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Dynamics of Giardia intestinalis encystation.
Vinopalová, Martina ; Doležal, Pavel (advisor) ; Verner, Zdeněk (referee)
Giardia intestinalis is an anaerobic parasite, that colonizes the small intestine of humans and other vertebrate hosts. This cosmopolitan parasite, which causes diarrhoea, is transmitted by contaminated water or food via a resistant stage, the cyst. The encystation process involves a number of events that lead to a complete reconstruction of the cell into the form of infectious cyst. The aim of this work was to visualize these modifications in vivo by means of enzymatic labelling of proteins. For the purposes of this work, enzymatic tags Y-FAST and HaloTag were chosen, as they enable visualizing live cells under anaerobic conditions. Chimeric protein constructs were created to visualize the dynamics of the encystation vesicles, the structures of endoplasmic reticulum, the adhesive disc and mitosis. Using the developed constructs, we successfully followed the dynamics of the encystation vesicles and the adhesive disc in vivo. Finally, this work has provided novel molecular tools, which will be used to follow the overall redesign of the parasite cell during encystation.
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Investigation of newly discovered protein GL50803_16424 in Giardia intestinalis.
Pelc, Josef ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
The anaerobic unicellular eukaryotic organism Giardia intestinalis is a worldwide parasite. Giardiasis, the intestinal disease caused by Giardia, is one of the most common parasitic disease in the developed part of the world, that causes health problems not only to humans but also to animals. This organism is also interesting for its many unique cellular features. One of them is the presence of mitosomes - the organelles derived from mitochondria. Analogously to mitochondria, mitosome is limited by two membranes and shares the mode of the protein transport. However, mitosome does not have its own genome and as far as we know, there is only one pathway of the iron-sulfur cluster biosynthesis in this organelle. Using the in vivo enzymatic tagging technique, several novel mitosomal proteins were identified, including GL50803_16424. The protein GL50803_16424 attracted our attention by interacting with components of all mitosomal subcompartments: the outer membrane, the membrane and the matrix. In addition, the expression of HA-tagged GL50803_16424 resulted in the formation of peculiar structures near the mitosomes never seen before in G. intestinalis. Bioinformatic approaches revealed that the GL50803_16424 has domain similar to the myelodysplasia- myeloid leukemia factor 1-interacting protein. Our...
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Biogenesis of Giardia intestinalis mitosomes
Voleman, Luboš ; Doležal, Pavel (advisor) ; Faso, Carmen (referee) ; Dawson, Scott C. (referee)
7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...
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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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Cellular Protein Transport and Its Role in Patogenesis
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Uzlíková, Magdalena (referee)
The main topic of this thesis are the protein secretion processes in several important human parasites - Toxoplasma gondii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. and Giardia intestinalis. Described here are the parasite's and the host proteins which participate in the pathogenic processes involving the protein secretion. As shown here, the protein secretion into the host environment is one of key tools serving the parasite to survive within and manipulate the host organism. Interestingly, different parasitic organisms use functionally and evolutionary distinct strategies to fulfill this aim. Key words secretory pathway, translocon, signal sequence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinalis
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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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Protein translocase in the mitosomes of Giardia intestinalis.
Fixová, Ivana ; Doležal, Pavel (advisor) ; Zubáčová, Zuzana (referee)
During the transformation of the bacterial endosymbiont into current mitochondria the protein import apparatus had to be created de novo. The reduced mitochondria (mitosomes) of the parasitic protist Giardia intestinalis represent unique cellular model for the examination of these fundamental transport processes. As the main objective of this project I will try to characterize the motor complex, which propels the protein transport, and also the translocation channel in the inner mitosomal membrane. To this aim I will exploit the presence of two membrane components Pam16 and Pam18, which were discovered in our laboratory, and which constitute the functional core of the motor complex. Based on the information from the analogous systems of yeast and mammalian mitochondria, these two components should physically interact with so far unknown translocation channel. In all other eukaryotes this channel is formed by a conserved protein Tim23. The absence of this protein in the genome of G. intestinalis suggests presence of completely novel, or maybe the original-bacterial protein. Having in hand this simplified mitochodrial model the project has potential to bring not only new data in parasite biology but also generate new information on the function and evolution of mitochondrial protein import.
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Genotyping of \kur{Giardia intestinalis} isolates
ŠRÁMOVÁ, Eliška
The aim of this work was assemble isolates of Giardia intestinalis from humans and other mammals. Stools samples were examined for presence of cysts by concentration settling method. Consequently sequencing of 532 bp parts of the TPI gene after previous amplification by the nested PCR was performed. In vitro cultures of selected isolates were established using experimental model hosts, gerbils.
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Prevalence of parasites of alimentary system in young cattle.
ŽIDKOVÁ, Marcela
In biennial following (spring 2005, autumn 2005, spring 2006, autumn 2006) we're rasitology examine 288 samples dropping from heifers and 288 samples dropping from bulls. Exhibits we're examined floatation in Sheather´s sugar solution. In examinate dropping we're proved present cysts Giardia intestinalis and oocysts Cryptosporidium andersoni and family Eimeria. In heifers was most frequent parasites coccidia Eimeria sp. (28,5 %). Flagellata Giardia intestinalis occur in 17 % of all designs. At least we're open up Cryptospordium andersoni (9,4 %). In bulls we're noted highest occurrence coccidia Eimeria sp. (19,1 %). Almost same occurrence we're found out near Giardia intestinalis (18,8 %). Very low prevalence we're have observed near Cryptosporidium andersoni (1,4 %).
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Prevalence of gastrointestinal parasites in calves reared indors and in hutches.
MOTTLOVÁ, Markéta
On two farms in a two-year period (spring 2005,autumn 2005, spring 2006, autumn 2006) calves faecal samples from the rectum or from the floor were obtained for parasitologic examination. A total of 560 samples from 209 calves were examined using the floatation method in Sheather's sugar solution. In positive samples the presence of Giardia intestinalis cysts, Cryptosporidium parvum, C. andersoni and Eimeria spp. oocysts was found. The infection by C. parvum was the highest from the 1st to the 3rd week and the infection by C. andersoni was the highest from the 7th to the 8th week of the calves age. In both breedings Coccidia Eimeria spp. was the most diagnosed, followed by G. intestinalis. With calves bred in stables by two a higher probability of parasitic infection was detected (46,9 %) compared to breeding of calves in outer individual boxes (17,6). The excrements of the calves were mainly of pasty or even of mushy consistence and the prevalence of the parasites was in most cases diagnosed in single infections.
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