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Proteolytic enzymes of the blood fluke Schistosoma mansoni: pathobiochemistry and their use in biomedicine
Leontovyč, Adrian ; Mareš, Michael (advisor) ; Kašný, Martin (referee) ; Mikeš, Libor (referee)
Blood flukes of the genus Schistosoma are causative agents of the disease schistosomiasis, which affects more than 250 million people worldwide and together with malaria represents the most important parasitic infection. There is a high risk of resistance development against the only drug in use, therefore novel therapeutic approaches for schistosomiasis are intensively researched. Proteolytic enzymes of schistosomes are crucial for their survival in the host and thus are promising drug and vaccine targets. This thesis is focused on two proteases of the human blood fluke Schistosoma mansoni, which were produced as recombinant proteins and functionally characterized. The first one is serine protease SmSP2, which is localized at the surface of the adult worms and secreted into the blood of the host. It was identified as a vasodilatory and fibrinolytic agent, and its modulatory role in host-parasite interactions was proposed. The second one is cysteine cathepsin SmCL3, which is involved in the digestion of host blood proteins serving schistosomes as nutrients. Potent peptidomimetic inhibitors of SmCL3 were identified, and their antischistosomal activity was demonstrated in an assay with live parasites. The thesis provides new important information about S. mansoni proteases, their pathobiochemistry...
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Kunitz-type inhibitors in Eudiplozoon nipponicum
Černíková, Markéta ; Mikeš, Libor (advisor) ; Jedličková, Lucie (referee)
Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...
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Synthesis of Neuraminidase binders suitable for theranostics
Berenguer Albiñana, Carlos ; Machara, Aleš (advisor) ; Cibulka, Radek (referee) ; Soural, Miroslav (referee)
Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...
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Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance
Poráč, Jakub ; Čečková, Martina (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Poráč Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance P-gp and BCRP are transmembrane proteins that form part of a large family of ABC transporters. These are ATP-driven transporters, which main task is to eliminate exogenous and endogenous substances and their metabolites from cells of both, healthy and tumour tissues. This activity is often associated with the expulsion of administered therapeutics and multiple drug resistance (MDR) in tumour cells. A promising therapy of cancer represents a newer class of drugs target the tyrosine kinase (TK), and cyclin-dependent kinases (CDK), which are cell enzymes responsible for the processes of proliferation, apoptosis and differentiation. Cyclin- dependent kinase inhibitors (CDKI) are used in the treatment of breast cancer, but at the same time they form a new group of drugs with the potential for use in hematological malignancies. In the treatment of AML, a new successful approach is TK inhibitors (TKI), which target the mutated FLT3 receptor, specifically the recently approved drugs midostaurin and...
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Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance
Poráč, Jakub ; Čečková, Martina (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Poráč Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance P-gp and BCRP are transmembrane proteins that form part of a large family of ABC transporters. These are ATP-driven transporters, which main task is to eliminate exogenous and endogenous substances and their metabolites from cells of both, healthy and tumour tissues. This activity is often associated with the expulsion of administered therapeutics and multiple drug resistance (MDR) in tumour cells. A promising therapy of cancer represents a newer class of drugs target the tyrosine kinase (TK), and cyclin-dependent kinases (CDK), which are cell enzymes responsible for the processes of proliferation, apoptosis and differentiation. Cyclin- dependent kinase inhibitors (CDKI) are used in the treatment of breast cancer, but at the same time they form a new group of drugs with the potential for use in hematological malignancies. In the treatment of AML, a new successful approach is TK inhibitors (TKI), which target the mutated FLT3 receptor, specifically the recently approved drugs midostaurin and...
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Effect of cholinesterase inhibitors on monoaminergic system and energic metabolism
Nowak, Tereza ; Mladěnka, Přemysl (advisor) ; Štěpánková, Šárka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...
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Effect of cholinesterase inhibitors on monoaminergic system and energic metabolism
Kalinová, Tereza ; Mladěnka, Přemysl (advisor) ; Štěpánková, Šárka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...
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Effects of NMDA receptor modulators on cell death in models of excitotoxicity in vitro.
Strnadová, Lenka ; Smejkalová, Tereza (advisor) ; Skřenková, Kristýna (referee)
NMDA receptors are ionotropic glutamate receptors (iGluR) activated by the amino acid glutamate and mediating signal transmission in the central nervous system. Their proper activity is essential for synaptogenesis, neuronal plasticity and synaptic transmission. However, excessive activation of NMDAR causes strong influx of calcium ions into neurons which triggers several destructive effects, eventually ending with cell death. This so-called excitotoxicity is present not only in many neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, but also in acute pathophysiological conditions, such as stroke or traumatic brain injury. General NMDAR inhibitors that could potentially prevent neuronal excitotoxicity have shown severe negative side effects in models in vivo. On the other hand, selective inhibitors of NMDA receptors with the ability to block the unwanted excessive activity while preserving NMDAR physiological function have shown to be therapeutically useful. This work is going to briefly summarize the knowledge of structure, activation and localization of NMDA receptors, then it is going to describe their rule in mediating neuronal toxicity and a few methods we can use to study excitotoxicity in vitro. Finally, this work will compare the effects of several known NMDAR...
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