|
|
Metabarcoding and environmental sequencing projects
Kandaurova, Ekaterina ; Hampl, Vladimír (advisor) ; Poláková, Kateřina (referee)
The thesis explores the use of metabarcoding in projects uncovering biological diversity and its applications in various fields of biology. The thesis provides an overview of the principles and workflow of metabarcoding, including the choice of specific molecular markers for various groups of organisms and the bioinformatics aspect of data analysis. Limitations of metabarcoding, such as lack of standardization, primer bias and targeting metabolically inactive organisms are discussed along with potential strategies for overcoming these obstacles. The practical application of metabarcoding is demonstrated on examples of its use in sequencing projects, such as Human Microbiome Project or TARA Oceans expedition. The potential of using metabarcoding for research purposes is discussed from the perspective of paleontology, diet analysis, and conservation biology. Overall, this work aims to assess the potential of metabarcoding as a powerful tool and could serve as an initial guidance for researchers interested in utilizing metabarcoding for their scientific investigations. Keywords: metabarcoding, eDNA, molecular markers, NGS, bioinformatics, biodiversity.
|
|
|
Investigation of the genome sizes, karyotype and ploidy within the genus Monocercomonoides
Kornalíková, Martina ; Hampl, Vladimír (advisor) ; Čepička, Ivan (referee)
Oxymonads are a group of anaerobic flagellated protists living mainly in the gut of insects and vertebrates. Here, we invetigate the ploidy and karyotype of various strains of Monocercomonoides using Fluorescence in situ Hybridization (FISH). For ploidy we used probes against the SufDSU gene, which is known to be present in a single copy in the genome of M. exilis. For karyotype investigations we used FISH probes against the TTAGGG telomeric repeat sequence. The results showed that all the investigated strains are haploid with a single clear signal from our SufDSU probes. The karyotype analyses showed that the average number of signals per nucleus varied between 9 to 17 in most of the investigated strains, indicating number of chromosomes similar to M. exilis. A notable exception from this are the strains of M. mercovicensis, where we observed a much higher number of signals, suggesting much higher number of chromosomes. For estimating of the DNA content in the nuclei of these oxymonads we used the flow cytometry, with M. exilis as a standard. Our results indicate that two of the investigated strains, Monocercomonoides sp. strain OEV and Mural1, have smaller genome sizes than M. exilis, while the rest of the strains have genomes sizes larger than M. exilis. We observed an unusually large variation...
|
|
|
Intermediate filament proteins of Preaxostyla flagellates
Švagr, Ezra ; Hampl, Vladimír (advisor) ; Varga, Vladimír (referee)
5 Abstract Monocercomonoides exilis and Paratrimastix pyriformis are protists from within the group Preaxostyla (Metamonada), they possess an excavate morphology that is presumed to be an ancestral cytoskeletal organisation of eukaryotic cells. A significant part of eukaryotic but specifically excavate morphology is fibers composed of unidentified proteins. The hypothesis on which this thesis builds upon is that these fibers are composed of Intermediate Filament proteins (IF proteins). IF proteins are a polyphyletic group of proteins involved in the assembly of mechano-elastically important fibers in eukaryotes. The most widespread group of these proteins is a family called SF-assemblins, homologues of which were identified first in Chlamydomonas reinhardii, and giardins a protein family first discovered in Giardia intestinalis, which were also found to be related to this group. Nested into the bigger hypothesis is an idea, that SF-assemblins are present in M. exilis and P. pyriformis, further strengthening their position as a universally present eukaryotic feature. The goal of this work was to find support for the morphological hypothesis that LECA possessed an excavate morphology in protein composition of the cytoskeleton. Two approaches were employed. First, identifying proteins in cytoskeletal fraction...
|
|
|
Quantitative Analysis of Gene Ancestry in Euglenoidea
Soukal, Petr ; Hampl, Vladimír (advisor) ; Burki, Fabien (referee) ; Horák, Aleš (referee)
Genes, units of genetic information, are passed from one individual to another, typically within the same species from parents to offspring. Occasionally, genes can be transferred to different species through a process called horizontal gene transfer (HGT). Endosymbiosis is a process in which one organism engulfs another organism and the genes flow between the two symbionts in a process called endosymbiotic gene transfer (EGT), which is a subtype of HGT. Eventually, the endosymbiont may become an organelle such as a mitochondrion or a plastid. Euglenids (Euglenoidea) are unicellular eukaryotes with diverse modes of nutrition - phagotro- phy (e. g., Peranema), osmotrophy (e. g., Rhabdomonas), mixotrophy (phagotrophy and phototrophy in Rapaza), and phototrophy (possessing plastids; e. g., Eutreptiella). The history of endosymbioses of green algae (especially Chlorophyta) and ancestors of Euglenophyceae (including Rapaza) is complex. In my thesis, I performed the quantitative analysis of gene ancestry (QAGA) based on transcripto- mic data of Euglena gracilis showing contribution of closely related Kinetoplastea (1,420 genes, 3.88 % of transcripts inherited vertically), and contribution of multiple "unrelated" (mainly) phototrophic organisms, e. g. Viridiplantae (572, 1.57 %), or Haptophyta (234, 0.64...
|
|
|
Characterization of Euglena gracilis and their role in metaboly movement
Šmída, Adam ; Hampl, Vladimír (advisor) ; Pergner, Jiří (referee)
Euglena gracilis is a free-living freshwater flagellate from the class Euglenida (Euglenozoa, Discoba, Excavata). The synapomorphy of Euglenida group is their pellicle, a firm layer underneath the surface of the cell which is composed of proteinaceous stripes (pellicle strips). The strips are locked into each other and run along the cell`s surface underneath the plasma membrane. In E. gracilis these strips can move by parallel sliding, therefore contracting the cell and changing its surface which results in a characteristic peristaltic euglenoid movement called metaboly. The mechanism behind this movement is still unresolved, however, it is expected that the force is generated by the cytoskeletal structures beneath the pellicle strips which consist of microtubules and other unknown cytoskeletal fibers. This thesis tests a hypothesis which proposes that centrins are involved in the pellicle contraction. Centrins are small proteins capable of binding calcium ions, they are very conserved throughout the eukaryotic tree and serve many purposes in the cell, of which one is to form contractile structures in ciliates species e.g., Vorticella or Paramecium. Sequences of centrins were searched for in the transcriptome of E. gracilis and after comparison with the available proteomic dataset of the isolated...
|
|
|
Evolution of the genetic code and classification of oxymonads
Šrámová, Eliška ; Hampl, Vladimír (advisor) ; Čepička, Ivan (referee)
Oxymonads are a group of heterotrophic flagellates living in low oxygen environment. These protists inhabit mainly the gut of xylophagous insects (cockroaches, termites), with an exception of the genus Monocercomonoides, which was described from the intestinal contents of many vertebrates. On the basis of molecular data, Oxymonadida are classified into the supergroup Excavata (Cavalier-Smith, 2002; Simpson et al., 2006, Hampl et al. 2009). This thesis was focused on the diversity of genus Monocecomonoides from the morphologically simplest family Polymastigidae. The main goal of our work was to gather sequence data from strains isolated from a wide spectrum of hosts. We have obtained 26 partial sequences of the gene for the SSU rDNA in total, of which two belonged to another oxymonad, apparently genus Oxymonas. Our phylogenetic analysis indicated that the representatives of the genus Monocercomonoides form one group, however with a low bootstrap support. On the basis of published data about the presence of non-canonical genetic code in some oxymonads (Keeling and Leander, 2003; de Koning et al., 2008), we decided to explore this rare phenomenon in representatives of the genus Monocercomonoides. For this part of the study we gathered 9 partial sequences of α-tubulin gene. In these sequences we have not...
|
|
|
Mitochondrion of Trimastix pyriformis
Novák, Lukáš ; Hampl, Vladimír (advisor) ; Kolísko, Martin (referee)
2013 DIPLOMOVÁ PRÁCE Lukáš Novák Abstract Free-living microaerophilic protist Trimastix pyriformis is closely related to oxymonads which are the largest eukaryotic group without any known mitochondrion. In contrast to oxymonads, an enigmatic reduced mitochondrion has been found in the cell of T. pyriformis. In EST data of T. pyriformis, a number of genes has been identified whose products are putatively localized in the mitochondrion. Among these are genes for all the components of the glycine cleavage system, [FeFe]hydrogenases and the mitochondrial marker Cpn60. We performed experiments in order to determine the cellular localization of these proteins. Our results show that the glycine cleavage system is localized in the mitochondrion. Results of the experiments carried out in order to localize two hydrogenases suggest also the mitochondrial localization but are not fully convincing. The attempt to localize Cpn60 has failed. We have also identified a set of new genes in transcripts of T. pyriformis and Monocercomonoides sp. (Oxymonadida). These genes code for some components of the SUF system of FeS cluster synthesis and a peroxidase rubrerythrin. Key words: Trimastix, Monocercomonoides, mitochondrion, hydrogenosome, mitosome, hydrogenase, glycine cleavage system, SUF system.
|
|
|
Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
|
|
|
Transfer of genetic information between parasite and its host
Soukal, Petr ; Hampl, Vladimír (advisor) ; Votýpka, Jan (referee)
Horizontal gene transfer (HGT) is considered a rare evolutionary event. It can take place between unrelated organisms that coexist in an intimate symbiotic relationship. Such relationship have some parasites with its host. HGT between eukaryotic parasite and its host was identified in some holoparazitic and hemiparazitic plants, the most important human protozoan parasites, microsporidias, human blood-flukes, parasitoids and fruit flies.
|
|
| |
guest ::
