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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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Diagnostic and therapeutic approaches to infections caused by free-living amoebae pathogenic to humans
Štofaniková, Petra ; Nohýnková, Eva (advisor) ; Stejskal, František (referee)
Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. are free-living amoebae which rarely cause human infections. N. fowleri induces primary amoebic meningoencephalitis mostly among children and young adults. B. mandrillaris is the causative agent of Balamuthia amoebic encephalitis and skin lesions with complicated healing. Amoebas of the genus Acanthamoeba are the ethiologic agent of focal granulomatous encephalitis. Its clinical presentation is almost the same as in Balamuthia encephalitis, but it occures mainly in immunocompromised patients. It can also affect lungs and skin. The infection of central nervous system usually has a fatal end. In immunocompetent people Acanthamoeba keratitis can occur. Unproper use of contact lenses or trauma of the cornea represents increased risk level. Without correct treatment Acanthamoeba keratitis can lead to sight loss. In all infections caused by free-living amoebae, early diagnosis and initialisation of effective therapy are essential for better prognosis. This thesis discusses contemporary diagnostic and therapeutic approaches to these infections.
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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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Live cycle of the free-living amoeba. Differentiation of amoebae of the genera Acanthamoeba and Balamuthia
Klieščiková, Jarmila ; Nohýnková, Eva (advisor) ; Ondriska, František (referee) ; Walochnik, Julia (referee)
Free-living pathogenic amoebae Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of important diseases of human: rarely occurring but highly fatal granulomatous amoebic encephalitis (both) and keratitis (Acanthamoeba). One of the reasons for the problematic therapy is differentiation into highly resistant cysts often found in affected tissues. In our study we have found that correct encystation in Acanthamoeba requires apart from others, the presence of functioning Golgi apparatus transporting the cyst wall material to the cell surface; glycogen phosphorylase degrading glycogen into glucose which seems to be further used for cellulose synthesis and two non-constitutive cellulose synthases. Acanthamoeba cellulose synthases seem not to be inhibited by known herbicides. In the cyst wall of acanthamoebae we detected cellulose, -mannan, and -1, 3-1, 4-linked glucan [lichenin or mixed-linkage glucan (MLG)]. Cellulose is present in the inner (endocyst) and the outer (exocyst) layers of the cyst wall, whereas-mannan and MLG are found in the endocyst. In a protozoan organism, MLG was detected for the first time. The MLG of Acanthamoeba has a similar composition to that found in barley with high amount of cellobiosyl and cellotriosyl followed by cellotetraosyl units. In contrast, with...
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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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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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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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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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