Grenoble), 1990. http://www.theses.fr/1990GRE18005.

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Cardoso, Fernanda Gomes. "Vias alternativas mitocondriais: estudos moleculares e bioquímicos de uma UCP-like de Aspergillus fumigatus". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-08012016-124834/.

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A. fumigatus é um patógeno oportunista que causa infecções invasivas em hospedeiros imunocomprometidos. Estudos de respiração mitocondrial sugeriram a presença de componentes alternativos em sua cadeia respiratória envolvidos com processos de adaptação a ambientes adversos, como a proteína desacopladora (UCP). UCPs são proteínas mitocondriais cuja atividade dissipa o potencial de membrana gerado durante o transporte de elétrons. Um gene contendo características das três assinaturas moleculares das Proteínas Transferidoras de Energia foi clonado e sequenciado. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois íntrons que, após o splicing, codifica uma proteína contendo 341 aminoácidos, com uma massa molecular de 37 kDa e um pI de 10,02. A fim de se avaliar as propriedades bioenergéticas da UCP-like, essa sequência foi clonada no vetor pYES2 e leveduras S. cerevisiae foram transformadas. Esferoplastos foram preparados e o potencial elétrico transmembrana mitocondrial foi estimado. Os resultados mostraram que o potencial de membrana de esferoplastos de leveduras expressando a proteína UCP-like foi ligeiramente menor e que o decréscimo momentâneo do potencial associado com a fosforilação do ADP foi mais lento quando comparado com o controle, indicando desacoplamento da respiração. Além disso, esse comportamento dos esferoplastos recombinantes foi similar ao controle quando GDP foi adicionado ao meio de reação, sugerindo uma inibição da proteína por esse composto. Para sua caracterização funcional em sistemas reconstituídos, a sequência foi clonada no vetor pET SUMO. A expressão foi realizada em E. coli e a proteína recombinante, purificada por cromatografia em resina de níquel, foi analisada por Western blot com anticorpos anti-(His)6-tag e anti-UCP2 e por espectrometria de massas. A formação dos lipossomos foi confirmada através de medidas de distribuição de partícula por espalhamento de luz dinâmico, as quais sugeriram a formação de vesículas estáveis. Em adição, foi investigada a participação da UCP-like na proteção do A. fumigatus contra danos oxidativos. O nível de mRNA foi determinado por PCR em tempo real na presença de paraquat e menadiona. Em A. fumigatus, a presença dessas drogas pró-oxidantes resultou em um aumento no nível de mRNA desse gene, sugerindo que essa proteína possa também fazer parte de um sistema de defesa antioxidante do fungo.
A. fumigatus is an opportunistic pathogen that causes invasive infections in immunocompromised hosts. Mitochondrial respiration studies suggested the presence of alternative components on its respiration chain, which are involved with the adaptation to hostile environments, such as the uncoupling protein (UCP). UCPs are mitochondrial proteins whose activity dissipates the membrane potential generated during electron transport. A gene containing features of three molecular signatures of Energy Carrier Protein was cloned and sequenced. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 341 amino acids protein with a molecular mass of 37 kDa and a pI of 10.02. In order to study bioenergetics properties of UCP-like, the cDNA sequence was cloned into pYES2 vector and transformed in S. cerevisiae. Spheroplasts were prepared and the mitochondrial electrical transmembrane potential was estimated. The results showed that, compared with control cells, mitochondrial electrical transmembrane potential of transformant spheroplasts was slightly smaller and the transient potential decrease associated with ADP phosphorylation was longer, indicating uncoupling of respiration. Moreover, this behavior of recombinant spheroplasts was similar to control cells when GDP was added to the reaction medium, suggesting the inhibition of uncoupling protein. For its functional characterization in reconstituted systems, the cDNA sequence was cloned into pET SUMO vector. The expression was carried out in E. coli and the recombinant protein, purified by chromatography on a nickel-chelating resin, was analyzed by Western blot using anti- (His)6-tag or UCP2 antibodies and by mass spectrometry. Liposome formation was confirmed by light scattering, suggesting the formation of stable vesicles. In addition, the participation of UCP-like in A. fumigatus protection against oxidative damage was investigated. mRNA level was determined by real time PCR in the presence of paraquat and menadione. In A. fumigatus, the presence of these pro-oxidants drugs resulted in increased mRNA level of this gene, suggesting that this protein might also be part of an antioxidant defense system of this fungus.

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41

Soriani, Frederico Marianetti. "Caracterização de uma cálcio ATPase PMR1 de \'Aspergillus fumigatus\'". Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-02102008-163023/.

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Os conhecimentos sobre a regulação dos níveis de cálcio e manganês no Aspergillus fumigatus são bastante limitados, sendo que a homeostase destes íons pode ser diretamente controlada pela ação de ATPases específicas, dentre elas as cálcio ATPases da subfamília PMR1. Desta forma, o objetivo do presente estudo foi a expressão, caracterização e validação como alvo quimioterapêutico do gene Afpmr1 de A. fumigatus. Inicialmente, foi realizada a complementação funcional, de uma cepa de S. cerevisiae nocaute para a PMR1, em meios de cultura suplementados com EGTA ou manganês, revertendo o fenótipo da cepa nocute. Além disto, após expressão do gene Afpmr1, foi verificada uma reversão na intensa distribuição de quitina na parede celular da cepa nocaute. Paralelamente, para a RNAi, um fragmento do gene Afpmr1 apresentando baixa identidade com outros genes de cálcio ATPases de diferentes espécies foi clonado em vetor de expressão em A. fumigatus (pALB1). Após indução da expressão, a construção de RNA dupla fita para RNAi silenciou tanto o gene alb1 isoladamente (clone controle), quanto o duplo silenciamento com o gene de interesse Afpmr1, conferindo à ambas construções coloração branca às colônias. Uma vez confirmado o silenciamento gênico, por técnicas de RT-PCR quantitativo, os clones selecionados foram utilizados em ensaios de fagocitose e killing de macrófagos. O clone com o gene Afpmr1 silenciado apresentou diminuição na porcentagem de fagocitose, no número médio de conídios fagocitados e na eficiência de eliminação destes conídios quando comparados com seus controles. Estes resultados mostram que o gene Afpmr1 pode ser expresso funcionalmente em sistemas heterólogos e seu silenciamento, em A. fumigatus, influencia processos celulares que podem estar relacionados à manutenção da estrutura e composição da parede celular, além de desencadear alterações na fagocitose e killing de macrófagos.
The knowledge about the regulation of Aspergillus fumigatus calcium and manganese levels are very limited, while these ions homeostasis could be directly controlled by the function of specific ATPases, like the PMR1 calcium ATPase. In this way, the aim of the present work was the expression, characterization e validation, as chemotherapeutic target, of the A. fumigatus Afpmr1 gene. Initially, the functional complementation of a PMR1 knock-out strain phenotype was analyzed in EGTA or manganese supplemented culture media. Besides, after Afpmr1 expression, an intense distribution of chitin through the cell wall of the knock-out strain was reversed. At the same time, a fragment of the Afpmr1 gene, showing low identity values for another calcium ATPase genes, was cloned in an A. fumigatus expression vector (pALB1) for RNAi. After the induction of gene expression, a double strand RNA construct for RNAi has properly silenced either the alb1 gene alone (control clone), or the double silencing with the gene of interest Afpmr1, leading to both constructions white colored colonies. After confirmation of the gene silencing by quantitative RT-PCR techniques, the selected clones were used in macrophages killing and phagocytosis assays. The Afpmr1 silenced clone showed a decrease in the phagocytosis percentage, in the mean number of internalized conidia and in the killing percentage when compared with control groups. These results show that the Afpmr1 gene can be functionally expressed in eukaryotic heterologous systems and its silencing, in A. fumigatus, alters cellular processes that can be related with the maintenance of the cell wall structure and composition, as well as promote alterations in the macrophages phagocytosis and killing.

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42

Vieira, Fabíola Giovanna Nesello. "Otimização da produção de β-xilosidase por Aspergillus fumigatus". Universidade Estadual do Oeste do Parana, 2014. http://tede.unioeste.br:8080/tede/handle/tede/187.

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Made available in DSpace on 2017-05-12T14:47:00Z (GMT). No. of bitstreams: 1 Fabiola G_ Nesello Vieira.pdf: 1352690 bytes, checksum: dde9077ce35994c9a408a58112929a03 (MD5) Previous issue date: 2014-06-10
The abundant lignocellulosic biomass in agro-industrial waste can be reused as an inexpensive substrate for inducing the production of enzymes such as β-xylosidases. The purpose of this study was to analyze the production of β-xylosidase from Aspergillus fumigatus (PC-7S-2 M), isolated from the Atlantic Forest of the Dog Head State Park (Paraná, Brazil) and later identified by morphological and molecular (ITS) methods. The mesophilic fungus was grown at 28 °C in liquid culture media containing Czapeck and 1% of different agroindustrial residues (w/v): passion fruit peel, Ponkan peel, barley brewing residue, soy flakes and ripe banana peel. Inoculants of 105 conidia ml-1 were incubated for 7 days, filtered and assayed for β-xylosidase intracellular activity obtaining a maximum value of 15 U ml-1 of the enzyme in the presence of barley brewing residue after 4 days of cultivation. Then, it was used a Central Composite Rotational Design (CCRD) to optimize the production of β-xylosidase, using barley brewing residue as carbon source at a significance level of p<0.10 which generated a predicted model of 245.04 U ml-1. Model validation provided an average optimized result equal to 229.06 U ml-1 for the enzyme. Thus, the production of β-xylosidase increased in 1,500% over the initially obtained for A. fumigatus in the presence of the barley brewing residue, therefore, achieving 93.47% of the predicted model. This finding emphasizes the availability of A. fumigatus β-xylosidase production with possible applications in several biotechnological process.
A biomassa lignocelulósica abundante nos resíduos agroindustriais, pode ser reutilizada como substrato barato para induzir a produção de enzimas, como β-Xilosidases. O objetivo deste trabalho foi analisar a produção de β-Xilosidase de Aspergillus fumigatus (PC-7S-2 M), isolado da Mata Atlântica do Parque Estadual Cabeça do Cachorro (Paraná, Brasil) e posteriormente identificado por métodos morfológicos e moleculares (ITS). O fungo mesofílico foi cultivado à temperatura de 28 °C em meios líquidos de cultura Czapeck, contendo 1% de diferentes resíduos agroindustriais (w/v): casca de maracujá, casca de pokan, bagaço de cevada, flocos de soja e casca de banana madura. Inóculos de 105 conídios mL-1 foram incubados durante 7 dias, filtrados e submetidos a dosagem de β-Xilosidase intracelular, obtendo-se um valor máximo de 15 U ml-1 para a enzima na presença de bagaço de cevada com 4 dias de cultivo. Assim, utilizou-se um delineamento composto central rotacional (DCCR) para otimizar a produção de -Xilosidase, usando o bagaço de cevada como fonte de carbono em um nível de significância p < 0,10, o qual gerou um modelo predito de 245,04 U ml-1. A validação do modelo forneceu um resultado otimizado médio igual a 229,06 U ml-1 para a enzima. Assim, a produção de β-Xilosidase aumentou em 1.500% em relação à obtida inicialmente para o fungo A. fumigatus na presença de bagaço de cevada como fonte de carbono (15 U ml-1), permitindo, deste modo, alcançar 93,47 % do modelo predito. Este achado ressalta a viabilidade de produção de β-Xilosidase de A. fumigatus com possíveis aplicações em vários processos biotecnológicos.

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Vieira, Fabíola Giovanna Nesello. "Otimização da produção de β-xilosidase por Aspergillus fumigatus". Universidade Estadual do Oeste do Parana, 2014. http://tede.unioeste.br:8080/tede/handle/tede/2646.

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The abundant lignocellulosic biomass in agro-industrial waste can be reused as an inexpensive substrate for inducing the production of enzymes such as β-xylosidases. The purpose of this study was to analyze the production of β-xylosidase from Aspergillus fumigatus (PC-7S-2 M), isolated from the Atlantic Forest of the Dog Head State Park (Paraná, Brazil) and later identified by morphological and molecular (ITS) methods. The mesophilic fungus was grown at 28 °C in liquid culture media containing Czapeck and 1% of different agroindustrial residues (w/v): passion fruit peel, Ponkan peel, barley brewing residue, soy flakes and ripe banana peel. Inoculants of 105 conidia ml-1 were incubated for 7 days, filtered and assayed for β-xylosidase intracellular activity obtaining a maximum value of 15 U ml-1 of the enzyme in the presence of barley brewing residue after 4 days of cultivation. Then, it was used a Central Composite Rotational Design (CCRD) to optimize the production of β-xylosidase, using barley brewing residue as carbon source at a significance level of p<0.10 which generated a predicted model of 245.04 U ml-1. Model validation provided an average optimized result equal to 229.06 U ml-1 for the enzyme. Thus, the production of β-xylosidase increased in 1,500% over the initially obtained for A. fumigatus in the presence of the barley brewing residue, therefore, achieving 93.47% of the predicted model. This finding emphasizes the availability of A. fumigatus β-xylosidase production with possible applications in several biotechnological process.
A biomassa lignocelulósica abundante nos resíduos agroindustriais, pode ser reutilizada como substrato barato para induzir a produção de enzimas, como β-Xilosidases. O objetivo deste trabalho foi analisar a produção de β-Xilosidase de Aspergillus fumigatus (PC-7S-2 M), isolado da Mata Atlântica do Parque Estadual Cabeça do Cachorro (Paraná, Brasil) e posteriormente identificado por métodos morfológicos e moleculares (ITS). O fungo mesofílico foi cultivado à temperatura de 28 °C em meios líquidos de cultura Czapeck, contendo 1% de diferentes resíduos agroindustriais (w/v): casca de maracujá, casca de pokan, bagaço de cevada, flocos de soja e casca de banana madura. Inóculos de 105 conídios mL-1 foram incubados durante 7 dias, filtrados e submetidos a dosagem de β-Xilosidase intracelular, obtendo-se um valor máximo de 15 U ml-1 para a enzima na presença de bagaço de cevada com 4 dias de cultivo. Assim, utilizou-se um delineamento composto central rotacional (DCCR) para otimizar a produção de -Xilosidase, usando o bagaço de cevada como fonte de carbono em um nível de significância p < 0,10, o qual gerou um modelo predito de 245,04 U ml-1. A validação do modelo forneceu um resultado otimizado médio igual a 229,06 U ml-1 para a enzima. Assim, a produção de β-Xilosidase aumentou em 1.500% em relação à obtida inicialmente para o fungo A. fumigatus na presença de bagaço de cevada como fonte de carbono (15 U ml-1), permitindo, deste modo, alcançar 93,47 % do modelo predito. Este achado ressalta a viabilidade de produção de β-Xilosidase de A. fumigatus com possíveis aplicações em vários processos biotecnológicos.

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44

Morley, Joseph Peter. "Studies of clinical and environmental isolates of Aspergillus fumigatus". Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39567.

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Aspergillus fumigatus is a fungus of great environmental importance as well as being the most common filamentous fungal respiratory pathogen. Airborne A. fumigatus spores can reach potentially dangerous concentrations, particularly in the air around industrial composting sites. Drug resistant A. fumigatus infections are an increasingly common problem and the limited range of drugs available for the treatment of Aspergillus infections means that any resistance developing is a major cause for concern. Mycoviruses may offer an alternative approach to conventional treatment and exist in many fungal populations, including A. fumigatus, but there is little understanding of their importance or the influence they may have on their hosts. Different air samplers used in bioaerosol studies were evaluated. An enrichment protocol for the isolation of mycoviruses from environmental samples was assessed as well as further characterisation and genome sequencing of a mycovirus of the A. fumigatus type strain NCPF7367. For clinical and environmental A. fumigatus collections drug susceptibility was assessed using the EUCAST protocol, virulence compared using a Galleria model and the mating type of the fungi determined by PCR. Significant differences in sampler efficacy were observed. Putative mycoviruses were obtained from environmental samples and sequencing of the NCPF7367 virus genome suggests that this is a capsid-less virus. Increased azole resistance and virulence was found amongst compost-derived A. fumigatus relative to clinical isolates. This study highlights the importance of sampler selection in bioaerosol studies. A novel enrichment procedure for mycovirus isolation from environmental samples appears promising and the genome sequencing sheds new light on the lifecycle of an A. fumigatus mycovirus. The study also suggests that compost-derived A. fumigatus may be less susceptible to azole drugs and more virulent than clinical populations. Drug resistance may be more geographically variable than previously thought and intermediate-level resistance appears not to be mediated by cyp51-A mechanisms.

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Rodriguez, de Francisco Borja. "Self-assembly into functional amyloids of Aspergillus fumigatus hydrophobins". Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS332.

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Les hydrophobines sont des protéines fongiques qui s’assemblent aux interfaces hydrophobes/hydrophiles ou air-eau (IHH) pour former des fibres amyloïdes fonctionnelles qui s’organisent en couches dont le caractère amphiphile détermine leurs fonctions. Les spores du champignon pathogène opportuniste Aspergillus fumigatus sont couvertes d’une couche de bâtonnets constitués de fibres amyloïdes formés par l’hydrophobine RodA. Ce revêtement hydrophobe facilite la dispersion des spores et les rend inertes vis à vis du système immunitaire humain. Deux proches homologues de RodA, RodB et RodC, sont aussi présents dans la paroi cellulaire des spores. Nous avons réalisé une étude comparative de l’auto-assemblage de ces trois protéines avec un accent particulier sur RodC. Nous avons montré que RodA-C nécessitent d’une interface IHH pour s’assembler en fibres et mis en évidence l’importance de la nature de l’interface dans la morphologie de leurs assemblages. Nous avons observé une auto-inhibition de la fibrillation avec la concentration et montré que celle-ci est due à la saturation de l’interface air-eau. En étudiant l’effet de mutations ponctuelles sur les cinétiques de fibrillation de RodC, nous avons établi des similitudes et des différences par rapport aux régions importantes pour la formation de fibres pour RodC et RodA, étudiée au préalable. La transition du monomère à l’état amyloïde est accompagnée d’une perte de régions désordonnées et un gain de feuillets β intermoléculaires en accord avec les analyses par mutagénèse dirigée, qui indiquent que des résidus hydrophobes dans des régions flexibles du monomère sont impliqués dans le squelette β-croisé des fibres
Hydrophobins are fungal proteins that spontaneously self-assemble at hydrophobic/hydrophilic or air/water interfaces to form functional amyloids that associate laterally into layers. The amphiphilic character of these layers is at the origin of the hydrophobin biological roles. The spores of the airborne fungal pathogen Aspergillus fumigatus are covered by an amyloid layer with rodlet morphology made up by the RodA hydrophobin. This hydrophobic coat facilitates air-dispersal of the spores and renders these inert relative to the human immune system. Two close homologs of RodA, named RodB and RodC, are also present in the spore cell wall. We have performed a comparative study on the self-assembly of the three proteins with particular emphasis on RodC. We have shown that RodA-C require an interface to form amyloids and revealed the importance of the nature of the interface in determining the morphology of hydrophobin assemblies. We have observed that the fibrillation of RodA-C is auto-inhibited (slower at higher concentrations) and shown that this phenomenon can be explained by saturation of the air-water interface. The analysis of the effect of single point mutations on the fibrillation kinetics of RodC revealed the regions that are important for fiber formation, which showed differences and similarities relative to the previously studied RodA. The transition from monomers to amyloids is accompanied by a loss of unordered regions and a gain in intermolecular β-sheets, in agreement with the mutational analyses of RodA and RodC that indicated that hydrophobic residues in flexible loops are involved in the cross-β core of the fibers

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46

Rowley, Jessica. "The interaction of Aspergillus fumigatus with the respiratory epithelium". Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/the-interaction-of-aspergillus-fumigatus-with-the-respiratory-epithelium(0fc10449-977d-4f14-a169-172e8204fee4).html.

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Aspergillus fumigatus is a filamentous fungus and the main pathogen responsible for the often fatal respiratory condition, aspergillosis. Airway epithelial cells (AECs) are likely to be the first line of host defence that come into contact with the inhaled conidia of A. fumigatus. Recent evidence strongly suggests that the response of the airway epithelium to inhaled pathogens is pivotal in orchestrating immune responses by inducing phagocytic-like reactions and the secretion of inflammatory cytokines and antimicrobial peptides. However, the majority of previous work investigating A. fumigatus-host interactions has been performed using macrophages and neutrophils, thereby neglecting the epithelium. AECs have been shown to secrete inflammatory cytokines in response to A. fumigatus although these studies predominantly used transformed AEC lines that lack tight junctions and do not fully differentiate. Furthermore, most studies used culture filtrate or extract of A. fumigatus rather than live, whole organism and as a result, the direct interaction of the germinating fungus and the airway epithelium has been overlooked. During the early germination and growth period, the cell wall composition of A. fumigatus is dynamic, with various antigens exposed at different morphological stages. The aim of this thesis was to determine whether AECsare able to alter the germination and growth rate of A. fumigatus, and, conversely, if A. fumigatus affects AECs in terms of the secretion of inflammatory mediators. These studies used live, germinating A. fumigatus, and human primary differentiated AECs to obtain a more realistic in vitro model than those used in previous studies. Data showed that AECs are able to significantly inhibit the germination and growth of A. fumigatus, although this effect was less pronounced in differentiated primary AEC than in transformed AEC lines. A. fumigatus also significantly inducedthe expression and secretion of the inflammatory cytokines, IL-6 and IL-8, probably via the interaction of fungal cell wall β-glucans, and as of yet unidentified AEC receptor. The A1160pyrG+ strain of A. fumigatus secreted factors capable of inducing cytokine secretion whereas Af293 strain did not, highlighting diverse mechanisms of action for different strains. Upregulation of both cytokines was dependent on the stage of A. fumigatus growth with induction synchronous with germination. Despite being associated with fungal sensitisation in asthmatics, AEC-derived cytokines associated with this disease, namely TSLP, IL33 and IL25,did not appear to be upregulated by transformed AECs in response to A. fumigatus. Similarly, A. fumigatus did not seem to induce synthesis and secretion of the acute phase response protein, fibrinogen above baseline levels. The data presented in this thesis confirms the importance of the airway epithelium in directing anti-A. fumigatus immunity and the involvement of complex ligand-receptor interactions.

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Safari, Maryam. "Aspergillosis : interactions of Aspergillus fumigatus and Human Airway Cells". Thesis, University of Westminster, 2013. https://westminsterresearch.westminster.ac.uk/item/8z158/aspergillosis-interactions-of-aspergillus-fumigatus-and-human-airway-cells.

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Aspergillus fumigatus is a filamentous fungus that colonises the lungs of immunosuppressed patients and causes aspergillosis. Despite recent medical advances, infectious diseases caused by opportunistic pathogens such as A. fumigatus are still one of the main causes of morbidity and mortality among immunosuppressed patients. Gliotoxin is a mycotoxin and a secondary metabolite of A. fumigatus. This toxin possesses immusuppresive activity affecting cells of immune system. This research shows that human adenocarcinoma alveolar epithelial cells (A549) are highly susceptible to gliotoxin- induced cell death. Wild type strains of A. fumigatus cause death in these cells whereas mutant strains (gliotoxin deficient) do not. Gliotoxin induced cell death is mostly via apoptosis programmed cell death rather than necrosis. Long term incubation of A549 cells with gliotoxin-deficient strain causes morphology changes (swelling/partial damage) in these cells. This research proposes that gliotoxin induced death/ inflammation of pneumocytes by programmed apoptosis may contribute to the pathogenesis of aspergillosis, particularly invasive aspergillosis. In addition, other produced metabolites (e.g. proteases or other toxins) also contribute (to a lesser extent) to the lung tissue damage in the absence of gliotoxin. One of the key risk factors in developing aspergillosis is corticosteroid therapy. This is due to the effect of glucocorticoids on suppressing body’s defence mechanism. As hydrocortisone is present during Aspergillus infection, understanding the ability of the fungus to perceive this host-factor enhances the understanding of A. fumigatus interaction with the host within the host environment. Previous reports have shown that physiological and pharmacological concentrations of hydrocortisone enhance the growth-rate of A. fumigatus in vitro. The present work demonstrates the effect of hydrocortisone on cultures of A. fumigatus at both morphological and molecular levels. Hydrocortisone causes an early onset of sporulation and seems to play a role in regulation of haemostasis and stress response in this fungus. Expression of stress-related proteins (hsp70, catalase), proteases (e.g. serine protease, metalloprotease), and carbohydrate metabolism-related proteins increased after supplementation of A. fumigatus cultures with hydrocortisone. Stimulation studies investigating the effect of secondary metabolites of A. fumigatus on A549 cells with and without pre-treatment of the fungal cultures with hydrocortisone indicated subtle increases in the number of dead cells after hydrocortisone pre-treatment. This study shows further that hydrocortisone treatment of the lung alveolar cells enhances the binding of A. fumigatus spores to these cells. The obtained data suggests that presence of hydrocortisone may contribute to the survival of this pathogen within the host environment. This study provides additional knowledge at molecular and cellular level where complications exist as a result of infection by A. fumigatus fungus and during treatment with glucocorticoids.

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48

Fischer, Sandra Elena. "Vergleich von konventionellen PCR Methoden mit den Aspergillus fumigatus spezifischen Primern TS2 und AfLC2 und dem Panfungal-Primer für die Aspergillus fumigatus Diagnostik bei Risikopatienten". [S.l. : s.n.], 2007.

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49

Guazzelli, Luciana Silva. "Estudo etiológico, clínico, laboratorial e epidemiológico da bola fúngica pulmonar por Aspergillus spp". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/30929.

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Descrição: Bola fúngica é definida como uma macrocolônia composta por emaranhado de hifas, células inflamatórias, fibrina, muco e fragmentos de tecidos. Aspergillus fumigatus é o agente etiológico mais frequente, responsável por cerca de 90% dos casos, seguido de A niger e A flavus, respectivamente. O antecedente mais comum para o desenvolvimento da bola fúngica é cavidade secundária à tuberculose e a manifestação clínica mais presente e causadora de óbitos nesses pacientes é a hemoptise. Objetivos: Investigar as espécies de Aspergillus causadoras de bola fúngica pulmonar, determinar as condições predisponentes e/ou associadas e a comprovação laboratorial para o diagnóstico etiológico e observar a resposta as diferentes medidas terapêuticas dos pacientes com bola fúngica pulmonar. Delineamento: Foram analisados retrospectivamente, prontuários de pacientes para a caracterização da bola fúngica pulmonar por Aspergillus. Local do estudo: Laboratório de Micologia da Santa Casa Complexo Hospitalar, no período de 1980 a 2009. Pacientes e métodos: Foram incluídos neste estudo todos os pacientes com diagnóstico de bola fúngica pulmonar aspergilar de uma população de 750 casos de aspergilose, de 1980 a 2009. Os critérios para o diagnóstico foram os seguintes: isolamento da espécie de Aspergillus proveniente do material de cavidade pulmonar associado à imagem radiográfica compatível; isolamento da espécie de Aspergillus em outros materiais do trato respiratório, excluindo material da cavidade, associado ou não ao exame direto positivo; imunodifusão radial dupla positiva para Aspergillus associada ao exame de imagem compatível. Resultados: Foram incluídos 391 pacientes com bola fúngica pulmonar aspergilar, a idade variou de 18-78 anos, sendo 67,3% do gênero masculino. O diagnóstico foi baseado nos achados clínicos, radiológicos e laboratoriais. Em todos os pacientes foram detectados achados característicos de bola fúngica tanto no radiograma quanto na tomografia de tórax e bola fúngica complexa foi detectado em 97,4% da casuística. Tuberculose curada foi a principal condição predisponente (89%). Hemoptise foi manifestação clínica mais frequente (89%). A espécie A. fumigatus foi o agente etiológico mais isolado, 89,3% dos casos, seguido de A niger 7,1% e menos frequente A flavus 3,3%. A positividade no cultivo foi de 84,7% nos espécimes clínicos e a imunodifusão radial dupla de 81,6% dos pacientes. A principal medida terapêutica foi ressecção cirúrgica apresentando desfecho favorável em 88,3%. A eliminação da bola fúngica por lise espontânea ocorreu em 2,3% dos casos. Mortalidade foi atribuída à cirurgia e a hemoptise em 32,3 e 13,8%, respectivamente. Conclusões: Tuberculose curada e hemoptise é a primeira hipótese diagnóstica de bola fúngica pulmonar. O sinal radiológico indicativo de bola fúngica é cavidade contendo produto patológico com densidade de partes moles, espessamento da parede da cavidade e da pleura circunjacente. A detecção de anticorpos séricos por imunodifusão radial dupla, e o cultivo de espécimes do trato respiratório inferior determinaram A. fumigatus como o principal agente etiológico da bola fúngica pulmonar. A medida terapêutica mais utilizada nos pacientes do presente estudo foi ressecção cirúrgica, e a metade da ocorrência de óbito esteve presente nestes casos.
Background: Pulmonary fungus ball is defined as a conglomeration, within a cavity of intertwined Aspergillus hyphae, inflammatory cells, fibrin, mucus and cellular debris. Aspergillus fumigatus is the most frequent etiologic agent, about 90% of cases, followed by A niger and A flavus, respectively. The most common condiction to develop fungus ball is residual cavities of healed tuberculosis, and the most prevalent clinical manifestation and cause of death is hemoptysis in these patients. Objectives: To investigate the species of Aspergillus causing pulmonary fungus ball, we compared underlying conditions, laboratory evidence to the etiological diagnosis, and response of the different therapy, and outcome of patients with pulmonary fungus ball. Design: We analyzed retrospectively the medical records of patients for the characterization of pulmonary Aspergillus fungus ball. Settings: A university-based tertiary care hospital in Porto Alegre, Rio Grande do Sul, Brazil. Patients and methods: The study included patients diagnosed with pulmonary Aspergillus fungus ball in a population of 750 cases of aspergillosis, from 1980 to 2009. The criteria for the diagnosis were: isolation of Aspergillus species from the material of the pulmonary cavity associated with the compatible radiographic image; isolation of Aspergillus species from other materials of the respiratory tract, excluding cavity material, with or without direct examination positive; double immunodiffusion positive for Aspergillus associated with compatible image. Results: We included 391 patients with pulmonary Aspergillus fungus ball, age ranged from 18 to 78 years, 67.3% were male. The diagnosis was based on clinical, radiological, and laboratory findings. In all patients we detected the characteristic findings of fungal ball, on X-ray and tomography; and complex fungal ball, on their radiological appearance, was detected in 97.4% of cases. Healed tuberculosis was the commonest pre-existing disease (89%). Hemoptysis was the major symptoms (89%). The species A. fumigatus was the most common etiologic agent, 89.3% of cases, followed by 7.1% A niger and A flavus less frequent in 3.3%. Culture was positive in 84.7% specimes, and immunodiffusion in 81.6% patients. The main treatment was surgical resection in 88.3% that had a favorable outcome. Spontaneous lysis was obtained in 2.3% of cases. Mortality was attributed to the surgery and hemoptysis in 32.3 and 13.8%, respectively. Conclusions: Patient with healed tuberculosis and hemoptysis is the first hypothesis diagnostic fungus ball. The most frequent radiological signs were rounded dense opacity surrounded with a halo of air in a thick cavity wall and thickening of the pleura over cavity. The detection of serum antibodies by double immunodiffusion, and the cultive of the lower respiratory tract specimens determined A. fumigatus as the main agent of pulmonary fungal ball. The detection of serum antibodies by double immunodiffusion, and the cultivation of the lower respiratory tract specimens determined A. fumigatus as the main agent of pulmonary fungal ball. The measure most commonly used therapy in patients of this study was to surgical resection, and half of the patients who died were in these cases.

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Hündling, Dörte. "Caractérisation biochimique et structurale de lectines d'Aspergillus fumigatus". Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV055/document.

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L'objectif de cette thèse a été de contribuer à la compréhension des stratégies d'infection du pathogène opportuniste Aspergillus fumigatus. Ce pathogène est une cause émergente de morbidité et de mortalité chez les patients dont l'immunité est compromise et dans les milieux hospitaliers. Une infection avec Aspergillus est en général appelée une Aspergillose et ellepeut se développer dans un certain nombre d'organes, le plus fréquemment dansl'appareil respiratoire (poumons et sinus). Outre les infections (Aspergillosis invasive), la colonisation par ce champignon peut causer des réactions allergiques (Aspergilloses broncho pulmonaire allergique) et de l'asthme. Le nombre de patients immunodéprimés augmente régulièrement à cause des avancées des traitements du SIDA, du cancer, de la mucoviscidose ainsi que par le nombre grandissant de transplantation d'organes. De nouveaux médicaments antifongiques et des médicaments préventifs sont nécessaires pour venir en soutienaux soins médicaux des patients. Bien que plusieurs fongicides existent déjà sur le marché, l'Aspergillosisinvasive reste souvent fatale. Ceci est lié d'une part à la difficulté d'établir le diagnostic, et d'autre part au fait que des résistances émergent rapidement. La motivation de cette thèse est de comprendre les mécanismes impliqués dans le premier contact entre les conidies et le tissu de l'hôte. Ces mécanismes d'adhésion initiaux sont souvent réalisés par des liaisons entre les lectines et les carbohydrates. Le tissu épithélial et la surface muqueuse du système respiratoire sont couverts de structures possédants des carbohydratestels que les glycoprotéines, les glycolipides et les glycosaminoglycanes. L'identification des lectines d'A. fumigatus et leurs caractérisations devraient dorénavant contribuer à la compréhension de la glycostratégie de ce pathogène opportuniste ainsi que des mécanismes impliqués dans l'adhésion et l'infection. L'analyse détaillée de la structure des lectines permettra d'établir le rôle de cesprotéinesdans la virulence et de guider la conception de glycomimétiques, afin d'inhiber le phénomène d'adhésion. Cettenouvelle approche consistant à bloquer l'adhésion de l'agent pathogène plutôt que sa prolifération, vise à diminuer les résistances par une réduction de la pression évolutive. Deuxstratégies différentes ont été utilisées pouridentifier de nouvelleslectines. Tout d'abord une purification des lectinesà partir d'extraits fongiques brutsa été tentée et d'autre part un criblage pour trouver des séquences similaires avec les protéines codées par A. fumigatusa été réalisé parmi une banque de lectines fongiques connues
The aim of this thesis was to contribute to the understanding of infection strategies of the opportunistic pathogen Aspergillusfumigatus. This pathogenic mould is an emerging cause of morbidity and mortality in immuno-compromised patients and hospital environments. An infection with Aspergillus is generally referred to as Aspergillosis; it can develop in a variety of organs but the most common sites are the respiratory apparatus i.e. lungs and sinuses. Besides infections (invasive aspergillosis), colonization with the fungus can cause allergic reactions (allergic broncho pulmonary aspergillosis) and asthma. The number of immuno-suppressed patients is steadily increasing due to advancement in the HIV, cancer and cystic fibrosis medical care, as well as an increasing number of organ transplantations. Needless to say that new antifungal drugs and preventive medication is desperately needed to support medical care for those patients. Even though several fungicides already exist on the market, invasive aspergillosis remains to be often fatal. On one hand, this is due to difficulties in diagnosis and on the other hand, resistances are emerging rapidly. The motivation behind this thesis is to understand the underlying mechanisms that are involved in the first contact between conidial spores and host tissues. Initial adhesion steps often involve carbohydrate binding proteins, called lectins. They recognize glycoconjugates such as glycoproteins, glycolipids and glycosaminoglycans which cover the epithelial tissue and mucosal surface of the respiratory tract.. Identification and characterization of the lectins from A. fumigatus will therefore contribute to the understanding of the glycostrategy of this opportunistic pathogen and of the mechanisms involved in adhesion and infection. Detailed structural analysis of the carbohydrate-protein interactions will allow ascertaining the lectins role in virulence and guide the design of glycomimetics, as adhesion inhibitors. With this novel approach of targeting the pathogen adhesion rather than its proliferation, resistances are believed to be less frequent due to the lack of evolutionary pressure. In this work, two different strategies were employed to obtain novel lectins. Firstly, lectins were purified from crude fungal extracts and secondly the A. fumigatus genome was screened for encoded proteins showing sequence similarity with known fungal lectins. While lectin purification from the crude extracts was inconclusive due to low lectin activity in the starting material, genome screening showed that several putative lectins were present. One of these lectins, named AFL6, belonged to the cyanovirin-N homolog (CVNH) family and it was recombinantly expressed and purified. Glycan array and micro calorimetry techniques were carried out to investigate its carbohydrate binding specificityand the three dimensional structure was determined using X-ray crystallography. The structure showed an overall similarity with other CVNHs with slight differences in the presumed carbohydrate binding sites. Unlike other family members, it shows a low affinity for mannosides and an apparent affinity for lactosamine containing glycan structures

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