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Effect of pathogens on the vaginal microbiome
Procházková, Jana ; Tachezy, Jan (advisor) ; Smutná, Tamara (referee)
The human body is heavily populated with a wide variety of microorganisms. These microorganisms form coherent communities, which in contact with specific human organs form microbiomes. Each microbiome is unique in its composition. The study of microbiomes enables us to describe their composition and to understand the complex interactions that occur between microorganisms and humans. Methods of microbiome analysis are constantly evolving. For example, next-generation sequencing is currently the most widely used method for the characterization of microbiomes. Bacteria of the genus Lactobacillus are the major component of a healthy vaginal microbiome in most women. A healthy vaginal microbiome is essential for maintaining the natural homeostasis and to assure a proper functioning of the female urogenital tract. Lactobacillus is also important for the protection of the vagina from pathogenic agents such as viruses, bacteria, archaea and eukaryotes. In this bachelor's thesis the major pathogens of the Bacteria and the Eukaryota domain are described. Hereby a particular focus is on Trichomonas vaginalis, which is the most prevalent pathogenic agent of the female urogenital tract worldwide. Studying the immunity of the vaginal microbiome to this parasite is therefore essential. Furthermore, the mechanisms of...
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Double expression systems with respect to parasitic protozoa
Gromyko, Anastasiia ; Verner, Zdeněk (advisor) ; Kraeva, Natalia (referee)
Protozoan parasite infections continue to pose a significant health challenge in developing countries, resulting in hundreds of thousands of deaths each year. These parasites exhibit a complex multi-stage life cycle and possess unique cellular structures. However, many of their biological processes remain poorly understood. Multigene expression is a promising approach to address this knowledge gap, as it enables the expression of functional protein complexes in vivo, the addition of fluorescent protein tags for visualization of protein localization within the cell, and the study of protein-protein interactions. This bachelor's thesis reviews the current knowledge on available systems and approaches for studying key model parasitic protozoan species. Keywords: expression systems, Trypanosoma brucei, Leishmania tarentolae, Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Toxoplasma gondii, Plasmodium falciparum, pET-Duet, CRISPR-Cas9
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Effect of endosymbionts on composition and properties of Trichomonas vaginalis exosomes
Zdrha, Alois ; Tachezy, Jan (advisor) ; Černý, Jan (referee)
Trichomonas vaginalis is a parasite of the human reproductive tract and the causative agent of trichomoniasis, the most spread non-viral sexually transmitted disease. Although trichomoniasis usually has mild symptoms, it can lead to serious adverse effects. The course of the disease is influenced by the host immune system, microbiome and virulence of the parasite. Importantly, the virulence of T. vaginalis is extremely variable, and it depends on secreted and surface molecules. Among these are proteases and adhesins, which can be secreted as part of the secretome or through extracellular vesicles (EVs). Further potential virulence factors are the endosymbionts of T. vaginalis: Trichomonas vaginalis virus (TVV) and Mycoplasma hominis. Although extensively studied, no consensus on whether TVV exacerbates trichomoniasis has been reached. The aim of this thesis is to analyse the effect of TVV on the protein and RNA exosomal cargo and to assess the cytokines induced by exosomes of TVV-positive T. vaginalis. Key words endosymbionts, dsRNA virus, TVV, exosome, parasite, Trichomonas vaginalis, bioinfor- matics
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The role of extracellular vesicles in transmission of dsRNA viruses in parasitic protist Trichomomas vaginalis.
Horáčková, Jana ; Rada, Petr (advisor) ; Voleman, Luboš (referee)
Trichomonas vaginalis is a parasitic protist found in the human urogenital tract, where it adheres to the epithelial cells. During infection, T. vaginalis cells release extracellular vesicles that allow them to communicate with each other and modulate the host immune response. Most studied T. vaginalis strains contain dsRNA Trichomonasviruses (TVVs). These viruses belong to the Totiviridae family, which includes other dsRNA viruses from various parasitic protists. TVV is transmitted vertically to the daughter cells during cell division. Horizontal transfer of TVV between T. vaginalis cells has not yet been proven. TVV particles were found inside small extracellular vesicles that release infected T. vaginalis into the extracellular space. In this thesis, it was shown that TVV could be transmitted between trichomonads during co-cultivation of infected and uninfected trichomonads. However, stable TVV infection of trichomonads was not observed. It was also proven that extracellular vesicles from infected trichomonads could transmit TVV among other trichomonads. Nevertheless, this way of transmission did not lead to the stable TVV infection of trichomonads either.
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Organelle proteomics of parasitic protists
Jedelský, Petr ; Tachezy, Jan (advisor) ; Kolářová, Libuše (referee) ; Půta, František (referee)
Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles in G. intestinalis mitosomes, notparticipating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one and twodimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...
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Pyruvate:ferredoxin oxidoreductase homologues from Trichomonas vaginalis hydrogenosomes
Zedníková, Věra ; Hrdý, Ivan (advisor) ; Doležal, Pavel (referee)
Oxidative decarboxylation of pyruvate is a fundamental reaction of living organisms in general, leading to energy conservation. In some anaerobic or microaerophilic eukaryotic or prokaryotic organisms pyruvate decarboxylation is carried out by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFO). PFO contains Fe-S clusters and thiamin pyrophosphate cofactor (TPP). In the reaction catalyzed by PFO, from pyruvate and Co-A arise acetyl-CoA, CO2, and two electrons are released. Those electrons are accepted by low molecular carrier proteins. Most frequently these proteins are ferredoxins or flavodoxins such as in nitrogen fixating bacteria. PFO can perform a reversible reaction. Trichomonads are mostly parasitic or endosymbiotic organisms with mitochondria-like organelles, hydrogenosomes. These organelles possess PFO which is one of the key enzymes in the metabolism of Trichomonas vaginalis hydrogenosomes. PFO of T. vaginalis, a sexually transmitted pathogen of man, plays also a role in a term of medical importance. PFO is, by a universally accepted concept, one of the key proteins acting in the activation of antimicrobial drugs against trichomoniasis 5-nitroimidazoles, including metronidazole. In the genome of T. vaginalis seven PFO genes were identified. They were named PFO A, B1, B2, C, D, E and...
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Protein translocation into hydrogenosomes of "Trichomonas vaginalis"
Radhakrishna Makki, Abhijith ; Tachezy, Jan (advisor) ; Hashimi, Hassan (referee) ; JACKSON, Catherine Lynn (referee)
Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...
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Analysis of lysosomes of Trichomonas vaginalis
Zimmann, Nadine ; Tachezy, Jan (advisor) ; Walochnik, Julia (referee) ; Field, Mark (referee)
Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode...
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Protein translocation into hydrogenosomes of "Trichomonas vaginalis"
Radhakrishna Makki, Abhijith ; Tachezy, Jan (advisor) ; Hashimi, Hassan (referee) ; JACKSON, Catherine Lynn (referee)
Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...
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Organelle proteomics of parasitic protists
Jedelský, Petr ; Tachezy, Jan (advisor) ; Kolářová, Libuše (referee) ; Půta, František (referee)
Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles in G. intestinalis mitosomes, notparticipating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one and twodimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...
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