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Notch Interaction with Glycogen Synthase Kinase-3β in Neurodegenerative Disease
Traub, Teodor ; Mašek, Jan (advisor) ; Rozbeský, Daniel (referee)
Glycogen synthase kinase-3βeta (GSK-3β) is a serine/threonine protein kinase involved in a number of signaling processes. Pharmacological inhibition of GSK-3β has been shown to have neuroprotective effects, and its dysregulation is present in a variety of neurodegenerative, neuromuscular, developmental, and psychiatric disorders. GSK-3β is an essential component of the canonical Wnt pathway, which is involved in nervous system development. Notch signaling, like Wnt, plays a key role in development, but its relationship to GSK-3β remains unclear. The existing literature indicates that GSK-3 phosphorylates Notch intracellular domain but contradicts whether GSK3β affects Notch positively or negatively. Thus, such molecular "cross-talk" is highly complex and interactions may exist at multiple levels beyond simple phosphorylation with other components being involved. Myotonic dystrophy is a genetic neuromuscular disease that causes dysregulated protein expression of many proteins, including GSK-3β, and features developmental, muscle, and neurological symptoms. Studying molecular interactions in the context of myotonic dystrophy may help uncover effects that GSK-3β and Notch have on development and disease of nervous and muscular systems. This thesis presents a review of previous studies concerning the...
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Growth factors in cultivated meat production
Janoušek, Jiří ; Rozbeský, Daniel (advisor) ; Mašek, Jan (referee)
The rapid expansion of global meat production poses severe environmental and ethical challenges, necessitating alternatives with reduced impacts. Cultivated meat, produced from animal cells in vitro, emerges as a promising solution. However, common cell culturing techniques using fetal bovine serum (FBS) are not sufficiently scalable for this purpose. The critical role of growth factors in replacing FBS is emphasized, with a focus on summarizing existing literature regarding their importance and potential for economically viable mass adoption. The described growth factor requirements of various cultivated meat production relevant cell types, as well as the potential improvements to the production processes, activity, or stability of the most important growth factors identified, are analyzed. Finally, alternative approaches to the use of growth factors are briefly explored. This work should provide a comprehensive overview of recombinant growth factor production and optimization as it pertains to the area of cultivated meat.
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The effect of variations in GRIN genes on the biogenesis and functional properties of the NMDA receptor
Kuchtiak, Viktor ; Balík, Aleš (advisor) ; Rozbeský, Daniel (referee) ; Ladislav, Marek (referee)
The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the central nervous system. The major class are the calcium-permeable NMDA receptors that are fundamental for the various forms of synaptic plasticity, a key mechanism in the process of learning and memory formation. NMDA receptors are heterotetrameric and are represented by three types of subunits: GluN1, GluN2A-D, and GluN3A-B. Each subunit consists of four domains, with the intracellular C-terminal domain accounting for up to half of the entire NMDA receptor subunit (GluN2A/2B). A body of evidence indicates that the hypofunction of the NMDA receptor plays an important role in the pathogenesis of several neuropsychiatric disorders, including schizophrenia. Schizophrenia is characterised by a high degree of heritability, but its genetic background is not yet fully understood. Previous studies have identified in the human genome several individual loci that contribute to disease susceptibility, including the GRIN genes encoding NMDA receptors. Using a sequencing approach, we identified and annotated genetic variations across all GRIN genes in a cohort of schizophrenia patients and control subjects. The submitted doctoral thesis focuses on the functional analysis of the genetic...
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Studies on structure and biological functions of NKR-P1 receptors
Rozbeský, Daniel ; Novák, Petr (advisor) ; Konvalinka, Jan (referee) ; Drbal, Karel (referee)
Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...
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Molecular interaction of developing axons with the surface ectoderm
Matějček, Miroslav ; Machoň, Ondřej (advisor) ; Rozbeský, Daniel (referee)
The complex of cranial nerves consists of twelve pairs of nerves, which connect organs of the head with the central nervous system. One of the organs, which are innervated by cranial nerves are whiskers (also known as vibrissae). Whiskers are special type of mammalian hairs, which are innervated by the infraorbital branch of trigeminal nerve. These hairs provide information necessary for orientation in terrain, localisation of prey and locomotion. The potential role of axons in whiskers development has been discussed for many years. However, this hypothesis has not yet been confirmed. In this research we have used murine strains Neurog1 KO and Meis2 cKO with either abnormal or completely missing trigeminal nerve. Thanks to the analysis of embryonic development in these strains we have denied our hypothesis, that axons of trigeminal nerve serve a role in initiation of whisker follicles. In Meis2 cKO embryos we observed malfunction in formation of whisker follicles. The expression of Meis2 during the development of whiskers has an influence on various signalling cascades including Fgf, Shh and Bmp. In regards to changes in mentioned signalling cascades we suggest that the whisker follicle development is altered between the first dermal and first epidermal signal.
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Molecular logic of the Notch ligands in development and disease
Trampotová, Eliška ; Mašek, Jan (advisor) ; Rozbeský, Daniel (referee)
The Notch signalling pathway is one of the key signal transduction pathways in the embryonic development of all metazoa. In mammals, the "canonical" signalling occurs on four Notch receptors, and it is triggered by five ligands varying in both their structures and functions. The importance of the pathway for embryonic development is reflected by the fact that mutations of either receptors or ligands result in various congenital disorders. A prime example is the Alagille syndrome - a rare multisystemic condition caused by mutations of the Notch ligand Jagged1 in 94 % of cases. Much of the research effort so far was concentrated into elucidating how the Notch receptors operate, however one could argue the importance of ligand activity regulation is even higher. This thesis thus aims to provide a comprehensive comparison of the five mammalian Notch ligands' structures and roles in developmental processes. The main focus of the thesis is the variability of ligand-receptor interactions and biophysical mechanisms of signalling, highlighting the importance of the Notch ligands' intracellular domains' interactions.
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Structural and functional studies of MICAL signalling in cytoskeletal dynamics
Rožová, Dominika ; Rozbeský, Daniel (advisor) ; Sulimenko, Vadym (referee)
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis...
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Structural and functional studies of signaling molecules in axon guidance
Knapp, Kryštof ; Rozbeský, Daniel (advisor) ; Novák, Petr (referee)
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a sig- naling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interac- tions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity...
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Stability study of the transcription factor and dsDNA complex in the gas and the liquid phases
Kadavá, Tereza ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee)
Integrative structural biology employs not only high-resolution techniques for the determination of the structure of biomolecules. Information about dynamical and heterogeneous assemblies is hardly achievable with conventional approaches. Therefore, low-resolution techniques are being utilized. As mass spectrometry (MS) is sensitive, specific, and versatile, there are a lot of structural techniques based on MS established. Native mass spectrometry is an approach that uses non-denaturing solvents for nanoelectrospray ionization, thus preserving tertiary and quaternary structures in the gas phase. Therefore, information on the composition of native biomolecules is obtained. Moreover, employing ion mobility (IM-MS) also provides structural insight. Using MS, we can directly analyze biomolecules perturbation, such as by variable-temperature nanoelectrospray ionization (vT-nESI). The aim of this master's thesis was to characterize the thermal stability of the protein-DNA complexes of TEAD1 and FOXO4 transcription factors DNA-binding domains. Complexes were studied using native MS and IM-MS employing an in-house developed vT-nESI source. First, vT-nESI was used to determine the melting temperatures of double-stranded DNA, which were in correspondence with those theoretically calculated and experimentally...
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Structural characterization of interactions between subunits of the GBAF chromatin remodeling complex
Naušová, Karolína ; Veverka, Václav (advisor) ; Rozbeský, Daniel (referee)
Epigenetics investigates heritable phenotypic changes that are not caused by alterations in DNA sequence. One of the major epigenetic tools is chromatin remodeling mediated by ATP-dependent chromatin remodeling complexes, which fundamentally affect gene expression and thus cellular fate. A mammalian variant of the ATP-dependent chromatin remodeling SWI/SNF complex is the BAF complex, which is involved in both activation and repression of gene expression. There have been identified three major variants of BAF complex one of which is non-canonical BAF, also known as GLTSCR1 containing BAF, GBAF. Each complex consists of up to 15 subunits, some of which are specific only to one of the complex variants. Mutations in genes coding subunits of BAF lead to several genetic disorders or to the cancer development. The GBAF complex contains specific subunits, BRD9 (Bromodomain containing protein 9), GLTSCR1 (Glioma tumor suppressor candidate region 1) and GLTSCR1L (GLTSCR1-like), which are essential for its formation. Although the exact function of the GBAF complex has not been elucidated yet, it is often associated with synovial sarcoma and rhabdoid tumors. Two of the three complexes are impaired in those tumors and gene expression is maintained only due to the GBAF complex. If GBAF would additionally loose...
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