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Genomics of Preaxostyla flagellates
Novák, Lukáš ; Hampl, Vladimír (advisor) ; Hirt, Robert (referee) ; Van der Giezen, Mark (referee)
Protists inhabiting oxygen-depleted environments have evolved various adaptation to thrive in their niches, including modified mitochondria to various degrees adapted to anaerobiosis. The most radically altered forms of these organelles (Mitochondria-Related Organelles, MROs) have completely lost their genomes and other defining features of canonical aerobic mitochondria. Anaerobic protists are often found as endobionts (parasites, mutualists, etc.) of larger organisms. The endobiotic lifestyle combined with anaerobiosis poses another source of evolutionary pressure forcing unique adaptations in the endobionts. Here we present new insights into the adaptations of an anaerobic protistan phylum Preaxostyla, especially with regard to the reductive evolution of mitochondria, which, uniquely among all known eukaryotes, led to a complete loss of the organelle in the oxymonad Monocercomonoides exilis. We have obtained M. exilis genomic assembly of good quality and completeness, as well as genomic and transcriptomic data of varying quality and completeness from 9 other Preaxostyla species. Based on extensive, thorough gene searches and functional gene annotation on these datasets, as well as phylogenetic analyses and protein localization experiments, we conclude: 1) M. exilis has completely lost the...
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Evolution of euglenid plastid proteome
Novák Vanclová, Anna ; Hampl, Vladimír (advisor) ; Gruber, Ansgar (referee) ; Maier, Uwe (referee)
Endosymbiotic gain and transfer of plastids is a widespread evolutionary phenomenon and a major driving force of eukaryotic evolution. The integration of a new organelle is accompanied by changes in its structure, gene content, molecular mechanisms for biogenesis and transport, and re-wiring of the host and organelle metabolic pathways. To understand the course and underlying mechanisms of plastid evolution, it is important to study these processes in variety of secondary algae and notice their differences and similarities. Euglenophytes gained their plastids from green eukaryotic algae after a long history of heterotrophic lifestyle. In my thesis, I participated in analyses of newly generated sequence datasets: transcriptomes of Euglena gracilis and Euglena longa and mass spectrometry-determined proteome of E. gracilis plastid with especial regard to the potential novelties associated with plastid gain and incorporation. In the resulting publications we particularly focus on plastid protein import machinery and targeting signals and report extremely reduced TIC and completely absent TOC in euglenophyte plastid. Using the proteomic dataset, we predict potential novel plastid protein translocases recruited from ER/Golgi and re-analyze plastid signal domains, characterizing previously overlooked...
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Analyses of Monocercomonoides genome sizes, ploidies and karyotypes
Kornalíková, Martina ; Hampl, Vladimír (advisor) ; Krylov, Vladimír (referee)
Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...
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Bacterial populations in mucosal tissues of the house mouse
Ptáčníková, Aneta ; Stopka, Pavel (advisor) ; Hampl, Vladimír (referee)
Microbiota becomes one of the most important subjects in biological research and numerous studies revealed that microbiota plays a broad spectrum of essential roles in different organisms. This master thesis focuses on the bacterial part of microbiota contained in mucosal tissues of wild house mice (Mus musculus musculus). Male and female samples were collected by nasal and oral cavity lavages, vaginal mucosa lavages and from urine and stool. We aimed to detect quantitative, qualitative and sex-specific differences in bacterial populations between mucosal tissues with particular focus on bacterial cycling in vaginal mucosa during the estrous cycles. Bacterial abundances were estimated by qPCR whilst bacterial diversity was detected by targeted metagenomic sequencing of the hypervariable region of the 16S rRNA gene. Significant differences were detected in bacterial abundances and alpha diversity between particular mucosal tissues. Stool samples contained the highest number of bacteria, while samples from the nasal mucosa and urine contained low amount of bacteria. The highest alpha diversity was discovered in stool samples, the least alpha diversity was found in the urine. Mucosal tissues also varied based on the bacterial composition on the level of particular genera. Detailed analysis of estrous cycles...
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Bioinformatic methods of detection of protein coevolution
Pařízková, Hana ; Schneider, Bohdan (advisor) ; Hampl, Vladimír (referee)
The term coevolution describes the situation when two or more species or biomole- cules reciprocally affect each others' evolution. On the protein level, it is thought to be the main mechanism ensuring correct folding, interactions and function of a protein, and it can be observed both on the level of interacting protein families and individual amino acid residues. Coevolution studies have been proved to be a powerful tool for prediction of protein structure, function, interaction partners, etc. In this thesis, different algorithms used for detection of protein coevolution are described, as well as their applications and limitations. Keywords: coevolution, protein family, protein structure prediction, interac- tion partners, correlated mutations, mirrortree, mutual information, direct cou- pling analysis
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Iron-sulphur cluster synthesis in anaerobic protists
Zelená, Marie ; Hampl, Vladimír (advisor) ; Mach, Jan (referee)
Iron-sulpur clusters are small ubiquitous cofactors of proteins present in all cells. These proteins participate in many important processes such as gene expression or respiratory chain. In vivo synthesis of iron-sulphur clusters requires complex biosynthetic pathways. In eukaryotic cells these pathways are localized inside mitochondria and plastids and in the cytosol. Cytosolic synthesis depends on a product of the mitochondrial pathway. Mitochondria of anaerobic protists in most cases went through reductive evolution, which is associated with the reduction of mitochondrial iron-sulphur cluster biosynthetic pathway. In many cases, these organisms acquired alternative pathways via lateral gene transfer, which act either as a complement to the mitochondrial pathway, or as its complete functional substitution. Replacement of the mitochondrial pathway might have been a key event for secondary loss of mitochondria. Key words: Iron-sulphur clusters, anaerobic protists, ISC, CIA, SUF, mitochondrion
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Losses of mitochondria and plastids in the evolution of eukaryotes
Trokšiar, David ; Hampl, Vladimír (advisor) ; Hadariová, Lucia (referee)
- 5 - Abstract: Mitochondria and plastids were acquired by endosymbiotic event, where prokaryotic organism was engulfed by ancestors of extant eukaryotes. There are more known endosymbiotic events in plastid evolution. In primary endosymbiosis cyanobacterium cell was engulfed by heterotrophic eukaryotic organism. In following secondary, tertiary and quaternary endosymbiotic events eukaryotic cell was engulfed by another eukaryote. Mitochondria originated by engulfment of α-proteobacteria. In the evolution of eukaryotes, reduction of mitochondria occurred in many lineages, making living under anaerobic conditions possible. The least reduced form is anaerobic mitochondria, which together with aerobic mitochondria and hydrogen producing mitochondria, possess genome. Hydrogenosomes and the most reduced form mitosomes, does not possess genome. Plastid reductions led to loss of photosynthetic ability. In last years, more examples of organisms that lost entirely their semi- autonomous organelle, are coming. Loss occur at two parasitic representatives of the Alveolata group, and one endobiotic oxymonad. Parasites Cryptosporidium parvum and Hematodinium lost nonphotosyntetic plastid, whereas Monocercomoides lost its mitochondria. Semi-autonomous organelles were dispensable, because all representatives have access to...
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Prokaryotic symbionts of free-living anaerobic protists
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Anaerobní prvoci jsou organismy obývající ředí bez přítomnosti kyslíku. Najdeme je anoxických habitatech jako jsou mořské a sladkovodní sedimenty, komunální skládky, nesmíme opomenout zástupce žijící v bachoru přežvýkavců, trávicím traktu švábů a dalších živočichů. Většinou mají anaerobní deriváty mitochondrií, často hydrogenosomy, organely produkující vodík. Metabolismus anaerobních prvoků je ve srovnání s aerobními organismy méně efektivní Časté interakce anaerobních ický my však mohou Symbiózy mezi anaerobními běžné a vznikly u zástupců mnoha anaerobních linií. žít buď endosymbioticky, uvnitř buňky hostitele, nebo ektosymbioticky, na povrchu hostitelské buňky. Dvě ekologicky významné skupiny prokaryot si dokázaly osvojit život symbiotickém vztahu volně žijícími anaerobními prvoky anogenní archebakterie, osídlující hlavně , využívají vodík nické sloučeniny k anu a síran redukující bakterie, žijící hlavně na povrchu buněk, využívají vodík a různé organické sloučeniny k redukci síranu na sulfan. Velmi málo se ví o bližším charakteru těchto vztahů druhové identitě a hostitelské specifitě prokaryotických symbiontů. Další výzkum je nutný pro pochopení fenoménu symbióz v anoxickém světě. Klíčová slova: anaerobní prvoci symbióza anogenní archebakterie síran redukující bakterie
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Prokaryotic symbionts of protists living in the intestine of wood eating cockroaches and termites
Kubánková, Aneta ; Hampl, Vladimír (advisor) ; Rotterová, Johana (referee)
Termites and wood-eating cockroaches are worldwide successful especially because of their ability to digest lignocellulose efficiently. After a long period of coevolution a complex ecosystem of diverse microorganisms was established in their hindgut. A lot of lineages of flagellates live solely within this unique environment and it is impossible to cultivate them in vitro. As termites are unable to survive without their eukaryotic endosymbionts, also protists are strongly linked to prokaryotes colonizing their cytoplasm and cytoplasmic membrane. Numerous phyla of bacteria participate in nitrogen fixation and efficient nitrogen utilization, synthesis of amino acids, cofactors and vitamines. Some of them take part in the degradation of lignocellulose. Representatives of another domain of prokaryotes, Archea, are important in the final stages of lignocellulose fermentation, because they utilize the produced hydrogen gas in a process of methanogenesis. Large part of our knowledge about the composition and function of the termite gut biota was acquired thanks to metagenomic studies.
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FISH method and its use in protistology
Frenclová, Martina ; Hampl, Vladimír (advisor) ; Zubáčová, Zuzana (referee)
Fluorescence in situ hybridization (FISH) is a technique that allows the localization and identification of specific sequences of nucleotides in DNA or RNA, which is subsequently visible under the microscope. FISH involves first denaturing the nucleic acids, either using high temperatures or by treatment with denaturing agents such as formamide.After subsequent induction of reasociation, the examined DNA or RNA pairs according to the complementarity rules with the short molecule called the probe, this process is called hybridization. Hybridization occurs in situ, that is within the examined specimen. Probes can be labeled either directly using fluorophores, or indirectly with a hapten, which is a substance having antigenic properties, which is subsequently detected using labeled antibodies or streptavidine. FISH has a large number of applications in molecular biology and medical science. In laboratory research in protistolgii FISH can be used for example to map the chromosomal genes to study the evolution of genome, analysis of nuclear organization or to confirmation of the origin of DNA sequence. Key words: FISH, fluorescence, probe, cell identification, environmental studies
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