This thesis deals with the Biology Olympiad and remaking of selected tasks of region round. The remaked tasks can be used in highschool education. The thesis includes theoretical and practical part. The theoretical part provides general information about the Biology Olympiad (BiO), charts the achievements of Czech students in International Biology Olympiad (IBO), includes literary review of selected topics. The practical part encompass four remaked tasks of different topics: Eucaryotic cell as a mosaic, Water in life of animals, Animals' areas and their distribution, History of the flora in Czech Republic. The tasks are processed in the form of worksheets. The worksheets were tested at schools. Knowledge of students was tested by using of created pretests and posttests. The practical part also presents the result of research based on a questionnaire survey focused on the involvement of school in BiO and teachers' opinions on these topics.

Laterale Gentransfer wurde zuerst von Schwartz und Dayhoff (1978) entdeckt, die es aber als eine Exzentrizität werteten und als solche ignorierten. Später, als mehrere DNS- und Eiweißsequenzen sequenziert und raffiniertere Phylogenien rekonstruiert wurden, hat die Rolle an Relevanz gewonnen, die der laterale (oder horizontale) Gentransfer in der evolutionären Geschichte von lebendigen Organismen gespielt hat. Außerdem existiert auch zwischen Endosymbionten und Zellkernen statt. Ich habe ein theoretisches Modell entwickelt, das den lateralen Gentransfer zwischen Endosymbionten und dem Zellkern repräsentiert. Das Modell erforscht die Bedeutung des Fehlens von Rekombination in den Organellen (Muller’s Ratchet) sowie Abweichungen von Muller’s Ratchet in Form der non-symmetrical homologous recombination in Gentransfermechanismen. Ich habe zum einen Zellkern-Inkompatibilitäten, die aus der Übertragung eines Gens resultieren, und zum anderen Zyto- und Zellkern-Inkompatibilitäten zwischen den mutierten endosymbiotischen Genomen und dem modifizierten Zellenkern untersucht. Die Ergebnisse zeigen, dass unter bestimmten Bedingungen die Existenz oder Nicht-Existenz von Rekombination die gleiche Wirkung haben können. Es zeigte sich auch, dass Rekombination, wenn sie vorkommt und wenn sie nicht symmetrisch ist, starke Auswirkungen auf die Allelenfrequenz einer Population haben kann. Es wurde auch klar, dass es eine starke Beziehung zwischen dem Zellkern und endosymbiotischen Genomen gibt, und dass das evolutionäre Schicksal des einen größtenteils von den evolutionären Kräften abhängig ist, die das andere beeinflussen. Wenn man Zellkern- und Cyto-Zellkerninkompatibilitäten in das Modell einführt, dann zeigen die Ergebnisse, dass die Inkompatibilitäten, die der laterale Gentransfer produziert hat, möglicherweise eine ähnliche Rolle im Speziationsmechanismus spielen könnten wie die Inkompatibilitäten zwischen Mitochondrien und Zellkernen in verschiedenen Nasonia-Arten. / Lateral gene transfer has played a key role in the evolution of living beings. This process was first acknowledged in 1978 by Schwartz and Dayhoff but considered a relatively infrequent eccentricity and ignored. Later on, as DNA and protein sequences accumulated and more refined phylogenies were reconstructed, the contribution of lateral (or horizontal) gene transfer to the evolutionary history of living organisms gained relevance. Besides, gene transfer is known to occur not only between independent organisms but also, and more frequently between endosymbionts including eukaryotic organelles. I developed a theoretical model to study the lateral gene transfer process between cell organelles (but extendible to other endosymbionts) and the cell nucleus. The model explores the role of the lack of recombination in the organelles (Muller''s ratchet) as well as deviations from Muller''s ratchet in the form of non-symmetrical homologous recombination in relation with the gene transfer process. Also, nuclear incompatibilities resulting from the inclusion of a transferred gene, and cyto-nuclear incompatibilities between the mutant endosymbiotic genomes and the modified nuclear genome are investigated. The results obtained show that under certain circumstances the existence recombination or its non-existence produce the same results, and that deviations from symmetry in the recombination process might have important effects on the frequency of different alleles. It is also clear that there is a strong relation between nuclear and endosymbiotic genomes, and that the evolutionary fate of one largely depends on the forces affecting the other. When nuclear and cyto-nuclear incompatibilities are introduced in the model, the results show that lateral gene transfer-induced incompatibilities could potentially play a role in the speciation process similar to the one produced by mitochondria in the Nasonia species.

La symbiose est un phénomène ubiquitaire dans la nature et joue un rôle évolutif majeur. Alors que la communauté scientifique reconnaît désormais l’importance des associations symbiotiques dans de nombreux processus biologiques et pathologiques chez les animaux, la compréhension des mécanismes de contrôle, de tolérance et de modulation des populations symbiotiques est un enjeu majeur. Pour aborder ces questions, j’ai étudié l’association entre le charançon Sitophilus et la bactérie intracellulaire Sodalis pierantonius. Sitophilus héberge son endosymbiote dans des cellules spécialisées, les bactériocytes, regroupées en un organe, le bactériome. En retour, S. pierantonius fournit à son hôte des nutriments présents en faibles quantités dans son alimentation, les céréales. S. pierantonius étant immunogène pour son hôte, dans un premier chapitre, j’ai étudié les régulations immunitaires spécifiques au bactériome assurant le maintien de l’homéostasie immunitaire. J’ai dans un premier temps montré que la compartimentalisation des endosymbiotes, limitant les contacts immunitaires avec l’hôte, repose sur l’expression IMD-dépendante d’un peptide antimicrobien, une régulation similaire aux réponses immunitaires aux pathogènes. Ensuite, j’ai montré comment l’immunogénicité des endosymbiotes, via son peptidoglycane, est limitée par des Protéines de Reconnaissance du PeptidoGlycane (PGRP). Le peptidoglycane symbiotique ne semble pas être reconnu dans le bactériome, et sa reconnaissance systémique est contenue par l’action locale de PGRP-LB. Cette protéine clive le peptidoglycane symbiotique, empêchant ainsi l’activation chronique du système immunitaire systémique. Dans un deuxième chapitre, j’ai étudié comment, au cours de la métamorphose, le bactériome se réorganise complètement. Le bactériome larvaire se dissocie, les bactériocytes migrent le long de l’intestin et forment de multiples nouveaux bactériomes. Une approche de dual-RNAseq nous a permis de révéler l’implication à la fois de l’hôte et du symbiote dans ce remodelage morphologique. Les résultats obtenus durant cette thèse montrent l’impact incommensurable des bactéries sur des processus immunitaires et développementaux, et sur l’évolution des animaux en général. / Symbiosis is ubiquitous in nature and plays a crucial role in evolution. As the scientific community is becoming increasingly aware of the importance of such associations in both biological and pathological processes in animals, understanding how symbiotic populations are controlled, tolerated, and modulated, is becoming a major stake. To address these questions, I studied the mutualistic association between the weevil Sitophilus and the intracellular bacterium Sodalis pierantonius. Sitophilus houses S. pierantonius in specialized host cells, the bacteriocytes, which group together in an organ, the bacteriome. In return, S. pierantonius provides its host with nutrients scarecely present in its cereal-based diet. S. pierantonius being immunogenic for its host, I studied in a first chapter how specific bacteriome immune regulations ensure the maintenance of host immune homeostasis. In a first part, I showed that endosymbiont compartmentalization, which limits host-endosymbiont immune contacts, relies on the IMD-dependent expression of one antimicrobial peptide, a regulation similar to that of immune responses in pathogenic conditions. Then, I showed how endosymbiont immunogenicity, via its peptidoglycan, is tamed by PeptidoGlycan Recognition Proteins (PGRPs). While symbiotic peptidoglycan would not be recognized within the bacteriome, its systemic recognition is circumscribed by PGRP-LB local action. PGRP-LB cleaves symbiotic peptidoglycan, thereby preventing a chronical and detrimental activation of the host systemic immunity. In a second chapter, I studied how, during metamorphosis, the bacteriome is completely remodeled. The larval bacteriome dissociates, bacteriocytes migrate along the midgut, and settle in multiple new bacteriomes. A dual-RNAseq approach allowed us to pinpoint both host and symbiont implication in this drastic morphological reorganization. The results obtained during this PhD show the immeasurable impact bacteria bear on host immune and developmental processes, and more generally on animal evolution.

Les amibes libres sont des microorganismes unicellulaires eucaryotes dont l'écologie au sein des réseaux d'eau potable est mal connue. Les amibes libres représentent un enjeu de santé publique, du fait de leur capacité à favoriser la présence de bactéries potentiellement pathogènes, parmi lesquelles des mycobactéries.Une campagne de prélèvement menée sur le réseau d'eau potable de Paris a permis d'évaluer la diversité des amibes libres et de leur microbiome bactérien, par pyroséquençage ciblant les gènes ribosomaux (16S et 18S). Ces analyses ont suggéré la prédominance des genres Acanthamoeba, Vermamoeba, Echinamoeba et Protacanthamoeba. Le microbiome des amibes a révélé une grande diversité bactérienne, dominée par Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas et Pseudoxanthomonas. L'intégration des paramètres physicochimiques a permis de suggérer l'importance de l'origine de l'eau, la température, le pH et la concentration en chlore dans la dynamique des populations amibiennes. Une endosymbiose originale entre V. vermiformis et des bactéries du phylum TM6 a également été mise en évidence.Les amibes ont été fréquemment co-isolées avec des mycobactéries dans le réseau, principalement les espèces M. llatzerense et M. chelonae. Des expériences d'infection chez A. castellanii ont permis d'observer la capacité de ces mycobactéries à survivre et croitre en présence d'amibes. Par génomique comparative et analyses transcriptomiques, plusieurs facteurs de virulence, conservés entre M. llatzerense, M. chelonae et M. tuberculosis, ont été identifiés et sont surexprimés au cours de l'infection. Ces données suggérent leur implication dans la résistance à la prédation amibienne.L'ensemble de ces travaux a permis d'améliorer la connaissance des populations amibiennes et de leur microbiome au sein du réseau d'eau potable, apportant des éléments supplémentaires concernant leur implication dans la survie et la persistance des mycobactéries. / Free-living amoebae are unicellular eukaryotes whose ecology in drinking water networks remains poorly understood. They may represent a public health concern, because of their ability to favour the presence of potentially pathogenic bacteria, among which are mycobacteria.A sampling scheme based on Paris drinking water network allowed identifying the diversity of both freeliving amoebae and their bacterial microbiome, using ribosomal RNA targeted pyrosequencing. These analyses indicated the major presence of Acanthamoeba, Vermamoeba, Echinamoeba and Protacanthamoeba genera. The microbiome was highly diverse and dominated by Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas and Pseudoxanthomonas. The coupling of physicochemical parameters to this analysis allowed underlining the importance of water origin, temperature, pH and chlorine concentration in shaping amoebal populations. Also an original endosymbiosis between V. vermiformis and a bacterium of the TM6 phylum was described. Free-living amoebae were frequently co-isolated with mycobacteria in the water network, mainly M. llatzerense and M. chelonae species. Infection experiments on A. castellanii illustrated the capacity of these species to resist and grow in presence of amoebae. Through genomics and transcriptomics approaches, several virulence factors, conserved between M. llatzerense, M. chelonae and M. tuberculosis were identified, and found to be upregulated during infection experiments. These results suggest their involvement in mycobacterial resistance to amoebal predation.Altogether, this work helped to better understand the ecology of free-living amoebae and their microbiome in drinking water networks, as well as the role of free-living amoebae in the survival and persistence of mycobacteria in such environments.

Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...

Plusieurs insectes se développant dans des milieux nutritionnellement déficients vivent en symbiose durable avec des bactéries intracellulaires (endosymbiotes) qui complémentent leur alimentation et améliorent leur pouvoir adaptatif. Alors que ces associations ont été largement étudiées sur les plans physiologiques et évolutifs, peu de travaux se sont consacrés à l’étude des mécanismes impliqués dans la tolérance et le contrôle des endosymbiotes par l’hôte. L’objectif de cette thèse est d’étudier, chez les charançons des céréales du genre Sitophilus, les particularités moléculaires et immunitaires du bactériome, un organe que l’insecte développe pour héberger les symbiotes et les isoler de sa réponse immunitaire systémique. Le bactériome du charançon exprime une réponse immunitaire modulée : des études transcriptomiques ont montré que les effecteurs de l’immunité sont peu exprimés dans cet organe, à l’exception d’un gène codant un peptide antimicrobien, la coléoptéricine A. Cette dernière interagit avec les endosymbiotes et participe à leur confinement intracellulaire. Dans une première partie, j’ai montré avec une approche d’interférence à l’ARN que l’expression du gène colA serait contrôlée par un système original qui impliquerait les gènes relish et tollip. Cette régulation « interne » au bactériome semble assurer le maintien des endosymbiotes et l’homéostasie de l’organe. Afin de comprendre comment le bactériome répond à une infection par les bactéries exogènes, j’ai suivi par RT-qPCR l’expression de gènes effecteurs de l’immunité dans le bactériome après injection systémique de bactéries à Gram positif ou négatif. Ceci a mis en évidence une réponse « externe », induite en cas d’infection, et qui aurait un rôle de protection des endosymbiotes contre les bactéries exogènes. Enfin, je me suis consacré à l’étude des changements de régulation accompagnant le passage du stade larvaire au stade adulte, marqué par une symbiose très dynamique. Le nombre d’endosymbiotes augmente fortement pendant les premiers jours de vie imaginale, puis diminue jusqu’à leur élimination complète par recyclage autophagique. Une analyse RNAseq a permis d’identifier les voies de signalisation dont l’activité accompagne cette dynamique. Une approche de RT-qPCR a également montré que l’immunité du bactériome est maintenue à un faible niveau d’activation pendant tout le processus de recyclage. Ce travail montre qu’au cours de leur évolution, les insectes ont sélectionné plusieurs stratégies pour assurer le maintien et l’ajustement de leur charge endosymbiotique en fonction de leurs besoins physiologiques : une signalisation immunitaire assurerait le confinement intracellulaire des endosymbiotes, et un ensemble de processus cellulaires incluant l’apoptose et l’autophagie semble être en associé aux voies métaboliques pour assurer le contrôle de la dynamique bactérienne et garantir le compromis bénéfice/coût de la symbiose. / Many insect species living on nutritionally unbalanced media depend on intracellular mutualistic bacteria, called obligatory endosymbionts, for their development and reproduction. Endosymbionts are housed in specialized host cells called bacteriocytes, that group together to form the bacteriome organ. Although such associations have been widely investigated on a physiological and evolutionary point of view, little is known about the mechanisms involved in the tolerance and the control of endosymbionts by the host. This work aims at deciphering the molecular and immune specificities of the bacteriome using the model system Sitophilus oryzae, the cereal weevil, and its obligate endosymbiont Sodalis pierantonius. The weevil bacteriome expresses a modulated immune response: transcriptomic studies showed that immune effector genes were lowly expressed despite the massive bacterial presence, with the exception of colA, a gene encoding for Coleoptericin A, an antimicrobial peptide. Coleoptericin A interacts with endosymbionts and participates in their intracellular seclusion. In a first chapter, I used RNA interference to demonstrate that colA gene expression may be controlled by an original system involving the genes relish and tollip. This “internal” regulation for endosymbiont control seems to maintain bacteriome homeostasis. In a second chapter, in order to understand how the bacteriome responds to an infection by exogenous bacteria, I followed up by RT-qPCR the expression of immune effector genes in the bacteriome after injection of Gram positive and Gram negative bacteria. This highlighted an “external” immune response, inducible upon infections, which may enable endosymbiont protection against exogenous intruders. In a third and last chapter, I focused on the regulation changes that accompany the switch from the larval stage to the imaginal stage, the latter being characterized by a very dynamic symbiosis. Endosymbiont load drastically increases during the first days of imaginal life, then rapidly decreases until complete elimination of the bacteria by autophagic recycling. RNAseq analysis allowed the identification of signaling pathways linked to this dynamic. A complementary RT-qPCR approach also showed that bacteriome immunity was laid low during the whole recycling process. This work shows that several strategies have been selected during host-symbiont coevolution to ensure the maintenance of the endosymbionts and the adjustment of their population depending on the insects physiological needs: immunity allows the intracellular seclusion in the bacteriocytes, and cell processes including autophagy and apoptosis are associated to metabolic pathways to control the endosymbiotic dynamics and secure the cost and benefit trade-off of symbiosis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta dissertação é apresentada uma modelagem analítica para o processo evolucionário formulado pela Teoria da Evolução por Endossimbiose representado através de uma sucessão de estágios envolvendo diferentes interações ecológicas e metábolicas entre populações de bactérias considerando tanto a dinâmica populacional como os processos produtivos dessas populações. Para tal abordagem é feito uso do sistema de equações diferenciais conhecido como sistema de Volterra-Hamilton bem como de determinados conceitos geométricos envolvendo a Teoria KCC e a Geometria Projetiva. Os principais cálculos foram realizados pelo pacote de programação algébrica FINSLER, aplicado sobre o MAPLE. / This work presents an analytical approach for modeling the evolutionary process formulated by the Serial Endosymbiosis Theory represented by a succession of stages involving different metabolic and ecological interactions among populations of bacteria considering both the population dynamics and production processes of these populations. In such approach we make use of systems of differential equations known as Volterra-Hamilton systems as well as some geometric concepts involving the KCC Theory and the Projective Geometry. The main calculations were performed by the computer algebra software FINSLER based on MAPLE.

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta dissertação é apresentada uma modelagem analítica para o processo evolucionário formulado pela Teoria da Evolução por Endossimbiose representado através de uma sucessão de estágios envolvendo diferentes interações ecológicas e metábolicas entre populações de bactérias considerando tanto a dinâmica populacional como os processos produtivos dessas populações. Para tal abordagem é feito uso do sistema de equações diferenciais conhecido como sistema de Volterra-Hamilton bem como de determinados conceitos geométricos envolvendo a Teoria KCC e a Geometria Projetiva. Os principais cálculos foram realizados pelo pacote de programação algébrica FINSLER, aplicado sobre o MAPLE. / This work presents an analytical approach for modeling the evolutionary process formulated by the Serial Endosymbiosis Theory represented by a succession of stages involving different metabolic and ecological interactions among populations of bacteria considering both the population dynamics and production processes of these populations. In such approach we make use of systems of differential equations known as Volterra-Hamilton systems as well as some geometric concepts involving the KCC Theory and the Projective Geometry. The main calculations were performed by the computer algebra software FINSLER based on MAPLE.

Our understanding of microbial evolution is largely dependent on available genomic data of diverse organisms. Yet, genome-sequencing efforts have mostly ignored the diverse uncultivable majority in favor of cultivable and sociologically relevant organisms. In this thesis, I have applied and developed cultivation independent methods to explore microbial diversity and obtain genomic data in an unbiased manner. The obtained genomes were then used to study the evolution of mitochondria, Rickettsiales and Haloarchaea. Metagenomic binning of oceanic samples recovered draft genomes for thirteen novel Alphaproteobacteria-related lineages. Phylogenomics analyses utilizing the improved taxon sample suggested that mitochondria are not related to Rickettsiales but rather evolved from a proteobacterial lineage closely related to all sampled alphaproteobacteria. Single-cell genomics and metagenomics of lake and oceanic samples, respectively, identified previously unobserved Rickettsiales-related lineages. They branched early relative to characterized Rickettsiales and encoded flagellar genes, a feature once thought absent in this order. Flagella are most likely an ancestral feature, and were independently lost during Rickettsiales diversification. In addition, preliminary analyses suggest that ATP/ADP translocase, the marker for energy parasitism, was acquired after the acquisition of type IV secretion systems during the emergence of the Rickettsiales. Further exploration of the oceanic samples yielded the first draft genomes of Marine Group IV archaea, the closest known relatives of the Haloarchaea. The halophilic and generally aerobic Haloarchaea are thought to have evolved from an anaerobic methanogenic ancestor. The MG-IV genomes allowed us to study this enigmatic evolutionary transition. Preliminary ancestral reconstruction analyses suggest a gradual loss of methanogenesis and adaptation to an aerobic lifestyle, respectively. The thesis further presents a new amplicon sequencing method that captures near full-length 16S and 23S rRNA genes of environmental prokaryotes. The method exploits PacBio's long read technology and the frequent proximity of these genes in prokaryotic genomes. Compared to traditional partial 16S amplicon sequencing, our method classifies environmental lineages that are distantly related to reference taxa more confidently. In conclusion, this thesis provides new insights into the origins of mitochondria, Rickettsiales and Haloarchaea and illustrates the power of cultivation independent methods with respect to the study of microbial evolution.