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Synthesis and characterization of binding ligands for the study of targeted lysosomal protein degradation
Sidej, Natan ; Konvalinka, Jan (advisor) ; Baszczyňski, Ondřej (referee)
Targeted protein degradation is a novel concept of chemical biology that has been formulated about 20 years ago. Its central postulate is based on the fact that instead of suppressing protein activity with low-molecular inhibitors, we can instead use molecular tools to hijack the host organism's own degradation pathways and force it to degrade chosen proteins by itself. This diploma thesis revolves around the preparation of biocompatible polymeric conjugates called "iBodies" that will be used to induce targeted lysosomal degradation of two model enzymes - Fibroblast activation protein α, and Glutamate carboxypeptidase II. First, a total of four low-molecular ligands were prepared and fully characterized by standard methods of organic synthesis. The first two are mannose-6-phosphonate derivatives that serve as the inducers of protein degradation via the cellular endosomal-lysosomal degradation pathway. The remaining two are known potent inhibitors of the chosen model enzymes that will serve as their targeting-ligands. The prepared compounds were then used to prepare a total of eight poly-N-(2-hydroxypropyl)methacrylamide conjugates called iBodies, after which the polymeric conjugates were fully characterized by standard means of macromolecular chemistry. Afterwards, the obtained conjugates will be...
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Secreted proteases of the liver fluke and their interaction with endogenous inhibitor
Buša, Michal ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)
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Evaluation of antifungal activity of protease inhibitors from potato (Solanum tuberosum L.)
REISEROVÁ, Jana
This diploma thesis is concerned on protease inhibitors isolated from potato (Solanum tuberosum L.) tubers and evaluation of their antifungal properties. Theoretical part of the thesis deals with protease inhibitors which have an antifungal effect. Tubers of potato cultivars Adéla, Ornella, Eurostarch - were used for protease inhibitors isolation. Antifungal activity of isolated protein fractions were evaluated versus fungi from genus Rhizoctonia and Fusarium that are important pathogens in agriculture. Their activity was also evaluated statistically.
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Separation of selected acidic proteins fractions from potato (Solanum tuberosum L.) tubers by chromatographic techniques
LORENC, František
This diploma thesis deals with acidic proteins contained in the potato (Solanum tuberosum) tubers, or rather about their separation with chromatography techniques. For the analysis were chosen tubers of two czech potato varieties, Adéla and Westamyl. From the concentrated potato juice were eliminated basic and most of the patatin proteins with the gravity column chromatography. In the next step were applied the chromatography techniques on the anion and hydroxyapatite columns by the fast protein liquid chromatography (FPLC) system. On the anion column were separated protein fractions which contained proteins of molecular weight in the range of 15 kDa to 60 kDa (Adéla) and 15 kDa to 100 kDa (Westamyl). The most abundant were the proteins with molecular weight 15 kDa a 20 kDa. In the last step was used the mass spectrometry for the identification of chosen protein fractions.
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Evaluation of antifungal activity of selected peptide fractions isolated from potato tubers against \kur{Fusarium }spp. fungi
REISEROVÁ, Jana
The bachelor thesis deals with evaluation of antifungal activity of selected peptide fractions isolated from potato tuber against Fusarium spp. fungi. Literature review contains information about isolated potato tuber proteins from Solanum tuberosum L., short description of genus Fusarium, species F. solani and F. oxysporum, information about plant defense mechanisms against fungi and at least information about antifungal proteins. The experimental part deals with evaluation of antifungal activity of selected peptide fractions from potato tuber of Solanum tuberosum L. against F.solani and F. oxysporum which were prepared by separation techniques.
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