Linhagens de Escherichia coli enterotoxigênica (ETEC) são consideradas como importante agente de diarréia, principalmente entre crianças e turistas em países em desenvolvimento. Entre os fatores de virulência expressos por ETEC, as enterotoxinas termo-lábil (LT) e termo-estável (ST) representam os mais relevantes fenótipos. Evidências preliminares sugerem que a severidade da diarréia associada a linhagens de ETEC deve refletir a diversidade natural de linhagens selvagens quanto à produção de enterotoxinas e/ou à ocorrência de variantes naturais com efeitos tóxicos reduzidos. No presente trabalho, investigamos diversidade genética do óperon etx, que codifica para a toxina LT, e da capacidade de produção e secreção de LT por linhagens de ETEC isoladas de humanos ou suínos em diferentes regiões geográficas. Os resultados mostraram considerável variabilidade na produção de LT com valores variando de 2 a 2.525 ng de toxina por mL de cultura. Secreção de LT foi também variável com valores variando de menos que 0,04% a 49,5% do total de LT produzida pelas diferentes linhagens de ETEC. Adicionalmente, experimentos de alça ligada em coelho mostraram uma boa correlação entre a quantidade de LT secretada sob condições in vitro e a capacidade de causar acúmulo de fluidos in vivo. Nós determinamos ainda diversidade de ETEC pela obtenção das seqüências dos óperons etxAB de 50 linhagens (LT+ or LT+/ST+) pertencentes a diferentes sorotipos com ênfase para as linhagens produtoras apenas de LT e isoladas de crianças assintomáticas. As seqüências de nucleotídeo completas dos genes etxAB revelaram 23 alterações de aminoácidos nas subunidades A (18) e B (5), as quais geraram 16 variantes de LT. Entre estes variantes de LT, um mostrou efeito tóxico reduzido em comparação à toxina de referência LT1. A forma de LT atenuada (LT4) tem atividade enzimática reduzida devido à troca de aminoácido. / Enterotoxigenic Escherichia coli (ETEC) strains represent an important etiological agent of diarrheal disease, particularly among children and travelers in developing countries. Among the virulence factors expressed by ETEC strains the heat-labile (LT) and heat-stable (ST) enterotoxins represent the most revelevant phenotypes. Indirect evidences suggest that the severity of diarrhea associated to ETEC strains might reflect the natural diversity of wild strains to produce enterotoxins and/or the occurrence of variants endowed with reduced toxic effects. In the present study, we investigated both the genetic diversity of the etx operon, encoding the heat-labile toxin, and the capability to produce/secrete LT by ETEC strains isolated from humans or porcine in different geoghrafic areas. The results showed a remarkable variability on the production of LT with values ranging from 2 to 2,525 ng of toxin per ml of culture. LT secretion was also variable with values ranging from less than 0.04% to 49.5% of total LT produced by the different ETEC strains. Additionally, rabbit ileal loop experiments showed a good correlation between the amounts of secreted LT under in vitro conditions and fluid accumulation in vivo. We determined also the diversity of the etxAB operon of 50 ETEC strains (LT+ or LT+/ST+) belonging to different serotypes with emphasis to LT+-only producing strains isolated from asymptomatic children. The complete nucleotide sequences of the etxAB genes revealed 23 amino acid changes at the A (18) or B (5) subunits, which generated 16 variant forms of LT. Among these LT variants, one of them showed reduced toxic effects in comparison to the reference toxin LT1. The attenuated LT form (LT4) had decreased enzymatic activity due to an amino acid replacement (K4R) at the A1 subunit. LT4 retains its immunogenic and adjuvant properties following nasal immunization. Additionaly, the LT4 variant showed altered immune modulatory features and promoted a more biased Th1 response, which favor activation of effector CD8+ T lymphocytes, to co-administred antigen with regard to LT1. Taken together, our results demonstrate that ETEC strains isolated from human subjects express natural genetic variability leading to a remarkable polymorphism of the etx operon as well as production and secretion of LT. Such natural genetic diversity observed in ETEC strains may affect the host-pathogen relationships and, consequently, contribute to the severity of the disease among infected subjects.

Bactérias endofíticas e epifíticas podem conferir ao seu hospedeiro características como maior resistência a condições de estresse, alterações nas condições fisiológicas, fixação de nitrogênio atmosférico, suprimento de nutrientes, produção de reguladores de crescimento vegetal, entre outros. Desta forma, o presente trabalho teve por objetivos estudar a composição da comunidade bacteriana associada à soja e avaliar diferentes mecanismos de interação bactéria-planta hospedeira. Para isso, bactérias endofíticas e epifíticas de folhas, caules e raízes de duas cultivares de soja, crescidas em solo com e sem aplicação pré-plantio do herbicida glifosato, foram amostradas em três estádios de desenvolvimento do hospedeiro, durante as safras de 2000/01 e 2001/02. Foram observadas diferenças significativas na diversidade e densidade bacterianas em relação às fases de crescimento da soja e tecidos da planta. Os principais grupos foram identificados como pertencentes aos gêneros Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium e Methylobacterium. Além da avaliação de populações cultiváveis, análise por DGGE revelou que a comunidade bacteriana endofítica de raiz de soja pode ser influenciada pelo tratamento do solo com o herbicida glifosato. O potencial destas bactérias para a promoção de crescimento vegetal por bactérias associadas à soja foi avaliado, sendo possível observar que populações endofíticas e epifíticas de soja apresentam características relacionadas à promoção de crescimento vegetal e que fatores como cultivar e estádio fenológico do hospedeiro podem influenciar as freqüências destas populações. A análise da variabilidade genética destas populações bacterianas revelou que diferentes fatores ambientais também podem influenciar a diversidade de grupos bacterianos. Além disso, populações endofíticas com capacidade de crescer na presença do herbicida glifosato foram caracterizadas e identificadas como pertencentes às espécies Burkholderia gladioli e Pseudomonas oryzihabitans, enquanto que Methylobacterium spp. colonizam ativamente a superfície e os tecidos internos de soja após inoculação via semente. Os resultados obtidos podem oferecer uma contribuição para a melhor compreensão da interação microrganismossoja e, conseqüentemente, de sua aplicação na cultura deste vegetal. / Endophytic and epiphytic bacteria may increase the fitness of the plant host by increasing resistance to stress conditions, alterations in the physiologic conditions, fixation of atmospheric nitrogen, nutrient supplying and plant growth regulators production. The aims of the present work were to study the composition of soybean-associated bacterial community and to evaluate different mechanisms for bacteria-host plant interaction. For that, endophytic and epiphytic bacteria from leaves, stems and roots of two soybean cultivars, planted in soil with and without pre-planting application of the glyphosate herbicide, they were colleted in three development stages of the host, during two crops. Significant differences were observed in the bacterial diversity and population density in relation to the soybean growth stages and plant tissues. The principal groups were identified as belonging to the genera Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium and Methylobacterium. Besides the evaluation of cultivable populations, analyses by DGGE revealed that the endophytic bacterial community from soybean roots may be influenced by the treatment of the soil with the glyphosate herbicide. Other analyzed aspect was the potential for plant growth promotion by soybean-associated bacteria; revealing that soybean's endophytic and epiphytic populations presented characteristics related to the plant growth promotion; factors such as cultivar and developmental stage of the host may influence the frequency of these populations. Environmental factors may affect the genetic variability of these bacterial populations. Besides, endophytic populations able to growth in medium containing glyphosate were characterized and identified as belonging to Burkholderia gladioli and Pseudomonas oryzihabitans species. Methylobacterium spp. were reintroduced in soybean seeds and superficial and endophytic colonization were evaluated by scanning electronic microscopy. The obtained results could offer a contribution for a better understanding of the interaction microorganism-soybean and, consequently, their possible use to improve soybean productivity.

Introduced populations of Eichhornia crassipes (Pontederiaceae) possess extremely low levels of genetic diversity due to severe bottleneck events and clonal reproduction. While populations elsewhere have been well studied, North American populations of E. crassipes remain understudied. We used Amplified Fragment Length Polymorphism markers to assess genetic diversity and population structure in North American E. crassipes populations. Patterns of diversity over the past fifty years were analyzed using herbarium specimens. Furthermore, we sampled populations across the Gulf Coast of the United States throughout a year to determine contemporary genetic diversity and assess potential seasonal effects. Genetic diversity was found to be scant in the United States without population structure, agreeing with previous studies from other regions. Genetic diversity has remained consistently low over the past fifty years despite significant changes in selection pressure. However, evidence for and against population structure between seasons was found and the consequences of this are discussed.

<p>In this thesis, I combined comparative and experimental approaches to examine selection on reproductive traits and population differentiation in the insect-pollinated, self-incompatible, perennial herb <i>Primula farinosa</i>. More specifically, I (1) determined whether the effects of floral display and interactions with pollinators and seed predators, and plant reproductive success were frequency-dependent and affected by surrounding vegetation context, (2) examined the consequences of intermittent drought years on population dynamics using numerical simulations based on demographic data collected over seven years, (3) analyzed among-population differentiation in flowering phenology and reproductive allocation, and its relationship to soil-depth at the site of origin. </p><p>A field experiment suggested that conspicuous plants facilitate inconspicuous plants in terms of pollinator attraction, and that the facilitation effect is contingent on the height of the surrounding vegetation. Further experiments revealed that both mutualistic and antagonistic interactions can result in frequency-dependent selection on floral display. Among inconspicuous plants, both fruit initiation, and damage from seed predators increased with the proportion of the conspicuous morph. The relative strength of these effects, and therefore their net outcome on the relationship between morph ratio and seed production varied among years. </p><p>I combined information on vital rates and their relation to environmental conditions in simulations to predict future population viability in changing environments. Simulated stochastic population growth rate decreased with increasing frequency of drought years. </p><p>Reproductive allocation varied significantly among populations both in the field and in a common-garden experiment, but was correlated with soil depth at the site of origin only in the field. The results suggest that among-population variation in reproductive effort in the field mainly reflects plastic responses to environmental conditions, and that this plasticity may be adaptive. The common-garden experiment suggested that the study populations have diverged genetically in flowering time.</p>

La phylogéographie intègre l’information géographique et génétique pour inférer l’histoire démographique et les processus évolutifs des espèces. La présente étude recherche à travers les patrons de différenciation de l’ADN chloroplastique (ADNcp) au sein de Santiria trimera (Oliv.) H.J.LAM ex AUBR. [Emend. ONANA] la reconstitution d’une histoire des végétations des écosystèmes de forêts tropicales humides d’Afrique. Le modèle S. trimera est un arbre dioïque endémique des forêts humides d’Afrique dont les drupes sont dispersées par les primates et les oiseaux. Les formes morphologiques de ce modèle sont très variables et suscitent la délicate question de délimitation des espèces. Trois régions de l’ADNcp (l’intergène trnL-F, une portion du gène rbcL et l’intron rpl36-infA-rps8) ont été séquencées chez 377 individus issus de 42 populations de l’île de São Tomé, du Haut- et Bas-Guinéen pour étudier la phylogéographie. Des arbres phylogénétiques ont été réalisés sur des séquences d’un intron nucléaire du gène Phosphoenolpyruvate Carboxylase (72 individus) et sur les trois régions chloroplastiques. Une analyse morphométrique a été réalisée sur des données collectées sur des arbres en fruits. A l’aide de 10 locus microsatellites nucléaires, nous avons déterminé la structure génétique entre trois morphotypes sympatriques et analysé la structure génétique spatiale au sein de chaque groupe génétique. Le séquençage de l’ADNcp a mis en évidence des doubles pics sur les chromatogrammes de séquences. L’analyse des séquences clonées de produits PCR, la distribution des sites à doubles pics dans les séquences et dans les populations et les états ancestraux des positions à doubles pics nous ont permis d’interpréter les doubles pics comme résultant d’une co-amplification d’une copie chloroplastique et de copies nucléaires des régions séquencées. Les pics majeurs ont été considérés comme les nucléotides d’ADNcp d’origine maternelle et les pics mineurs ont été exclus de notre jeu de données. Les domaines phytogéographiques de São Tomé, du Haut- et Bas-Guinéen ne partagent aucun haplotype chloroplastique. Le Bas-Guinéen montre une plus grande diversité génétique. Les zones de distribution des haplotypes rares coïncident avec les refuges forestiers hypothétiques. L’analyse morphométrique et la phylogénie des séquences d’ADNcp suggèrent conjointement la reconnaissance de deux espèces bien différenciées. La structure génétique au sein d’une même population présumée suggère que les trois morphotypes en sympatrie dans les populations du Gabon constituent deux réservoirs génétiques différenciés sans individus hybrides. Selon le Concept Biologique de l’Espèce, S. trimera est probablement un mélange de deux espèces. On peut définir une espèce constituée d’individus avec des racines échasses et petites folioles coriaces (SRsl) et une seconde espèce constituée à la fois d’individus avec des racines échasses et de grandes folioles papyracées (SRll) et d’individus sans racine échasse avec de grandes folioles coriaces (NSR). Au vu de ces résultats, la classification taxonomique de S. trimera nécessite une révision. La confusion de ces deux espèces dans les forêts du Gabon explique une plus forte divergence de lignées chloroplastiques sympatriques par rapport aux lignées issues des régions biogéographiques isolées. L’un des deux haplotypes principaux de l’espèce à grandes folioles (SRll + NSR) est distribué dans le nord du Gabon et l’autre est distribué dans le sud. Au sein de l’espèce à petites folioles (SRsl), les zones d’endémisme de lignées chloroplastiques se situent dans l’Ouest du Cameroun qui est considéré comme une zone de fort endémisme et de forte diversité en espèces. Globalement, les patrons phylogéographiques mis en évidence entre lignées chloroplastiques de S. trimera sont compatibles avec les hypothèses biogéographiques basées sur les patrons de diversité et d’endémisme des espèces. / Phylogeography combines geographic and genetic information to infer demographic history and evolutionary processes. The present study of the spatial structure of chloroplast DNA (cpDNA) within Santiria trimera H.J.LAM ex AUBR. [Emend. ONANA], a primate- and bird-dispersed dioecious tree typical of African rainforests, provides insights into African vegetation history. This tree displays striking morphological variation which poses the problem of species delineation. Three regions of cpDNA (intergene trnL-F, a portion of rbcL gene and intron rpl36-infA-rps8) were sequenced in 377 individuals from 42 populations from São Tomé island and from Upper- and Lower-Guinean forests to study phylogeography. To study genetic divergence among morphotypes of S. trimera, phylogenies of a nuclear intron of Phosphoenolpyruvate Carboxylase from 72 individuals and concatenated sequences of the three cpDNA sequences were compared to morphological data from fruit-bearing trees. Using ten nuclear microsatellite markers, we defined the genetic structure among three sympatric morphotypes and analysed the spatial genetic structure within each genetic group. CpDNA sequences revealed double-peaks on sequence chromatograms. Sequences of cloned PCR products, the distribution of double peaks on sequences and in populations and ancestral nucleotides inferred from different taxa of the Burseraceae family enabled us to deduce that double peaks were due to the co-amplification of chloroplast and nuclear copies of the cpDNA region. Major peaks were considered as originating from maternal cpDNA. Subordinated peaks corresponding to the nuclear copies were excluded from our data set. The phytogeographic domains of São Tomé, Upper and Lower Guinea did not share any haplotype. Lower Guinea was the most diversified and the most divergent haplotypes were found in Gabonese forests. Endemism areas of haplotypes coincide with hypothetic forests refuges. Morphometric analyses and phylogenies cpDNA converge to delineating two well-differentiated species within S. trimera. Likewise, the genetic structure within one assumed population suggests that the three sympatric morphotypes constitute two genetically isolated populations without any hybrid. Following the Biological Species Concept, S. trimera is probably a mixture of two species in Lower Guinean forests. The first species is composed of all individuals with stilt roots and small leaflets (SRsl) and the second one is composed of both the morphotype with stilt roots, large and thin leaflets (SRll) and the morphotype without stilt roots with large and tough leaflets (NSR). In the view of our results, the taxonomical classification of S. trimera requires a revision. The confusion of both species in Gabonese forests explains that the highest divergence among chloroplast lineages was found in sympatric populations instead of among isolated biogeographic regions. One of the two major haplotypes of the second species (NSR + SRll) was distributed in the north of Gabon while the other haplotype was distributed in the south. Within the species with small leaflets (SRsl), areas of elevated haplotype endemism in West Cameroon coincided with hypothetic forest refuges. Overall, phylogeographic patterns within our model were in accordance with biogeographic hypotheses based on species endemism and diversity patterns.

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

En este trabajo de Tesis se evalúo de manera extensiva la diversidad genética presente en los maíces nativos de Latinoamérica, combinando datos fenotípicos y genotípicos, con dos propósitos diferentes. El primero, un estudio donde la diversidad genética, las relaciones interpoblacionales y la estructura poblacional de los materiales nativos permitieron ligar aspectos históricos, antropológicos y arqueológicos del maíz para crear una historia consolidada de la migración del maíz desde su centro de origen en Mesoamérica hacia el Caribe y Sudamérica. El segundo, es explorado el potencial de las razas de maíz para proporcionar nuevos alelos favorables para mejoramiento. La evaluación fenotípica y genotípica conjunta de accesiones del Banco de Germoplasma con material mejorado por CIMMYT permitió identificar fuentes de variación alélica importante para algunos caracteres de interés como tolerancia a la sequía. La caracterización de la diversidad genética de los materiales nativos para la conservación y explotación con fines de mejoramiento es un enfoque muy prometedor en particular con el desarrollo constante de nuevas herramientas genéticas. / In this thesis work, the genetic diversity of Latin American maize landraces was evaluated extensively combining phenotypic and genotypic data, for two different purposes. First, to study the genetic diversity, relationships, and population structure of native materials allowing the linkage of historical, anthropological and archaeological data to create a consolidated history of maize migration from its origin center in Mesoamerica towards The Caribbean and South America. Second, to explore the potential of maize landraces to provide new elite alleles for breeding, the phenotypic and genotypic characterization of gene bank accessions compared to improved material from CIMMYT allowed the identification of important sources of allelic variation for traits of interest including drought tolerance. The characterization of native genetic diversity for conservation and exploitation towards breeding is a very promising approach, especially considering on going development of new genetic tools and methodologies

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