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Neofabraea is a genus of an important plant pathogenic fungi having worldwide appearance. Four Neofabraea species are responsible for bull's eye rot of pome fruits. The aim of this thesis was to investigate which of these species occur in the Czech Republic. 81 isolates were collected during a two- year period and they were identified using PCR fingerprinting (primers ERIC 1R and M13-core) and DNA sequencing (ITS, mtSSU and tub2). The results showed that species N. alba, N. perennans and Cryptosporiopsis kienholzii occur in the Czech Republic. According to available information, this is the second record of C. kienholzii in Europe. One isolate (KP4) failed to be identified as any of the species. KP4 is very close to C. kienholzii, but it can be distinguished both biologically and genetically. Furthermore, the aggressiveness of individual species was compared.N. perennans and strain KP4 proved to be most aggressive, the least aggressive is C. kienholzii. Two N. alba strains (KP36 and KP37) isolated from healthy apple fruit and leaf are pathogenic for apple fruits. Keywords: aggressiveness, Cryptosporiopsis kienholzii, Dermateaceae, Helotiales, apple tree, Malus, PCR fingerprinting, postharvest diseases Powered by TCPDF (www.tcpdf.org)

Made available in DSpace on 2014-06-12T15:53:07Z (GMT). No. of bitstreams: 2 arquivo5192_1.pdf: 788582 bytes, checksum: 9c493ee2ba6857656c0a20883bb05c1f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A relação biológica de leveduras industrialmente importantes do complexo Saccharomyces sensu stricto causa equívoco na correta identificação das leveduras. Por isso, este trabalho apresenta a técnica de PCR - fingerprinting com diferentes marcadores que identificaram com eficiência as verdadeiras espécies do complexo Saccharomyces sensu stricto e indicaram os híbridos naturais isolados de processos industriais. Linhagens tipo, linhagens comerciais de vinho e leveduras isoladas de vinho artesanal foram submetidas à análise de PCR - fingerprinting usando oligonucleotídeos únicos (SPAR) e análise de restrição de genes ribossomais e estruturais. Todas as seis espécies reconhecidas do complexo Saccharomyces sensu stricto foram discriminadas e erros na taxonomia anterior das leveduras de vinho foram observados. Além disso, estes marcadores SPAR mostraram a complexidade da constituição híbrida dos isolados industriais. O uso de marcadores SPAR pode ajudar na identificação de leveduras isoladas da indústria e do meio ambiente dentre uma das seis espécies do complexo Saccharomyces sensu stricto e seus híbridos interespecíficos, e na reclassificação de linhagens depositadas em coleções de leveduras. Embora este conjunto de marcadores falhe em determinar a contribuição de parentais no processo de hibridização, pode eficientemente traçar a dispersão de leveduras híbridas que foram originadas em eventos simples de hibridização. O padrão único gerado pelos marcadores SPAR foi bastante eficiente no monitoramento da população de leveduras durante o processo de fermentação industrial e pode detectar a aproximação de leveduras híbridas em cada meio ambiente

The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.

Die vorliegende Studie wurde in einer Gruppe von Dörfern im Ostsudan, Gedaref State, durchgeführt. Bei 100 Patienten mit der Verdachtsdiagnose Kala Azar- oder Post-Kala Azar- Leishmaniose war der Erregernachweis mit der PCR direkt in klinischen Proben, die auf Filterpapier aufgebracht worden waren, ohne vorherige Kultivierung erfolgreich. In dieser PCR wurden die ribosomalen, internal transcribed spacer (ITS1 & ITS2) amplifiziert, weil sie sehr variabel sind, eine klare Speziesidentifizierung gestatten und bei weiterführenden Analysen der PCR-Produkte auch der Nachweis stammspezifischer Unterschiede erwartet werden konnte. Für die Analyse der Diversität von Leishmania donovani-Isolaten aus dem Sudan wurden 4 verschiedene PCR-basierte Methoden eingesetzt: das PCR-Fingerprinting mit unspezifischen Einzelprimern, die RFLP- Analyse des amplifizierten ITS-Locus, ,,single strand conformation polymorphism (SSCP)- Analysen der amplifizierten ITS-Region, des Gens, welches für die Hauptoberflächenprotease (gp63) kodiert, und anonymer DNA- Fragmente sowie Sequenzanalysen der entsprechenden Zielregionen. Das PCR-Fingerprinting und die Restriktionsanalyse der ITS-Region lieferten weitgehend übereinstimmende Fragmentmuster für alle untersuchten L.donovani-Stämme. 12 unterschiedliche Profile wurden bei der SSCP-Analyse des ITS1-Locus für 86 Isolate aus dem Sudan erhalten, während der ITS2-Locus bei diesen Stämmen hochkonserviert war und nur ein Stamm ein unterschiedliches SSCP-Muster aufwies. L. Donovani -Stämme anderer geographischer Herkunft hatten unterschiedliche ITS2-Profile in der SSCP. Für den gp63 - Locus waren 3 polymorphe SSCP-Muster bei 31 untersuchten sudanesischen Isolaten nachweisbar. Für die meisten der anonymen DNA-Fragmente, L510, L413, LK413, L0308 UND L0114, konnten leider nur von 8 kultivierten Stämmen gute PCR-Produkte erhalten werden. Lediglich das Fragment L0110 konnte erfolgreich von 31 auf Filterpapier aufgebrachten Proben direkt amplifiziert werden. Die Suche nach Polymorphismen mit der SSCP ergab keine Unterschiede in diesen anonymen DNA-Regionen, mit Ausnahme des Fragments L0114, das zwei verschiedene Muster aufwies. Die Ergebnisse der SSCP-Analysen und der DNA- Sequenzierung stimmten gut überein, wodurch bestätigt wurde, dass die SSCP genetische Unterschiede auf dem Niveau einzelner Basenaustausche nachweisen kann. Die SSCP- Technik hat Vorteile gegenüber den anderen Methoden, die für die Untersuchung von Sequenzvariationen innerhalb der Spezies L. donovani angewandt wurden. Es konnten keine Korrelationen zwischen der Form der klinischen Manifestation und den Ergebnissen des PCR- Fingerprinting, der ITS-RFLP- und ITS-SSCP- Analysen festgestellt warden. Diese Studie ist von besonderem Nutzen in epidemiologischen Feldstudien, bei denen die Kultivierung der Erreger besonders in Entwicklungsländern extrem schwierig sein kann. / This study was carried out in clusters of villages that represent an endemic focus of visceral leishmaniasis (VL). These villages were located in Gedaref state, eastern Sudan. For diagnostic purposes polymerase chain reaction (PCR) was performed successfully, directly from clinical samples spotted on filter papers with no prior cultivation from 100 patients suspected of having kala-azar or post kala-azar dermal leishmaniasis. Mainly the ribosomal internal transcribed spacer (ITS1 & ITS2) were targeted in PCR because this region is more variable and allows clear species identification and also strain differences could be expected by further analysis of these PCR products. PCR was found to be more sensitive compared to the gold standard microscopic method. Four PCR based approaches were used to analyse diversity within Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single non-specific primers, restriction analysis of the amplified ITS locus (RFLP), single-stranded conformation polymorphism (SSCP) of the ITS region, major surface protease (gp63) gene, anonymous DNA fragments and sequencing of these targeted regions. When PCR fingerprinting and restriction analysis of ITS region were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave 12 different SSCP profiles among the 86 Sudanese isolates, where as the ITS2 locus was highly conserved among the 86 samples with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. The gp63 locus gave 3 polymorphic patterns among 31 Sudanese isolates. Concerning most of the anonymous DNA fragments namely, L510, L413, LK413, L0308 and L0114 unfortunately, we succeeded to get good PCR products only from DNA extracted 8 successful cultures. Only for the fragment L0110 we were able to get good PCR products from 31 samples spotted on filter papers. When these PCR products were investigated for polymorphisms using SSCP no differences were observed with exception of L0114 region, which showed 2 patterns. SSCP analysis correlates well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigating of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics. This study is beneficial particularly in epidemiological studies based on field-work where obtaining cultures can be extremely difficult especially in developing countries.

Des études ont récemment mis en évidence le rôle des lipopolysaccharides (LPS) des bactéries à Gram négatif de la flore intestinale dans le processus de l’inflammation conduisant à l’obésité et au diabète de type 2.Le présent travail est réalisé dans le cadre d’une collaboration entre les équipes du Dr. Caroff (U. Paris-Sud, Orsay) et du Pr. Zhao (U. Jiao Tong, Shanghai). Les expériences présentées ont été réalisées lors de séjours dans les deux laboratoires.Il a été démontré en Chine que des bactéries Sulfato-réductrices (SRB) à Gram négatif étaient présentes en plus forte proportion dans la flore intestinale chez les souris suivant un régime gras. Les mêmes résultats ont été observés chez l'homme. L’hypothèse selon laquelle des SRB seraient à l’origine de grandes quantités d’endotoxines chez les obèses et les patients diabétiques a été émise. Plusieurs souches de SRB isolées de la flore intestinale humaine d’un sujet sain et d’un sujet diabétique ont été cultivées en Chine. Des études de relation structure/activité des LPS isolés de ces bactéries ont été réalisées dans le laboratoire Français pour déterminer leur rôle dans le développement des maladies métaboliques. Les souches isolées des deux sujets ont pu être classées dans le genre Desulfovibrio. Les LPS correspondants ont été extraits et purifiés par des méthodes mises au point dans l’équipe d’Orsay. La structure chimique a été élucidée par les méthodes suivantes : Electrophorèse, Chromatographie sur couche mince, Chromatographie en phase gazeuse et Spectrométrie de masse MALDI. C’est ainsi que des spectres de masse ont été obtenus et que la structure des lipides A, principes actifs des LPS, isolés de SRB a été décrite pour la première fois. Les activités biologiques testées (TNFα, IL-6) varient en fonction du nombre d’acides gras présents. Les LPS de SRB du patient sain ont une structure variable (Smooth versus Rough) en fonction de la quantité de fer présent dans le milieu, et ceux isolés du patient diabétique présentent des structures atypiques qui ne sont pas toutes inflamogènes. Une molécule membranaire inconnue, que nous avons nommée « Glycosyl’X » était co-extraite avec les LPS. Elle joue apparemment un rôle important dans la croissance des SRB et a été étudiée après des étapes de purification complexes. Les structures et le pouvoir inflammatoire de ces molécules dont la structure varie avec les souches, et qui chélatent le fer, ont été étudiées. Elles sont de nature principalement osidique et fixées à la membrane. La proportion de ces molécules par rapport aux LPS varie avec la quantité de fer disponible dans le milieu. Un milieu riche en fer favorise la croissance des Desulfovibrio portant les Glycosyl’X qui n’ont pas de pouvoir inflammatoire eux-mêmes, mais entrent en compétition avec les LPS, modulant ainsi indirectement l’activité de ces derniers. L’augmentation du nombre de Desulfovibrio conduisant à l’augmentation des molécules Glycosyl’X pourrait aussi moduler positivement (par présentation) ou négativement (par élimination des bactéries) l’adsorption du fer dans les intestins dont l’équilibre est essentiel pour l’homéostasie métabolique.Par ailleurs, la croissance des Desulfovibrio augmente la production d’Hydrogène Sulfuré connu pour son action délétère sur les cellules. Nous favorisons l’hypothèse selon laquelle son action sur la disjonction des cellules épithéliales permettrait le passage des différents LPS relargués par la flore Gram-négative intestinale, et même des bactéries entières, vers la circulation sanguine. / Recent studies have highlighted the role of lipopolysaccharide (LPS) in the intestinal flora (gut microbiota) which could contribute to the inflammation process leading to obesity and type 2 diabetes. This thesis is part of a collaborative project between the laboratories of Dr. Caroff (U. Paris -Sud, Orsay, France) and Prof. Zhao (U. Jiao Tong , Shanghai, China). It has been shown by Pr.Zhao’s team in 2010 that the Sulfate -Reducing Bacteria (SRB) were presented in greater proportion in the intestinal mice flora following a fat diet compared to mice following a normal diet. The same results were observed in humans. The starting hypothesis was that SRB could produce a large amount of endotoxin in obese and diabetic patients and play a role in the development of metabolic diseases. Several SRB strains isolated from the human intestinal flora of a healthy subject and of a diabetic subject were grown in the Chinese laboratory. Studies of their LPS structure / activity relationships were carried out in the French laboratory. The aim of this study was to determine their roles in the development of metabolic diseases.Strains isolated from the two subjects could be classified in the Desulfovibrio genus. The corresponding LPS were extracted and purified by the methods developed in the French laboratory. The chemical structure was elucidated by the following methods: Electrophoresis, Thin layer chromatography, Gas chromatography and MALDI mass spectrometry. The mass spectra were obtained and the structure of lipid A, the active part of LPS isolated from SRB was described here for the first time. The biological activities test (TNFα, IL-6) vary depending on the number of fatty acids present in their lipid A structure. The LPS of SRB isolated from the healthy patient had a variable structure (Smooth versus Rough) depending on the amount of iron present in the medium, and those isolated from diabetic patients had atypical structures are not all inflamogenic .An unknown membrane molecule, which we named "Glycosyl'X" was co-extracted with the LPS. It apparently plays an important role in the growth of SRB was investigated after complex purification steps. The structures and the inflammatory power of these molecules variying with strains chelating iron were studied. They are mainly of glycosidic nature and linked to the bacterial membrane.The proportion of these molecules relatively to LPS varies with the amount of iron in the medium. An environment rich in iron promotes the growth of Desulfovibrio Glycosyl'X, molecules but competes with LPS and indirectly modulates the activity of the latter. The increase number of Desulfovibrio leading to increased Glycosyl'X molecules may also modulate positively (by presentation) or negatively (by killing bacteria) the absorption of iron in the intestines which balance is essential for metabolic homeostasis.Furthermore, the growth of Desulfovibrio increasing the production of Hydrogen Sulfide is known for its deleterious effects on the cells. We favor the hypothesis that its action on the separation of epithelial cells favors the passage of different LPS released by the Gram- negative of intestinal flora and even whole cell bacteria into the bloodstream.