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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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The role of untranslated mRNA regions in Giardia intestinalis.
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Zubáčová, Zuzana (referee)
Giardia intestinalis is an anaerobic protozoan pathogen, agent of the disease known as giardiasis. The regulation of gene expression during giardia cell- and life-cycle has been poorly studied so far, with the exception of variable surface proteins, which constitute the immunoprotective coat of the cell. In this diploma thesis, we focus on the possible role of the 3' untranslated region (3'UTR) of mRNA that mediate stability and localization of mRNA transcripts. We use RNA binding proteins of PUF family, which control the function of the target transcripts by their repression, activation or sequestration, to monitor and verify the role of 3'UTRs. These only eukaryotic proteins are highly evolutionarily conserved. Each of them contain highly conserved C-terminal domain, which specificly binds to 3'UTR of mRNAs. We have identified five different PUF proteins in the genome of G. intestinalis (GiPUF), cinfirmed their expression in G. intestinalis trophozoites and located all five proteins in the cytoplasm. GiPUF2, GiPUF3 and GiPUF5 show an additional affinity to the surface of the endoplasmic reticulum. We have identified the C-terminal binding domain in protein sequences of all GiPUF. The most conserved GiPUF4 contain eight binding sites, nearly identical to the binding site of human Pum1 protein,...
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FISH method and its use in protistology
Frenclová, Martina ; Hampl, Vladimír (advisor) ; Zubáčová, Zuzana (referee)
Fluorescence in situ hybridization (FISH) is a technique that allows the localization and identification of specific sequences of nucleotides in DNA or RNA, which is subsequently visible under the microscope. FISH involves first denaturing the nucleic acids, either using high temperatures or by treatment with denaturing agents such as formamide.After subsequent induction of reasociation, the examined DNA or RNA pairs according to the complementarity rules with the short molecule called the probe, this process is called hybridization. Hybridization occurs in situ, that is within the examined specimen. Probes can be labeled either directly using fluorophores, or indirectly with a hapten, which is a substance having antigenic properties, which is subsequently detected using labeled antibodies or streptavidine. FISH has a large number of applications in molecular biology and medical science. In laboratory research in protistolgii FISH can be used for example to map the chromosomal genes to study the evolution of genome, analysis of nuclear organization or to confirmation of the origin of DNA sequence. Key words: FISH, fluorescence, probe, cell identification, environmental studies
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FISH method and its use in protistology
Frenclová, Martina ; Hampl, Vladimír (advisor) ; Zubáčová, Zuzana (referee)
Fluorescence in situ hybridization (FISH) is a technique that allows the localization and identification of specific sequences of nucleotides in DNA or RNA, which is subsequently visible under the microscope. FISH involves first denaturing the nucleic acids, either using high temperatures or by treatment with denaturing agents such as formamide.After subsequent induction of reasociation, the examined DNA or RNA pairs according to the complementarity rules with the short molecule called the probe, this process is called hybridization. Hybridization occurs in situ, that is within the examined specimen. Probes can be labeled either directly using fluorophores, or indirectly with a hapten, which is a substance having antigenic properties, which is subsequently detected using labeled antibodies or streptavidine. FISH has a large number of applications in molecular biology and medical science. In laboratory research in protistolgii FISH can be used for example to map the chromosomal genes to study the evolution of genome, analysis of nuclear organization or to confirmation of the origin of DNA sequence. Key words: FISH, fluorescence, probe, cell identification, environmental studies
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Regulation of gene expression in anaerobic parasitic protist and practical application of knowledge
Brzoň, Ondřej ; Zubáčová, Zuzana (advisor) ; Horváthová, Lenka (referee)
Because of its importance for every single cell, regulation of gene expression is often studied at all levels today. On the other hand, rather sparse amount of information is available for protists and our understanding of their gene expression is nearly limited to several model organisms. Three anaerobic human parasites have been studied in details: Trichomonas vaginalis, Entamoeba histolytica and Giardia intestinalis. Most recently, some others anaerobic protists (e.g. Entamoeba invadens or some species of Spironucleus genus) have been researched as well. This work is focused on the structure of promoter regions in protein coding genes and application of this knowledge in construction of transfection systems as one of the most effective and powerful tools in molecular and cell biology.
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The role of untranslated mRNA regions in Giardia intestinalis.
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Zubáčová, Zuzana (referee)
Giardia intestinalis is an anaerobic protozoan pathogen, agent of the disease known as giardiasis. The regulation of gene expression during giardia cell- and life-cycle has been poorly studied so far, with the exception of variable surface proteins, which constitute the immunoprotective coat of the cell. In this diploma thesis, we focus on the possible role of the 3' untranslated region (3'UTR) of mRNA that mediate stability and localization of mRNA transcripts. We use RNA binding proteins of PUF family, which control the function of the target transcripts by their repression, activation or sequestration, to monitor and verify the role of 3'UTRs. These only eukaryotic proteins are highly evolutionarily conserved. Each of them contain highly conserved C-terminal domain, which specificly binds to 3'UTR of mRNAs. We have identified five different PUF proteins in the genome of G. intestinalis (GiPUF), cinfirmed their expression in G. intestinalis trophozoites and located all five proteins in the cytoplasm. GiPUF2, GiPUF3 and GiPUF5 show an additional affinity to the surface of the endoplasmic reticulum. We have identified the C-terminal binding domain in protein sequences of all GiPUF. The most conserved GiPUF4 contain eight binding sites, nearly identical to the binding site of human Pum1 protein,...
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Chromosomes of parasitic protist Trichomonas vaginalis
Zubáčová, Zuzana ; Tachezy, Jan (advisor) ; Alsmark, Cecília (referee) ; Vanáčová, Štěpánka (referee)
Charles University in Prague Faculty of Science Programme of study: Parasitology Abstract of Ph.D. thesis Chromosomes of parasitic protist Trichomonas vaginalis Zuzana Zubáčová, MSc. Supervisor: Prof. RNDr. Jan Tachezy, Ph.D. Praha, 2011 Abstract Genome sequencing and associated proteome projects has revolutionized research in the field of molecular parasitology. Progress in sequencing of parasite as well as free-living species enables comparative and phylogenetic studies and provides important data sets for understanding of basic biology and identification of new drug targets. The genome sequencing of Trichomonas vaginalis revealed surprisingly large genome size of this organism (~160 Mb) that resulted from expansion of various repetitive elements, specific gene families and large scale genome duplication. I studied genome sizes of other nine selected species from Trichomonadea group to find whether other trichomonads have undergone similar genome expansion. The measurement of nuclear DNA content by flow cytometry revealed relatively large DNA content in all tested species although within a broad range (86-177 Mb). The largest genomes were observed in the Trichomonas and Tritrichomonas genera while Tetratrichomonas contains the smallest genome. The genome sizes correlated with the cell volume however no...
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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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Cellular functions of tail-anchored proteins.
Martincová, Eva ; Zubáčová, Zuzana (referee) ; Doležal, Pavel (advisor)
This work reviews recent studies on the biogenesis of tail-anchored proteins. These proteins form a distinct class of integral membrane proteins which are intensively studied nowadays. Although this class of proteins is defined by the structure and membrane topology, individual proteins do not share any functional similarities. The basic cellular functions, structure and localisation are reviewed there. The work is focused mainly on the unique transport mechanisms and the determination of the target cellular compartments - endomembrane system and mitochondrial outer membrane. A separate part of the work also summarizes existing knowledge about VAP protein family which belongs to the class of tail-anchored proteins and which is conserved across all eukaryotic species. The last chapter presents results and goals of the research of these proteins in the human parasite Giardia intestinalis in the laboratory of organellar biogenesis.
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