National Repository of Grey Literature 52 records found  beginprevious15 - 24nextend  jump to record: Search took 0.00 seconds. 
Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
High-throughput screening for the discovery of small molecules modulating cell fate
Ribeiro Pombinho, António José ; Bartůněk, Petr (advisor) ; Bařinka, Cyril (referee) ; Jiráček, Jiří (referee)
The discovery of chemical compounds able to modify the way cells proliferate, differentiate or die can lead not only to the formulation of new drugs for disease treatment or prevention but also to their use as biological probes in the study of the molecular pathways involved in these processes. In order to test thousands of these small molecules in cellular assays, instrument automation and assay miniaturization are necessary. In this thesis, applications of High-Throughput Screening campaigns are described. The Hypoxia and Wnt pathways involved in stem and cancer cell proliferation; the differentiation of hematopoietic, neural and mesenchymal stem cells; and the TRAIL pathway leading to selective cancer cells death were the main subjects chosen. With this approach, it was possible to test the effect of small molecules in eukaryotic cells and in unicellular organisms as exemplified by the search of compounds leading to the death of the protozoan parasite Leishmania. Several chemical compounds were identified as active in modulating cell fate. Of remark were: Monensin that inhibits the Wnt pathway and prevents the growth of tumors in a mouse model of colorectal cancer; Homoharringtonine that, only in combination with TRAIL, induces the death of cancer cells implanted in immunodeficient mice; and...
Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography
Vaněk, Ondřej ; Bezouška, Karel (advisor) ; Hrabal, Richard (referee) ; Bařinka, Cyril (referee)
Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...
Study of the factors affecting the binding specificity of the 14-3-3 proteins.
Veisová, Dana ; Obšilová, Veronika (advisor) ; Bařinka, Cyril (referee) ; Krůšek, Jan (referee)
113 11. Summary The 14-3-3 proteins are dimeric molecules with a characteristic shape and molecular mass about 30 kDa found in all eukaryotes. They are playing a key role in a variety of biological processes such as signal transduction, cell differentiation and apoptosis. The C- terminal segment of human 14-3-3ζ plays an important role as an autoinhibitor which can occupy the ligand binding groove in the absence of binding partner and blocks the binding of inappropriate ligand. The C-terminal segment structure has not been identified for any of the known crystallographic structures. Unlike the helical region α1-α9, the C-terminal segment shows the highest sequence variability. It is believed that the C-terminal segment is the most flexible region and can exist in a lot of conformations. The yeast isoforms of the 14-3-3 proteins Bmh1 and Bmh2 possess a distinctly variant C-terminal segment which is longer and contains a polyglutamine stretch of unknown function. The role of this C-terminal part has been studied with many of different biophysical methods. Dynamic light scattering, sedimentation velocity, time resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that an apparent size of the molecules Bmh1 and Bmh2 is significantly bigger compared to the 14-3-3 isoforms....
The crystal structure of PI4 kinase
Bäumlová, Adriana ; Bouřa, Evžen (advisor) ; Obšil, Tomáš (referee) ; Bařinka, Cyril (referee)
Phosphatidylinositol 4-kinases (PI4K/PI4-kinases) catalyse the production of phosphatidylinositol 4-phosphate (PtdIns4P), the first step in the generation of higher phosphoinositides. PtdIns4P is an essential precursor in the production of second messengers, Ins(1,4,5)P3 and diacylglycerol, in a receptor activated phospholipase C signalling pathway. Moreover, PtdIns4P itself regulates conserved compartment-specific biological processes, mainly via recruiting a broad spectra of effector proteins. Because PI4-kinases have a central position in PtdIns4P synthesis on a surface of intracellular membranes, they are implicated in a wide range of PtdIns4P-induced processes such as lipid transport and metabolism, intracellular trafficking processes and cargo sorting, membrane and cytoskeleton remodelling events, signal transduction and many others. In mammals, two types of PI4-kinases were identified: type II and type III. Both types do not bear high sequence similarity to each other and, therefore, they possess diverse biochemical properties. In order to elucidate their structural relationship to other lipid kinases, structural analysis is highly demanded. The structural characterisation of individual PI4-kinases could also clarify the catalytic mechanism of PtdIns4P synthesis. Furthermore, information...
Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
Structure-assisted development of a continuous carboxypeptidase assay
Rakhimbekova, Anastasia ; Bařinka, Cyril (advisor) ; Bouřa, Evžen (referee)
Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...
The use of phage display to investigate Leishmania mexicana surface antigens
Krylová, Anna ; Spitzová, Tatiana (advisor) ; Bařinka, Cyril (referee)
Leishmania is a protozoan parasite of vertebrates transmitted by the bite of infected phlebotomine sandflies. In humans, it causes a disease called leishmaniasis, which ranks as one of the most serious neglected tropical diseases. In the vectorial part of the life cycle, the crucial moment is when the flagellate forms (promastigotes) attach to the midgut epithelium of the sandfly. For most leishmania species, little is known about which types of phlebotomine receptors and leishmania surface antigens participate in the binding. Phage display was used to screen for Leishmania mexicana peptide ligands which may play a role in such binding. By affinity selection of phages incubated with promastigote cells, 16 unique peptides were identified. Fluorescent labelling of peptide-bearing phages indicated their putative binding sites on the leishmania surface. Based on the hypothesis that the identified peptides may be a part of receptors found in the phlebotomine midgut, experiments were performed where the sandflies were infected with promastigotes whose binding sites were blocked by two different peptide-bearing phages. The extent of the infection was different between the two cases. However, no statistically significant difference from the control group was observed. Despite unsuccessful attempts to identify a...
Molecular basis of interactions between Dishevelled 3 (Dvl3) and Protein Regulator Of Cytokinesis 1 (PRC1)
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Macůrková, Marie (referee)
Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...
Substrate specificity of histone deacetylases
Ustinova, Kseniya ; Bařinka, Cyril (advisor) ; Bumba, Ladislav (referee) ; Obšil, Tomáš (referee)
In the cell, tubulin undergoes post-translational modifications that create functionally distinct microtubules and mark them for specialized functions. Acetylation of Lys40 of α-tubulin is one of such post-translational modifications controlled by the activity of histone deacetylase 6 (HDAC6). The Lys40 acetylation is a hallmark of stable microtubules, it protects them from mechanical aging, influences cell motility as well as axonal branching and maintenance of neuronal processes. Tubulin stands out as the most prominent physiological substrate for HDAC6. Being a multidomain cytosolic protein, HDAC6 is involved in the myriad of cellular processes and is a promising target for the treatment of cancer and neurodegenerative diseases. The understanding of the mechanisms of HDAC6 interactions with its substrates, especially with tubulin, can open avenues for the development of new treatment strategies exploiting highly selective HDAC6 inhibitors. In this thesis, we have investigated the molecular basis of tubulin recognition by HDAC6. We provided a detailed kinetic analysis showing the HDAC6 deacetylation rate of free tubulin is 1500-fold faster than microtubules. Additionally, we have shown that amino acids of the flexible Lys40 loop (except P1 and P-1) make a minor contribution to the substrate...

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