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Diversity of prasinophyte algae related to the euglenid plastid
Lukešová, Soňa ; Hampl, Vladimír (advisor) ; Horák, Aleš (referee)
Euglenophyceae represent a group of unicellular eukaryotic organisms that have gained during their evolution the ability to photosynthesize. They aquired plastids via secondary endosymbiosis with a green alga as the plastid donor. Phylogenetic studies searching for the origin of this organelle revealed the green alga Pyramimonas parkeae from Prasinophytes as the closest known relative to euglenid plastids. Pyramimonas parkeae and Euglena share several genes clusters with unique order of genes in their plastid genomes, which also point to the Pyramimonadales as the donor of the plastids. However, it is posible, that organisms more closely related to euglenid plastids than P. parkeae, occur in the environment. In my diploma thesis I focused on the exploration of diversity of Pyramimonadales and Euglenophyceae in environmental samples. I used several approaches to perform this task. I amplified parts of the plastid genomes in environmental samples by using specific PCR and determined their position in the phylogenetic tree. I also made large-scale phylogenetic analyses based on 16S rRNA and 18S rRNA sequences from representatives of the groups Euglenophyceae, Prasinophytes and environmental samples. The results revealed the presence of a large number of environmental sequences relative to the...
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Type II secretion system in the mitochondria of Naegleria gruberi.
Krupičková, Alžběta ; Doležal, Pavel (advisor) ; Hampl, Vladimír (referee)
Several types of the transport (secretion) systems can be found in the membranes of gram-negative bacteria. These systems export proteins into the extracellular milieu of bacteria. One of them, type II secretion system (T2SS), exports the folded proteins through the outer bacterial membrane. The core subunits of T2SS were discovered in the genomes of several protists belonging to the Discoba and Malawimonada groups, including Naegleria gruberi and Malawimonas sp. str. 249. Our experiments suggest that these subunits are localized in the mitochondria of these protists. The discovery of these genes in eukaryotes is surprising. If the T2SS was active in these mitochondria, it would represent an evolutionary intermediate stage of the organelle, which can export and import the proteins. In experimental part of the thesis, we studied how the proteins of N. gruberi and M. sp. str. 249 interact. We demonstrated several interactions, which correspond to the function of bacterial T2SS. To this aim, we employed bacterial and yeast two-hybrid systems. Further, we explored the import of the core subunit of T2SS, pseudopilin, of N. gruberi into the inner mitochondrial membrane. And we also investigated the import of M. sp. str. 249 secretin into the outer mitochondrial membrane.
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Analysis of gene regulatory regions in the genome of oxymonad Monocercomonoides
Brzoň, Ondřej ; Hampl, Vladimír (advisor) ; Vopálenský, Václav (referee)
iv Abstract Regulation of gene expression is a key ability of every single cell in its development, differentiation and homeostasis. On the other hand, rather sparse amount of information is available for protists and our understanding of regulation of gene expression in eukaryotes is limited to a few model organisms. Our research is aimed at oxymonads, poorly studied group of anaerobic protists, which inhabit digestive tract of some animals. In this study we focus on the genus Monocercomonoides. Gene expression is modulated at multiple levels by many mechanisms. This thesis is focused on structure of promoter regions, 5' untranslated regions and basal transcription and translation initiation factors. Our results are compared to the closest studied relatives of Monocercomonoides - Trichomonas vaginalis and Giardia intestinalis. We have identified several conserved motifs in promoter regions of Monocercomonoides, including TATA box and TATA-like motif. These motifs potentially play a role in the transcription regulation. 5' untranslated regions are relatively short (typically 20 - 30 nucleotides) and GC content in these regions is low compared to model organisms. In selected genes, the quality of the automatic prediction of UTR was verified by RACE. We have annotated sets of basic transcription (23 proteins)...
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Diversity of the genus Monocercomonoides
Vlasáková, Jitka ; Hampl, Vladimír (advisor) ; Kostka, Martin (referee)
Oxymonads (Excavata, Preaxostyla) are a group of anaerobic endobiotic flagellates living primarily in guts of xylophagous insects (cockroaches and termites). Some representatives of the genus Monocercomonoides belonging to the morphologically simplest family Polymastigidae have been described also from the guts of vertebrates. Oxymonads are a group of protist in which mitochondrion has not been proven yet. In this work, we have sequenced gene fo SSU rRNA of two strains of Monocercomonoides and performed phylogenetic analysis of oxymonads. Five selected strains Monocercomonoides isolated from different hosts and distantly related on the phylogenetic tree were studied by using light (DIC and protargol staining) and transmission electron microscopy. The aim was to find differences between these strains. We have focused primarily on the morphology of the nucleus, appearance of the endoplasmic reticulum and structure of the fibre R1. We were able to distinguish the strains by the position of karyosome (central and parietal) and the number of microtubules in the R1 fibre (6-12) and we assume that they represent separate species. The phylogenetic position and appearance of the strains NAU3 were so different that it may represent a new genus. Mitochondria or double-membrane bounded organelles have not been...
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Pathogenic trichomonads in cats and dogs
Vobořilová, Pavlína ; Tachezy, Jan (advisor) ; Hampl, Vladimír (referee)
Trichomonads are anaerobic flagellated protists that are either parasites or commensals. They frequently inhabit digestive, respiratory, and urogenital tracts of vertebrates, including domestic cats and dogs. In these hosts, four trichomonad species has been described: Tetratrichomonas canistomae and Tetratrichomonas felistomae that are commensals of the host oral cavity; Pentatrichomonas hominis, a commensal of intestinal tract that could be found in dogs and cats but also in other mammals including humans; and pathogenic Tritrichomonas foetus that causes, in addition to cattle infection, feline intestinal trichomonosis. Although, trichomonads in dogs and cats are probably of cosmopolitan distribution we have no information about their presence in Czech Republic. The first aim of this study was to distinguish types of trichomonads present in the oral cavity of dogs and cats and to get preliminary epidemiological data. The second aim was to demonstrate the presence of Tritrichomonas foetus in cats and dogs in the Czech Republic and to identify potential risk factors for the disease. Cultivation and nested PCR were used to determine the presence of trichomonads in dogs and cats. Sequencing and phylogenetic analysis based on ITS1-5.8rRNA-ITS2 gene sequence was used to identify species of isolated...
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Membrane proteome of euglenid plastid
Vanclová, Anna ; Hampl, Vladimír (advisor) ; Füssy, Zoltán (referee)
Euglenophyta are monophyletic group of euglenids defined by presence of green, three membrane- bound plastid which has been aquired via secondary endosymbiosis with chlorophyte alga. Mechanism of transport of nuclear-encoded proteins into this plastid is not yet completely understood. It was observed that the proteins are transported to the outermost plastid membrane in vesicles passing through ER and Golgi, but the mechanism of their recognition and fusion with the target membrane remains unclear. Translocation system of inner two membranes is still completely unknown, regarding the situation in other plastids, it has been proposed that homologues of TOC and TIC complexes are present. In this work we analyzed sequence data from proteome of isolated plastid membranes of model organism Euglena gracilis and transcriptome of E. gracilis and its distant relative Eutreptiella gymnastica. We studied whether they contain proteins potentially involved in transport and homologues of proteins of transport systems known from plastids in other organisms (TOC/TIC, ERAD-like transport, SNARE). However, all our results are negative. It is hard to determine whether these findings indicate the possible absence of TOC and TIC complexes in euglenid plastid, or rather the insufficiency of our data. Powered by TCPDF (www.tcpdf.org)
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The complicated evolution of methionine adenosyltransferase in euglenids and eukaryotes in general
Szabová, Jana ; Hampl, Vladimír (advisor) ; Krajčovič, Juraj (referee) ; Sanchez-Perez, Gabino (referee)
Many eukaryotic genes do not follow vertical inheritance pattern. In the present work, we have chosen as a model the gene for methionine adenosyltransferase (MAT), in which we have decided to examine in detail the evolutionary history. MAT is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogs: MAT and MATX. Both paralogs have punctate distributions across the tree of eukaryotes and, except for a few cases, they are mutually exclusive. This points to the complicated evolutionary history of this gene couple, which may be caused by either differential loss of old paralogs or the spread of one of these paralogs by horizontal gene transfer (HGT). We have focused on the evolution of this enzyme particularly within one of the best-known groups of flagellates, the euglenids, because it was hypothesized that MATX evolved in photosynthetic euglenids before it spread to other lineages. We gained 26 new sequences from 23 euglenid lineages and one prasinophyte alga Pyramimonas parkeae. MATX was found only in photoautotrophic euglenids. Both, mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae, the closest known relative of the euglenid plastid ancestor, only displayed the MAT paralog. In contrast, both paralogues were found in two euglenid species...
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The use of environmental sequencing in the studies on eukaryotic diversity
Lukešová, Soňa ; Hampl, Vladimír (advisor) ; Škaloud, Pavel (referee)
The bachelor's thesis focuses on environmental sequencing method and its usage in examining the diversity of microbial eukaryotic organisms. It describes the method, its mechanism, application and the problems associated with it. The work describes the current view of the phylogeny of the major eukaryotic lineages with their closer characteristics. Particular attention is paid to newly discovered groups. The work addresses the relationships inside these groups, summarizes findings of their ecology, food resources, and morphology. A separate chapter is devoted to the occurrence of the protist groups in extreme environmental conditions. Key words: Diversity of eukaryotes, environmental DNA, sequencing, rRNA, phylogenetics.
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Morphological and phylogenetic characterization of tetratrichomonads of group A
Krížová, Kateřina ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Trichomonads is a group of anaerobic single-celled eukaryotes currently classified in the kingdom Excavata (phylum Parabasalia), although does not share any of typically excavate features (the characteristic arrangement of the cytoskeleton and the ventral feeding groove). The taxonomy is based only on the molecular-phylogenetic analyses. Genus Tetratrichomonas contains probably the highest number of known species within the phylum Parabasalia, parasites and commensals of numerous vertebrate and invertebrate hosts. According to phylogenetic methods the genus has been divided into group A and B. This thesis is focused on the group A, which is split into 12 lineages (lineage 1 - 10, novel lineage 2, lineage LP). During our survey we discovered a new lineage - called sheep lineage, related to this group. Its position in group A is still unclear. Morphological differences between lineages have more or less confirmed the position of other lineages in phylogeny based taxonomy tree, which has been published previously. In this work, the differences between lineages have been described detailly. It is obvious that almost all lineages or groups of lineages are distinguishable from each other on the basis of morphological characters (size and shape of the body, the number of anterior flagella, parabasal body size,...
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Search for the remnant of plastid in the cell of Rhabdomonas sp.
Soukal, Petr ; Hampl, Vladimír (advisor) ; Oborník, Miroslav (referee)
Phylum Euglenozoa contsists of four groups - Euglenoidea, Kinetoplastea, Symbiontida and Diplonemea. Phototrophic euglenids, which constitute a clade, possess green plastid acquired via secondary endosymbiosis from green alga related to recent genus Pyramimonas. According to generally accepted plastid-late hypothesis, the endo- symbiosis took place after split between phototrophic euglenids and all other euglenozo- ans. Endosymbiotic event is always associated with gene transfer from endosymbiont to nuclei of host. Even if the endosymbiont is completely lost we should be able to observe enrichment of the host genome with the genes derived from endosymbiont. Some recent phylogenetic analyses uncovered genes related to green algae in trypanosomas (Kineto- plastea: Trypanosomatida). Based on this observation, authors postulated a hypothesis that the plastid was present already in common ancestor of kinetoplastids and euglenids and was lost in kinetoplastids and some euglenids including osmotrophic Rhabdomonas costata. During analysis of transcriptome of R. costata we found 63 genes, which could originated from green (24 genes) or other (49 genes) algae. In phylogenetic trees only one was robustly related to green and four were robustly related to other algae. Since the number of genes related to...
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