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Proteomic Analysis of Trichomonas vaginalis hydrogenosone
Campo Beltran, Neritza ; Tachezy, Jan (advisor) ; Nohýnková, Eva (referee) ; Yarlett, Nigel (referee)
Trichomonas vaginalis is a human pathogen that affects annually approximately 258 million people worldwide. This parasite possesses organelles of mitochondrial origin called hydrogenosomes, which generate ATP under anaerobic conditions. The identification of the protein content at the subcellular level may provide new targets for antiparasitic drugs developments as well as it contributes for our understanding of the organelles function and evolution. The availability of protocols for organelles purification and the complete genome sequence allow the study of the organellar proteomes using mass spectrometry and bioinformatics, providing a powerful strategy that combine cell biology and proteomics. In our research, we used several approaches to identify the protein composition in hydrogenosomes and mitosomes. We performed transcriptomic and proteomic analysis to investigate the molecular responses of Trichomonas vaginalis upon iron availability. Furthermore, the changes in the proteome during the development of metronidazole resistance were also studied. The organelles separated by differential and Optiprep-sucrose gradient centrifugation were analyzed with nano- RP-HPLC/MALDI-TOF/TOF. We also used Triton X-114 phase partitioning to separate membrane proteins and iTRAQ technique to label the peptides...
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Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.
Voleman, Luboš ; Doležal, Pavel (advisor) ; Nohýnková, Eva (referee)
SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...
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Nuclei of Giardia intestinalis
Krížová, Kateřina ; Nohýnková, Eva (advisor) ; Jiráková, Klára (referee)
4 Abstract Giardia intestinalis is a protozoan parasitic organism early diverging from common ancestor of all eukaryotes. That is why many distinctions of structure and ongoing processes occure in Giardia. Although the current findings suggest that a number of features and simplicity may be given to adaptation of the parasitic way of life, Giardia still remains a unique model, which may indicate something about many of the "core" processes that define eukaryotic cells. This paper summarizes a lot of known information related to two morphologically similar nuclei of Giardia. The nuclei have been compared both with each other and with the general knowledge of the eukaryotic nucleus. Although initially the nuclei were considered to be exactly the same, more recent studies and researches have brought plenty of evidence that they differ from each other. The main difference is basically the unequal content of chromosomes in each nucleus. Other differences consist in the number and deployment of nuclear pores or in the synchronization of the cell cycle. Special type of mitosis occurs there as well. Even though many claims have been clarified and refuted since the first study confirming the identity of nuclei was published in 1990, a lot of information needed for a complete understanding of the functioning of the...
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Giardia intestinalis karyotypes
Hudosová, Lenka ; Nohýnková, Eva (advisor) ; Král, Jiří (referee)
Giardia intestinalis is a parasitic protist that causes one of the most common diarrheal disease of parasite origin. The cell of Giardia contains two nuclei with unknown number of chromosomes until recently. Karyotype was determined five years ago using conventional cytogenetic method by Tůmová and collaborators. In my work, I assessed karyotype of four isolates, six lines and three clonal lines by the same method. It was confirmed, that two nuclei within one cell could differ in chromosome number, the differences found were 1, 2 or 6 chromosomes. Aneuploid number of chromosomes was found too. In case that both nuclei within single cell contained the same number of chromosomes, there were 10 chromosomes indentified in each nucleus. It was also revealed, that karyotype is not specific feature for different genetic groups (in this work assemblages A and E). Karyotype can be different even among lines and clonal populations derived from the same isolate. Changes in karyotype in the course of in vitro cultivation were detected within three populations. Results are discussed in relation to known facts.
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Leishmania infantum in south Turkey
Bublíková, Jitka ; Nohýnková, Eva (referee)
Leishmaniasis is a serious disease caused by protozoans of the genus Leishmania. It occurs in different levels of severity from mild, where the parasite causes ulcers on the skin to severe, where the internal organs are attacked and can result in death of the patient. It is transmitted small bloodsucking insects of the genus Phlebotomus. Our research was focused on outbreak of cutaneous leishmaniasis in the Cukurova region in the south Turkey. For the causative agent, there was largely regarded Leishmania tropica. In our study, we demonstrated as the causative agent Leishmania infantum, which is often described in visceral form. The negative result of rK39 test confirmed non-visceral form of the disease. L. infantum was also detected in the most abundant sand fly species, Phlebotomus tobbi. We have shown that some P. tobbi were engorged with human blood and any sand fly did not contain dog's blood. Therefore, we assume anthroponotic cycle in the focus, even L. infantum is usually a zoonosis. In the rigorous thesis, I tried to summarize the current knowledge about the outbreak of cutaneous leishmaniasis in the Cukurova region. Follow-up studies confirmed the abundance of P. tobbi through the season, but also revealed the high incidence of P. perfiliewi during the season. It could be also involved in...
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Encystation and life cycle of free living amoebae of the genus Acanthamoeba spp.
Bínová, Eva ; Nohýnková, Eva (advisor) ; Dyková, Iva (referee) ; Ondriska, František (referee)
Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...
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Cell cycle and differentiation in Giardia Intestinalis
Jiráková, Klára ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee) ; Müller, Norbert (referee)
Giardia is a unicellular parasitic organism; it is a worldwide cause of human diarrhea. It has minimalistic genome equipment and simplified molecular and metabolic pathways. In this respect, it is a suitable model organism for studying cell cycle regulation and to define the minimal genetic and protein equipment required for the functional reproduction of the eukaryotic cell. Its life cycle comprises of two stages; a pathogenic trophozoite and an infective cyst, which can survive in outer environment. New knowledge about encystation can be therapeutically important because this process is a target for vaccine and drug development. Since cell cycle analysis requires a synchronized population, we studied the effect of the synchronization drug aphidicolin on individual cell characteristics during the cell cycle of Giardia trophozoites. Our results showed that aphidicolin caused inhibition of DNA synthesis and trophozoites were aligned according to their DNA content in G1/S border. Subsequent inhibition of entry into mitosis and cytokinesis indicates, that Giardia has functioning DNA damage checkpoint. Extensive treatment with aphidicolin causes side effects. We detected positive signals for phosphorylated histone H2A which, in mammalian cells, is involved in a signaling pathway triggered as a reaction...
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Current treatment options of intestinal and extra-intestinal amebiasis.
Pastyříková, Aneta ; Nohýnková, Eva (advisor) ; Stejskal, František (referee)
Amoebiasis is a parasitic infection caused by the pathogenic intestinal amoeba Entamoeba histolytica, which multiplies as a minute form in the human lumen of the colon. Under not yet defined conditions, the minute form can be transformed into a magna form that attacks the colon wall and is able to spread out of the intestine by blood and infect internal organs, especially the liver. Amoebiasis is primarily treated with chemotherapeutics, which are divided into luminal and tissue depending on the site of action. Luminal drugs target the stages of E.histolytica in the lumen of the intestine and are therefore used to treat asymptomatic and symptomatic forms of intestinal amoebiasis. Tissue drugs, especially the 5-nitroimidazole derivative, metronidazole, which is the drug of choice in the treatment of amoebiasis, penetrate the tissues and are used to treat invasive intestinal and extra-intestinal infections. If chemotherapy is ineffective or there is a risk of rupture of abscess and sepsis, surgical treatment is performed. This thesis summarizes therapeutic approaches to individual clinical forms of amoebiasis, possibilities of chemotherapy at present, but also before the use of 5-nitroimidazole derivates, and alternatives to current drugs. Key words Intestinal amebae of humans, Entamoeba histolytica,...
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Maintenance of chromosomes integrity in Giardia intestinails as a model organism.
Uzlíková, Magdalena ; Nohýnková, Eva (advisor) ; Lalle, Marco (referee) ; Stejskal, František (referee)
Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...
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Free living amoebae as causative agents of human infections.
Markovičová, Ivana ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee)
Free living amoebae as causative agents of human infections are world wide occuring organisms with the ability of dual lifestyle both in nature, which is their original habitat, so within host tissue. The aim of this theses is to summarize the basic available knowledge of the pathogenic species Naegleria fowleri and Balamuthia mandrillaris and of the pathogenic genus Acanthamoeba which invade CNS and are causative agents of rare but serious human infections, often leading to death of a patient. The exception of their deathly consequences is an Acanthamoeba keratitis infection in which non curing cases lead to a significant visual impairment and even to visual loss. In each of the chapters, the work is tend to summarize transmision ability, mechanisms of pathogenesis and pathophysiology of these three free living amoebae in order to describe their ability to cause infection.
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