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Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni
Jílková, Adéla ; Mareš, Michael (advisor) ; Obšil, Tomáš (referee) ; Mikeš, Libor (referee)
Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...
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Nutritional biology of synanthropic mites (Acari: Acaridida)
Erban, Tomáš ; Smrž, Jaroslav (advisor) ; Grubhoffer, Libor (referee) ; Šustr, Vladimír (referee)
Ph.D. THESIS TITLE Nutritional Biology of Synanthropic Mites (Acari: Acaridida) ABSTRACT Several attempts to describe the nutritional biology of acaridid mites were undertaken, however full understanding of these processes remains incomplete. The objective of this Ph.D. thesis was to expand our knowledge concerning digestive physiology of stored product and house dust mites and to apply this knowledge to their nutritional biology. The research approach adopted in this Ph.D. thesis includes in vitro characterization of enzymatic activity in whole mite extracts (WME) and spent growth medium extracts (SGME), evaluation of the enzyme activities with respect to the gut physiological pH, enzyme inhibition experiments, in vivo localization of enzyme activities in the mite gut, determination of effects of nutrient or antifeedant additives in experimental diets on mite population growth and determination of the feeding preferences of synanthropic mites as assessed by in vitro and in vivo analyses. The gut contents of twelve species of synanthropic acaridid mites were determined to be within a pH range of 4 to 7 and showed a pH gradient from the anterior to the posterior midgut. The pH in digestive tract of synanthropic acaridid mites corresponds to the activity of proteases, α-glucosidases, α-amylases and...
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Inhibitors of mouse serine racemase
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
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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Study of the cleavage kinetics of Gag polyprotein from HIV-1 virus by the viral proteinase
Krištofičová, Ivica ; Ingr, Marek (advisor) ; Martínek, Václav (referee)
Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...
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Role of STAT3 signalling in oncogenesis and cancer therapy
Machalová, Veronika ; Hodný, Zdeněk (advisor) ; Brábek, Jan (referee)
STAT3 (Signal Transducer and Activator of Transcription 3) is considered to be one of the possible targets of cancer treatment. The ability of STAT3 constitutive activation to form tumors is a foundation of such theories. Additionally, constitutively activated STAT3 is present in many types of cancer with high occurrence, such as breast and prostate carcinoma. This protein is required in normal body cells as well. STAT3 is a transcription factor targeting many genes that are essential for the cell. STAT3 is activated by phosphorylation of its tyrosine residue and homodimerization. Proteins transcribed with help of STAT3 function in cell cycle progression, cell growth, replication, negative regulation of apoptosis, and other roles, typical for cancer. Moreover, STAT3 is modulating mitochondrial function and maintaining ROS production in mitochondria, but in form of transcriptionally inactive monomers. The purpose of this Thesis is to review known data about STAT3 in oncogenesis and by that, to show STAT3 has great potential to become the target of cancer treatment. This Thesis contains a short overview of known STAT3 inhibitors as well. Key words: Signal Transducer and Activator of Transcription 3 (STAT3), JAK/STAT3 pathway, constitutive activation, cancer, tumor, inhibitor, mitochondria, apoptosis
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Examination of the activity and possibility verification ability of various types of corrosion inhibitors
Kroča, Michal ; Švehla, Vladimír (referee) ; Drochytka, Rostislav (advisor)
The aim of the work is to describe the degradation of concrete structures from the perspective of corrosion of steel reinforcement. The basis of a detailed survey and analysis of additives containing corrosion inhibitors, designed to improve the concrete matrix and increased protection of steel reinforcement. The essential part is a summary of the detection methods designed to verify the presence of inhibitors in reinforced concrete (direct method) or anti - corrosion effect on steel reinforcement (indirect method). Subsequently, to compare the available products in the market based on their characteristics and parameters, including prices and environmental point of view. From the data obtained is drawn optimization calculation, the output of which is to determine nevhodnějšího product for protection of steel reinforcement embedded in concrete structures, especially at a dosage rate specified by the manufacturer.
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A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, M. ; Hradílek, M. ; Lepšík, M. ; Konvalinka, J.
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance developoment. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permits to assess the donor/acceptor realtions of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired ihnibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
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