The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

Thèse de doctorat : Parasitologie. Mycologie : Paris 12 : 2003. / Titre provenant de la thèse originelle.

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.

Thèse de doctorat : Sciences de la vie et de la santé. Parasitologie, mycologie : Paris 12 : 2006. / Titre provenant de l'écran-titre. Pagination : 186 f. Bibliogr. f. 97-107.

Thesis (Ph. D.)--Oregon State University, 2005. / Printout. Includes bibliographical references (leaves 128-149). Also available on the World Wide Web.

Background: Asthma affects 5.4 million people in the UK. Asthma subgroups are also susceptible to inhalation of fungal spores (Aspergillus fumigatus) and development of pulmonary fungal aspergilloma; presenting a life threatening but poorly understood condition. NHS costs for corticosteroids, bronchodilators and antifungal agents that are only partially effective continue to rise. Allergy immunotherapy development is of great interest as it is specific to the allergen and can harness key adaptive immune T-cells to down-regulate inflammatory responses. Immunotherapy has been used with varying degrees of success for treatment of grass, pollen, venom, cat and dog allergens however to date has not been directed to fungal allergens. The study aims were: 1) to further understand the A. fumigatus allergens and the protein epitopes responsible for generating immune responses. 2) To genotype participating ABPA/SAFS patients to observe any HLA associations. Methods: 37 subjects with fungal sensitivity were recruited to the study which received permission from the local ethics committee (UHSM LREC). Computer bioinformatic predictions using Propred software identified several potential fungal T cell peptide epitopes; of which 8 peptides were soluble and tested in vitro for specific T-cell proliferation responses by flow cytometric analysis. Skin prick tests determined subject responses to fungal allergens including A. fumigatus, and DNA analysis determined subject HLA type. Results: 5 of 8 soluble peptides were Aspergillus fumigatus derived and 3 from Alternaria alternata. All 8 peptides induced higher CD4 proliferative responses in ABPA/SAFS patients, compared to healthy controls from highest significance to lowest as follows: peptide 1.1 > 9.1 > 8.1 > 2.1 > 9.1.1 > 4.1 > 4.1.1 and 10.1.1. 73% subjects elicited skin responses to A. fumigatus. DNA HLA typing identified alleles associated with ABPA/SAFS but not all allele sub types. Discussion: The ABPA/SAFS group consistently raised T-cell responses to fungal peptides compared to controls. This demonstrates peripheral CD4s retain memory for fungal specificity and clearly respond when challenged with fungal epitopes in vitro. This concept underpins the rationale to further characterize the responding CD4 cells and pursuing bioinformatics approaches for immunotherapy investigations for fungal allergy.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-05-02T12:12:25Z No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-04T12:25:29Z (GMT) No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-04T12:25:37Z (GMT) No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Made available in DSpace on 2017-05-04T12:40:10Z (GMT). No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) Previous issue date: 2016-06-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Aspergillus fumigatus is an opportunistic pathogenic filamentous fungus, and the causative agent of aspergillosis, including the invasive pulmonary aspergillosis in immunocompromised individuals, most lethal form of the disease. As in all eukaryotes, the plasma membrane of A. fumigatus is composed of sterols, glycolipids and sphingolipids. The sphingolipids molecules are structural elements that participate in the regulation of the actin cytoskeleton endocytosis, and cell growth. In Saccharomyces cerevisiae, the sphingolipid synthesis is regulated by proteins such as the "AGC kinases" known as Ypk1p and Ypk2p that inactivate Orm1p and Orm2p, which are inhibitory proteins of the serine palmitoyltransferase (SPT) enzyme, the first enzyme that participates in biosynthetic pathway of sphingolipids. Thus, Ypk1/2p are responsible for stimulating the production of sphingolipids in response to several cellular stress factors, such as cell wall stress and heat stress. Here, we aimed to expand the study of sphingolipid biosynthesis in A. fumigatus by functional characterization of the ypkAYPK1 gene by establishing its role in the regulation of sphingolipid synthesis and in the maintenance of cell integrity. To accomplish this goal, one null mutant strain ΔypkAypk1 and a conditional mutant strain niiA::ypkA of A. fumigatus were obtained. The mutant strains were characterized by phenotypic tests aimed at understanding the role of this gene in sphingolipid synthesis, virulence and pathogenicity of this fungus. Subsequently real time RT-PCR experiments were performed to quantify the expression of this gene in conditions of heat stress. The results indicated that deletion of the gene ypkA promotes changes in the development of the fungus, which presents severe defects in the vegetative growth and absence of conidia. In addition, the conditional mutant shows increased sensitivity to lipid synthesis inhibitory drugs. The results also indicate that there is no interaction between genetic ypkA and pathway genes of the cell wall. Thus, we propose that protein kinase YpkA in A. fumigatus is related to vegetative growth and participates in the sphingolipid biosynthesis in A. fumigatus, contributing to the development and integrity of the fungal cell. / Aspergillus fumigatus é um fungo filamentoso patogênico oportunista, e o agente etiológico da aspergilose, que em imunocomprometidos pode vir a ocasionar a forma mais letal da doença, a aspergilose pulmonar invasiva. Como em todos os eucariotos, a membrana plasmática de A. fumigatus é composta por esteróis, glicolipídios e esfingolipídios. As moléculas de esfingolipídios são elementos estruturais que participam da regulação da endocitose, do citoesqueleto de actina e do crescimento celular. Em Saccharomyces cerevisiae, a síntese de esfingolipídios é regulada pelas proteínas do tipo “AGC quinases” conhecidas como Ypk1p e Ypk2p, que inativam Orm1p e Orm2p, as quais são proteínas inibidoras da enzima serina palmitoil-transferase (SPT), a primeira enzima que participa da via de biossíntese dos esfingolipídios. Dessa forma Ypk1/2p são responsáveis por estimular a produção de esfingolipídios na membrana plasmática em resposta a diversos fatores de estresse celular, como por exemplo o estresse de parede celular, estresse térmico entre outros. Diante disso, este trabalho teve como objetivo ampliar o estudo da biossíntese de esfingolipídios em A. fumigatus através da caracterização funcional do gene ypkAYPK1, de forma a estabelecer sua função na regulação da síntese de esfingolipídios e na manutenção da integridade celular. Para cumprir esse objetivo, uma linhagem mutante nulo ΔypkAypk1 e uma linhagem mutante condicional niiA::ypkA em A. fumigatus foram obtidas. As linhagens mutantes foram caracterizadas através de ensaios fenotípicos visando a compreensão do papel desse gene na síntese de esfingolipídios, na virulência e patogenicidade desse fungo. Posteriormente, foram realizados experimentos de RT-PCR em tempo real para a quantificação da expressão desse gene em condições de estresse térmico. Os resultados indicaram que a deleção do gene ypkA promove alterações no desenvolvimento vegetativo do fungo, o qual cresce na forma de colônias pequenas as quais não formam conídios. Ainda, o mutante condicional, quando reprimido, apresenta sensibilidade a drogas inibidoras da síntese de lipídeos e esfingolipídios. Os resultados indicam também que não há interação genética entre ypkA e os genes da via da integridade da parede celular. Assim, podemos propor que a proteína quinase YpkA de A. fumigatus está relacionada com o crescimento vegetativo e participa da biossíntese de esfingolipídios em A. fumigatus, contribuindo com o desenvolvimento e integridade da célula fúngica.

Allergen proteases from a number sources including the filamentous fungus A. fumigatus, are thought to be important in the development of severe asthma through protease dependent interactions with the respiratory epithelium. The first aim of the thesis was to determine the effect of a variety of growth substrates on the secretion of proteases from different strains of A. fumigatus. The second aim was to investigate the effects of recombinant allergen proteases Asp f 5 and Asp f 13 expressed in the P. pastoris protein expression system and crude A. fumigatus culture supernatants on airway epithelial cells and determine whether protease induced interleukin 8 (IL-8) release from airway epithelial cells was dependent on the activation of protease activated receptor 2 (PAR-2).Results demonstrated that the AF293 strain of A. fumigatus secreted serine proteases during growth on pig lung homogenate medium and metalloproteases during growth on a casein based medium but suppressed protease secretion in Vogel's minimal medium. Analysis of the secretion and RNA levels of proteases in A. fumigatus showed that the matrix metalloprotease, Asp f 5 and the serine protease, Asp f 13 were up-regulated and secreted during growth in pig lung medium and that the matrix metalloprotease, Lap1 was up-regulated and secreted along with Asp f 5 and Asp f 13 in casein medium. This finding was confirmed using protease inhibitors and by using strains of A. fumigatus in which Asp f 5 and Asp f 13 genes were disrupted. These results suggest that A. fumigatus was able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. Furthermore, in several strains of A. fumigatus, protease activity was not suppressed by growth in Vogel's medium, suggesting differences in the regulation of protease secretion between strains. Both A. fumigatus culture supernatants and recombinant Asp f 5 and Asp f 13 produced in P. pastoris caused epithelial airway cell desquamination, and IL-8 release in a protease and dose-dependent manner. In addition, both recombinant Asp f 5 and Asp f 13 were both shown to cleave PAR-2 at a site that resulted in receptor activation.In conclusion, differences in the secretion of proteases between A. fumigatus strains and during growth of A. fumigatus on different media suggests a requirement for the standardisation of the preparation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in vitro and in vivo research studies. Furthermore, it is proposed that allergen proteases secreted by A. fumigatus may interact with a variety of host proteins including, matrix molecules, enzymes and receptors which may exacerbate allergic airway diseases.

Diese Arbeit beschäftigt sich mit den antimykotischen Eigenschaften von NK-Zellen und dient der Charakterisierung der Immunantwort gegenüber A. fumigatus in Abhängigkeit der MOI (Multiplizität der Infektion). Klinisch interessant ist dies bei immunsupprimierten Patienten mit invasiver Aspergillose. Anhand von Oberflächenmarkern konnten eine an die Pilzkonzentration angepasste Bindung und Aktivierung von NK-Zellen demonstriert werden. Daneben kam es zu einer Modulation der Freisetzung ausgewählter Zytokine nach Konfrontation mit steigenden Mengen von A. fumigatus. Besonders deutlich war der Effekt bei den Chemokinen CCL3 und CCL4, deren Zusammenhang mit Pilzinfektionen bereits gezeigt wurde. Die Ergebnisse zum MOI-abhängigen Verhalten von NK-Zellen gegenüber A. fumigatus bestätigen die Relevanz bei der antimykotischen Immunantwort und verdeutlichen, weshalb ihnen zunehmende diagnostische und therapeutische Bedeutung zukommt. / The thesis focuses on the antifungal activity of NK-cells and characterizes the immune response towards A. fumigatus depending on the MOI (multiplicity of infection). This is of clinical interest regarding immunosuppressed patients suffering from invasive aspergillosis. Surface markers were used to demonstrate the binding and activation of NK cells adapted to the fungal concentration. In addition, the release of selected cytokines was modulated after confrontation with increasing amounts of A. fumigatus. The chemokine CCL3 and CCL4 that have already been described to be important during fungal infections showed the most impressive results. This study confirms the role of NK cells within the antifungal immune response and the increasing relevance for diagnosis and therapy.

Die invasive Aspergillose ist eine gefürchtete Erkrankung besonderes bei immunsupprimierten Patienten, die eine Stammzelltransplanation erhalten sollen. Diese Patienten leiden in der Regel an einer hämatologischen Grunderkrankung wie einer Leukämie oder einem Lymphom. Das Prinzip der Stammzelltransplanatation ist es, das kranke Knochenmark mittels Chemotherapie und Bestrahlung zu zerstören und es dann mit gesunden Stammzellen zu ersetzen. Während dieser Konditionierung ist der Patient also ohne eigene Abwehrzellen. In dieser Phase gestaltet sich die rechtzeitige Diagnose häufig als schwierig, weil die konventionellen Diagnosemethoden von der Anwesenheit von neutrophilen Granulozyten abhängen. Häufig ist die Infektion bei der Entwicklung von unspezifischen Symptomen wie Fieber oder Dyspnoe schon weit fortgeschritten. Hat sich der Pilz in der Lunge ausgebreitet, so kommt es zur Ausbildung einer Hypoxie und im Verlauf auch zu einer Hypoxämie aufgrund einer Diffusionsstörung in den entzündeten Lungenabschnitten. Ziel dieser Arbeit war es, den Einfluss von Hypoxie auf die Interaktion zwischen dendritischen Zellen und T-Zellen besser zu verstehen. Dendritische Zellen spielen eine entscheidende Rolle bei der Immunabwehr von Pilzinfektion und bilden das Verbindungsstück zwischen angeborenen und erworbenen Immunsystem. T-Zellen übernehmen ebenfalls wichtige Aufgaben bei der Abwehr von Pilzinfektionen, indem sie Interferon gamma produzieren. Zudem sollte die Funktion verschiedener Botenstoffe in Signalkaskaden näher untersucht werden. Es konnte gezeigt werden, dass die Infektion von dendritischen Zellen mit Aspergillus fumigatus unter Hypoxie, die dendritischen Zellen anschließend unter Normoxie dazu befähigt, eine verstärkte adaptive Immunantwort hervorzurufen. Hypoxie könnte somit als zusätzlicher Faktor verwendet werden, um dendritische Zellen als effektive Adjuvantien in einer Impfung gegen Aspergillus fumigatus zu verwenden. Ursächlich für die verstärkte Fähigkeit naive T-Zellen zu aktivieren, scheint zum einen die Apoptose und zum anderen eine verstärkte Produktion von Interleukin 12p70 zu sein. / The invasive pulmonary aspergillosis is a dreaded disease particularly in immunocompromised patients. These patients often receive a stem cell transplantation because they suffer from leukemia or lymphoma. If patients receive a stem cell transplantation, the sick bone marrow is destroyed by chemotherapy and radiation and replaced with healthy stem cells. During this conditioning, the patients have no cells against pathogen organism. When the patient shows symptoms like fever or dyspnea, in most cases the infection has already progressed and is at an advanced stage. In case of the spreading of the mold, a hypoxia occurs as reason of the diffusion impairment in the infected sections of the lungs. Aim of the present study was to understand the influence of hypoxia on the interaction between dendritic cells and t cells in a better way. Dendritic cells play a crucial role in the immune defense against fungal infections and form the link between the innate and adaptive immune system. T cells also have a main part in the defense against such infections. In addition, the function of different messengers in signal cascades should be further explored. We could show that an infection of dendritic cells by Aspergillus fumigatus under hypoxic conditions leads to an enhanced adaptive immune response. Thus, hypoxia could be an additive factor to use dendritic cells as an effective adjuvant in a vaccination against Aspergillus fumigatus. Apoptosis and an increased production of interleukin 12p70 appears to be the cause of the increased ability to activate naïve t cells.