The purpose of this study was to examine the direct and indirect effects of ultraviolet-B (UV-B) radiation on freshwater protists. Laboratory experiments were conducted in order to determine the importance of photoenzymatic repair (PER) of UVBinduced DNA damage in the heterotrophic nanoflagellate Paraphysomonas sp. Investigations into the combined effects of UV-B and chromophoric dissolved organic matter (CDOM) were conducted in laboratory experiments in which protist cultures were exposed to UV-B radiation in the presence and absence of water amended to a higher CDOM concentration in order to determine the ability of CDOM to act as a UV-B filter and as a potential nutrient source. Field experiments examined the responses of natural communities of protists and bacteria to ultraviolet radiation (UVR) in the presence of high and low concentrations of CDOM. Ultraviolet-B radiation (UV-B; 280 – 320 nm) negatively affects many aquatic organisms, including heterotrophic flagellates, by directly damaging DNA. The quantity of UV-B reaching the surface of a lake varies with atmospheric chemistry, including stratospheric ozone, and the presence of large holes (Alldredge 1977) in this ozone shield during the last decade resulted in historically high UV-B levels. In aquatic systems, the nature of the damage to organisms depends on the intensity and duration of solar radiation, plus its attenuation in the water column. The amount of UV-B damage is highly dependent on the concentration of CDOM in the water column because CDOM strongly absorbs UV-B radiation. This protective role of CDOM is likely to be reduced in areas where warmer, drier climate decreases watershed runoff, which ultimately results in acidification and increased CDOM photodegradation. However, CDOM also may also act as an organic carbon source for bacteria and stimulate growth of the microbial food web, including bacterivores like heterotrophic flagellates. The effect of UV and CDOM interactions on the microbial food web is not well understood, but climate-related increase in CDOM in an oligotrophic lake could increase the heterotrophic microbial food web impact by reducing UV-B damage and increasing available resources. Since aquatic organisms, including protists, are differentially susceptible to UV-B radiation, climate change effects on CDOM and UV-B attenuation are likely to alter the ecology and community structure of aquatic systems. This thesis describes investigations into the direct and indirect effects of UV-B radiation on freshwater protozoa. The role of PER of direct UV-B induced DNA damage was examined in laboratory experiments that compared the survival and population growth of the heterotrophic nanoflagellate Paraphysomonas sp. at two environmentally relevant temperatures. The results from these experiments demonstrated the reliance of Paraphysomonas sp. on PER, with 100% mortality in the absence of the photorepair radiation that activates photoenzymatic repair enzymes. The ability of Paraphysomonas sp. to recover from exposure to UV-B radiation declined in flagellates adapted to 15°C relative to the same exposures at 20°C. Experiments examining the direct and indirect effects of UV-B radiation and CDOM on freshwater protists conducted in the laboratory and in an oligotrophic lake in the Pocono Mountains showed that potential DNA damage resulting from UV exposure is reduced and microbial growth may be enhanced with an increase in CDOM concentration. / Biology

La diversité des protistes marins planctoniques, après avoir été historiquement étudiée sur des critères morphologiques, est depuis récemment sujette à une intense recherche à l’aide d’approches moléculaires. Notamment, les études basées sur l’amplification directe de marqueurs moléculaires à partir d’ADN environnemental ont révélées une exceptionnelle diversité. L’objectif central de ce travail est d’améliorer notre compréhension sur le lien existant entre la connaissance classique des eucaryotes unicellulaires et leur diversité estimée à partir des données moléculaires, en particulier pour une meilleure interprétation des processus évolutifs et écologiques. Pour cela, nous avons utilisé comme système modèle l’ordre des ciliés tintinnidés (Tintinnida) qui constituent un groupe riche en espèces, aisément identifiables au microscope grâce à leur coquille externe (lorica) et communément rencontrés dans l’ensemble des eaux marines et lacustres du monde. Un suivi approfondi sur deux ans des tintinnidés de la Baie de Villefranche-sur-Mer (Mer Méditerrannée, France), couplant des analyses moléculaires de la diversité à partir de cellules individuelles et à partir d’ADN environnemental, a permis de caractériser la composition de ces communautés et leur dynamique temporelle aux échelles macro- et micro-évolutives. La première partie de ce travail a été destinée à la réalisation d’une phylogénie moléculaire de référence pour les tintinnidés en incorporant les séquences de 62 individus de morphologies diverses pour lesquelles des données moléculaires n'étaient pas disponibles. Nous avons amplifié et séquencé les gènes codant pour les ARN ribosomiques (ARNr 18S, 5.8S et 28S) et les espaces intergéniques correspondants (ITS1 et ITS2). La classification taxonomique a été réévaluée d’après les données moléculaires. Dans un deuxième temps, nous avons testé l’efficacité des approches moléculaires conventionnelles (amplification, clonage et séquençage Sanger du gène de l'ARNr 18S) et plus récentes (amplification et pyroséquençage de régions de l'ARNr 18S et de l’ITS), pour décrire la composition des communautés des tintinnidés dans des échantillons environnementaux en les comparant avec des estimations de la diversité par observation morphologique sur les mêmes échantillons. Si il existe de légères incongruences entre les approches et/ou les différents marqueurs employés, les approches cultureindépendantes s’avèrent efficaces pour décrire la diversité morphologique. En revanche, afin de ne pas surévaluer artificiellement le nombre d’espèces estimées à partir des données de pyroséquençage, il faut que des méthodes de débruitage et de regroupement en unités taxonomiques opérationnelles (UTOs) contraignantes soient appliquées. La troisième partie de ce travail a été dédiée au suivi temporel des communautés de tintinnidés à différentes profondeurs dans la baie de Villefranche, basé sur le clonage et le séquençage du gène de l'ARNr 18S et des régions ITS. Il apparaît des différences de distribution au cours de l’année à une même profondeur, en particulier en termes d’abondance de séquences pour une UTO donnée. Malgré un cadre phylogénétique solide et assez enrichi, l’approche moléculaire révèle des séquences éloignées des espèces déjà séquencées. La découverte de ces clades environnementaux souligne potentiellement l’importance écologique d’espèces encore mal connues. Enfin, le séquençage direct du gène de l'ARNr 18S et de l'ITS2 à partir des cellules individuelles de l'espèce <Undella claparedei> a offert l’opportunité d’une étude populationnelle sur une période de deux ans. La diversité intra-spécifique mesurée met à jour des phénomènes d’hybridation entre variantes génétiques. Une structuration génétique temporelle a également été observée pour le gène de l'ARNr 18S. Les implications de ces différentes recherches sont discutées dans le cadre de l’étude de la diversité et de l’écologie des tintinnidés, et plus largement, des protistes marins. / The marine protistan diversity has been historically studied based on morphological characterization but has recently been the object of intense research using molecular approaches. Studies based on the amplification of molecular markers from environmental DNA revealed an outstanding diversity, partly new and uncharacterized. However, the actual extent of this diversity remains poorly known and highly debated. The main goal of this work was to improve our knowledge on protistan diversity to bridge the gap between molecular environmental surveys and classical protistology to better understand the ecology and evolution of unicellular eukaryotes. For this purpose, we used as a model the species-rich order of the tintinnid ciliates (Tintinnida, Ciliophora), which are easily distinguishable because of their secreted shell, the lorica, and commonly found in marine waters all around the globe. A two-year monitoring of the tintinnid populations in the Bay of Villefranche-sur- Mer (Mediterranean Sea, France), combining molecular analyses of the diversity based on single-cells and environmental DNA, gave us the opportunity to describe the tintinnid community composition and its temporal dynamics. In the first part of this work, we constructed a reference molecular phylogeny for the tintinnids including new sequences from 62 specimens of diverse morphologies, for which we amplified and sequenced the ribosomal coding genes (18S, 5.8S and 28S rRNA) and the corresponding intergenic spacers (ITS1 and ITS2). The taxonomic classification of the Tintinnida has been revised based on these molecular data. In the second part, in order to assess the accuracy of molecular-based approaches to describe the natural species assemblages of tintinnids, we compared the morphology-based diversity estimates with those derived from classical (amplification, cloning and Sanger sequencing of the 18S rRNA gene) and more recent (direct pyrosequencing of amplified 18S rRNA genes and ITS regions) molecular approaches. Even if there are still some disagreements between the different methods and/or molecular markers, the culture-independent approaches were efficient to describe the morphological diversity. However, a careful and rigorous analysis of pyrosequencing datasets, including sequence denoising and stringent sequence clustering in Operational Taxonomic Units (OTUs) with well-adjusted parameters, is necessary to avoid overestimating the species number. The third part of the thesis is dedicated to the study of the genetic diversity of tintinnids over a one-year survey in the Bay of Villefranche at five different depths by combining community fingerprinting analysis using denaturing gradient gel electrophoresis (DGGE) with direct PCR amplification and sequencing of 18S, 5.8S, and 28S rRNA genes and ITS regions. These analyses revealed marked seasonal changes, in particular in the sequence abundances of certain OTUs. In addition, despite an enriched phylogenetic reference sequence dataset for the tintinnids, we retrieved two abundant phylotypes without any closely related known species, highlighting the possible ecological relevance of unidentified species. Finally, we studied the intra-specific diversity of populations of the species <Undella claparedei> based on 18S rDNA and ITS direct sequencing of single-cells collected over a period of two years. We detected signals of hybridization and sexual recombination among different genetic variants. We also found genetic structuring of the 18S rRNA gene data differentiating populations collected at different times. The implications of all these results are discussed in the framework of the diversity and ecology of tintinnid ciliates and, more generally, of marine protists

Trichomonas vaginalis is a causative agent of the most common non-viral sexually transmitted disease with approximately 275 mil new cases annually. Virulence of this parasitic depends on at least four factors: cell shape transformation, cytoadherence, secretion of cysteine proteases, and presence of endosymbionts. Over the past decades, extracellular vesicles appeared being another important player in the host-parasite interaction. It was discovered that T. vaginalis is one of the protists that can shed the extracellular vesicles such as exosomes and ectosomes. These vesicles are possibly involved in host-parasite communications, however limited information is available about their function. To investigate a possible role of exosomes in T. vaginalis virulence, we first selected suitable strain, which is free of endosymbionts (TV 17-2MI). Next we prepared six clones of TV 17-2MI strain to test whether the strain is homogenous concerning the virulence, or there are differences in virulence among individual cells. Mouse intraperitoneal virulence tests revealed that the clones displayed significant differences in virulence level, particularly in abscess formation and mortality of infected animals. Thus, for the first time we demonstrated heterogeneity of cells derived from a single T. vaginalis strain...

Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...

Mes travaux de recherche ont eu pour but d’analyser la diversité des microorganismes des trois domaines du vivant présents dans des biofilms phototrophes exposés à l’air, se développant sur des substrats minéraux divers, afin d’essayer, d’une part, de répondre à des questions de diversité et de biogéographie et, d’autre part, d’étudier le processus de colonisation par le biais d’expériences d’exposition contrôlées.J’ai ainsi caractérisé, essentiellement par des approches moléculaires basées sur l'analyse des banques des gènes d'ARNr de la petite sous-unité (SSU rDNAs) et sur des analyses d'empreintes communautaires, la diversité microbienne (procaryote et eucaryote) formant des biofilms matures (exposés depuis plusieurs années) dans plusieurs sites géographiques en Irlande du Nord, en France et en Ukraine, dans la région de Chernobyl. Dans ces biofilms soumis à forte pression sélective, nous avons mis en évidence beaucoup de microorganismes hétérotrophes et phototrophes, mais avec une diversité relativement restreinte en comparaison à d’autres milieux comme les sols ou les systèmes aquatiques. Les archées étaient absentes. Les conditions environnementales auxquelles ce type de biofilm est constamment exposé comme l’irradiation, la dessiccation et la limitation des nutriments sélectionnent des microorganismes qui développent des stratégies pour s’adapter comme, entre autres, la production de pigments. Ce sont des microorganismes fréquemment retrouvés dans des milieux désertiques extrêmes et résistants aussi aux radiations ionisantes qui ont ainsi été identifiés, notamment des Deinococcales et des Actinobacteria, ou encore des champignons ascomycètes (Ascomycota). Parmi les organismes phototrophes, nous avons dénombré des Cyanobacteria, des algues vertes (Chlorophyta) et des Streptophyta. Nous avons mis en évidence que les facteurs environnementaux influencent la composition des biofilms. Toutefois, tandis que la composition de la communauté bactérienne est fortement dépendante de la nature du substrat ou elle se développe, la composition des communautés microbiennes eucaryotes dépend de la distance géographique. Nous avons également mené des expériences de colonisation en exposant un même substrat minéral dans trois sites géographiques en Irlande du Nord et en France. L'analyse de la diversité microbienne lors du processus de colonisation a révélé des changements importants dans la composition des communautés, que ce soit pour les procaryotes ou pour les eucaryotes avec, cependant, des comportements différents de ces deux groupes de microorganismes. Dans le cas des bactéries, on observe une transition des Gammaproteobacteria, qui dominent les temps 0-6 mois et qui correspondent vraisemblablement aux cellules inactives en dispersion, vers des Betaproteobacteria, Bacteroidetes, Alphaproteobacteria et Actinobacteria dans des phases successives de formation du biofilm. Par contre, dès leur détection sur le substrat minéral, les eucaryotes sont massivement dominés par des champignons ascomycètes et basidiomycètes, des algues vertes ainsi que d'autres composantes minoritaires comme des ciliés, étant détectées dans des stades plus tardifs. Nos résultats montrent que les organismes hétérotrophes sont pionniers dans la formation de ces biofilms, ce qui permet d'émettre l'hypothèse qu'ils facilitent l'installation des cyanobactéries et surtout des algues vertes. Ils montrent aussi que le processus d'assemblage des communautés bactériennes dépend du temps de colonisation, alors que le site géographique détermine celui des microorganismes eucaryotes. Ces différences majeures de comportement pourraient être expliquées par des modes de vie différents entre les organismes de ces deux grands groupes. / The major objective of my PhD work was the analysis of the diversity of microorganisms from the three domains of life associated with phototrophic biofilms developing on different mineral substrates exposed outdoors. These studies aimed at answering questions about microbial diversity and biogeography and also at studying the colonization process through controlled exposure experiments. I have thus characterized, essentially by molecular methods based on small subunit (SSU) rRNA gene libraries and fingerprinting analyses the diversity of prokaryote and eukaryote microorganisms forming mature biofilms (exposed for several years) in various geographic sites in Northern Ireland, France and Ukraine, in the Chernobyl area. In these biofilms, subjected to strong selective pressure, we found many heterotrophic and phototrophic microorganisms, but their diversity was limited when compared to that of other environments such as soils or aquatic systems. Archaea were absent from all biofilms. The environmental conditions to which these biofilms are constantly exposed, such as irradiation, desiccation and nutrient limitation select for organisms that develop particular adaptive strategies including, among others, pigment production. The microorganisms identified in these biofilms are also frequently found in extreme, desert environments and are known for their resistance also to ionizing radiation, such as Deinococcales and Actinobacteria or ascomycete fungi (Ascomycota). Among phototrophic lineages, we identified Cyanobacteria, Chlorophyta (green algae) and sometimes Streptophyta. We showed that environmental parameters influenced biofilm microbial communities. However, whereas the bacterial community composition depends on the nature of the substrate, the microbial eukaryotic community composition depends on the geographic distance. We also carried out colonization experiences exposing outdoors the same mineral substrate in three different sites in Northern Ireland and France. The analysis of microbial diversity along the colonization process revealed important changes in community composition both for prokaryotes and eukaryotes, although the behavior of the two groups was different. In the case of bacteria, we observed a transition from Gammaproteobacteria, which dominated the initial 0-6 months and which likely corresponded to inactive dispersive cells, towards Betaproteobacteria, Bacteroidetes, Alphaproteobacteria and Actinobacteria in successive steps of biofilm formation. By contrast, since their detection on mineral substrates, eukaryotes were massively dominated by ascomycete and basidiomycete fungi, green algae and other minor components such as ciliates were detected in later stages of biofilm formation. Our results show that heterotrophic organisms are pioneers in the formation of these biofilms, leading to the hypothesis that they facilitate the settlement of Cyanobacteria and, especially, of green algae. They also show that the process of bacteria community assembly depends on colonization time whereas the geographic site determines that of eukaryotic microorganisms. These major differences might be explained by different lifestyles between organisms of the two groups

Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...