A classificação de fungos microscópicos é limitada pela escassez de detalhes morfológicos e incertezas na caracterização bioquímica. Nesse trabalho desenvolvemos um método simples, baseado na amplificação de um segmento de cerca de 2.800 bares de bases seguida de digestão com DdeI e análise do padrão após eletroforese em agarose. Demonstramos que o método discrimina tão bem quanto a sequência de DNA do segmento após analisar mais de 400 amostras que foram classificadas em 33 espécies. Essa tecnologia facilita a busca por novos representantes da imensa diversidade existente, espécies que podem ser exploradas quanto ao seu valor potencial para a medicina e a indústria. Outras metodologias moleculares foram desenvolvidas para discriminar entre espécies próximas e na identificação de novas espécies. Em seguida, o trabalho esteve voltado para o desenvolvimento de um ensaio laboratorial que nos permitisse estudar a transição dimórfica em Candida albicans. Desenvolvemos dois novos ensaios, um biológico e outro sobre membrana artificial, para estudar a indução ou inibição da produção de hifas. A expressão de genes associados ao processo foi acompanhada de maneira quantitativa. Fracionando cromatograficamente o soro fetal bovino, obtivemos resultados indicativos de frações estimuladoras e inibidoras da transição morfológica. / Fungal identification is limited by few morphological details and uncertainty in the biochemical tests. In the present work we developed a simple procedure, based upon DNA amplification of a 2,800 base pairs region followed by the amplicon digestion with DdeI and electrophoretic analyzes in agarose gels. After analyzing more than 400 samples encompassing 33 species, we demonstrate that this method discriminates as well as DNA sequencing of the region. This technology simplifies the search for new representatives among the great diversity of fungi of medical and industrial interest. We devised also molecular methods to discriminate between closely related species and described a new putative species. Next our work was dedicated to develop an in vitro assay for the study of the dimorphic transition in Candida albicans. We devised two biological assays and one that used an artificial membrane to investigate the induction and inhibition of hyphal production. The expression of genes associated with the morphological transition was examined in a quantitative way. We fractionated bovine fetal serum by column chromatography and obtained results pointing to fractions that stimulate or inhibit hyphal production.

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The thesis deals with the effect of arbuscular mycorrhiza (AM) on the growth of selected plant species at a locality in České středohoří. This locality is characterized by close neighborhood of a semi-natural dry grassland and a former field abandonned in the 1990s, typical for the mosaic of biotopes in the region. The study is based on previous findings that some plant species, which are common at the semi-natural dry grasslands, do not colonize the abandoned fields. As AM is an important factor affecting diversity and productivity of plant communities we hypothesized that this phenomenon could be related to changes in AM fungal communities at the abandoned field. The hyphothesis was tested in a greenhouse pot experiment with three taxonomically related pairs of plant species, always one species growing at the abandoned field and the second one not. Growth and phosphorus uptake of the plants was followed in soils of both biotopes after factorial inoculation with AM fungal communities from both biotopes. The experiment was complemented by description of AM fungal communities in the roots of six plant species pairs from the locality using terminal restriction fragment length polymorphism (T-RFLP). The greenhouse experiment revealed positive mycorrhizal response in all plant species, but the origin...

The thesis focuses on studying changes of microbial communities living in the soil contaminated by heavy metals. Two sites with different degree of contamination were selected in the Příbram area. Respiration was measured in vitro in the soil samples supplemented with various carbon sources and different concentration of cadmium. The respiration showed that even at cadmium concentration of 1000 mg.kg-1 the community is viable and capable of utilization of substrates while increasing the respiration rate. Enviromental DNA from soil samples was isolated and 16S rRNA gene of actinobacteria was amplified. The terminal restriction fragment length polymorphism analysis showed a clear difference between the profiles of both sites. The shifts in the community profiles were observed also after the addition of substrates. The quantification of total bacteria and actinobacteria was performed by quantitative PCR based on amplification of part of the 16S rRNA gene. The more contaminated site contained slightly more bacteria, but almost twice the actinobacteria than the less contaminated one. The sequencing of amplicons of a part of 16S rRNA gene by Illumina showed an increase in proportion of actinobacteria and changes of their community structure in the more contaminated site. The conclusion was made that, high...

The diploma thesis is focussed on understanding relationships between soil chemical characteristics, actinobacterial communities of agricultural field soils and occurrence of potato common scab, a disease caused by members of the genus Streptomyces. The aim of monitoring study, on thirty-three sites covering main potato- growing regions in the Czech Republic, was to find relationships suitable for prediction of common scab severity. The second part of the thesis compared actinobacterial communities and incidence of Streptomyces harboring a pathogenic determinant, gene txtA (gene of biosynthetic pathway of phytotoxin thaxtomin A), in soils differing in occurrence of common scab. In the screening study, analysis of terminal restriction fragment length polymorphism (T-RFLP) was employed to compare composition of soil actinobacterial communities. Real-time PCR was used to quantify total actinobacteria and streptomycetes harboring txtA gene in soils differing in scab incidence. The screening study revealed negative correlations between the scab severity and (i) available phosphorus in soil and (ii) diversity of actinobaterial community. The results were used to design a model for scab prediction. A qPCR analysis showed difference in numbers of total actinobacteria and the strains harboring txtA gene in...

In dieser Arbeit wurde die systematische Suche nach krankheitsassoziierten Genen bei periodischer Katatonie fortgeführt. Für diese Erkrankung war die klinische Abgrenzbarkeit und die familiäre Häufung signifikant und ließ aufgrund der vertikalen Transmission und dem Auftreten über mehrere Generationen und hinweg auf einen Hauptgeneffekt schließen. Nach der Durchführung von Kopplungs-Analysen kristallisierten sich zwei koppelnde Regionen auf den Chromosomen 15 und 22 heraus. Mittels Haplotypanalyse konnte der Genort auf Chromosom 22q13 auf einen knapp 5 Mbp großen Bereich eingeschränkt werden. Im kodierenden Bereich des MLC1-Genes segregierte im mit periodischer Katatonie assoziierten Haplotyp eine Variante (p.Leu309Met). Da Mutationen im MLC1-Gen bereits im Zusammenhang mit Megalenzephaler Leukoenzephalopathie beschrieben worden waren, wurden in dieser Arbeit zunächst fünf Patienten mit dieser Erkrankung auf Mutationen in kodierenden Bereichen von MLC1 systematisch untersucht. Daran schloss sich eine Analyse dieses Gens bei 140 Patienten mit periodischer Katatonie an. Ein Zusammenhang zwischen Mutationen in MLC1 und dem Auftreten von Megalenzephaler Leukoenzephalopathie wurde untermauert, wohingegen die Ergebnisse eindeutig gegen eine Assoziation mit periodischer Katatonie sprachen. Ein weiteres im Gehirn exprimiertes Kandidatengen (KIAA0767/DIP) wurde in dieser Arbeit untersucht. Dabei wurden sechs SNPs im exonnahen intronischen Bereich entdeckt sowie eine Variante im Exonbereich (p.Glu156Asn). Dies ist eine seltene Normvariante, eine Assoziation zur periodischen Katatonie wurde in einer Fall-Kontroll-Studie ausgeschlossen. Insgesamt wurde durch die systematische Mutationsanalyse die Kandidatenregion auf Chromosom 22q13.3 weiter eingeengt. Gegen einen Zusammenhang zwischen MLC1 und periodischer Katatonie sprechen die vorgestellten validen Ergebnisse. / The present dissertation has carried on the systematic screening for genes involved in periodic catatonia. The clinical definition and the familial clustering of this disorder were of statistical significance. Its vertical transmission and the occurrence across generations led to the conclusion of it being a major gene effect. Linkage scans discovered two linking regions on chromosome 15 and chromosome 22. Using haplotype analysis it was possible to restrict the candidate region on chromosome 22q13 to region of 5 Mbp. In the coding region of the MLC1 gene a variant (p.Leu309Met) segregated in the haplotype associated with periodic catatonia. As detailed descriptions of mutations in the MLC1 gene in connection with megalencephalic leucoencephalopathy already exist, for the present dissertation five patients with the aforesaid disorder were systematically screened for mutations in the coding regions of MLC1. This was followed by an analysis of the according gene in 140 patients with periodic catatonia, which substantiated a correlation between mutations in MLC1 and the occurrence of megalencephalic leucoencephalopathy, whereas the results definitely did not support an association with periodic catatonia. Additionally, a further brain-expressed candidate gene (KIAA0027/DIP) was screened in this dissertation discovering six single nucleotide polymorphisms in the intronic exon-near region and a variant in the exonic region (p.Glu156Asn). This is a rare normal variant, an association with periodic catatonia was excluded in a case-control study. Altogether the systematic mutation analysis enabled a further reduction of the candidate region on chromosome 22q13.3. The research results, which meet the criterion of validity, do not support a correlation between MLC1 and periodic catatonia.

O melão-de-São-Caetano (Momordica charantia L) é uma cucurbitácea que ocorre em todas as regiões geográficas brasileiras. A espécie apresenta duas \"variedades\" distintas, comumente conhecidas por \"variedade silvestre\" e \"variedade cultivada\", esta última caracterizada por plantas de maior tamanho. Plantas da \"variedade selvagem\" são hospedeiras de um fitoplasma do grupo 16SrIII-J, que causa superbrotamento de ramos, nanismo generalizado, clorose, redução no tamanho de folhas e frutos. No entanto, não há informação da \"variedade cultivada\" ser hospedeira de fitoplasmas. Assim, no presente estudo, plantas sintomáticas desta \"variedade\" foram analisadas quanto à presença de fitoplasmas em seus tecidos, visando associar este tipo de patógeno à doença. Neste mesmo trabalho, os isolados do fitoplasma encontrados nas plantas sintomáticas da \"variedade cultivada\" foram molecularmente analisados em relação ao gene secY, atualmente usado como um marcador que permite identificar variações genéticas sutis. Buscou-se, com isto, identificar possíveis variantes entre os isolados. Amplificações de fragmentos de DNA a partir dos genes 16S rRNA (1,2Kb) e secY (1,6Kb) demonstraram a associação de fitoplasma do grupo 16SrIII com as plantas sintomáticas de melão-de-São-Caetano da \"variedade cultivada\". Nestas plantas, corpúsculos pleomórficos, típicos de fitoplasmas, foram visualizados no floema dos tecidos doentes, confirmando os resultados dos testes moleculares. Análises de RFLP virtual, conduzidas com o fragmento genômico correspondente ao gene secY e diversas enzimas de restrição, permitiram identificar o referido fitoplasma como pertencente ao subgrupo 16SrIIIJ. A análise filogenética evidenciou que o fitoplasma padrão do subgrupo 16SrIII-J emergiu do mesmo ramo que o fitoplasma identificado no melão-de-São-Caetano, confirmando que ambos estão estritamente relacionados. O emprego do gene secY como marcador para diferenciação fina entre fitoplasmas não apontou variabilidade genética entre os isolados do fitoplasma detectado tanto nas plantas da \"variedade cultivada\" como naquelas pertencentes à \"variedade selvagem\". Como conclusão, a \"variedade cultivada\" de M. charantia é hospedeira do mesmo fitoplasma encontrado na \"variedade selvagem\", o qual é afiliado ao subgrupo 16SrIII-J; por sua vez, o gene seY não distinguiu geneticamente os isolados do fitoplasma pertencente ao subgrupo 16SrIII-J, presentes nas plantas de melão-de-São- Caetano. / Momordica charantia is a species of the family Cucurbitaceae that occurs in all Brazilian geographic regions. This species presents two distinct \"varieties\", commonly called \"wild variety\" and \"cultivated variety\", whose plants are bigger than those belonging to the \"wild variety\". Plants of the \"wild variety\" are hosts of a phytoplasma of the group 16SrIII-J, which is associated with shoot proliferation, stunting, chlorosis, and small leaves and fruits. However, there is no information about the occurrence of phytoplasmas in plants of the \"cultivated variety\". Thus, in the present study, symptomatic plants belonging to this \"variety\" were analysed in order to associate phytoplasmas with the disease. In addition, strains of the phytoplasma found in symptomatic plants of the \"cultivated variety\" were molecularly analyzed in relation to the gene secY, which is currently used as a marker for identification fine of genetic diversity, aiming provide elements for construction of new schemes for classification of phytoplasmas. The objective was to identify distinct strains among the strains present in plants of the wild and cultivated varieties. Amplifications of DNA fragments from the genes 16Sr rRNA (1.2Kb) and secY (1.6Kb) demonstrated the association of a phytoplasma of group 16SrIII with the symptomatic plants belonging to \"cultivated variety\". These plants revealed the presence of pleomorphic bodies, typical of phytoplasmas, which was visualized in the phloem vessel from diseased tissues, confirming the results obtained by molecular assays. Virtual RFLP analysis, performed with the genomic fragment corresponding to the gene secY and various restriction enzymes, allowed to identify this phytoplasma as a member of the subgroup 16SrIII-J. Phylogenetic analysis revealed that the phytoplasma used as reference for 16SrIII-J subgroup and the phytoplasma identified in plants of M. charantia emerged from the same branch, confirming that both phytoplasmas are closely related. The gene secY, used as marker for fine differentiation of phythoplasmas, did not show genetic variability among the strains of the phytoplasma detected both plants belonging to \"cultivated variety\" and \"wild variety\". In conclusion, plants of the \"cultivated variety\" are hosts of the same phytoplasma found in the \"wild variety, which is affiliated with the 16SrIII-J subgroup; in addition, the gene secY did not distinguish isolates of the phytoplasma belonging to 16SrIII-J subgroup, present in plants of M. charantia.

Estudou-se Microsporum gypseum de diferentes fontes regiões geográficas do Brasil.Os fungos foram isolados do solo, de animais e obtidos em coleções de cultura. Em 692 amostras de solo e a taxa de recuperação foi de 19,2%. A atividade queratinolítica e elastinolítica foi avaliada quantitativamente em 138 amostras de M. gypseum. A biotipagem molecular foi realizado através da técnica de PCR-RFLP com a enzima MvaI. O seqüenciamento da região ITS1 do rDNA foi realizado em amostras representativas. M. gypseum de amostras clínicas e do solo mostraram diferenças quantitativas significantes na expressão das enzimas. A PCR-RFLP para a região ITS1 não mostrou diferença. Pelo seqüenciamento foram obtidas duas espécies sexuadas (A. gypseum e A. incurvatum). As atividades enzimáticas sugerem um importante papel na patogenicidade e uma provável adaptação desta espécie de fungo ao parasitismo animal. A análise dos resultados pode auxiliar a identificação e o conhecimento de mecanismos de virulência destes fungos. / Microsporum gypseum from different geographical sources and regions of Brazil were studied. The fungi were isolated from the soil, animals and obtained in collections of culture. In 692 samples of soil, the recovery rate was 19.2%. The keratinolytic and elastinolytic activities were quantitatively performed on 138 samples of M. gypseum. The molecular biotyping was carried out by the technique of PCR-RFLP with the enzyme MvaI. The sequencing of the region ITS1 rDNA was carried out on representative samples. Samples of M. gypseum from clinical isolates and from soil showed significant quantitative differences in the expression of enzymes. The PCR-RFLP for the ITS1 region showed no difference. For the sequencing was obtained two sexual species (A. gypseum and A. incurvatum). The enzyme activities suggest an important role in pathogenicity and a likely adjustment of this kind of parasitic fungus to feed. The results may help the identification and knowledge of mechanisms of virulence of these fungi.

La microflore des raisins est importante d’un point de vue technologique car elle conditionne en partie la qualité du vin. Or, la diversité des flores fongiques présentes sur baies de raisin ainsi que leur potentiel de contamination du produit final ne sont pas encore pleinement connus. Dans ce cadre, la caractérisation des flores fongiques cultivables présentes sur baies de raisin a été réalisée par PCR ITS-RFLP. 41 espèces de moisissures différentes sur les 43 étudiées appartenant à 11 genres différents ont été caractérisées de façon fiable. Seules les espèces Penicillium thomii et Penicillium glabrum ont présenté le même profil. Ainsi 96.3% des souches étudiées ont été caractérisées avec au maximum 4 enzymes de restriction et 41.5% des souches ont pu l’être avec seulement 2 enzymes de restriction. Ces résultats ont permis d’enrichir les bases de données, moyennement pourvues en séquences ITS caractéristiques de genres ou d’espèces de moisissures présentes sur baies de raisin. De plus, une étude exhaustive des moisissures présentes sur baies de raisin en Bourgogne a permis, par PCR ITS-RFLP, d’identifier 199 souches au niveau de l’espèce et ce quelque soit le genre. Penicillium spinulosum est l’espèce majoritaire isolée pour le millésime 2008 en Bourgogne. Parallèlement, la quantification de Botrytis cinerea, choisi comme micro-organisme modèle, a été réalisée par qPCR. La technique qPCR décrite dans ce travail présente (i) une bonne sensibilté avec une limite de détection de 6.4 pg d’ADN correspondant à 540 spores, (ii) l’originalité de travailler en échantillons naturellement contaminés et la fiabilité d’utiliser un standard interne. L’évaluation de l’efficacité de différentes stratégies de traitements anti-Botrytis a confirmé l’importance de la prophylaxie (effeuillage) dans la lutte contre Botrytis cinerea. / Microbial population of grapes is important from a technological point of view because it determines the quality of wine. But few studies have focused on fungal populations of grapes. A better knowledge of the fungal diversity on grapes, particularly as concerns species responsible for wine defects, may help efforts to control their development.We report the development of a PCR ITS-RFLP method as a fast and easy technique for identifying species of fungal genera present on grapes. By this methode, 41 different fungal species among 43 studied species belonging to 11 genera were characterized at the species level. Only P. thomii remained indistinguishable from P. glabrum. Using this PCR-ITS-RFLP, 96.3% strains tested could be differentiated to the species level with only four enzymes and 41.5% only with two enzymes. Moreover this work has contributed to the enriching of the database of fungal ITS sequences.Thus 199 isolated strain were on grapes in Burgundy vineyard were chacacterized at species level indepdantly of the genus by this method. P. spinolusum was the most frequently isolated species of Penicillium in Burgundy for 2008 vintage. Paralelly, the quantification of Botrytis cinerea, used as model, was developped by qPCR. The assay contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes, had high efficiency and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). This method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard and demonstrates the importance of the prophylactic method.