Parareptilien stellen ein artenreiches Monophylum paläozoischer und früh-mesozoischer Amnioten dar; sie bilden innerhalb der Reptilien die Schwestergruppe zu den Eureptilien. Pareiasaurier, eine Untergruppe der Parareptilien, sind eine bedeutende Komponente spät-permischer …kosysteme. Jüngst wurde ein Schwestergruppenverhältnis zu den Nycyeroletern entdeckt, mit welchen sie die "Pareiasauromorpha" bilden. Diese Arbeit befasst sich mit der Beschreibung einiger dieser bisher schlecht untersuchten Parareptilien und deren Verwandtschaftsverhältnisse.Der Pareiasaurier Deltavjatia vjatkensis und der Nycteroleter Emeroleter levis aus der russischen Lokalität Kotel''nich werden mithilfe neu entdeckten Materials im Detail beschrieben. Das gut erhaltene Deltavjatja Materials umfasst mehrere Grössenstadien und erlaubt eine Analyse der Wachstumsraten. Eine geometrisch-morphometrische Analyse des Schädeldachs lässt eine allometrische Zunahme von Schnauzenlänge und Postorbitalregion erkennen. Auch wird der historisch erste Pareiasaurier, Parasaurus geinitzi, aus dem oberpermischen Kupferschiefer von Deutschland neu beschrieben. Zudem ergibt eine Neuanalyse der russischen Nycteroleter eine Synonymie von Tokosaurus perforatus und Macroleter poezicus. Eine phylogenetische Analyse unter Verwendung von Parsimonie und bayesischen Methoden ergibt eine basale Stellung von Parasaurus und eine Monophylie der Nycteroleter; letztere wird aber nur durch die Parsimonie unterstützt. Alle phylogenetischen Methoden ergeben jedoch eine Monophylie der Pareiasauromorpha. Eine strato-kladistische Analyse zeigt zudem eine ähnliche Topologie. Eine biogeographische Analyse der Pareiasauromorpha ergibt mehrfache Verbreitungsereignisse nach Russland und China. Diese stimmen überein mit denen anderer oberpermischer Gruppen, jedoch werden nur wenige dieser Ereignisse komplett bei allen Taxa gefunden. / Parareptilia is a diverse, yet enigmatic clade of fossil amniotes, sister-group to Eureptilia within Reptilia. Pareiasauria, a widely distributed and speciose parareptile group, was a prominent part of Late Permian ecosystems, yet few taxa have been examined in detail. This work describes some poorly known parareptiles, using this data to assess interrelationships and evaluate evolutionary trends within the clade. A reassessment of a group of derived parareptiles, the Russian ''nycteroleters'', results in the synonymy of Tokosaurus perforatus with Macroleter poezicus. A phylogenetic analysis of parareptilian relationships recovers a ''nycteroleter'' monophyly in the parsimony analysis, but not with Bayesian, with the genus Bashkyroleter paraphyletic in both. A monophyletic clade consisting of the nycteroleters and pareiasaurs, termed Pareiasauromorpha, is supported by all methods. Parasaurus geinitzi von Mayer, 1857 is redescribed and confirmed to be a pareiasaur based on the anatomy of the type specimens and fragmentary material that has subsequently been assigned to the taxon. Well-preserved cranial and postcranial material of the pareiasaur Deltavjatia vjatkensis from Kotel''nich, Russia, is described in detail for the first time, including specimens of radically different size classes. The first geometric morphometric analysis of a parareptile reveals an allometric increase in snout length along with an increase in postorbital length. A reassessment of the relationships of Pareiasauromorpha using different methods of phylogenetic inference recovers similar results. The topologies are not well supported, but are relatively consistent with the stratigraphic record. Biogeographic analysis of pareiasauromorph taxa recovers complex and reticulated patterns of dispersal and vicariance, including multiple dispersal events into both Russia and China; patterns that are consistent with those seen in other Late Permian groups, though few events are shared by all clades.

La production de vanille (Vanilla planifolia, Orchidaceae) est limitée par la pourriture des racines et des tiges (PRT) provoquée par des champignons telluriques du genre Fusarium contre lesquels la lutte chimique ou prophylactique est inefficace. Ce travail décrit finement l'interaction entre les vanilliers et les Fusarium dans le but de développer la lutte génétique. L'analyse moléculaire (gène EF1α et IGS) et la détermination du pouvoir pathogène de 365 souches de Fusarium isolées de vanilliers à La Réunion et à Madagascar ont démontré que les souches pathogènes sur vanillier appartiennent presque toutes au complexe d'espèce F. oxysporum et ont une origine polyphylétique. Les observations en microscopie à champ large et multi-photons démontrent que la colonisation des racines débute au niveau des poils absorbants puis gagne le cortex, mais que le système vasculaire est épargné. Cela conduit à renommer l'agent pathogène en F. oxysporum f. sp. radicis-vanillae (Forv). Dans les interactions incompatibles (souche pathogène et vanillier résistant), l'épaississement et la lignification des parois de l'hypoderme, constitutive et induite par le champignon, joue un rôle significatif dans le blocage de l'invasion mycélienne. Le rôle important des composés phénoliques dans la résistance des vanilliers a été confirmé par les analyses d'expression différentielle de transcrits obtenus de novo par RNA-seq Illumina. Enfin, un test robuste de détermination in-vitro de la résistance à Forv a été validé et de nouvelles sources de résistance génétique à la PRT ont été identifiées. Nos résultats ouvrent des perspectives prometteuses pour la sélection de variétés améliorées de vanilliers. / Vanilla is a high value cash crop that is continuously demanded by the agri-food and cosmetics industries for its incomparable flavor. Most of vanilla comes from the cured fruits of V. planifolia G. Jackson, a hemi-epiphytic climbing orchid cultivated in the humid tropics. In all the countries were it is cultivated, the vanilla vines suffer from a root and stem rot (RSR) caused by the soil borne fungus Fusarium oxysporum which dramatically reduces plant production and the durability of plantations. No efficient control method is currently available for this disease. Sources of genetic resistance to RSR exist in few vanilla relatives, but so far no commercial resistant variety has been produced. The purpose of this thesis was to better describe the diversity and histopathology of the causal agent of RSR and to evaluate the potential sources of genetic resistance that could be used in breeding programs. In a first step, a collection of 377 single-spored Fusarium isolates recovered from rotten roots and stems during surveys conducted in 52 vanilla plots from Reunion Island, Madagascar and French Polynesia were characterised. Representative subsets of isolates were genotyped using the Elongation Factor 1α and Intergenic Spacer gene sequences. Their pathogenicity was assayed by root dip inoculation on the susceptible V. planifolia accession pla0001. Results showed that F. oxysporum was the principal species responsible for the disease in the field, although a few F. solani isolates showing slight pathogenicity were also isolated. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Remarkably, their pathogenicity varied in gradient between non- pathogenic (about 42% of isolates) to highly pathogenic (14%). In a second step, 254 vanilla accessions comprising 18 species and six types of hybrids were assessed for resistance to RSR in the field (natural inoculum) and in the lab (in-vitro plants inoculated with Fo072). The strong resistance to RSR of all V. pompona accessions and hybrids of V. planifolia X V. pompona or V. phaeantha, were confirmed, and novel sources of resistance to RSR were added including, V. bahiana, V. costariciensis and V. crenulata. Most of the V. planifolia accessions, V. ×tahitensis and V. odorata were susceptible to RSR. However, three inbreeds of V. planifolia showed a high level of resistance to Forv. To our knowledge this is the first report of resistance to RSR in V. planifolia accessions. For the 26 accessions evaluated in both conditions, a strong correlation was observed between long term (9 years) evaluation in the field and ratings on in-vitro plants at 15dpi. Thirdly, we monitored by wide field and multiphoton microscopy the root infection process and the responses of one susceptible accession (V. planifolia pla0001) and two resistant accessions (V. planifolia pla0020 and V. pompona pom0018) to challenge inoculation with the severe isolate Fo072. In the compatible interaction (Fo072 – pla0001) invasion started from penetration of hyphae emitted from germinated conidia in the hairy region of root rapidly colonizing the cortex but never expanded to the vascular bundles up to the 9th dpi. It was therefore suggested to prefix the forma specialis name of the causal agent of RSR with radicis to point out its non-vascular pathogenicity in vanilla. In the two incompatible interactions, the important role played by hypodermis cells for impeding the invasion of the cortex by Fo072 was demonstrated by specific staining and spectral analysis of lignin precursors. Both constitutive and pathogen induced defense mechanism were described in pla0020 and pom0018. The mechanisms included the deposition of lignin in the hypodermal cell wall, entrapment of hyphae in specific hypodemal cells and polyphenolics secretion in intercellular spaces. Further, a de novo transcriptome analysis was experimentedon 8 pooled samples.

Os fungos entomopatogênicos dos gêneros Metarhizium, Beauveria e Isaria (Ordem Hypocreales), são comumente encontrados em solo onde sobrevivem de maneira saprofítica ou como endofíticos do sistema radicular das plantas. Informações sobre a composição destas espécies bem como sua diversidade, distribuição e associação com diferentes tipos de cultivos e vegetação nativa são escassas no Brasil. O presente estudo foi desenvolvido para comparar a abundância de fungos entomopatogênicos e a diversidade genética de isolados de Metarhizium spp. em amostras de solo de cultivos anuais, perenes e vegetação nativa, em cinco estados brasileiros que representam os biomas Amazônia, Cerrado, Caatinga, Mata Atlântica e Pampa, em duas estações (seca e úmida) nos anos de 2012 e 2013. O isolamento dos fungos foi realizado com meio seletivo e \"Insect bait\" utilizando Galleria mellonella e Tenebrio molitor. Nos estudos de diversidade genética de Metarhizium spp. foram utilizadas sequências de DNA da região MzIGS3. Representantes dos haplótipos revelados nesta análise tiveram a região 5\'-TEF sequenciada para identificação específica. Fungos entomopatogênicos foram isolados de 86% das 1.056 amostras de solo sendo Metarhizium o gênero predominante (66% das amostras de solo), seguido por Beauveria (41,9%) e Isaria (10,8%). Em geral, as maiores densidades de fungos entomopatogênicos foram obtidas nos biomas Amazônia e Cerrado e as menores densidades detectadas no bioma Caatinga. Metarhizium spp. foi detectado em maior número de amostras de solo em vegetação nativa e cultivos anual e perene do Cerrado. A frequência de Isaria spp. foi baixa nas amostras de solo, sendo detectado em maior número de amostras nos solos com cultivos anuais e vegetação nativa na Amazônia e Caatinga. Metarhizium spp. foi geralmente encontrado em um maior número de amostras coletadas na estação úmida em comparação com as coletas da estação seca, por outro lado Beauveria spp. foi superior na estação seca. A diversidade dos isolados de Metarhizium spp. provenientes de áreas de vegetação nativa foi maior do que dos isolados de cultivos anuais e perenes. Seis linhagens foram encontradas neste estudo; M. robertsii, M. anisopliae, M. pingshaense e três espécies indeterminadas. M. robertsii foi a linhagem predominante (65% dos isolados) sendo encontrado em áreas com vegetação nativa e cultivos anual e perene dos cinco biomas. Metarhizium sp. indet. 1 apresentou a maior diversidade haplotípica dentre as linhagens estudadas. Uma nova linhagem, não caracterizada taxonomicamente, Metarhizium sp. indet. 3, foi encontrada predominantemente na Caatinga. Somente na Amazônia foram encontradas todas as linhagens. O conhecimento da composição das populações de fungos entomopatogênicos nativos bem como sobre a filogenia, diversidade e distribuição dos haplótipos de Metarhizium spp. em solos brasileiros, gerado neste estudo, poderá servir como subsídio para o desenvolvimento de estratégias de conservação e maximização do controle biológico natural de pragas. / Entomopathogenic fungi of the genera Metarhizium, Beauveria and Isaria (order Hypocreales) are associated to the soil where they survive saprofitically or as endophytes of the plants root system. Information on the species composition and its diversity, distribution and association of these fungi with different types of crops and native vegetation are scarce in Brazil. The present study was carried out to compare the abundance of entomopathogenic fungi and the genetic diversity of Metarhizium spp. Isolated from soil samples from annual and perennial crops and native vegetation in five Brazilian states that represent the biomes Amazon, Cerrado, Caatinga, Atlantic Forest and Pampa, in two seasons (wet and dry) in the years 2012 and 2013. The isolation of fungi was performed with selective medium and \"Insect bait\" using Galleria mellonella and Tenebrio molitor. DNA sequences of the region MzIGS3 were used in genetic diversity studies of Metarhizium spp. Representatives haplotypes revealed in the diversity analysis had the 5\'-TEF region sequenced for species identification. Entomopathogenic fungi were isolated from 86% of 1,056 soil samples and Metarhizium was the predominant genus (66% of soil samples), followed by Beauveria (41.9%) and Isaria (10.8%). In general, the highest densities of entomopathogenic fungi were obtained in the Amazon and Cerrado biomes and the lowest densities were detected in the Caatinga biome. Metarhizium spp. was detected in a greater number of soil samples from native vegetation and annual and perennial crops of Cerrado. The frequency of Isaria spp. was low in soil samples being detected in a greater number of soils with annual crops and native vegetation in the Amazon and Caatinga. Metarhizium spp. was usually found in a greater number of samples collected during the wet season compared to the collections in the dry season. On the other hand, Beauveria spp. was higher in the dry season. The diversity of isolates of Metarhizium spp. from areas of native vegetation was greater than that obtained from annual and perennial crops. Six lineages were found in this study; M. robertsii, M. anisopliae, M. pingshaense and three indeterminate species. M. robertsii was the predominant (65% of isolates) found in areas with native vegetation and in the annual and perennial crops of the five biomes. Metarhizium sp. indet. 1 showed the greatest haplotype diversity among the strains studied. A new strain, not characterized taxonomically, Metarhizium sp. indet. 3, was found predominantly in the Caatinga. Only in the Amazon, all lineages were found. The knowledge on species composition of entomopathogenic fungi as well as about phylogeny, diversity and distribution of haplotypes of Metarhizium spp. in Brazilian soils, generated in this study, may be useful for the development of strategies for conservation and maximization of natural biological control of pests.

Para avaliar a circulação e freqüência dos genótipos de eritrovírus na região amazônica, foi analisado um total de 487 amostras de soros/plasmas colhidas de pacientes apresentando sintomas e sinais clínicos sugestivos de infecção pelos eritrovírus. O ensaio imunoenzimático foi utilizado para detecção de anticorpos específicos para B19, das classes IgM/IgG, e a reação em cadeia da polimerase/semi-nested PCR para detecção do DNA. Das 487 amostras examinadas, 117 (24%) mostraram a presença do DNA dos eritrovírus, sendo todas as 117 foram posteriormente seqüenciadas e genotipadas. A maioria dos isolamentos foi classificada como genótipo 1 (91% das amostras) e 3b (9% ). Também observamos três diferentes grupos dentro do genótipo 1 (A1, A2, B). Conseqüentemente, estas linhagens de B19 introduzidas em Belém apresentaram uma elevada taxa de mudanças dos aminoácidos, decorrência da pressão seletiva, gerando reinfecções consecutivas em uma pequena rede de transmissão, metaforicamente comparada a uma \"panela de pressão evolutiva\". / To assess the circulation and relative frequency of erythrovirus genotypes in clinical samples from patients living in the Amazon region we screened a total of 487 samples from patients suffering from different clinical manifestations suggestive of erythrovirus infections. Enzyme-linked immunosorbent assay was used to detect B19-specific IgM/IgG antibodies and polymerase chain reaction/ semi-nested PCR for viral DNA detection. Of the 487 samples 117 (24%) were positive for the erythrovirus DNA and all 117 isolates were sequenced and genotyped analyzing a fragment of 476 bp of VP1 and VP2 gene sequences of the erythrovirus. The majority of isolates was classified as genotype 1 (91% of the samples) and 3b (9% of the samples). We also reported three different clusters (A1, A2, B) within genotype 1. Our analysis revealed a strikingly different pattern of evolutionary change for those viral lineages introduced in Belém, which exhibited a higher rate of nonsynonymous substitutions compared to those viruses sampled from other locations.

Nesta tese apresentamos alguns resultados teóricos e práticos da modelagem de seqüências simbólicas com cadeias estocásticas parcimoniosas. As cadeias estocásticas parcimoniosas, que incluem as cadeias estocásticas de memória variável, constituem uma generalização das cadeias de Markov de alcance fixo. As seqüências simbólicas às quais foram aplicadas as ferramentas desenvolvidas são as cadeias de aminoácidos. Primeiramente, introduzimos um novo algoritmo, chamado de SPST, para selecionar o modelo de cadeia estocástica parcimoniosa mais ajustado a uma amostra de seqüências. Em seguida, utilizamos esse algoritmo para estudar dois importantes problemas da genômica; a saber, a classificação de proteínas em famílias e o estudo da evolução das seqüências biológicas. Finalmente, estudamos a velocidade de convergência de algoritmos relacionados com a estimação de uma subclasse das cadeias estocásticas parcimoniosas, as cadeias estocásticas de memória variável. Assim, generalizamos um resultado prévio de velocidade exponencial de convergência para o algoritmo PST, no caso de cadeias de memória ilimitada. Além disso, obtemos um resultado de velocidade de convergência para uma versão generalizada do Critério da Informação Bayesiana (BIC), também conhecido como Critério de Schwarz. / In this thesis we present some theoretical and practical results, concerning symbolic sequence modeling with parsimonious stochastic chains. Parsimonious stochastic chains, which include variable memory stochastic chains, constitute a generalization of fixed order Markov chains. The symbolic sequences modeled with parsimonious stochastic chains were the sequences of amino acids. First, we introduce a new algorithm, called SPST, to select the model of parsimonious stochastic chain that fits better to a sample of sequences. Then, we use the SPST algorithm to study two important problems of genomics. These problems are the classification of proteins into families and the study of the evolution of biological sequences. Finally, we find upper bounds for the rate of convergence of some algorithms related with the estimation of a subclass of parsimonious stochastic chains; namely, the variable memory stochastic chains. In consequence, we generalize a previous result about the exponential rate of convergence of the PST algorithm, in the case of unbounded variable memory stochastic chains. On the other hand, we prove a result about the rate of convergence of a generalized version of the Bayesian Information Criterion (BIC), also known as Schwarz\' Criterion.

O vírus respiratório sincicial humano (HRSV) é o principal agente viral causador de doença respiratória em bebês e crianças em idade pré-escolar. A fim de estudar a variabilidade genética de HRSV, grupo B, com inserção de 60 nucleotídeos no gene G, selecionamos amostras de aspirado de nasofaringe de crianças menores de 5 anos de idade, com doença respiratória aguda, admitidas no hospital universitário da Universidade de São Paulo. Testamos 521 amostras, das quais 35,3% foram positivas para HRSV. A região G2 da glicoproteína G foi utilizada para genotipar essas amostras. Todas as amostras do grupo B apresentaram a inserção de 60 nucleotídeos no gene da proteína G, como descrito anteriormente em Buenos Aires, em 1999. As modificações de aminoácidos e nucleotídeos dessas amostras foram comparadas com outras amostras com inserção de 2001-2005. A seqüência de nucleotídeos duplicados foi a cópia exata dos 60 nucleotídeos precedentes em vírus mais antigos, mas as cópias do segmento duplicado acumularam substituições de nucleotídeos em vírus mais recentes. / Human respiratory syncytial virus (HRSV) is the leading viral cause of respiratory illness in infants and young children. In order to study the genetic variability of HRSV group B, with 60-nucleotide duplication in the gene G, we selected nasopharyngeal aspirates samples of children less than five years of age, with acute respiratory illness admitted in the university hospital of São Paulo (USP). We tested 521 samples and the HRSV-detection test positivity rate was 35.3%. The G2 region of glycoprotein G was used as genotyping default. All type B HRSV had a 60-nucleotide duplication in the attachment protein gene like previously described in Buenos Aires, in 1999. Changes in aminoacids and nucleotides in these samples were compaired with other samples with duplication from 2001-2005. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but copies of the duplicated segment accumulated nucleotide substituions in more recent viruses.

A comparação de genomas, genes ou até sequências de nucleotídeos não condificantes é uma importante tarefa na qual a bioinformática pode ser aplicada, uma vez que ela auxiliar em diversas atividades, por exemplo, análises filogenéticas. Análise filogenética, por sua vez, busca analisar a relação evolutiva de cada espécie, considerando suas características genéticas. Esses processos e as técnicas que os implementam se baseiam em sequências de nucleotídeos sequenciadas e armazenadas em bancos de dados de genomas públicos. Com análise filogenética também é possível identificar possíveis origens de um gene. Essa tarefa é de grande importância, pois auxilia na identificação da origem de genes patogênicos, podendo auxiliar no combate e prevenção do surgimento de doenças. Um problema potencial dessas sequências é a possibilidade de haver erros nas anotações (marcações de sequências como genes). Esses erros são pouco explorados por pesquisadores atualmente. Outro tema pouco explorado é a análise filogenética de genes exclusivos, que são genes que se manifestam em apenas uma espécie, considerando um grupo de espécies próximas. A identificação de genes exclusivos de alguma espécie pode servir para a correta identificação de, por exemplo, a espécie que causa uma doença, de forma a permitir o uso do tratamento mais específico e adequado. A importância da descoberta de filogenias de genes exclusivos e a dificuldade de garantir a consistência nas anotações genéticas motivaram este trabalho, que teve como objetivo implementar ferramentas para interpretar dados de comparação genética, identificando potenciais erros em anotação de genes exclusivos e criando estratégias para identificar a origem desses genes. As origens de genes exclusivos exploradas neste trabalho envolvem a possibilidade dos genes exclusivos terem derivado de outras famílias de genes do próprio organismo, ou, os genes exclusivos se diferenciaram muito dos genes ancestrais. Essas hipóteses, juntamente com a hipótese da existência de erros de anotação, foram exploradas em experimentos utilizando as ferramentas desenvolvidas. Os experimentos visaram a analisar a aplicabilidade da estratégia desenvolvida. Foram utilizados genomas de bactérias do gênero Xanthomonas, que contém um grande grupo de bactérias que causam doenças em plantas. Os resultados obtidos demonstram que existe uma quantidade considerável de potenciais erros de anotação nos genomas considerados, provando a hipótese de que a inconsistência nas anotações genômicas possui grande influência para a dificuldade na identificação de filogenias (tanto de genes exclusivos como para não exclusivos). Os resultados também demonstraram que boa parte dos genes exclusivos possivelmente se originaram de outras famílias de genes do próprio genoma. Ou ainda, que esses genes sofreram modificações em relação aos genes ancestrais, mas ainda possuem certas semelhanças com sequências de nucleotídeos que não codificam genes em outras espécies mais distantes. Por fim, a estratégia desenvolvida se mostrou útil na análise filogenética das bactérias estudadas, sendo este um forte indício de que a mesma abordagem pode ser utilizada para problemas similares com outras espécies de seres vivos / Comparison of genomes, genes or even non-coding nucleotide sequences is an important task in which bioinformatics can be applied, since it allows the application of phylogenetic analyses. Phylogenetic analysis, in its turn, seeks to analyze the evolutionary relation of each species, considering its genetic characteristics. These processes and the techniques that implement them are based on nucleotide sequences sequenced and stores in databases of public genomes. With phylogenetic analysis it is also possible to identify possible origins of a gene. This task has a great importance, because it allows the identification of the origin of pathogenic genes, which may help to combat or prevent deseases. A potencial problem of these sequences is the possibility of having annotation errors (sequences marking as genes). These errors are little explored by researchers nowadays. Another unexplored topic is the phylogenetic analysis of exclusive genes, which are genes thaht manifest in only one species, considering a group of nearby species. The identification of exclusive genes of a species may serve to correctly identify, for example, a desease, in order to allow the use of a more especific and appropriate treatment. The importance of discovering phylogenies of exclusive genes and the difficulty of guaranteeing the consistency of genetic annotations motivated this work, whose objective was to implement tools to interpret data of genetic comparison, identifying annotation errors in exclusive genes and creating strategies to identify the origin of these genes.The origins of exclusive genes explored in this work involve the possibility of the exclusive genes have derived of other gene families of the organism itself, or, the exclusive genes differed a lot from the ancestral genes. Theses hypotheses, with the hypotesis of the existance of annotation errors, were explored in experiments using the developed tools. The experiments aimed to analyse the applicability of the developed strategy. Genomes of bacteria of the genus Xanthomonas were used, which contains a large group of bacteria that cause diseases in plants. The results show that there is a considerable amount of annotation errors on the genomes, proving the hypothesis that the inconsistency in genomic annotations has a great influence on the difficulty in identifying phylogenies (both exclusive and non-exclusive genes). The results also show that much of exclusive genes possibly originated from other gene families of the genome itself. Furthermore, these genes may have sufferedmodifications in relation to the ancestral genes, but still have certain similarities with nucleotide sequences that don\'t encode genes in other more distant species. Finally, the strategy developed proved useful on phylogenetic analysis of the studied bacteria, which is a strong indication that the same approach can be used for similar problems with other species of living beings

<p>Microorganisms, including two fungi, <i>Phytophthora cryptogea</i> and <i>Fusarium oxysporum</i> strain Fo-(IMI 386351), and one bacterium, <i>Pesudomonas</i> sp. strain MF30, were tested for their abilities to stimulate plant defence responses in tomato (<i>Lycopersicon esculentum</i> –L.) and to serve as effective biocontrol agents (<b>Bs</b>). The study included <i>in vivo</i> and <i>in vitro</i> characterization of biological attributes of the microorganisms, pertaining to their abilities to stimulate plant immunity against a fungal pathogen, <i>Fusarium oxysporum</i> f. sp. <i>lycopersici</i> (Fol), the causal agent of tomato wilt disease. Using <i>Lycopersicon esculentum</i> –L. as a model plant for examining some fundamental elements of the plant-microorganism interaction, the study reveals and clarifies some aspects of the close association and the complexity of such systems.</p><p>For each <b>B</b>, the results revealed a <b>B</b>-distinct plant-microorganism interaction, which included systemic induced resistance (SIR). A phylogenetic analyses of the partial sequences of two Fo-(IMI 386351) genes, a mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and the nuclear translation elongation factor 1α (EF-1α), provided phylogenetic trees confirming that Fo-(IMI 386351) might be a member of Fol or of <i>F. oxysporum</i> f. sp. <i>melonis</i>, which have polyphyletic evolutionary origins. RFLP analysis (mtDNA), suggested that Fo-(IMI 386351) probably belongs to Fol. For routine and accurate differentiation between two morphologically indistinguishable <i>F. oxysporum formae speciales</i> strains, <i>F. oxysporum</i> f. sp. <i>lycopersici</i> and <i>F. oxysporum</i> f. sp. <i>radicis-lycopersici</i>, a molecular method (mtDNA RFLP analysis) was developed, and its usefulness for such differentiation was compared with that of two other methods: isozyme analysis and an osmotic method, revealed with high performance liquid chromatography (HPLC). The HPLC-spectra of Fo-(IMI 386351) had an extra peak for the two tested fractions, indicating that activation of the observed plant defence mechanism could have been at least partially the result of one of the products of the eliciting microbe. Preliminary results obtained by nuclear magnetic resonance spectrometry of those fractions suggest that the extra peak probably represents an oligosaccharide, which may have acted as a mobile signal and triggered the plant defence mechanisms.</p><p>We concluded that (1) our three tested microorganisms are able to stimulate plant defence mechanisms by triggering SIR (plant immunity), (2) the complexity and elaborateness of evolved plant-microbe interactions involving plant defence can, at least in some cases, be observed and studied in the laboratory, and (3) molecular tools can be a powerful means for identifying fungal strains and for clarifying their taxonomical relationships.</p>

In this thesis relationships and the occurrence of reticulate evolutionary events in the club moss genus Diphasiastrum are investigated. Diphasiastrum is initially established as a monophyletic group within Lycopodiaceae using non recombinant chloroplast sequence data. Support is obtained for eight distinct parental lineages in Diphasiastrum, and relationships among the putative parent taxa in the hypothesized hybrid complexes; D. alpinum, D. complanatum, D. digitatum, D. multispicatum, D. sitchense, D. tristachyum and D. veitchii are presented. Feulgen DNA image densitometry data and sequence data obtained from three nuclear regions, RPB2, LEAFY and LAMB4, were used to infer the origins of three different taxa confirmed to be allopolyploid; D. zanclophyllum from South Africa, D. wightianum from Malaysia and an undescribed taxon from China. The two Asian polyploids have originated from two different hybrid combinations, D. multispicatum x D. veitchii and D. tristachyum x D. veitchii. Diphasiastrum zanclophyllum originates from a cross between D. digitatum and an unidentified diploid taxon. The occurrence of three homoploid hybrid combinations commonly recognized in Europe, D. alpinum x D. complanatum, D. alpinum x D. tristachyum and D. complanatum x D. tristachyum, are verified using the same three nuclear regions. Two of the three hybrid combinations are also shown to have originated from reciprocal crosses. Admixture analyses performed on an extended, dataset similarly identified predominately F1 hybrids and backcrosses. The observations and common recognition of hybrid species in the included populations are hence most likely due to frequent observations of neohybrids in hybrid zones. Reticulate patterns are, however, prominent in the presented dataset. Hence future studies addressing evolutionary and ecological questions in Diphasiastrum should emphasize the impact of gene flow between parent lineages rather than speciation as the result of hybridization.

Το σύστημα του Συμπληρώματος απαρτίζεται από μια ομάδα πρωτεϊνών του πλάσματος και κυτταρικών υποδοχέων που παίζουν σημαντικό ρόλο στην άμυνα του ξενιστή μέσω της αλληλεπίδρασής τους τόσο με συστατικά της φυσικής, όσο και της προσαρμοστικής ανοσίας. Παράλληλα, μια ομάδα από ρυθμιστικές πρωτεΐνες του πλάσματος και της κυτταρικής μεμβράνης προστατεύει τα κύτταρα του ξενιστή από την αυτόλογη δράση του Συμπληρώματος. Η σύνθεση του τελικού λυτικού συμπλόκου MAC, που οδηγεί σε κυτταρόλυση, ρυθμίζεται από τις ρυθμιστικές πρωτεΐνες : CD59, vitronectin και clusterin. Μέχρι σήμερα οι ρυθμιστικές πρωτεΐνες της λυτικής οδού του Συμλπηρώματος έχουν ελάχιστα χαρακτηριστεί σε σπονδυλωτά πέραν των θηλαστικών. Οι οστεϊχθύες αποτελούν ένα σημαντικό μοντέλο, καθώς είναι οι μόνοι οργανισμοί που εμφανίζουν ένα πλήρως αναπτυγμένο και διευρυμένο σύστημα συμπληρώματος, μέσω διπλασιασμού πολλών γονιδίων του Συμπληρώματος. Προκειμένου να μελετηθεί η παρουσία και η φυλογενετική εξέλιξη του ρυθμιστικού μορίου της clusterin, το οποίο παρεμποδίζει την πρόσδεση του συμπλόκου C5b-7 στην μεμβράνη του κυττάρου-στόχου, με αποτέλεσμα την αναστολή της κυτταρόλυσης, έγινε κλωνοποίηση και χαρακτηρισμός του γονιδίου της clusterin στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss irideus). Απομονώθηκαν δύο ισομορφές του γονιδίου της clusterin, clusterin-1 (TCLU-1) και clusterin-2 (TCLU-2) από cDNA βιβλιοθήκη ήπατος πέστροφας, με χρήση ειδικών ολιγονουκλεοτιδίων. Οι προκύπτουσες αμινοξικές αλληλουχίες των ισομορφών clusterin-1 και clusterin-2 παρουσιάζουν 89% ταυτοσημία μεταξύ τους, καθώς και 40 και 38% ταυτοσημία με την clusterin του ανθρώπου, αντίστοιχα. Η μοριακή δομή των συναγομένων πρωτεϊνών αντιστοιχεί στην α΄ αλυσίδα της clusterin και ομοιάζει με εκείνη των θηλαστικών. Γονιδιωματικοί κλώνοι απομονώθηκαν και για τις δύο ισομορφές και μελετήθηκε η οργάνωση εξωνίων-εσωνίων των αντίστοιχων γονιδίων. Η οργάνωση των γονιδίων clusterin-1 και clusterin-2, με τα μέχρι στιγμής δεδομένα, δείχνει αντιστοιχία εξωνίων - εσωνίων με το γονίδιο της clusterin του ανθρώπου, στην περιοχή ομολογίας τους. Ως προς το μέγεθος, τα αντίστοιχα εξώνια φαίνονται συντηρημένα, ενώ τα εσώνια ποικίλουν, εμφανίζοντας μικρότερο μέγεθος συγκριτικά με εκείνα της clusterin του ανθρώπου. Ανάλυση κατά Southern έδειξε ότι η clusterin απαντάται σε δύο τουλάχιστον αντίγραφα στο γονιδίωμα της πέστροφας. Τα γονίδια clusterin-1 και clusterin-2 εμφανίζουν διαφορική έκφραση στο επίπεδο του mRNA σε διάφορους ιστούς πέστροφας, όπως διαπιστώθηκε με RT-PCR. Παρατηρήθηκαν υψηλά επίπεδα mRNA του γονιδίου TCLU-1 στο ήπαρ και στο σπλήνα, ενώ η έκφραση του γονιδίου TCLU-2 παρατηρήθηκε αυξημένη στην καρδιά και στο έντερο, χαμηλή στο ήπαρ και στο σπλήνα δεν ανιχνεύτηκε. Τέλος, η φυλογενετική ανάλυση των πρωτεϊνών της clusterin από διάφορους οργανισμούς εμφάνισε την TCLU-1 να ομαδοποιείται με την TCLU-2, ενώ η όλη υποομάδα προηγείται φυλογενετικά των ορθόλογων πρωτεϊνών του pufferfish και του zebrafish και αυτές των ορθόλογων των άλλων οργανισμών που αναλύθηκαν. Συμπερασματικά, το γονίδιο της clusterin ανιχνεύεται στην ιριδίζουσα πέστροφα, σε δύο τουλάχιστον ισομορφές και παρουσιάζει ομολογία με την clusterin του ανθρώπου, τόσο στο επίπεδο οργάνωσης του γονιδίου, όσο και στο επίπεδο της αμινοξικής αλληλουχίας και της προκύπτουσας δομής. Η παρουσία ισομορφών από εναλλακτικό μάτισμα που ισχύει στην clusterin του άνθρωπου δεν έγινε δυνατόν να πιστοποιηθεί. / The complement system consists of a group of plasma proteins and cell receptors that play a critical role in host defense, by interacting with components of both innate and adaptive immunity. Α group of plasma and cell membrane regulatory proteins protects the host cells from the autologous complement activation. The conformation of the lytic complex MAC, which results to the cytolysis, is regulated by the regulatory proteins: CD59, clusterin και vitronectin. Up to date, the regulatory proteins of MAC complex are not well characterized in low vertebrates. Bony fish represent an attractive model for studies on complement system, as they are, the only organisms that reveal a completely developed and extended system, via duplication and functional differentiation of many genes of the complement system. In order to elucidate the presence and the evolution of the clusterin regulatory molecule, which impedes the binding of C5b-7 complex to the membrane of the target cell, inhibiting cytolysis, we report here the cloning and characterization of the clusterin gene in rainbow trout (Oncorhynchus mykiss irideus). Two isoforms of clusterin gene, clusterin-1 (TCLU-1) and clusterin-2 (TCLU-2) were isolated from a trout liver cDNA library, by PCR using specific oligonoucleotides. The deduced amino-acid sequences of the isoforms TCLU-1 and TCLU-2 show 89% identity to each other, as well as 40 and 38% identity to human clusterin, respectively. The domain structure of the deduced proteins corresponds to the α΄ chain of clusterin and resembles to that of mammalian. Genomic clones were isolated for the two isoforms and intron-exon organization of corresponding genes was clarified. The organization of the clusterin genes TCLU-1 and TCLU-2, as the partial data have shown, is in line with the intron-exon organization of the human clusterin gene, at their homology region. Regarding the length, the corresponding exons seem to be conserved, but the introns in trout clusterin genes are shorter than human clusterin. Southern blot analysis has shown that clusterin can be found as two copies at least, in the trout genome. Also, the genes TCLU-1 and TCLU-2 reveal differential expression, at mRNA level, among several trout tissues, as it was found out by RT-PCR analysis. High levels of TCLU-1 mRNA are detected in liver and spleen. On the other hand, TCLU-2 is expressed mainly in heart and intestine, while is not detected any expression in spleen. Finally, the phylogenetic analysis of the clusterin proteins from various organisms, showed grouping of TCLU-1 and TCLU-2, while this subgroup precedes evolutionary the clusterin orthologs of pufferfish, zebrafish and the other examined organisms, afterwards. Conclusively, the clusterin gene in rainbow trout is detected as two isoforms and shows homology with human clusterin, not only at the level of gene organization, but also at the level of amino-acid sequence and domain architecture. The alternative splicing of human clusterin was not able to be certified in trout.