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Biophysical Characterization of Coronaviral nsp14 Inhibition
Trembulaková, Pavla ; Bouřa, Evžen (advisor) ; Dejmek, Milan (referee)
RNA virus SARS-CoV-2 caused worldwide pandemic of severe disease COVID-19 which lasted more than a year. Repair mechanisms of this virus during replication process significantly reduce efficiency of nucleotide analog drugs, eg. remdesivir. Nonstructural protein (nsp) 14 and nsp10 form a complex which acts as an exonuclease enzyme and will be furthrer referred to as an ExoN complex. This complex can probably cause lower efficiency of incorporation of nucleotide analogs compared to viruses without exonuclease enzymes. The two-protein complex with active site on nsp14 containing two magensium ions seems like a good target for testing potent inhobitors. Among possible inhibitors of SARS-CoV-2 exonuclease complex are isobavachalcone and sofalcone. According to published studies, those small organic molecules chelate magensium ions in active site of exonulease part in nsp14. This results in inactivation of ExoN complex active site in nsp14 structure and disables the catalytic function which acts as repairing element in RNA synthesis process. This tesis focuses on characterization of inhibition of protein complex nsp14 and nsp10 in presence of small selected molecules, isobavachalcone and sofalcone. Exonuclease activity assays in presence of various RNA substrates were performed. Furthermore, there have been...
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Production and characterisation of human C1 inhibitor and Plasmodium falciparum PfMSP3.1 recombinant proteins for structural studies
Čápová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
PfMSP3.1 is one of the surface proteins of the intracellular parasite Plasmodium falciparum, which causes malaria. As one of the evasion strategies of the immunity system of the human host this protein interacts with one regulator of the complement system - C1 inhibitor. Determining the exact binding site and its structural assessment would help to better understand the interaction between the parasite and the host, which is necessary for the disease progression and thus for the development of a potential therapy. In the theoretical part of the thesis, the life cycle of Plasmodium falciparum, the role of the parasite stage called merozoite, the role of its surface proteins, including merozoite surface protein 3, in the attack of red blood cells by the parasite, are described in more detail. It also briefly describes the complement system, its activation pathways and the regulation of these pathways. The experimental part includes the cloning of plasmids to produce C1 inhibitor and various forms of merozoite surface protein PfMSP3.1, transfection of S2 insect cells with these plasmids, subsequent protein expression in S2 cells and their purification. In the second half of the experimental part, we tried to create complexes of C1 inhibitor with individual PfMSP3.1 forms and an attempt to crystallize...
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Properties of plant extracts
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Plants synthesize a wide variety of compounds called secondary metabolites. These compounds comprise products of the phenylpropanoid pathway (flavonoids, stilbens, anthocyanines and tannins), isoprenoids, and alkaloids. Since plant secondary metabolites seem to affect human health in a positive way, decrease the risk of some diseases such as cancer, diabetes type 2 and disorders of the cardiovascular system, their studies attract a lot of attention. The mechanism of the effect of individual compounds on human health has not been clarified yet, the positive effect is probably caused by their antioxidant capacity. In this work 22 extracts from different kinds of fruit and vegetables were prepared. The total content of phenolic compounds and flavonoids, the antioxidant capacity by methods FRAP (ferric ion reducing antioxidant power) and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6- sulphonic acid)] were determined. High content of phenolic compounds was established in the extract from strawberry, cherry and kiwi, the highest antioxidant capacity was found in the extract from strawberry. Another studied attribute of extracts from fruit and vegetables was their influence on the activity of digestive enzymes pancreatic lipase and α-amylase. The extracts from lemon, red currant, jostaberry, lime and...
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Properties of plant extracts
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Plants synthesize a wide variety of compounds called secondary metabolites. These compounds comprise products of the phenylpropanoid pathway (flavonoids, stilbens, anthocyanines and tannins), isoprenoids, and alkaloids. Since plant secondary metabolites seem to affect human health in a positive way, decrease the risk of some diseases such as cancer, diabetes type 2 and disorders of the cardiovascular system, their studies attract a lot of attention. The mechanism of the effect of individual compounds on human health has not been clarified yet, the positive effect is probably caused by their antioxidant capacity. In this work 22 extracts from different kinds of fruit and vegetables were prepared. The total content of phenolic compounds and flavonoids, the antioxidant capacity by methods FRAP (ferric ion reducing antioxidant power) and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6- sulphonic acid)] were determined. High content of phenolic compounds was established in the extract from strawberry, cherry and kiwi, the highest antioxidant capacity was found in the extract from strawberry. Another studied attribute of extracts from fruit and vegetables was their influence on the activity of digestive enzymes pancreatic lipase and α-amylase. The extracts from lemon, red currant, jostaberry, lime and...
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