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Metronidazole resistence in clinical cases of giardiasis.
Perglerová, Aneta ; Tůmová, Pavla (advisor) ; Uzlíková, Magdalena (referee)
Metronidazole (MTZ) targets a spectrum of anaerobic pathogens ranging from bacteria to eukaryotic parasites, including the protist Giardia intestinalis (Metamonada). Despite the long time and widespread use of MTZ, understanding giardiasis treatment failure (G-TF) is incomplete. Most of the knowledge was gained on laboratory-generated resistant lines or on clinical lines without data on the treatment outcome. In cooperation with Bulovka University Hospital in Prague, 70 giardiasis cases were followed between 2020-2023, of which 25.7% exhibited the G-TF. Giardia genotyping revealed that G-TF cases belonged to the same genetic group (assemblage B, mainly BIII); and G-TF cases were all imported, out of which 60% were from returning travellers from India. To test the hypothesis that resistance is genetically determined/associated with a transmissible genotype, genetic polymorphisms shared in Giardia G-TF samples in a set of genes involved in MTZ metabolism and detoxifications (NR1, NR2, prx1, hmp) were examined. The analysis revealed amino acid substitutions (K42E, K139R) in NR2 isolates of G-TF patients, which might affect protein structure. Also, a gene dosage effect as drug resistance co-variant/cause due to gene duplications or deletions of the hmp gene was tested. Results from dPCR revealed that...
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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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Diverzita endoparazitů lišek polárních
PAVLOVIČOVÁ, Markéta
The foxes represent a reservoir of zoonotic infections therefore it is important to be familiar with an occurrence of possible diseases. Arctic fox (Vulpes lagopus) faeces were collected between 2003 and 2011 from various locations on Bylot Island, Canada. These samples were consequently treated with microscopic and molecular methods to prove the presence of microsporidia, cryptosporidia and giardia. The presence of a specific DNA of Cryptosporidium was detected in two samples and specific DNA of Microsporidia was detected in 34 samples for genus Encephalitozoon and in five samples for genus Enterocytozoon. None of the animals showed any symptoms of a diarrhoea. Subsequent analyses showed the presence of the Cryptosporidium canis fox genotype, Encephalitozoon cuniculi II and III genotypes and Encephalitozoon hellem genotype 1A. None of the examined faeces samples were positive for the presence of specific G. intestinalis DNA. Results of the thesis show that Artic foxes are mainly infected by microsporidia of the genus Encephalitozoon, while the occurrence of other observed parasites was rare.
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Průzkum výskytu oportunního prvoka, \kur{Giardia intestinalis}, u zdravých lidí a jejich zvířat v ČR s použitím qPCR
BROŽOVÁ, Kristýna
The main aim of this study was to optimize the qPCR diagnostic protocol for determination of the prevalence of intestinal protist, Giardia intestinalis, in a volunteer group across the Czech Republic. A total of 428 samples were collected from gut-healthy humans (296) and from animals (132) with which these people were in close contact. The overall prevalence of G. intestinalis was 7 % in humans and 19 % in animals. In addition, we compared the sensitivity of two molecular methods for detecting of the presence of giardia, specifically qPCR and conventional PCR. Based on our results, we found out that qPCR is more sensitive method than PCR.
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Development of new molecular tools for altering gene expression in Giardia intestinalis .
Horáčková, Vendula ; Doležal, Pavel (advisor) ; Horváthová, Lenka (referee)
Giardia intestinalis is a widespread intestinal parasite that causes diarrhea in human and other vertebrate hosts. Although, fully sequenced genome of G. intestinalis has been published, very little is known about the regulation of gene expression. Together with the tetraploid genome, this complicates the use of many common reverse genetics methods. The aim of this thesis was to develop new molecular tools that can be used to alter gene expression in G. intestinalis. For the purposes of this work, new vectors for tetracycline-inducible gene expression including T7 promoter and endogenous oct promoter were designed. Furthermore, cwp1 gene knock-out was created using CRISPR-Cas9 technology. In order to modify mechanisms of double strand break repair, expression of two key components of bacterial NHEJ pathway - LigD and Ku - was introduced into cells of G. intestinalis.
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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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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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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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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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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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