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Recombinant cysteine peptidases of ticks
Srba, Jindřich
Recombinant cysteine peptidases of Ixodes ricinus Jindřich Srba Abstract Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In a tick, Ixodes ricinus, cysteine proteases, cathepsins B and L, are part of a gut-associated multi-peptidase complex. This thesis deals with preparation of recombinant procathepsins B and L and characterization of functional and biochemical properties of cathepsins B and L. After expression procathepsins B and L in E. coli and their purification from inclusion bodies by affinity chromatography, the basic strategy was searched that would enable to achieve successful refolding. A suitable type in both cases showed to be "basic" refolding. Both refolded procathepsins were auto-activated in the acidic environment of pH 4,0; cathepsin B (but not cathepsin L) also at pH 5,5. Activity was determined at pH dependence substrate specificity and inhibition. The pH optimums for hydrolytic activity of cathepsins B and L were in the range of 4,5 - 5,5 and 3,0 - 3,5, respectively. Both enzymes were blocked by an inhibitor of cysteine peptidase group and each of them by specific inhibitors designed for mammalian cathepsins B and L. The results of this thesis show that cathepsins B and L produced in the gut I. ricinus exhibit exo- and endo-peptidase...
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Pharmacovigilance:Spontaneous Reporting Systems
Srba, Jindřich ; Vlček, Jiří (advisor) ; Votava, Martin (referee) ; Petráček, Jan (referee)
Pharmacovigilance: Spontaneous reporting systems Srba J. Abstract Introduction: Pharmacovigilance system is consisted of several approaches used for drug safety surveillance. Spontaneous reporting systems in European countries have been established in the second half of the 20th century. Originally, spontaneous collection of adverse drug reactions was designed for healthcare professionals. Under-reporting was in general considered as the main weakness of spontaneous reporting system. To increase the reporting activity, direct patient reporting has been included in spontaneous reporting system at national competent authorities. Aims: The aim of this work was to characterize spontaneous reporting system. Subsequently, we focused on the evaluation of position of patient-reported outcomes of adverse events (PRO-AE) in established systems of the European countries. In particular, we i) characterized reporting activity within European countries, ii) evaluated the position of adverse drug reaction (ADR) reports directly submitted by patients to national competent authorities, and iii) overviewed of the processing of PRO- AEs. Methodology: Practical part was based on questionnaires distributed to national competent authorities and literature search of electronic databases with relevant published articles. Results:...
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Recombinant cysteine peptidases of ticks
Srba, Jindřich
Recombinant cysteine peptidases of Ixodes ricinus Jindřich Srba Abstract Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In a tick, Ixodes ricinus, cysteine proteases, cathepsins B and L, are part of a gut-associated multi-peptidase complex. This thesis deals with preparation of recombinant procathepsins B and L and characterization of functional and biochemical properties of cathepsins B and L. After expression procathepsins B and L in E. coli and their purification from inclusion bodies by affinity chromatography, the basic strategy was searched that would enable to achieve successful refolding. A suitable type in both cases showed to be "basic" refolding. Both refolded procathepsins were auto-activated in the acidic environment of pH 4,0; cathepsin B (but not cathepsin L) also at pH 5,5. Activity was determined at pH dependence substrate specificity and inhibition. The pH optimums for hydrolytic activity of cathepsins B and L were in the range of 4,5 - 5,5 and 3,0 - 3,5, respectively. Both enzymes were blocked by an inhibitor of cysteine peptidase group and each of them by specific inhibitors designed for mammalian cathepsins B and L. The results of this thesis show that cathepsins B and L produced in the gut I. ricinus exhibit exo- and endo-peptidase...
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