National Repository of Grey Literature 170 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Regulatory mechanism of the cyclin-dependent kinase 16 through the cyclin Y/14-3-3 complex
Janáčková, Zuzana ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
CDK16 is a cell-cycle-related cyclin-dependent kinase that functions primarily in neurite growth processes, axonal transport regulation and sperm development. It also plays a role in the progression of X-linked intellectual disability and, together with its activating partner cyclin Y, in the development and progression of various types of cancer. Consequently, CDK16 and cyclin Y could represent potential new therapeutic targets. Both CDK16 and cyclin Y are structurally atypical among similar proteins, and the mechanism of activation of CDK16 by cyclin Y is unclear. However, it is clear from recent studies that the interaction between CDK16 and cyclin Y requires the prior interaction of cyclin Y with 14-3-3 proteins. In addition, for cyclin Y to interact with 14-3-3 proteins, its phosphorylation on the S100 and S326 binding sites of 14-3- 3 is required. To investigate the mechanism of activation of CDK16 by cyclin Y, we decided to characterize the interaction between CDK16, cyclin Y and 14-3-3 proteins in detail. The interaction between CDK16, cyclin Y and 14-3-3 proteins was characterized using biophysical methods including fluorescence anisotropy measurements, native electrophoresis, size exclusion chromatography and protein crystallography. The for- mation of binary pCCNY-14-3-3 and ternary...
Characterization of protein-protein interactions between Forkhead box O (FOXO) and p53 transcription factors
Mandal, Raju ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Pavlíček, Jiří (referee)
The transcription factor p53 plays a key role in cell cycle arrest, DNA repair, apoptosis, tumor suppression, and maintaining cellular homeostasis. Under cellular stress, p53 directly interacts with the Forkhead box O (FOXO) 4 transcription factor, thereby upregulating the expression of the p21 gene, resulting in the induction of cellular senescence. However, the detailed molecular mechanism behind FOXO4-p53 interaction remains unclear due to the unavailability of structural data. Therefore, main goal of this doctoral thesis was the characterization of the interaction between FOXO4 and p53 using several biophysical techniques including sedimentation velocity analytical ultracentrifugation (SV AUC), nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled to mass spectrometry. Furthermore, we also investigated the DNA binding properties of both proteins with their respective consensus DNA sequences in the presence or absence of their binding partners by fluorescence anisotropy measurements along with the comparison of p53-binding surfaces of the Forkhead domain of three different FOXO proteins by NMR spectroscopy. In addition, we also optimized small molecule compounds for the inhibition of FOXO3-DNA interaction. Our results revealed that the p53 interacts with FOXO4 through...
Structural characterization of influenza A polymerase PA subunit domains in complex with novel inhibitors
Radilová, Kateřina ; Kožíšek, Milan (advisor) ; Rumlová, Michaela (referee) ; Obšil, Tomáš (referee)
Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the life cycle of the virus. It is responsible for viral replication and transcription. One of its subunits, the polymerase acidic protein, interacts with the PB1 subunit via a crucial protein- protein interaction at its C-terminal domain. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly. The N-terminal domain carries the endonuclease active site with two manganese ions. Both domains are considered promising drug targets. Current strategies to fight the influenza virus are limited to seasonal vaccines, and there are only a few anti-influenza drugs targeting mostly other viral proteins. Many used antivirals are susceptible to rapid resistance mutations development or cause severe side effects. This thesis provides structural insights into the two domains of the PA subunit. The first part is devoted to the characterization and optimization of a PB1-derived minimal peptide interacting with the C-terminal domain. Results from this part may be considered as a starting point for the rational design of first-in-class anti-influenza inhibitors of the PA-PB1 protein-protein interaction. In the other half, we have explored the inhibitory potency of flavonoids and...
Is there a direct link between global value chain participation and economic growth? Analysis of EU member countries
Obšil, Tomáš ; Semerák, Vilém (advisor) ; Kábrt, Martin (referee)
This thesis studies the strength of correlation between Global value chain par- ticipation and economic growth in the Czech Republic, Slovakia, Slovenia, Es- tonia, Poland, Austria, Germany, and Hungary from 1995 to 2018. This rela- tionship is demonstrated by using traditional panel data methods, with forward and backward GVC participation as our main explanatory variables and GDP per capita as our response variable. Based on the graphs and initial models, the variables seemed to be correlated; nevertheless, our analysis did not confirm this hypothesis. Furthermore, we use the total value added of the food, textile, and electronic industries as proxy variables for sectoral growth. We found, in most cases, a strong and significant correlation between the growth of these industries and BP and FP. JEL Classification B22, C33, E23, F41, F63, L60, O47, O52 Keywords global value chains, economic growth, central and eastern European countries, forward partic- ipation, backward participation Title Is there a direct link between global value chain participation and economic growth? Analysis of EU member countries.
Characterization of the binding interface between transcription factors FOXO4 and p53
Brzezina, Adam ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
This work deals with the study of human transcription factors FOXO4 and p53. FOXO4 is a member of the "O" subfamily of FOX transcription factors. Genes encoding FOXO proteins are evolutionarily conserved across species. FOXO transcription factors regulate the expression of genes involved in the control of metabolism, cell cycle and cell proliferation, cell survival and stress resistance. They are considered tumour suppressors because of their ability to arrest the cell cycle and induce apoptosis. However, their function in tumorigenesis appears to be more complicated, as recent studies indicate a poorer prognosis for the development of tumours that express higher levels of FOXO4. The p53 protein is a thoroughly studied naturally occurring tumour suppressor. The cellular response after its activation is somewhat similar to that of FOXO4, it can also block cell cycle progression or induce apoptosis depending on the cell type and severity/type of cellular stress. Both FOXO4 and p53 appear to be key molecules affecting aging. Under stress conditions, p53 and FOXO4 interact with each other and together increase the expression of p21 protein, thereby inducing the transition of cells to a senescent state. The accumulation of senescent cells is recognised as one of the main causes of ageing and the...
Cloning, expression and characterization of the viral proteins
Pekárek, Matěj ; Šilhán, Jan (advisor) ; Obšil, Tomáš (referee)
Over the last twenty years three new coronavirus strains have appeared in human population, that cause dangerous infections of respiratory tract. At this moment no approved drug exists, that could mitigate the infection caused by these viruses. One of the possible targets of these drugs are proteins responsible for viral genome replication. Nsp13 is primarily a helicase, that unwinds double stranded nucleic acids in the direction of 5'-3', but it also increases activity of replication and transcription complex, it participates in the synthesis of 5' cap on viral RNA and it probably mediates the backtracking of replication and transcripton complex during the process of RNA repair. On top of that nsp13 is strongly conserved among all coronaviruses and it is possible target not only for COVID-19 treatment, but also infections caused by potential future coronaviruses. Theoretical part of the thesis briefly describes the life cycle of SARS-CoV-2, functions of individual proteins of this virus, that participate in replication and transcription of RNA and it summarizes current knowledge about function of nsp13 and its potential inhibitors. In experimental part we have prepared the expression vector for nsp13, that was used for the production of nsp13. Nsp13 was then isolated and purified. (In Czech) Key...
Modulation of DNA Binding Affinity of Transcription Factors FOXO and p53 Through Protein-protein Interactions
Hofmanová, Adéla ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract The forkhead box "O" (FOXO) proteins are a subclass of the Forkhead family of transcription factors that play a critical role in a variety of cellular processes such as response to cellular stress, gluconeogenesis, cell cycle control, apoptosis, senescence, and repair of DNA damage. They are generally considered to be tumor suppressors. However, it has been shown that they can promote tumorigenesis and induce resistance to the chemotherapeutic agents. Despite many years of research into the biological role of FOXO proteins, a number of questions remain to be answered. For example, whether the slight structural differences observed in the otherwise highly homologous DNA-binding domains of individual FOXO transcription factors affect their DNA binding affinity. Furthermore, it is unclear how protein-protein interactions affect DNA binding affinity of FOXO proteins. Recent study has described the interaction of FOXO transcription factors with the p53 protein. Protein p53 is called the guardian of the genome due to its ability to mediate the response to acute DNA damage. The interaction of FOXO and p53 proteins appears to have a major effect on the DNA binding affinity of both these proteins. Based on this, DNA-binding domains of the human transcription factors FOXO1, FOXO3 and FOXO4 (FOXO1(144-270),...
Studying protein interactions with expanded genetic code
Tekel, Andrej ; Obšil, Tomáš (advisor) ; Fuertes Vives, Gustavo (referee)
Nature, using proteins composed of approximately 20 amino acids repertoire, is able to perform a large number of admirable things, many of which we are still not quite able to mimic. However, it has to be said, that the set of chemical scaffolds available to the nature is somewhat limited. Therefore, it seems plausible to assume, that we could make things better simply by introducing novel scaffolds and exploring chemical space so far unavailable. It is exactly this idea upon which expanded genetic code is based on. To set ourselves free from limitations imposed by standard genetic code. This thesis will aim to provide an overview of various attempts to this problem and will try to do so in a distinct context of studying protein interactions. I will thus try to highlight methods by which we are currently able to expand genetic code, specific chemistries and physical properties of non-canonical amino acids and biophysical methods which can profit from genetic code expansion. Keywords: expanded genetic code, non-canonical amino acid, amber codon, fluores- cence, magnetic resonance, crosslinking, click chemistry
Dynamic saturation optical microscopy using photoswitchable proteins
Kolářová, Marie ; Benda, Aleš (advisor) ; Obšil, Tomáš (referee)
Fluorescence microscopy is an essential technique for live cell imaging. One of its drawbacks is a rather low diffraction limited spatial resolution, which is described by Abbe diffraction law. Therefore, in the last decade a lot of new methods improving spatial resolution were developed. One of them is dynamic saturation optical microscopy (DSOM) that is based on spatial monitoring of reversible transition kinetics between bright and dark states of fluorophores. The dark state is possible to obtain for example by using reversibly photoswitchable fluorescent proteins such as Dronpa and its variants. These proteins undergo reversible transition from fluorescent to nonfluorescent state after irradiation by blue and ultraviolet light. In my work I focus on employing the kinetics of controllable photoswitching of Dronpa in improving the overall image quality, including the spatial resolution. The experiments were performed on yeasts expressing selected proteins labelled with Dronpa. Firstly, photoswitching behaviour of Dronpa was confirmed. Secondly, experimental conditions were optimized by studying dependence of switching rate on laser intensities and on excitation wavelength and by studying protein photostability. Experiments were performed on different timescales and for various proteins. Using the optimal...
Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.

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