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Molecular mechanisms of Bardet-Biedl Syndrome
Mašková, Kristýna ; Huranová, Martina (advisor) ; Doubravská, Lenka (referee)
Bardet-Biedl Syndrome (BBS) is a rare genetic disease categorized under ciliopathies; a group of diseases linked to primary cilia dysfunction. Primary cilia, also known as cellular antennae, play a vital role in sensing extracellular stimuli and transducing them through various signalling cascades. Proper cilia function and signalling depends on multiple ciliary proteins, with eight of them forming a BBSome complex. The BBSome is involved in the transport of proteins into and out of the cilia. Mutations in genes encoding BBSome complex lead to BBS. Among these genes, BBS1, which encodes the BBS1 subunit of the BBSome, is notably highly mutated compared to others. This thesis focuses on the BBS1 subunit and aims to investigate the molecular mechanisms underlying three specific BBS patients' mutation located in BBS1: M390R, E224K, R160Q. In the first part, we validated the expression of these selected BBS1 variants and examined their effects on the expression of other BBSome subunits. We observed decreased expression levels of BBS4 and BBS5 subunits in the presence of M390R and E224K mutations. Secondly, we assessed BBSome assembly in the context of these mutations, showing that R160Q mutation did not impair BBSome assembly, whereas assembly was severely disrupted in the presence of M390R variant,...
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Left-right asymmetry specification in vertebrates
Vrúbel, Matěj ; Soukup, Vladimír (advisor) ; Fabian, Peter (referee)
The left-right body axis, along with the dorso-ventral and antero-posterior axis, is certainly very important, but at the same time the most neglected body axis of bilaterally symmetrical animals. The asymmetrical distribution of visceral organs along this body axis is vital for many animals. In vertebrates, this asymmetry becomes established at early embryonic stages. An essential role in this process is played by the organizer, which is responsible for the correct establishment of the left-right axis. Among vertebrates, organizers of left-right asymmetry are found in different parts of the embryonic body, and with few exceptions, they are composed of cells with motile cilia, which create an unidirectional leftward flow of extracellular fluids in the organizer. The flow is probably sensed by the cells that surround the left side of the organizer. These cells respond to the aforementioned mechanical stimulus by triggering the Nodal signaling pathway. This signaling cascade results in the left-sided expression of the Pitx2 gene, which specifies the left side of the embryonic body. It appears, that the role of Nodal signaling pathway in determining left-right asymmetry is not only present in all vertebrates, but also is probably ancestral to bilaterally symmetrical animals. Another mechanism ensuring...
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Left-right organizer of body asymmetries in ray-finned fishes
Kupková, Anežka ; Soukup, Vladimír (advisor) ; Krylov, Vladimír (referee)
Left-right asymmetry of the body occurs across a number of organisms from invertebrates to vertebrates, and is mostly exhibited by the asymmetry of internal organs. These asymmetries are established at early stages of embryonic development due to the action of temporary structures called organizers of left-right asymmetry. In ray-finned fishes, the most-studied organizer is the so-called Kupffer's vesicle present in the teleosts. It is a hollow structure composed of monociliary cells. The cilia of these cells rotate and generate a leftward flow of extracellular fluid. The flow subsequently triggers the induction of the Nodal signalling cascade, which is responsible for left-right organ orientation and is considered evolutionarily conserved in vertebrates. The main participants in this pathway are the Nodal, Pitx2 and Lefty factors. In contrast to teleosts, the left-right organiser of non-teleost ray-finned fishes resembles the gastrocoel roof plate present in amphibians, which is apparently ancestral for ray-finned fishes. This bachelor thesis evaluates the origin and function of Kupffer's vesicle, describes the Nodal signalling cascade triggered by this organizer, and compares Kupffer's vesicle with the organizer of non-teleost ray-finned fishes.
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Searching for the common function of the BBSome across the evolution and development
Mašková, Kristýna ; Štěpánek, Ondřej (advisor) ; Čajánek, Lukáš (referee)
The BBSome is a protein complex whose function is associated with ciliary trafficking. It has been found that the BBSome is evolutionarily conserved among ciliated organisms. The disruption of the BBSome leads to cilia dysfunctions and affects many signalling pathways. In humans, genetic defects in the BBSome are the cause of Bardet-Biedl Syndrome, which is a pleiotropic disease. The BBSome is studied separately in various model organisms and cell lines to understand the molecular functions of the BBSome. These studies have not been compared to see if there is a common BBSome function, which could be why the BBSome is evolutionarily conserved among ciliated organisms. In this bachelor's thesis, the knowledge about the BBSome was summarized and compared to identify a putative common function of the BBSome among various model organisms or cell types. It seems that there has not been found any BBSome function that could be identified as the common function because the BBSome has many specialized functions in different organisms and tissues. The only distant similarity is BBSome-dependent ciliary retrograde transport, which has been described in most of the studied model organisms and cell types.
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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