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Determination of binding constants of human insulin complexes with serotonin, dopamine, arginine, and phenol by pressure assisted partial filling affinity capillary electrophoresis
Šolínová, Veronika ; Žáková, Lenka ; Jiráček, Jiří ; Kašička, Václav
A new method, pressure assisted partial filling affinity capillary electrophoresis (PF-ACE), has been developed to study noncovalent interactions of the hexamer of human insulin (HI) with cationic ligands, such as phenolic neurotransmitters serotonin and dopamine, and amino acid arginine, or with anionic ligand phenol, in alkaline aqueous solutions. The apparent binding constants, Kb, of the HI-ligand complexes were determined from the dependence of the effective migration time changes of the above ligands on the variable zone lengths of HI dissolved in the background electrolyte and hydrodynamically introduced into the bare fused silica capillary close to the UV detector. The HI interactions with the above ligands were found to be moderately strong, with Kb values in the range 385-1314 L/mol.
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Synthesis and characterization of new insulin analogs with a triazole bridge at the C-terminus of the B chain
Kuntová, Vendula ; Jiráček, Jiří (advisor) ; Ryšlavá, Helena (referee)
Insulin is a peptide hormone responsible for maintaining glucose homeostasis in the circulation. Insulin interacts with two isoforms of the insulin receptor, IR-A and IR-B, which have different tissue distribution. IR-A is supposed to have rather mitogenic function and IR-B rather metabolic function. The goal of this study was to develop insulin analog, which will be more selective for IR-B than human insulin. We prepared three new insulin analogs with a 1,2,3-triazole bridge at the positions B26 and B29. The triazole bridge was formed by Cu(I)- catalysed cycloaddition between side chains of azidopentanoic acid (N3Pent) at B26 and propargylglycine (Prg) at B29. The analogs differed in configurations on C carbons of unusual amino acids at the positions B26 and B29. Specifically, we prepared insulin analog 1 with D- N3PentB26 and D-PrgB29, insulin analog 2 with D-N3PentB26 and L-PrgB29 and insulin analog 3 with L-N3PentB26 and D-PrgB29. New analogs were tested for their binding to both isoforms (IR-A and IR-B) of the insulin receptor. Analogs 1 and 2 were less potent in binding than human insulin and had no selectivity for receptor isoforms. Analog 3 was 4-times more potent in binding to IR-B and 2-times more potent in binding to IR-A than human insulin. However, the binding selectivity of the...
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Analogues of IGF-1 for the study of interactions of the hormone with the receptors for IGF-1 and insulin
Macháčková, Kateřina ; Jiráček, Jiří (advisor) ; Obšilová, Veronika (referee) ; Šulc, Miroslav (referee)
Insulin/IGF system is a complex network of three similar hormones (insulin, IGF-1 and IGF-2) and their three similar receptors (IR-A, IR-B and IGF-1R,), which play important roles in maintaining basal energy homeostasis of the organism, in growth, development, life-span but also in development of diseases such as diabetes mellitus, cancer, acromegaly or Laron dwarfism. Despite structural similarities between family members, each member have its unique role in the system. Identification of structural determinants in insulin and IGFs that trigger their specific signalling pathways is important for rational drug design for safer treatment of diabetes or for more efficient combating of cancer or growth-related disorders. In this thesis, we focused on identification of such structural determinants in IGF-1. Comparison of our data with parallel studies with IGF-2 and insulin could give a more complex picture of the problem. First of all, we developed necessary methodologies for the preparation of IGF-1 analogues. We developed a new methodology for the total chemical synthesis of IGF-1 analogues based on the solid-phase synthesis of fragments and their ligation by a CuI -catalyzed cycloaddition of azides and alkynes. In parallel, we developed a procedure for a recombinant production of IGF- 1 and its...
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Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Jiráček, Jiří (referee) ; Schneider, Bohdan (referee)
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
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Preparation of insulin analogs for the study of interactions with insulin receptors.
Hanková, Kateřina ; Jiráček, Jiří (advisor) ; Dračínská, Helena (referee)
Insulin jako jeden z důležitých hormonů lidského organismu se podílí na řadě metabolických procesů. Jednou z funkcí insulinu je pak regulace hladiny glukosy v krvi a jejího vstupu do tkání, ale i stimulace růstu buněk. Insulin má velmi podobnou primární i terciální strukturu s růstovými faktory IGF-1 a IGF-2, jejichž primární funkcí je regulace růstových procesů organismu. Zprostředkování účinku těchto tří hormonů na buněčné úrovni je zajištěno díky jejich specifickým receptorům (mitogenní isoformě receptoru insulinu IR-A, metabolické isoformě IR-B a receptoru pro IGF-1), což může, díky podobnosti těchto receptorů i hormonů, vést ke křížovým interakcím mezi nimi. Poruchy tohoto systému mohou vést k závažným onemocněním. Nejrozšířenějším z těchto nemocí je diabetes mellitus, ale závažné jsou i poruchy růstu a rakovinné bujení. Vytvoření takového analogu insulinu, který bude selektivní pouze pro isoformu IR-B receptoru insulinu, by mohlo vést zejména k bezpečnější léčbě diabetu. Tato práce se zaměřuje na lepší pochopení významu jednotlivých aminokyselin na daných pozicích v A-řetězci insulinu, u kterých je předpokládáno, že jsou součástí vazebného místa hormonu pro receptory. Tyto znalosti by mohly vést k následnému lepšímu porozumění významu těchto aminokyselin ve vazbě na receptory IR-A, IR-B a...
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Synthesis and characterization of new insulin derivatives with altered selectivity for insulin and IGF-1 receptors.
Halamová, Tereza ; Jiráček, Jiří (advisor) ; Dračínská, Helena (referee)
Insulin receptor (IR) exists in two isoforms (IR-A and IR-B), which differ in the tissue distribution and probably also in their function, i.e. in their response to insulin binding. It is supposed that IR-A activates mainly mitogenic processes and that IR-B triggers mainly metabolic effects resulting in the uptake of glucose by muscle and fat cells. Insulin can also weakly bind to the receptor for IGF-1 (IGF-1R), a growth factor involved in the regulation of growth and development. Insulin derivatives selectively binding only to one of the receptors would be interesting for the study of the receptors but also potentially for the treatment of diseases such as diabetes or cancer. Here we used our experience in the structure-activity studies of insulin for the design, synthesis and biological characterization of 4 new insulin derivatives in order to modify their selectivity towards the individual receptors. We systematically modified insulin by amidation of the C-terminus of its B-chain or by prolongation of the B-chain by 1-3 carboxyamidated glycine residues. Binding affinities of all new analogues for IR-A and IR-B were determined and for some of the analogues binding affinities for IGF-1R as well. Finally, abilities of analogues to activate autophosphorylation of intracellular subunits of IR-A and...
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Vaclav Dobias (1909-1978), between music and politics
Jiráček, Jiří ; Randák, Jan (advisor) ; Kopeček, Michal (referee)
The presented thesis deals with the topic of Czechoslovak cultural politics in music during the Socialist Realist era. The author based the study on the personal history of Václav Dobiáš, a prominent composer, cultural organiser and a pedago- gue, who serves as a typical representation of an artist, who bestowed all his artistic talents to the cause of the Communist Party of Czechoslovakia. The first chapter briefly describes Socialist Realism as a movement in music in the Soviet Union, the place of its origin. The main emphasis is put on the introduction to Zhdanov's po- litical speech, which consequently became the mandatory norm in the countries of the Eastern bloc. The next chapter is focused on Czechoslovak reflexion of Socialist Realism and its reception. This chronological development aims to expose the spe- cifics of each periods' reception of this artistic movement. Following chapters are dedicated to Václav Dobiáš. The first chapter of this part of the thesis deals with the beginning of the artist's journey which was strongly influenced by the avantgarde movement of 1930's. Next chapters depict his adoption of communist ideology in 1945, explore the motives behind this ideological conversion and characterize his po- litically-engaged work. This part is followed by a portrayal of Dobiáš as a...
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The Study of the Immune Response of Larvae of the Fleshfly Sarcophaga Bullata
Mášová, Alice ; Jiráček, Jiří (advisor) ; Žurovec, Michal (referee) ; Bezouška, Karel (referee)
Conclusion lnsect immune response is a complicated process. Antimicrobiď peptides fiequently act synergistically, some proteins have transporter functions, peptide inhibitors can block processing enzyrnes involved in activating cascades or different enzymes can activarc antimicrobially active proteins or peptides. In this study we presented nvo different isolation protocols, which resulted in the identification of several already known and two novel antirnierobial proteins or peptides from the hemolymph of the larvae of flesbfty. sareophaga bullan. We are the first group to monitor the time<ourse of tissue-specific expÍession patlems of eight genes in Sarcophaga bullan |arvae ďter different immune challenges using qPCR. we show similarities, as well as differences in insect immune responses. we are also the first group to analyze the expression pattoms of the genes that encode sBp and sarcocystatin, proteins that are mainly connect€d with larvat development and metamorphosis. Using 2D- electrophoresis we analyzď the time.dependent immune response in larval fat bodies and hemocytes. We detect€d 9 up.regulated proteins in hemocyŮes and 15 differentially expressed proteins in fat body cells. We hope that our study will shed more light into ttre complex processes of immune responses in Sarcophaga bullan larvae. 20
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Preparation of a new insulin analog in order to study the interaction of the hormone with insulin receptor isoforms,
Halamová, Tereza ; Jiráček, Jiří (advisor) ; Obšilová, Veronika (referee)
Insulin acts as a key hormone in the blood glucose levels maintaining mechanisms. Outside this metabolic function it also has a growth hormone functionality. The interaction of insulin with the two existing insulin receptor isoforms - IR-A and IR-B, which are variously represented in the human body is determining insulin. IR-A, supposed to be mainly responsible for the mitogenic function of insulin, is located in the brain or lymphatic cancer and fetal tissue, whereas IR-B, performing metabolic function is located in adipose and muscle tissue. Present aim is to design such insulin analogs that would preferentially bind to IR-B, and could thus more efficiently carry out physiological metabolic function of insulin necessary for patients with diabetes. Based on the recently solved 3D structure of insulin bound to IR, it was found that the C-terminus of the B-chain of insulin must undergo conformational change bending it in about 90ř, for efficient binding to IR. The aim of this thesis was the preparation and characterization of two insulin analogs with bridging C-terminus of the B-chain in positions B26-B29 and B27-B29 using disulfide bridge. This could fix a bended structure of the B chain end and could help to increase the affinity of IR and specificity for IR-B. The preparation was carried out...
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