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Role of glycosylation of ionotropic glutamate receptors in mammalian neurons
Danačíková, Šárka ; Horák, Martin (advisor) ; Kriška, Ján (referee)
Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. There are two distinct types of glutamate receptors, ionotropic and metabotropic, present in the mammalian excitatory synapses. My thesis is focused on the ionotropic glutamate receptors, which play critical roles in learning and memory formation. The main subtypes of ionotropic glutamate receptors are α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. All types of the ionotropic glutamate receptors, which are assembled as tetramers, contain many glycosylation sites, which can be modified by glycans or monosaccharides. The glycans and monosaccharides attached to the ionotropic glutamate receptors have been shown to regulate key processes such as folding of the subunits, transport to the cell surface as well as their functional properties. Recent literature also suggests that many neurological and psychiatric disorders such as schizophrenia exhibit abnormal glycosylation of ionotropic glutamate receptors. Thus, understanding of the molecular mechanisms, which regulate the glycosylation of the ionotropic glutamate receptors, may be important for developing new therapies for the patients with altered functioning of the glutamatergic synapses in the...
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Wnt signaling in intestinal homeostasis and tumorigenesis
Janečková, Lucie ; Kořínek, Vladimír (advisor) ; Macůrek, Libor (referee) ; Truksa, Jaroslav (referee)
The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....
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Molecular mechanisms of Wnt signalling in mammalian cells
Lukáš, Jan ; Kořínek, Vladimír (advisor) ; Kohoutová, Milada (referee) ; Macůrková, Marie (referee)
Wnt signalling represents an important mechanism participating in control of cellular and developmental processes, including establishment of cell polarity, cell fate specification, stem cell self-renewal, tissue patterning and organogenesis, homeostasis maintenance and regeneration. Misregulation of the Wnt signalling during embryogenesis leads to developmental defects while aberrant activation later in development is associated with degenerative diseases and a number of cancers. The presented PhD thesis is based on four original publications that deal with the post-translational modifications of Wnt ligands and molecular mechanisms contributing to the regulation of a transcriptional profile of the so-called canonical Wnt pathway. Wnt signalling pathway is used repetitively both in time and different cellular contexts throughout development of multicellular organisms. Inevitably, in each single situation -catenin/TCF complexes, the downstream effectors, induce only subsets of all potential target genes. How this differential tissue- and stage-specific control over various subsets of target genes is achieved with such a limited number of nuclear effectors is not fully understood. Along with the expression of specific LEF/TCF family members or their variants containing different functional domains...
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Molecular mechanisms and components controlling the Wnt signaling pathway output
Krausová, Michaela ; Kořínek, Vladimír (advisor) ; Drbal, Karel (referee) ; Bryja, Vítězslav (referee)
Beyond its essential roles in embryonic development, the Wnt-mediated signal transduction cascade is critically implicated in homeostasis of adult tissues. In the gastrointestinal epithelium, the threshold of active Wnt signaling is kept in a physiological range by a spectrum of regulatory networks and loops, thereby balancing the opposing processes of cell fate determination, proliferation and stem cell self-renewal. Furthermore, compelling evidence undoubtedly link an aberrant Wnt activity to the onset of bowel cancer. Understanding the principle causes and effects secondary to excessive Wnt signaling can provide valuable insights into the pathology of the malignant transformation of the colorectum. The proposed thesis attempts to focus on novel modes of the Wnt pathway modulation; both general and context-specific nuances of the Wnt level adjustment are thereby delineated. The results are presented in three distinct research publications and one review article. The first study examines the contribution of the distinct post-translational modifications, which the Wnt proteins undergo, to their proper processing, secretion and signaling activity. First, we investigated the sequential order and mutual interdependence of cysteine and serine-linked fatty acylation and N-linked glycosylation of murine...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Regulatory mechanisms of WNT signalling
Pospíchalová, Vendula
AB T mu hom dise β sign mu liga stab tran sign T the focu disc cate of t cell targ the inte con sec I sign BSTRACT The Wnt si lticellular o meostasis. A eases, most β-catenin is nalling). In ltiprotein c ands when t bilized and nscription f nalling is tig This thesis knowledge uses on seq cusses the enin signall the Wnt pa ls of intest geted mous thesis des eraction wit nditional Hi retory cell t In conclusi nalling path T ignalling pa organisms Accordingly notably can s a central m n unstimula complex and they engage d transloca factors and ghtly regula is based on e of the reg quential po positive ro ling outcom athway whic tinal epithe e strains th scribes unp th members ic1 deletion types and en ion, our fin hway in dev athway is o ensuring s y, mutations ncer. mediator of ated cells d degraded e their recep tes to the to drive th ated at vario n four origin gulation of sttranslation ole of nucle me. The third ch reduces lium. Final at enable st ublished da s of the Wn n in the inte nhanced tum ndings contr velopment an one of the m successful s in the pat f canonical W β-catenin d in the pro ptors, degrad nucleus t he transcrip ous levels b nal articles f the Wnt s nal process ear protein d study repo the levels o lly, the las tudying the ata on the nt pathway, estinal epith mourigenesi ributed to t...
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Regulatory mechanisms of WNT signalling
Pospíchalová, Vendula ; Kořínek, Vladimír (advisor) ; Trka, Jan (referee) ; Bryja, Josef (referee)
AB T mu hom dise β sign mu liga stab tran sign T the focu disc cate of t cell targ the inte con sec I sign BSTRACT The Wnt si lticellular o meostasis. A eases, most β-catenin is nalling). In ltiprotein c ands when t bilized and nscription f nalling is tig This thesis knowledge uses on seq cusses the enin signall the Wnt pa ls of intest geted mous thesis des eraction wit nditional Hi retory cell t In conclusi nalling path T ignalling pa organisms Accordingly notably can s a central m n unstimula complex and they engage d transloca factors and ghtly regula is based on e of the reg quential po positive ro ling outcom athway whic tinal epithe e strains th scribes unp th members ic1 deletion types and en ion, our fin hway in dev athway is o ensuring s y, mutations ncer. mediator of ated cells d degraded e their recep tes to the to drive th ated at vario n four origin gulation of sttranslation ole of nucle me. The third ch reduces lium. Final at enable st ublished da s of the Wn n in the inte nhanced tum ndings contr velopment an one of the m successful s in the pat f canonical W β-catenin d in the pro ptors, degrad nucleus t he transcrip ous levels b nal articles f the Wnt s nal process ear protein d study repo the levels o lly, the las tudying the ata on the nt pathway, estinal epith mourigenesi ributed to t...
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Assessing biochemical properties of PDE8A1: Design of experimental system in living cells"
Galica, Tomáš ; Černý, Jan (advisor) ; Mašek, Tomáš (referee)
4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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Studies of minor capsid proteins of the mouse polyomavirus
Vít, Ondřej ; Němečková, Šárka (referee) ; Forstová, Jitka (advisor)
Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique Nterminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1binding alpha-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to Nterminus of EGFP were prepared to determine the hydrophobic...
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