Le système hématopoïétique est un tissu en constant renouvellement et les cellules souches hématopoïétiques (CSHs) sont indispensables pour soutenir la production des cellules matures du sang. Deux fonctions définissent les CSHs; la propriété d’auto-renouvellement, soit la capacité de préserver l’identité cellulaire suivant une division, et la multipotence, le potentiel de différenciation permettant de générer toutes les lignées hématopoïétiques. Chez l’adulte, la majorité des CSHs sont quiescentes et l’altération de cet état corrèle avec une diminution du potentiel de reconstitution des CSHs, suggérant que la quiescence protège les fonctions des CSHs. La quiescence est un état réversible et dynamique et les réseaux génétiques le contrôlant restent peu connus. Un nombre croissant d’évidences suggère que si à l’état d’homéostasie il y a une certaine redondance entre les gènes impliqués dans ces réseaux de contrôle, leurs rôles spécifiques sont révélés en situation de stress. La famille des bHLHs (basic helix-loop-helix) inclue différentes classes des protéines dont ceux qui sont tissu-spécifiques comme SCL, et les protéines E, comme E12/E47 et HEB. Certains bHLHs sont proposés êtres important pour la fonction des cellules souches, mais cela ne fait pas l’unanimité, car selon le contexte cellulaire, il y a redondance entre ces facteurs. La question reste donc entière, y a-t-il un rôle redondant entre les bHLHs d’une même classe pour la fonction à long-terme des CSHs? Les travaux présentés dans cette thèse visaient dans un premier temps à explorer le lien encore mal compris entre la quiescence et la fonction des CSHs en mesurant leurs facultés suite à un stress prolifératif intense et dans un deuxième temps, investiguer l’importance et la spécificité de trois gènes pour la fonction des CSHs adultes, soit Scl/Tal1, E2a/Tcf3 et Heb/Tcf12. Pour répondre à ces questions, une approche cellulaire (stress prolifératif) a été combinée avec une approche génétique (invalidation génique). Plus précisément, la résistance des CSHs au stress prolifératif a été étudiée en utilisant deux tests fonctionnels quantitatifs optimisés, soit un traitement basé sur le 5-fluorouracil, une drogue de chimiothérapie, et la transplantation sérielle en nombre limite. Dans la mesure où la fonction d’un réseau génique ne peut être révélée que par une perturbation intrinsèque, trois modèles de souris, i.e. Scl+/-, E2a+/- et Heb+/- ont été utilisés. Ceci a permis de révéler que l’adaptation des CSHs au stress prolifératif et le retour à l’équilibre est strictement contrôlé par les niveaux de Scl, lesquels règlent le métabolisme cellulaire des CSHs en maintenant l’expression de gènes ribosomaux à un niveau basal. D’autre part, bien que les composantes du réseau puissent paraître redondants à l’équilibre, mes travaux montrent qu’en situation de stress prolifératif, les niveaux de Heb restreignent la prolifération excessive des CSHs en induisant la sénescence et que cette fonction ne peut pas être compensée par E2a. En conclusion, les résultats présentés dans cette thèse montrent que les CSHs peuvent tolérer un stress prolifératif intense ainsi que des dommages à l’ADN non-réparés, tout en maintenant leur capacité de reconstituer l’hématopoïèse à long-terme. Cela implique cependant que leur métabolisme revienne au niveau de base, soit celui trouvé à l’état d’homéostasie. Par contre, avec l’augmentation du nombre de division cellulaire les CSHs atteignent éventuellement une limite d’expansion et entrent en sénescence. / The hematopoietic system is constantly replenished by hematopoietic stem cells (HSCs) that are essential to sustain mature blood cells production. Two key functions characterize HSCs; their capabilities to self-renew, i.e. maintenance of cellular identity following cell division, and their multipotencies, i.e. their potentials to generate all hematopoietic lineages. In adults, most HSCs are quiescent and alterations to this state correlate with decreased reconstitution potential, thus suggesting that quiescence protects HSC functions. Quiescence is a reversible and dynamic state, and genetic networks controlling these characteristics are poorly described. Recent evidence suggests that during steady-state hematopoiesis, genes controlling HSC functions are highly redundant, whereas stress conditions may reveal their specific roles. Transcription factors of the basic helix-loop-helix (bHLHs) family include tissue-specific subclasses (e.g SCL) and more ubiquitous E proteins (e.g. E12/E47 and HEB). Several bHLH members have been described as important for HSC functions, however this question is still highly debated in the field due to functional redundancies. How different bHLHs from a same subclass can uniquely affect long term HSC functions is still an open question. The work presented in this thesis aimed to address the question how three bHLH transcription factors specifically Scl/Tal1, E2a/Tcf3 and Heb/Tcf12 control HSC functions after an important proliferative stress to eventually re-establish steady state conditions typified by quiescence in adult HSCs. . To this end, we used three converging approaches, at the cellular level, by imposing a proliferative stress on HSCs, a genetic approach, by deleting genes of interest and genome-wide transcriptomics. More precisely, HSC resistance to proliferative stress has been evaluated under two extreme conditions; i.e. by consecutive treatments with the chemotherapeutic drug 5-fluorouracil (5-FU), mimicking a clinical situation in cancer chemotherapy, and by serial transplantation assays with limited cell numbers. Moreover, to test if a genetic network regulates HSCs functions, we also used three mouse models, i.e. Scl+/-, E2a+/- et Heb+/-. Using these tools, we showed that HSC adaptation to proliferative stress and return to steady state is strictly regulated by Scl expression levels that restricts ribosomal gene expression. Moreover, despite some degree of redundancy within this network, Heb expression levels restrain the excessive proliferation of HSC upon stress conditions by inducing senescence, a function that cannot be compensated for by E2a. To conclude, our results show that HSCs can tolerate both proliferative stress and unrepaired DNA damages without affecting their primary function to replenish the hematopoietic system. This is especially true if their metabolism can come back to basal levels. However, with increased numbers of cell divisions, HSC will sooner or later reach their expansion limit and enter senescence.

Future climate scenarios in the Baltic Sea project increasing sea surface temperature, as well as increasing precipitation and river runoff resulting in decreased salinity. These changes can severely impact the dynamics and function of brackish water communities, specifically phytoplankton. Phytoplankton are a significant source of organic matter to other trophic levels, and some species can be toxic. Their response to future climate conditions is of great relevance for the health of humans and aquatic ecosystems. The aim of this thesis was to assess the potential for climate-induced changes, such as decreasing salinity, to affect phytoplankton dynamics, physiology and chemical profiles in the Baltic Sea.      Phytoplankton successional patterns in the Baltic Proper consist of a spring bloom where diatoms and dinoflagellates co-occur and a summer bloom dominated by filamentous/colonial cyanobacteria. The consensus is that future warmer conditions will promote filamentous/colonial cyanobacteria blooms. This thesis shows that phytoplankton biomass in the spring bloom was lower in years with milder winters compared with cold winters. This suggests that in terms of annual carbon export to higher trophic levels, loss of biomass from the spring bloom is unlikely to be compensated by summer cyanobacteria. High frequency sampling of phytoplankton performed in this thesis revealed a strong relationship between the dynamics of pico- and filamentous cyanobacteria. Large genetic diversity was found in cyanobacterial populations with high niche differentiation among the same species. At community level, high temperature and low salinity were the main factors shaping the summer cyanobacterial composition. These conditions may promote the predominance of opportunistic filamentous cyanobacteria, e.g. Nodularia spumigena. This species produces various bioactive compounds, including non-ribosomal peptides such as the hepatotoxin nodularin. In this work, N. spumigena subpopulations evolved different physiological strategies, including chemical profiles, to cope with salinity stress. This high phenotypic plasticity ensures survival in future climate conditions. Under salinity stress, some subpopulations displayed shorter filaments as a trade-off. This indicates that the future freshening of the Baltic Sea may promote grazing on filamentous cyanobacteria and modify carbon flows in the ecosystem. In this thesis, Baltic N. spumigena chemotypes and genotypes grouped into two main clusters without influence of geographical origin. Thus, chemical profiling can be used to explore conspecific diversity in closely genetically related N. spumigena subpopulations.      Overall, this thesis has significantly expanded the knowledge on phytoplankton community and population responses to short- and long-term environmental changes, relevant to project the impacts of future climate conditions in the Baltic Sea.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les cellules souches ont attiré l’attention du public ces dernières années, grâce non-seulement à leur utilisation comme thérapies visant à s’attaquer à certains types de cancers, mais aussi en relation avec leur potentiel dans le domaine de la médecine regénérative. Il est établi que le destin cellulaire des cellules souches embryonnaires (ESC) est régulé de façon intensive par un groupe de facteur clés agissant sur leur pluripotence. Il est néanmoins envisageable que certains déterminants influençant l’auto-renouvellement et la différenciation de ces cellules soient toujours inconnus. Afin de tester cette hypothèse, nous avons généré, en utilisant une méthode par infections virales, une collection de ESC contenant des délétions chromosomales chevauchantes que nous avons baptisée DelES (Deletion in ES cells). Cette librairie contient plus de 1000 clones indépendants dont les régions délétées couvrent environ 25% du génome murin. À l’aide de cette ressource, nous avons conduit un criblage de formation de corps embryoïdes (EB), démontrant que plusieurs clones délétés avaient un phénotype de différenciation anormal. Nos études de complémentation sur un groupe de clones ont par la suite permis l’identification de Rps14 - un gène codant pour une protéine ribosomale (RP) comme étant haploinsuffisant pour la formation de EB. Dans un deuxième temps, l’analyse approfondie des résultats de notre crible a permis d’identifier un groupe de gènes codants pour des RP qui semblent essentiels pour la différenciation des ESC, mais dispensables pour leur auto-renouvellement. De manière intéressante, les phénotypes anormaux de formation en EB les plus marqués sont associés à des délétions de RP qui se retrouvent au site de sortie des ARN messagers (ARNm) du ribosome, soit Rps5, Rps14 et Rps28. Étonnament, alors qu’un débalancement des RP conduit généralement à une réponse de type p53, l’haploinsuffisance de ces trois gènes ne peut être renversée par une simple réduction des niveaux d’expression de ce gène suppresseur de tumeurs. Finalement, nos études de profilage polysomal et de séquençage à haut-débit montrent une signature spécifique de gènes liés au mésoderme chez un clone hétérozygote pour Rps5, suggérant ainsi une explication au phénotype de différenciation p53-indépendant identifié chez ces ESC. Nos travaux rapportent donc la création d’une ressource intéressante de génomique fonctionnelle qui a permis de mettre à jour le rôle essentiel que jouent les RP dans le processus de formation de EB. Nos résultats permettent aussi de documenter une réponse p53-indépendante suite à un débalancement de RP dans un contexte opposant l’auto-renouvellement et la différenciation des ESC. / Stem cells have captured public’s attention in the last years, thanks to their involvement in cancer therapies and also their huge theoretical potential in the regenerative medicine field. In order to translate this new technology to the clinic, a better understanding of their regulatory mechanisms is still needed. It is well established that mouse embryonic stem cell (ESC) fate is highly regulated by core pluripotency factors. However, it is conceivable that novel self-renewal or differentiation regulators are not yet described. To investigate this possibility, we used a viral-based approach to generate a collection of ESC with nested chromosomal deletions called DelES (Deletion in ES cells). This library contains more than a thousand independent ESC clones highly enriched in chromosomal deletions which together cover ~25% of the mouse genome. Using this resource, we conducted an embryoid body (EB) differentiation screen and showed that several clones were having an abnormal EB formation phenotype. Complementation studies later identified Rps14-a ribosomal protein (RP) coding gene- as a novel haploinsufficient gene in EB formation from undifferentiated ESC. Further analyses of our screen results showed a strong bias for a subset of small subunit ribosomal protein genes which are critical for ESC differentiation but not for their self-renewal activity. Interestingly, the most severe differentiation phenotypes were found with ribosomal proteins associated to the ribosome’s mRNA exit site, namely Rps5, Rps14 and Rps28. While RP gene imbalance often leads to a p53 response that can be corrected by p53 suppression, ESC clones with decreased expression of mRNA exit site RP genes were surprisingly insensitive to p53 reduction, but were rescued by BAC or cDNA complementation, thus confirming the causative nature of these genes in the ESC phenotype. Finally, polysomal profiling and RNA-Seq studies showed that Rps5 deleted ESC exhibit an abnormal mesodermal gene signature. Together, our work presents a highly valuable resource for functional genomic studies in ESC and also highlights a novel p53-independent role linked to RP gene imbalance. Our results shed light on the relevance of these subunits for the developmental transition of ESC from a pluripotent to a differentiated state.

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs) together with the small ribosomal subunit to locate the mRNA's translational start and to properly decode the genetic message that it carries. The largest of these factors, eIF3, forms the scaffold for other initiation factors to promote their spatially coordinated placement on the ribosomal surface. It is our long-standing pursuit to map the 40S-binding site of the yeast multisubunit eIF3 and here we present three new mutual interactions between these two macromolecules (i) The C-terminal region of the eIF3c/NIP1 subunit is comprised of the conserved bipartite PCI domain and we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 in vivo. The extreme C-terminus directly interacts with small subunit ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of ASC1 impairs eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this protein fold in protein-RNA interactions. We conclude that the c/NIP1...

Les communautés du coralligène dominées par des gorgonaires ont été sévèrement affectées par des évènements de mortalités massives liés au réchauffement de la Méditerranée. Pour évaluer la contribution des bactéries associées à l’holobionte Paramuricea clavata à son fonctionnement et sa santé, il est apparu primordial de caractériser le compartiment microbien naturel de ce gorgonaire tempéré.Dans ce contexte, l’objectif général de cette thèse était de décrire les interactions existant entre la gorgone rouge P. clavata et ses bactéries associées en Méditerranée nord-occidentale. Les analyses entreprises par des techniques culture-indépendantes basées sur l’analyse des ADN ribosomiques 16S bactériens ont inclus (i) la caractérisation de la variation spatio-temporelle des communautés bactériennes, (ii) la localisation des bactéries dans les tissus de l’hôte, (iii) l’évaluation de la stabilité des associations gorgones-bactéries en conditions de stress et (iv) la détermination de la spécificité d’hôte des bactéries dominantes entre différentes espèces de gorgonaires sympatriques (Eunicella singularis, Eunicella cavolini et Corallium rubrum). Les résultats obtenus ont établi que P. clavata et son microbiote forment un holobionte au sein duquel hôte et bactéries vivent en étroite association, stable dans le temps et l’espace ou en conditions de stress. Les communautés bactériennes associées sont principalement endosymbiotiques et dominées par un ribotype bactérien appartenant à un genre nouveau de la famille des Hahellaceae qui semble présenter une forte spécificité d’hôte. Ces résultats suggèrent un rôle particulier de ce genre bactérien chez les holobiontes gorgonaires. / Coralligenous communities dominated by gorgonian species have been severely affected by diseases and mass mortality events linked to the current warming trends reported for the Mediterranean Sea. The characterization of the natural microbial compartment of this temperate gorgonian species becomes a crucial step in the evaluation of the bacterial contribution to health and functioning of the Paramuricea clavata holobiont.Under these circumstances, the global aim of this PhD work was to describe the interactions existing between the red gorgonian P. clavata and its associated bacteria in the Northwestern Mediterranean basin. The culture-independent analyses based on the bacterial 16S ribosomal DNA included (i) the characterization of spatiotemporal variation of the bacterial communities, (ii) the localization of the bacteria within host tissues, (iii) the evaluation of the stability of gorgonian-bacterial associations under stress conditions and (iv) the determination of the host-specificity of dominant bacteria in different sympatric gorgonian species (Eunicella singularis, Eunicella cavolini and Corallium rubrum).The results of this study highlighted that P. clavata and its microbiota form a holobiont in which host and bacteria live in close association. This association is spatiotemporally stable and maintained under stress conditions. Associated bacterial communities are mostly endosymbiotic and dominated by a bacterial ribotype belonging to a new genus within the Hahellaceae family that seems to be host-specific. These results suggest a particular role of this bacterial genus in the gorgonian holobionts.

a. Do ponto de vista metodológico dois problemas foram enfrentados neste trabalho: i) extração dos RNA\'s e ii) fracionamento das células das glândulas salivares de R. angelae. i) O processo prático elaborado para resolver o primeiro problema se constituiu numa variação da técnica clássica de extração com fenol. Jogando com a presença de detergente e variações de pH e de temperatura, foi possível se chegar a um método que garante a extração e um fracionamento parcial de, praticamente, todo RNA celular. Com extrações a baixa temperatura obtém-se preferencialmente rRNA e tRNA. O RNA refratário a extração com fenol a frio tem propriedades do RNA nuclear, mas o citoplasma, também parece conter classes de RNA somente extraíveis a quente. A dificuldade de extração do RNA nuclear com fenol frio, foi especulativamente interpretada como uma propriedade das RNP\'s que encerram esta classe de RNA. A grande vantagem deste método de extração está no fato de ter permitido isolar, com as extrações a alta temperatura, frações de RNA enriquecidas em produtos de transcrição específicos de determinados períodos do desenvolvimento larval. ii) O fracionamento celular das glândulas salivares não é alcançado pela aplicação das técnicas tradicionais de fracionamento de tecido. Em vista disso foi necessário procurar uma alternativa experimental para êste caso. Combinando um enzima proteolítico com a ação de detergentes não iônicos foi conseguido um método útil para o fracionamento das células das glândulas salivares. Êste método foi aplicado nesta tese para um estudo inicial das RNP\'s e da transferência do rRNA do núcleo para o citoplasma nas células das glândulas salivares. b. Foi possível verificar com êste trabalho que o rRNA é sintetizado inicialmente na forma de um precursor primário de 37s, o qual é processado no interior do núcleo para dar as espécies maduras de rRNA segundo o esquema: (Ver no arquivo) No 3º período do 4º estádio do desenvolvimento larval de R. angelae, a síntese de rRNA é intensa. Neste mesmo período observa-se claramente um nucleolo típico na base do cromossomo X. No período seguinte ocorre uma redução na síntese das formas maduras de rRNA. Esta queda de síntese é desencadeada por um bloqueio ao nível do processamento dos precursores nucleares de rRNA citoplasmático. Estudos citológicos tem revelado que neste período o nucleolo sofre uma aparente desagregação. c. Os resultados de incorporação de uridina-H3 mostraram que o 3º e 4º períodos tem uma velocidade de síntese de RNA muito à do 2º período. No 4º período quando a síntese de rRNA é inibida aumenta de importância a síntese de outras classes de RNA. Aparentemente é muito intensificada a síntese de RNA nuclear. Êstes resultados são interpretados como consequência da ativação de novos genes nesta época do desenvolvimento larval. Êstes fatos não são inesperados pois este estágio se caracteriza pelo desenvolvimento de \"puffs\" gigantes, os quais são específicos do tecido e do período de desenvolvimento. O 5º período parece representar uma fase de transição entre uma época de ativa síntese de RNA e outra onde a atividade transcritiva sofre uma redução drástica, provavelmente devido à histólise do tecido que vai ocorrer logo em seguida. d. Os dados apresentados nesta tese são considerados sob todos os aspectos significativos. Com isto faz-se uma tentativa de discussão crítica, no sentido de avaliar a importância do sistema glândulas salivares de R. angelae, como modelo de estudo para a genética molecular dos organismos superiores. / Not available

O efeito de alterações climáticas sobre os micro-organismos ainda é incerto, pois pouco se conhece sobre as espécies que habitam regiões extremas como o gelo, solo antártico, e o solo permanentemente congelado (permafrost). O permafrost tem como característica a preservação de material biológico por milhões de anos, servindo como fonte para estudos de evolução e biogeografia de micro-organismos. O objetivo deste trabalho foi estudar a diversidade microbiana em amostras de gelo, solo exposto por recuo de geleira e permafrost polares, e a diversidade funcional do gene alcano monoxigenase (alk). Métodos independentes de cultivo baseados no gene 16S rRNA foram utilizados, como DGGE, clonagem e pirossequenciamento. As geleiras da Ilha Rei George (Península Antártica) e do Pólo Sul Geográfico possuem cerca de 3.104 cél./mL e são compostas por micro-organismos diferentes, com predominância dos Filos Proteobacteria, Actinobacteria, Firmicutes e Cyanobacteria, muitos dos quais já descritos em outros ambientes criogênicos. O solo em frente à geleira Baranowski apresenta uma estrutura de comunidade diferente do gelo. O solo exposto por recuo de geleira apresenta uma sucessão ecológica, com predominância de heterotróficas durante todo o processo. Fixadores de nitrogênio no solo foram compostos por cianobactérias no início, e por Rhodopseudomonas e Rhodobacter no final da sucessão. Estes resultados foram melhor observados com o pirossequenciamento. As mudanças observadas podem estar relacionadas ao aumento de K, Mg+, NH4+, NO3- e/ou CO2 detectados após 15-20 anos de exposição do solo. A comunidade de permafrosts varia com o local e a idade de congelamento (de 5.000 a 8 milhões de anos). O gene alkM foi detectado em permafrosts do Ártico com 3 milhões de anos, e o gene alkB em amostras do Ártico com 15.000 e 120.000 anos, e em solos modernos da Antártica. Alguns clones indicam que podem representar novos genes para alcano monoxigenases. As contribuições deste projeto abrangem os objetivos do Ano Polar Internacional (IPY 2007-2009), sobretudo na avaliação da ecologia microbiana da Antártica. / The effect of climate changes on microorganisms is still unclear, because little is known about the species that inhabit the extreme regions as the glacial ice, antarctic soils and the permanently frozen soil (permafrost). The permafrost is able to preserve the sedimented biological materials by thousands or even millions of years, being an important source for microbiological studies. The objective was to study the microbial diversity in cryogenic samples: glacial ice, soil exposed by glacial retreat and polar permafrosts, as well as to study the functional diversity of alkane monooxygenase genes (alk) in the permafrost. Cultivationindependent methods based on the 16S rRNA gene were used, as DGGE, clone library and 454 Pyrosequencing. Analysis of the King George Island (Antarctic Peninsula) glaciers and the South Pole ice revealed about 3x104 cells/mL each, and different micro-organisms were detected, predominantly members from Proteobacteria, Actinobacteria, Firmicutes and Cyanobacteria, many of which already described in other cryogenic environments. The soil in front of the Baranowski Glacier has a different community structure compared with the ice. Soils exposed by glacier retreat revealed an ecological succession, and heterotrophic bacteria occurred all through the process. Nitrogen-fixing populations were composed by cyanobacteria at the early stages, and shifted to Rhodopseudomonas and Rhodobacter in the older soils. The observed changes may be related to an increase of K, Mg+, NH4 +, NO3- and/or CO2, detected after 15-20 years of soil exposure. The community of permafrosts varies by location and age (5,000 - 8 millions of years). The alkM gene was detected in old Arctic permafrosts (3 millions of years), while alkB genes were found on Arctic samples from 15,000 to 120,000 years, and in Antarctic modern soils. Some of these clones may represent new alk genes. The contributions of this project covers the goals of the International Polar Year (IPY 2007-2009), particularly in assessing the microbial ecology of Antarctica.

As florações de cianobactérias em reservatórios de águas utilizadas para consumo humano são freqüentes hoje em dia e normalmente são atribuídas à crescente eutrofização destas águas. Em anos recentes o aparecimento de linhagens de cianobactérias produtoras de toxinas tem preocupado bastante os responsáveis pelo monitoramento da qualidade das águas, uma vez que estas toxinas representam risco para a saúde pública. A detecção precoce da presença de linhagens tóxicas nesses reservatórios é essencial para que ações corretivas de controle das florações tenham sucesso. No presente trabalho, a diversidade de cianobactérias presentes em alguns locais das represas Billings e Guarapiranga foi avaliada utilizando a técnica de DGGE (eletroforese em gel de poliacrilamida com gradiente desnaturante) e/ou construções de mini-bibliotecas de amplicons do gene de rRNA 16S e da região do espaço intergênico da ficocianina (PC-IGS). A DGGE, utilizando os iniciadores CYA359F/CYA781R específicos para o gene de rRNA 16S de cianobactérias, mostraram que estes organismos estavam presentes nas onze amostras de água analisadas. Microcystis aeruginosa (94-97% de identidades), Geitlerinema (89% de identidade) e Synechococcus (89% de identidade) puderam ser identificadas. A mini-biblioteca construída com os amplicons de rRNA 16S obtidos usando os iniciadores 27F1/1494Rc produziu seqüências de outro grupo de bactéria (Actinobacteria), indicando a inespecificidade desses iniciadores. Entretanto, na mini-biblioteca construída com os amplicons de PC-IGS obtidos usando os iniciadores PCβF/PCαR, somente seqüências de cianobactérias foram geradas. Nessa mini-biblioteca foram identificadas várias linhagens de Microcystis aeruginosa (98-100% de identidades) e também de Anabaena (89% de identidade). Esses dois gêneros de cianobactérias conhecidos por produzirem microcistinas, foram detectados na amostra de água que continha alta concentração desta toxina (23,49 μg/L). Os oligonucleotídeos iniciadores OMETF/OMETR foram desenhados para amplificar uma região pequena e variável do domínio da N-metiltransferase do gene mcyA e foram testados em treze espécies de cianobactérias isoladas das represas Billings e Guarapiranga. Seqüências geradas por esses iniciadores foram isoladas, clonadas e sequenciadas com sucesso em duas espécies controle (M. aeruginosa NPJB1 e M. panniformis SPC702) e em dois isolados das represas (M. aeruginosa SPC777 e M. protocystis SPC697). A amplificação dessa região do mcyA a partir dos DNAs dos isolados de cianobactérias permitiu identificar linhagens do gênero Microcystis potencialmente produtoras da toxina microcistina. Essa técnica uma vez bem estabelecida para organismos isolados, poderá proporcionar base suficiente para uma melhor detecção de comunidades aquáticas de cianobactérias potencialmente produtoras de microcistinas em ambientes naturais. / Cyanobacterial blooms in water reservoirs used for human consumption are frequent nowadays and are usually attributed to the increasing water eutrophization. In recent years, the appearance of toxin-producing cyanobacterial strains has concerned managers of water quality since these toxins represent a public health risk. Early detection of the presence of toxic strains in these reservoirs is essential for the success of bloom control corrective actions. In the present work, cyanobacterial diversity was evaluated in several sites of Billings and Guarapiranga reservoirs using DGGE (denaturing gradient gel eletrophoresis) and/or mini-library constructions of both 16S rRNA gene and phycocyanin intergenic spacer (PC-IGS) region amplicons. The DGGE, using CYA359F/CYA781R primers, specific for cyanobacterial 16S rRNA gene, showed the presence of these organisms in the eleven water samples analyzed. Microcystis aeruginosa (identities of 94-97%), Geitlerinema (identity of 89%) and Synechococcus (identity of 89%) were identified. The mini-library constructed with 16S rRNA amplicons obtained using 27F1/1494Rc primers produced sequences of another group of bacteria (Actinobacteria), indicating the unespecificity of these primers. However, the mini-library constructed with PC-IGS amplicons obtained using PCβF/PCαR primers, generated cyanobacterial sequences only. In this mini-library several Microcystis aeruginosa strains (identities of 98-100%) and Anabaena (identity of 89%) were identified. These two cyanobacterial genera known to produce microcystins were detected in a water sample containing a high concentration of this toxin (23.49 g/L). The OMETF/OMETR primers were designed to amplify a small and variable region of the N-methyltransferase domain of mcyA gene and were tested in thirteen cyanobacterial strains isolated from Billings and Guarapiranga reservoirs. Sequences generated with these primers were successfully isolated, cloned and sequenced in two control species (M. aeruginosa NPJB1 and M. panniformis SPC702) and in two isolates from these reservoirs (M. aeruginosa SPC777 and M. protocystis SPC697). The amplification of this region of mcyA using cultured cyanobacteria DNAs allowed the identification of Microcystis strains with the potential to produce microcystin toxin. This technique, after it is well established for cultured organisms, will provide a basis for better detection of potential microcystin-producing cyanobacterial aquatic communities in natural environments.

Neste trabalho foram caracterizadas molecularmente e inferidas as relações filogenéticas de 14 isolados brasileiros de Acanthamoeba, provenientes de casos de ceratite, e 8 isolados da ATCC (4 de ceratite e 4 ambientais). Foram utilizados inicialmente os métodos de RAPD, RFLP de DNA genômico total e RFLP do SSU rDNA. Apesar de revelar a alta variabilidade genética em Acanthamoeba, estes métodos permitiram estabelecer grupos bem definidos de isolados mais similares geneticamente. O seqüenciamento do SSU rDNA permitiu a inferência da filogenia entre os isolados utilizados nesse estudo em relação àqueles presentes na literatura, que estão distribuídos em doze tipos de seqüência deste gene. Dentre os 17 isolados de ceratite presentes em nosso estudo, 16 apresentaram SSU rDNA tipo T4 (anteriormente já fortemente correlacionado à ceratite) e um deles constitui um novo tipo de seqüência. Dois dos 4 isolados ATCC (ambientais) cujas seqüências ainda não haviam sido determinadas também apresentaram novos tipos de SSU rDNA, enquanto outros 2 apresentaram o tipo T4. / In this work we performed the molecular phylogeny and characterization of 22 Acanthamoeba isolates, 14 Brazilian keratitis isolates and 8 from ATCC, 4 keratitis and 4 environmental isolates. In spite of the extensive genetic variability disclosed by RAPD, total genomic DNA RFLP and SSU rDNA RFLP techniques, these methods enabled us to group some isolates in well defined clusters of genetically more related organisms. Sequencing of SSU rDNA allowed inference of the phylogeny of our isolates with those present in the literature, which are distributed through 12 sequence types of this gene. Among the 17 keratitis isolates of our study, 16 presented SSU rDNA of type T4 (previously found to be strongly correlated to keratitis), and one was assigned to a new sequence type. Of the 4 isolates from ATCC whose sequences were previously undetermined, the two environmental isolates also constituted new sequence types, while the two keratitis isolates were assigned to type T4.