Les membres des familles Mimiviridae et Marseilleviridae, qui infectent et se répliquent dans Acanthamoeba spp. et d’autres protistes phagocytaires, ont été découverts au cours de la dernière décennie et rattachés à un groupe monophylétique de virus nommés les grands virus à ADN nucléocytoplasmiques (NCLDVs), qui infectent un large éventail d’eukaryotes y compris différents organismes unicellulaires. Récemment, il a été proposé de reclasser les NCLDVs dans un nouvel ordre viral nommé les Megavirales. Plusieurs dizaines de virus géants des amibes ont été isolés, mais le génome de peu d’entre eux a été étudié de façon approfondie. Nous avons étudié les génomes de ces virus géants d'amibe afin d’acquérir une meilleure compréhension de leur répertoire de gènes et leur importance évolutionnaire. L'analyse phylogénétique des virus géants d'amibe distingue clairement trois lignées, nommées A, B et C. Nous avons étudié en détail le génome de Acanthamoeba polyphaga moumouvirus, le membre fondateur de la lignée B et avons déchiffré son contenu en gènes et sa relation évolutive avec d'autres organismes. Nous avons également étudié les génomes de Terra1 virus et Terra2 virus, qui appartiennent respectivement aux lignées C et A, et ont été isolés à partir d'échantillons de sol alors que les mimivirus décrits aupravant ont été isolés à partir d'eau douce ou de mer. En outre, nous avons décrit le génome du virus Courdo11, qui appartient à la lignée C, et est étroitement lié au premier Mimivirus isolé d'un humain, qui présentait une pneumonie inexpliquée. / The members of families Mimiviridae and Marseilleviridae, which infect and replicate in Acanthamoeba spp. and other phagocytic protists, were discovered during the past decade and linked to a monophyletic group of viruses named the Nucleocytoplasmic Large DNA viruses (NCLDVs), which infect a broad variety of eukaryotes including diverse unicellular organisms. Recently, it has been proposed to reclassify the NCLDVs into a new viral order named the Megavirales. Several dozens of giant viruses of amoeba have been isolated but the genome of very few has been extensively studied. We studied the genomes of these giant viruses of amoeba to gain a better understanding of their gene repertoire and evolutionary importance. The phylogenetic analysis of giant viruses of amoeba clearly distinguished three lineages, named lineages A, B and C. We studied in detail the genome of Acanthamoeba polyphaga moumouvirus, the leader member of lineage B to decipher its gene content and its evolutionary relationship with other organisms. We further studied the genomes of Terra1 virus and Terra2 virus, which belong to lineages C and A, respectively, and were isolated from soil samples whereas previously described mimiviruses of amoeba were isolated from fresh or marine water. Furthermore, we described the genome of Courdo11 virus, which belongs to lineage C, and is closely related to the first mimivirus isolated from a human, who exhibited unexplained pneumonia.

4 Abstract The main goal of my thesis was to create our own proposals for laboratory work in biology of unicellular organisms (Protista). I designed the laboratory tasks, so that they can be mastered by the pupils of sixth year of primary school. Creating the proposals for laboratory exercises was preceded by the work in the laboratory at Charles University in Prague, Faculty of Education, the Department of Biology and Environmental Studies. In the laboratory, I verified the methods of cultivation of selected representatives of ciliates (Paramecium caudatum), amoebae (Amoeba proteus) and euglenids (Euglena gracilis) and selected methods of dyeing their different organels. Key words: protists, Paramecium caudatum, Amoeba proteus, Euglena gracilis, cultivation, dying

Les amibes sont des protozoaires unicellulaires ubiquitaires dans l'environnement qui se nourrissent des microorganismes qui les entourent. Cependant plusieurs études ont déjà montré que certains de ces microorganismes (bactéries et virus) sont capables d'entrer dans les amibes et de s'y multiplier au lieu d'y être digérés. Cette capacité et l'analogie entre les amibes et les macrophages fait de ces microorganismes de potentiels pathogènes humains. L'utilisation de la technique de coculture sur amibes a donc été développée afin de permettre l'isolement de ces microorganismes. En 2001 elle a permis l'isolement d'un petit coque Gram positif qui sera identifié en 2003 comme étant le premier virus géant isolé : Acanthamoeba polyphaga Mimivirus, pour « mimiking microbe » en référence à sa coloration positive au Gram. La découverte de ce virus associé aux amibes et qualifié de « géant » il y a moins d'une décennie a bouleversé les définitions même de virus. Une taille exceptionnelle de 500 nm, un génome qui dépasse 1 Mb, une particule comportant des ADN et des ARN, autant de caractéristiques originales qui l'ont rendu exceptionnel. Depuis, d'autres virus géants ont été isolés, notamment Marseillevirus, et il est probable que la famille s'étende rapidement maintenant que l'on sait que leur isolement était impossible à cause de leur caractère non filtrable. Des études en métagénomique dans l'environnement ont suggéré une grande ubiquité de ces virus dans l'environnement, notamment hydrique. / Free living amoebas are ubiquitous in the environment and feed on microorganisms. However, several studies have already shown that some of these microorganisms (bacteria and virus) are able to enter the amoeba and multiply within the cell. This capacity and the analogy between amoebas and macrophages allow to think that these microorganisms are potentially human pathogens. The use of the amoeba coculture method was thus developed to allow the isolation of these microorganisms. In 2001 it allow the isolation of a small Gram positive coccus which will be characterized in 2003 as the first isolated giant virus : Acanthamoeba polyphaga Mimivirus. The discovery of this virus less than a decade ago challenged the definition of viruses. An exceptional size of 500 nm, a genome which exceeds 1 Mb, a particle containing DNA and ARN, so many original characteristics which made it exceptional. Since, other giant viruses were isolated, in particular Marseillevirus, and it's likely that the family quickly extends now that we know that their isolation was impossible because of their not filterable character. Environmental metagenomic studies suggested a big ubiquity, particularly into hydric environment. Furthermore the description of virophages, small viruses capable of infecting Mimivirus and behaving towards him as a bacteriophage, contribute to debate on the nature of viruses as well as on their place in the evolution.

Le génome de Mycobacterium tuberculosis, l’agent causal de la tuberculose, code pour une protéine ayant la capacité de se fixer sur la cellulose (Rv1987), une cellulase potentielle (Rv1090), et une cellulase pleinement active (Rv0062). Cette observation est surprenante, car la cellulose est un composant majeur des parois des cellules végétales, tandis que M. tuberculosis est un pathogène humain sans contact connu avec des plantes. Nous avons émis l’hypothèse que ces protéines pourraient jouer un rôle dans les interactions entre les mycobactéries du complexe M. tuberculosis avec les kystes d’amibes libres, dont la paroi contient également de la cellulose. Dans notre travail de thèse, nous avons cherché par une analyse in silico la présence de ces trois gènes chez toutes les bactéries ayant un génome complètement séquencé présentes dans la base de données CAZy (accessible en ligne à l’adresse www.cazy.org). Cette étude a montré que seulement 2,5% des bactéries codent pour les trois gènes simultanément. Parmi ces bacteries, nous avons ensuite confirmé expérimentalement par PCR et séquençage la présence des gènes Rv0062, Rv1090 et Rv1987 chez les mycobactéries du complexe M. tuberculosis. Nous avons ensuite vérifié la transcription de ces trois gènes chez la souche de référence M. tuberculosis H37Rv, puis produit dans Escherichia coli des protéines de fusion Rv1090 et Rv1987 et montré qu'elles étaient capables d'hydrolyser la cellulose (Rv1090) et de s’y fixer (Rv1987). De plus, nous avons mis en place un model expérimental d’interaction entre les mycobactéries du complexe M. tuberculosis et les amibes libres dans le but de comprendre le rôle des gènes Rv0062, Rv1090 et Rv1987. Dans un premier temps nous avons montré que M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii ainsi que Mycobacterium avium utilisé ici comme un controle positif étaient capables de survivre dans le cytoplasme des amibes libres telles que Acanthamoeba polyphaga. Ensuite, nous avons montré que M. tuberculosis et M. bovis mais pas M. canettii étaient capables de survivre à l’intérieur des kystes d’amibes. Enfin nous avons montré que M. tuberculosis, M. bovis et M. canettii étaient capables de survivre dans le sol pendant au moins 6 mois. Les données établies dans cette thèse soutiennent le rôle des cellulases dans la survie environnementale des mycobactéries du complexe M. tuberculosis, et ouvrent la voie à l’étude de cette phase méconnue dans le cycle de ces organismes / The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a protein with the ability to bind to cellulose (Rv1987), one potential cellulase (Rv1090), and one fully active cellulase (Rv0062). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. We hypothesized that these genes could play a role in the interactions between M. tuberculosis complex organisms and amoebal cysts, whose wall contains cellulose.In our thesis work, we have searched by in silico analysis for the presence of these three genes in all bacteria with complete sequenced genomes present in the CAZy database (available online at www.cazy. org). This study showed that only 2.5% of bacteria encode the three genes simultaneously. Among these bacteria we have confirmed experimentally by PCR and sequencing the presence of Rv0062, Rv1090 and Rv1987 in the M. tuberculosis complex organisms. We have checked the transcript of the three genes in the reference strain M. tuberculosis H37Rv and we subsequently produced Rv1090 and Rv1987 fusion proteins in Escherichia coli and demonstrated that they were indeed able to hydrolyze (Rv1090) and to bind (Rv1987) cellulose. In addition, we have developed an experimental model of interaction between M. tuberculosis organisms and the free-living amoebae in order to understand the role of Rv0062, Rv1090 and Rv1987 genes. Initially we have shown that M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii and Mycobacterium avium used here as a positive control were able to survive in the cytoplasm of the free-living amoeba such as Acanthamoeba polyphaga. We have further shown that M. tuberculosis and M. bovis but not M. canettii were able to survive within the amoebal cysts. Finally we have shown that M. tuberculosis, M. bovis and M. canettii were able to survive in soil for at least 6 months. The data obtained in this thesis support the role of cellulase in the survival of M. tuberculosis complex organisms in the environment and pave the way for the study of this unknown phase in the cycle of these organisms.

La mycobactérie à croissance rapide Mycobacterium abscessus est un pathogène opportuniste de l’homme, particulièrement des poumons, capable de se multiplier au sein de macrophages (MФ) mais aussi d’amibes environnementales. Nous avons pu montrer que l’environnement amibien est propice à l’adaptation de M. abscessus à cette survie intramacrophagique. Par des analyses transcriptomiques comparant les adaptations de M. abscessus au sein d’amibes ou de MФ, nous observons des enrichissements de voies biologiques démontrant des adaptations au stress oxydatif et des adaptations métaboliques telle que la béta-oxydation des lipides et l’assimilation des sulfates. Ces adaptations ont également été observées chez Mycobacterium tuberculosis, mycobactérie pathogène pulmonaire stricte de l’homme responsable de la tuberculose. Parmi les gènes induits par M. abscessus au sein des amibes figurent des gènes impliqués dans le transport de polyamines, la biosynthèse du MoCo (molybdopterin cofactor) ainsi que l’assemblage des centres fer-soufre (Fe-S). L’induction de tels gènes, décrits comme des facteurs de virulence chez certaines bactéries intracellulaires, contribuerait à la virulence de M. abscessus en permettant sa survie intramacrophagique. Quarante-cinq gènes ont été identifiés comme fortement induits en amibes chez M. abscessus. Mycobacterium chelonae, appartenant au même complexe génomique que M. abscessus et responsable exclusivement d’infections cutanées, ne présente pas de telles inductions après analyse de son transcriptome intracellulaire, ce qui pourrait expliquer son absence de survie en MФ. Cinq opérons recouvrant 10 gènes ont été délétés au sein du génome de M. abscessus par recombinaison homologue. Ces gènes sont requis pour la survie de M. abscessus en amibes et en MФ. La surexpression chez M. chelonae de deux de ces gènes, MAB_1517c et MAB_2649, codant respectivement une protéine TcmP (tetracenomycin polyketide synthesis O-methyltransferase) et une MmpS (mycobacterial membrane protein small), a conféré une survie intra-macrophagique à M. chelonae, suggérant que l’induction de ces gènes en amibes favorise la survie intracellulaire de M. abscessus en MФ. Enfin, l’analyse du transcriptome de M. abscessus en MФ révèle des adaptations propres à la vie intramacrophagique. Différents gènes particulièrement induits sont impliqués dans le métabolisme de la proline, la sécrétion de protéine par le système de sécrétion de type II ou appartiennent la voie MEP (méthylérythritol phosphate), des voies biologiques contribuant à la virulence de pathogènes. De plus, parmi les gènes induits, certains correspondent à des activités de N-acétylation, d’oxydoréduction, de liaison à l’oxygène ou de détoxification de l’oxyde nitrique par des dioxygénases qui sont enrichies. Comparé à 5 autres opérons délétés, sélectionnés selon leur niveau d’induction et leur activité biologique, le gène eis2 (MAB_4532c), codant une N-acétyltransférase, est essentiel à la survie de M. abscessus en MФ.L’utilisation d’une approche complémentaire, le criblage d’une banque de mutants par transposition chez M. abscessus en amibes, a révélé le rôle essentiel du gène mmpl8 (mycobacterial membrane protein large 8) parmi la famille de protéines MmpL, impliquées dans le transport et/ou la synthèse de lipides mycobactériens. Chez M. abscessus, l’absence de production de cette protéine est corrélée à un défaut d’export d’un nouveau glycolipide (GDND, glycosyl diacetylated nonadecyl diol) et à un phénotype délétère pour la bactérie en MФ.En conclusion, notre travail a montré le rôle fondamental de l’amibe dans l’apprêtement de M. abscessus à la survie intramacrophagique. Trois gènes ayant fait l’objet d’études approfondies, mmpL8, eis2 ainsi que le gène eccB4 (MAB_3759c) mis en évidence par le crible amibien de la banque de mutants de transposition, participent à ce phénotype, confirmant le caractère pathogène de M. abscessus. / Mycobacterium abscessus is a rapidly growing mycobacterium, causing opportunistic infections in humans, and notably pulmonary infections. M. abscessus is able to multiply inside macrophages (MФ) and environmental amoebae. Here we demonstrate that M. abscessus undergoes adaptations in amoebae allowing its survival in MФ. Intracellular adaptations of M. abscessus to amoebae and MФ were assessed by RNAseq. We observed a significant enrichment of biological pathways reflecting adaptations to oxidative stress and metabolic adaptations illustrated by the consumption of fatty acids and activation of the sulfate assimilation pathway. These adaptations have been described in intramacrophagic Mycobacterium tuberculosis, a strictly pathogenic mycobacteria infecting the lung of humans and causing tuberculosis. Among the set of genes induced by M. abscessus during the amoebal co-culture are genes implicated in polyamine transport, MoCo (molybdopterin cofactor) biosynthesis and iron-sulfur (Fe-S) cluster assembly. The induction of such genes, described as virulence factors from intracellular bacteria, might enhance M. abscessus virulence and thus allow its survival in MФ. Forty-five genes are highly induced along the amoebal co-culture. In comparison, the amoebal co-culture with Mycobacterium chelonae, a mycobacterium that belongs to the same genomic complex as M. abscessus and causing solely extrapulmonary infections, does not elicit the same adaptations; potentially explaining M. chelonae inability to persist in macrophages. Five operons, representing a total of 10 genes, were deleted from M. abscessus genome by homologous recombineering. These genes are required for both M. abscessus survival in amoebae and MФ. Overexpression of two of these genes in M. chelonae, MAB_1517c and MAB_2649, encoding a TcmP (tetracenomycin polyketide synthesis O-methyltransferase) protein and an MmpS (mycobacterial membrane protein small) protein respectively, enhances M. chelonae survival in MФ, suggesting that the induction of these genes favors M. abscessus survival in MФ. Analyses of M. abscessus transcriptome in MФ also shed light on adaptations specific to the bacterium intramacrophagic life. Several genes highly induced in macrophages are implicated in biological pathways known to contribute to bacteria virulence, including proline metabolism, protein secretion by the type II secretion system and the MEP (methylerythritol phosphate) pathway. Among the set of induced genes selected according to their level of induction and their biological activity, N-acetylation and redox activities, bounding to oxygen and detoxification from nitric oxide by dioxygenases are significantly enriched. Among operons from this set of genes, it appears that M. abscessus eis2 gene (MAB_4542c), encoding a N-acetyltransferase, is essential for M. abscessus survival in MФ.In addition, a complementary approach to RNAseq, the screening of a transposon (Tn) mutant library of M. abscessus inside amoebae, revealed important roles of the mmpL8 gene encoding a mycobacterial membrane protein large belonging to a family of proteins implicated in lipid biosynthesis and export to the cell surface. When this protein was no longer produced by M. abscessus, a lower amount of a new glycolipd family (GDND, glycosyl diacetylated nonadecyl diol) was observed as well as a deleterious phenotype in MФ.To conclude, our work has shown a fundamental role of amoebae in triggering the virulence of M. abscessus, further allowing its survival in macrophages. Besides, three genes that have been studied more extensively – mmpL8, eis2 and eccB4 (revealed by the Tn library screening) – are required for M. abscessus survival in macrophages and confirmed its pathogenic behavior.

L'ulcère de Buruli, maladie négligée, pouvant entraîner déformations et incapacités permanentes, causée par Mycobacterium ulcerans, une mycobactérie associée aux écosystèmes aquatiques. Les sources et réservoirs de M. ulcerans ne sont pas précisément connus limitant la prophylaxie. Ma thèse contribue à démasquer les sources de contamination par M. ulcerans. Ma revue de la littérature répertoriant les sources potentielles, met en exergue les pièces manquantes pour la compréhension de l’épidémiologie de l’ulcère de Buruli. Mon étude de coculture M. ulcerans-Acanthamoeba griffini indique que cette amibe n’est pas un réservoir de M. ulcerans. Ensuite, mon étude du métabolisme des substrats carbonés indique que les bactéries, champignons, algues et mollusques peuvent combiner des sources carbonées au profit de M. ulcerans dans l’environnement. Capitalisant sur ce résultat, mon étude de l’effet des mycolactones secrétées par M. ulcerans sur les champignons, montre une attraction sur Mucor circinelloides. Ces observations démasquent une nouvelle activité de la mycolactone d’attraction d’organismes fungiques. Le mode de transmission de M. ulcerans à l’homme reste également un mystère. Dans ce sens, j’ai détecté des séquences d'ADN spécifiques de M. ulcerans sur la peau saine d’individus asymptomatiques en zones d’endémie. Ces données pourraient aider à promouvoir une prophylaxie fondée sur le port des habits de protection au contact des environnements à risques. En perspective à mon travail de thèse, une collaboration est mise en place avec le PNLUB et l’Institut Pasteur de Côte d’Ivoire pour étudier les réservoirs de M. ulcerans en Côte d'Ivoire. / Buruli ulcer is a dermis, epidermis and sometimes bone infection leading to deformities and permanent disabilities. It is caused by Mycobacterium ulcerans, a mycobacterium associated to the aquatic ecosystems but its sources and reservoirs are not yet defined. Therefore, no prophylaxis is established. This thesis contribute to unmask the sources of contamination of M. ulcerans. My review has identified potential sources of M. ulcerans in the environment and highlighted the missing pieces for understanding the epidemiology of Buruli ulcer. My study on the role of amoeba in the survival of M. ulcerans in the environment, install M. ulcerans as susceptible to amoeba rendering amoeba an unlikely host of M. ulcerans. Hereafter, i studied carbon substrates metabolized by M. ulcerans strains. Literature survey indicated that the environmental sources of carbon substrates metabolized by M. ulcerans were bacteria, fungi, algae and mollusks. I therefore studied the interactions of M. ulcerans with fungi by testing the effect of mycolactones on fungi. Mycolactones showed an attraction effect on Mucor circinelloides. This observation suggest a novel role for mycolactones as chemoatractants to fungi. The mode of transmission of M. ulcerans to humans remains unknown. I showed that M. ulcerans DNA can be detected on the healthy skin of asymptomatic persons, suggesting an asymptomatic carriage. These data could help promote prophylaxis based on wearing protective clothing in contact with risky environments. In perspective to my thesis work, we set up a collaboration with the Buruli Ulcer Program (PNLUB) and Institut Pasteur Côte d'Ivoire to study the reservoirs of M. ulcerans.

Arcella Ehrenberg 1832 is one of the most numerous testacean genera. Arcellinids are an aerobic lineage of testate amoeba that live in a wide variety of environments. Probably their ability to survive in such divergent conditions is related to some de- gree of metabolic flexibility. Anaerobic organisms have gained and lost a number of genes related to energetic metabolism. These processes modify classic mitochondria until loss of function and transformation in mitochondrial related organelles (mitosomes and hydrogenosomes). Here I propose that Arcella intermedia adaptation to microarophilic environments is related to the acquisition of new genes. There are two main modes of acquisition of new genes. The traditional view, where duplication is followed by mutations and neo-functionality of the duplicate. Or genes can be acquired from other species (lateral gene transfers). The second process has a major importance in prokaryotic evolution and is probably under considered in eukaryotic evolution. I also propose in this work that genes related to anaerobic metabolism in Arcella are acquired by lateral gene transfer. However, analysis of genomic and transcriptomic data are absent for A.intermedia. Characterizing genome-scale data from eukaryotes is essential for gene discovery and for inferring transitions over the tree of life. The transcriptome dataset from this work provides the first effort of characterization of expressed sequences in A.intermedia. We used single cell from different moments of growth and whole culture RNA extraction in order to increase the diversity of metabolic moments of the cells. Mapped sequences allowed us to identify functional pathways in A.intermedia cells. In general, it seems that metabolic processes are showing up more, followed by signaling and responses to stimuli. We describe functioning of carbohydrate and energy metabolism including even an anaerobic pathway to produce energy. We found ACS-ADP and PFO genes in A.intermedia. We describe amino acid metabolism, with at least 12 amino acids metabolizing pathways described and catabolism mainly related to TCA cycle intermediates. Calcium, Ras GTPases, PI3K-AK and AMPK-mTOR are the main signaling pathways represented in transcriptomes. We described important pathways for amoeba: endocytosis and phagocytosis and it seems to be similar with the ones already described for other amoeba with a dependence on F-actin and small GTPases of Rho subfamily. We couldn\'t find lots of information about programmed cell death in A.intermedia, however cell growth are similar to pathways described for dinoflagellates. We expect that upcoming genomes will finish the description of functioning of those organisms, but we believe our work already is a good starting point. In order to gain a clearer picture of the presence of anaerobic metabolism genes in Amoebozoa, we conducted BLAST searches in Amoebozoa and Arcellinida data bases for the presence/absence of ACS-ADP, PFO and [FeFe]-H2ase. Other Arcellinida species also presented these genes, Difflugia sp., Difflugia compressa and Cyclopyxis lobostoma. Besides these, the already known Mastigamoeba balamuthi, Entamoeba histolytica and Acanthamoeba castelanii. Amoebozoa sequences don\'t form a monophyletic group in any of the three genes. However, Arcellinida sequences always grouped together. As such distinct amoeba groups have those anaerobic metabolism genes, however, most of the Amoebozoa do not. It is more likely to think of lateral transfers occurring independently among these amoeba groups, generating the possibility of occupying a new niche. The main objective of this work was to start generating tools to understand the ability of some testate amoeba to resist environmental harsh conditions. We found lots of interesting questions but the one we focused on this dissertation was (1) the evolution of anaerobic related genes in testate amoeba lineages. The assembled and annotated sequence data will be available as reference sequences, making the work with this group easier. The results can also potentially be applied as biomonitoring markers for the management of water resources. This work will improve the general knowledge on the evolution and function of freshwater organisms. We expect also to make a contribution on the understanding of the impact of lateral gene transfers in Arcellinida diversity / Arcella Ehrenberg 1832 é um dos gêneros de tecamebas mais numerosos, perten- cente aos Arcellinida. Estas são uma linhagem aeróbia de amebas tecadas que vivem em uma grande variedade de ambientes. Provavelmente, sua capacidade de sobreviver em condições tão divergentes está relacionada a algum grau de flexibilidade metabólica. Os organismos anaeróbicos ganharam e perderam vários genes relacionados ao metabolismo energético. Este processo modifica mitocôndrias clássicas até a perda da função e transformação em organelas relacionadas (mitossomos e hidrogenossomos). Aqui proponho que a adaptação de Arcella intermedia a ambientes microaerófilos está relacionada à aquisição de novos genes. Existem dois modos principais de aquisição de genes. Na visão tradicional, a duplicação gênica é responsável por gerar diversidade, seguida por mutações e neofuncionalidade da duplicata. Alternativamente, os genes podem ser adquiridos de outras espécies (transferências laterais de genes). O segundo processo tem uma grande importância evolutiva e é ainda pouco considerado na evolução eucariótica. Por isso, também proponho neste trabalho que genes relacionados ao metabolismo anaeróbico em Arcella sejam adquiridos por transferência lateral de genes. Entretanto, a análise de dados genômicos e transcriptômicos é inexistente para A.intermedia. A caracterização de dados em escala genômica de eucariotos é essencial para a descoberta de genes e para a inferência transições sobre a árvore da vida. O conjunto de dados de transcriptoma deste trabalho fornece um primeiro esforço de caracterização de sequências expressas em A. intermedia. Utilizamos extrações de célula-única em diferentes momentos de crescimento e extração de RNA de cultura inteira, a fim de aumentar a diversidade de momentos metabólicos das células. Sequências mapeadas permitiram identificar vias funcionais em células de A. intermedia. Em geral, parece que genes relacionados a processos metabo?licos são os que aparecem mais frequentemente, seguidos dos de sinalização e respostas a estímulos. Nós descrevemos a função do metabolismo de carboidratos e energia, incluindo uma via anaeróbica. Encontramos em A.intermedia os genes ACS-ADP e PFO. Descrevemos o metabolismo de aminoácidos, com pelo menos 12 vias metabólicas de aminoácidos descritas e catabolismo relacionado a intermediários do ciclo de TCA. Cálcio, Ras GTPases, PI3K-AK e AMPK-mTOR são as principais vias de sinalização representadas nos transcriptomas. Descrevemos importantes vias para amebas, que são endocitose e fagocitose. Parecem ser vias semelhantes àquelas já descritas para outras amebas, com dependência de F-actina e pequenas GTPases da subfamília Rho. Não conseguimos encontrar muitas informações sobre a morte celular programada em A. intermedia, mas o crescimento celular é semelhante com as vias descritas para os dinoflagelados. Esperamos que os próximos genomas terminem a descrição da função desses organismos, mas acreditamos que nosso trabalho já é um bom ponto de partida. A fim de obter uma visão mais clara da presença de genes de metabolismo anaeróbico em Amoebozoa, realizamos buscas no BLAST em bancos de dados de Amoebozoa e Arcellinida, para a presença/ausência de ACS-ADP, PFO e [FeFe] -H2ase. Outras espécies de Arcellinida também apresentaram estes genes, Difflugia sp, Difflugia compressa e Cyclopyxis lobostoma. Além destes, os já conhecidos Mastigamoeba balamuthi, Entamoeba histolytica e Acanthamoeba castelanii. Sequências de amebozoários não formam um grupo monofilético em nenhum dos três genes. No entanto, as sequencias de Arcellinida sempre se agrupam. Como são grupos de Amoebozoa de tal maneira distintos que possuem estes genes de metabolismo anaeróbico, e sendo que a maioria não possui, é mais provável que sejam transferências laterais independentes entre esses grupos de ameba, gerando a possibilidade de ocupar um novo nicho. O objetivo principal deste trabalho foi gerar ferramentas para entender a capacidade de algumas amebas tecadas em resistir a condições adversas do meio ambiente. Encontramos muitas questões interessantes, mas a que teve nosso foco nesta dissertação foi (1) a evolução de genes relacionados ao metabolismo anaeró?bio em linhagens de amebas tecadas. Os dados da sequência reunidos e anotados estarão disponíveis como sequências de referência, facilitando o trabalho com esse grupo. Os resultados também podem ser aplicados aos marcadores de biomonitoramento para o gerenciamento dos recursos hídricos. Este trabalho irá melhorar o conhecimento geral sobre a evolução e função de organismos de água doce. Esperamos tambem contribuir para a compreensão do impacto das transferências laterais na diversidade de Arcellinida

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

This thesis consists of four papers and an introduction.  In Paper I we calculate the second order derivatives of the Ronkin function of an affine polynomial in three variables. This gives an expression for the real Monge-Ampére measure associated to the hyperplane amoeba. The measure is expressed in terms of complete elliptic integrals and hypergeometric functions.  In Paper II and III we prove that a certain semi-explicit cohomological residue associated to a Cohen-Macaulay ideal or more generally an ideal of pure dimension, respectively, is annihilated precisely by the given ideal. This is a generalization of the local duality principle for the Grothendieck residue and the cohomological residue of Passare. These results follow from residue calculus, due to Andersson and Wulcan, but the point here is that our proof is more elementary. In particular, it does not rely on the desingularization theorem of Hironaka. In Paper IV we prove a global uniform Artin-Rees lemma for sections of ample line bundles over smooth projective varieties. We also prove an Artin-Rees lemma for the polynomial ring with uniform degree bounds. The proofs are based on multidimensional residue calculus. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript. Paper 4. Manuscript.</p>

This thesis focuses on the analysis of the role of adhesion between substrate and cell and factors of Quorum sensing on the migration of Dictyostelium amoeba. Tools to automate the recordings of videomicroscopy and image analysis have been developed to work with very large samples of cells and toquantify cell migration. A microfluidic device for cell detachment in hydrodynamic flow combined witha motorized stage has allowed a statistical study of adhesion but also the dynamics of detachment. The analysis of the migration of Dictyostelium in non nutritive medium highlights the role of density on celldifferentiation and migration capacity. We observe the presence of a maximum speed of migration after6 hours of starvation. We show that the adhesion to glass is twice as low in deprivation buffer as inthe nutrient medium. The experiences of migration in growth medium revealed the presence of a factorof detection of density secreted by the cells and regulating their random migration. The diffusion coefficient, the persistence of the movement and morphology of cells vary depending on the concentrationof this factor. This factor does not affect cell adhesion but only the dynamics of detachment. Finally, the testing protocol developed allowed us to make a comparative study of migration by varying otherparameters such as surface or the chemical composition of experimental medium. This work concludesby outlining the possible role of adhesion to the migration of Dictyostelium in nutrient medium.