New Microbial Glycosidases

Sunrvranv Glycosidases from fungi are useful in the preparation of various glycosides mainly by transglycosylation or reversed glycosylation. The lack ofany requirement for protection- deprotection sequences, mild conditions and easier synthesis of thermodynamically not preferred glycosidic bonds are the main advantages of glycosidase-catalysed synthesis of glycosides. In ttris thesis we concentratedon exoglycosidases,mainly B.N.aceýlhexosaminidases, cr-galactosidases and cr-L-rhamnosidases. For the enzyme preparation taxonomically characterised fungal strains from public collections were used. A library comprising more than 200 various glycosidases was developed by modification ofcultivation conditions and by the use ofspecific inducers. The enrymes were used for screening ofsubstrate specificity and stability in organic solvents and subsequently for synthesis and modification of various substrates. Both B-N-acetylgalactosaminidase and B-N-acetylglucosaminidase activities in the series of B-N-acetylhexosaminidases were determined. Saccharides with strong immunomodulation activity $-o-GalpNAc-(i-+4)-o-GlcpNAc and B-o-GalpNAc-(1-+6)-o-GlcpNAc were synthesisedby transglycosylation using B-N-acetylhexosaminidase from Penicillium oxalicum CCF 2430' enryme having the highest B.N-acetylgalactosaminidase activiý....Sunrvranv Glycosidases from fungi are useful in the preparation of various glycosides mainly by transglycosylation or reversed glycosylation. The lack ofany requirement for protection- deprotection sequences, mild conditions and easier synthesis of thermodynamically not preferred glycosidic bonds are the main advantages of glycosidase-catalysed synthesis of glycosides. In ttris thesis we concentratedon exoglycosidases,mainly B.N.aceýlhexosaminidases, cr-galactosidases and cr-L-rhamnosidases. For the enzyme preparation taxonomically characterised fungal strains from public collections were used. A library comprising more than 200 various glycosidases was developed by modification ofcultivation conditions and by the use ofspecific inducers. The enrymes were used for screening ofsubstrate specificity and stability in organic solvents and subsequently for synthesis and modification of various substrates. Both B-N-acetylgalactosaminidase and B-N-acetylglucosaminidase activities in the series of B-N-acetylhexosaminidases were determined. Saccharides with strong immunomodulation activity$-o-GalpNAc-(i-+4)-o-GlcpNAc and B-o-GalpNAc-(1-+6)-o-GlcpNAc were synthesisedby transglycosylation using B-N-acetylhexosaminidase from Penicillium oxalicum CCF 2430' enryme having the highest B.N-acetylgalactosaminidase activiý....Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Proposal of Didactic Text for Subject Physics Designed to Elementary School

Tato práce pod názvem „Návrh učebního textu pro předmět fyzika kapitoly Kapaliny pro ZŠ“ je závěrečnou prací Doplňkového pedagogického studia Univerzity Pardubice. Tento učební text je věnován části učiva předmětu fyzika, kapitole o kapalinách, a je určený pro žáky 2. stupně základních škol. Rozsah učiva je dán rámcovým vzdělávacím programem. Cílem práce bylo vytvořit učební text srozumitelnou a přehlednou formou, kde jednotlivé části na sebe logicky navazují a žáci se v textu snadno orientují. Proto je učební text tvořen kromě textové části i obrazovou částí a odkazy na videa s vhodnými názornými pokusy. Pokusy jsou zde i popsány a vyobrazeny, platnosti zákonů a existence jednotlivých jevů jsou jejich prostřednictvím odvozovány.This thesis under the title “Proposal of Didactic Text for Subject Physics Chapter Liquids Designed to Elementary School” is the final thesis of the Supplementary Pedagogical Studies of the University of Pardubice. This didactic text is dedicated to the part of curriculum of the subject physics, to the chapter about liquids, and is stated for students of the 2nd Grade of Elementary School. The scope of the curriculum is given by the Framework Educational Plan. The goal of the thesis was to create didactic text in an understandable and clear form, where individual parts follow each other logically and students find the way around the text easily. Therefore, the text consists not only of the text but also of images and video links with suitable illustrative experiments. The experiments are described and pictured, and validity of the physical laws and the existence of particular phenomena are derived from them

Experience of English Teachers with Students with Learning Disabilities

The aim of this diploma thesis is to find what stance the particular teachers of primary and secondary schools (N=50) adopt on the problem of learning disability, especially dyslexia. Very important is to study their awareness and knowledge of this issue, their opinions and especially their personal experience. In the theoretical part of the work the history and characteristics of learning disabilities are introduced, then there is the focus on dyslexia particularly where causes, diagnostics and possibilities of reeducation are presented as well. The important part of these issues is a help of specialists which is also a component of the theoretical part. The next section is focused on teaching of foreign languages, especially English language, where the suitable methods and possible teaching aids for learning disabilities are mentioned. In the practical part of this thesis three case interpretations of pupils/students with learning disabilities are presented as well as the research - the questionnaire survey - itself. There is good knowledge of teachers in this topic which is obvious from the result of this research, at the same time their experience with these children is significant, their opinions on mixed classes are predominantly positive. On the other hand, there is a majority of answers..

New Microbial Glycosidases

Sunrvranv Glycosidases from fungi are useful in the preparation of various glycosides mainly by transglycosylation or reversed glycosylation. The lack ofany requirement for protection- deprotection sequences, mild conditions and easier synthesis of thermodynamically not preferred glycosidic bonds are the main advantages of glycosidase-catalysed synthesis of glycosides. In ttris thesis we concentratedon exoglycosidases,mainly B.N.aceýlhexosaminidases, cr-galactosidases and cr-L-rhamnosidases. For the enzyme preparation taxonomically characterised fungal strains from public collections were used. A library comprising more than 200 various glycosidases was developed by modification ofcultivation conditions and by the use ofspecific inducers. The enrymes were used for screening ofsubstrate specificity and stability in organic solvents and subsequently for synthesis and modification of various substrates. Both B-N-acetylgalactosaminidase and B-N-acetylglucosaminidase activities in the series of B-N-acetylhexosaminidases were determined. Saccharides with strong immunomodulation activity $-o-GalpNAc-(i-+4)-o-GlcpNAc and B-o-GalpNAc-(1-+6)-o-GlcpNAc were synthesisedby transglycosylation using B-N-acetylhexosaminidase from Penicillium oxalicum CCF 2430' enryme having the highest B.N-acetylgalactosaminidase activiý...

Magnetické částice s vázanými kinázami pro sekvenční in vitro fosforylace peptidů a proteinů

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer´s disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.Posttranslační modifikace včetně fosforylace významně ovlivňují fyziologickou funkci bílkovin, zvláště těch přirozeně nesbalených a zapojených do mnoha neurodegenerativních onemocnění. Nicméně strukturální a funkční studie takových proteinů vyžadují plně definovanou fosforylaci, včetně těch, které nejsou fyziologické. Proto byly kinázy ERK2 a GSK-3p imobilizovány na různé superparamagnetické částice s karboxylovými, aldehydovými, Ni2 + nebo Co3 + funkční skupiny, s cílem účinně fosforylovat peptidy a proteiny in vitro. Úplná fosforylace specifických syntetických peptidů potvrdila úspěšnou vazbu kináz. Enzymy kovalentně imobilizované na karboxylovaných částicích SeraMag zůstaly aktivní při opakovaném použití, přičemž po 10 použitích byla reziduální aktivita 99,5 ± 0,34% pro GSK-3β a 36,2 ± 2,01% pro ERK2. Částice byly také použity k postupné fosforylaci rekombinantního tau, který je v podmínkách in vivo biomarkerem Alzheimerovy choroby. Systém sestávající ze dvou plně aktivních kináz imobilizovaných na magnetických částicích je tak představen poprvé. Ve srovnání s rozpustnými enzymy jsou fosforylace s enzymy vázanými na částicích snadněji proveditelné, je možné je opakovaně použít a tedy s nízkými náklady. Důležité je, že tyto částice jsou také snadno odstranitelné z fosforylačních reakcí, a minimalizuje se tak kontaminace fosforylovaných produktů nebo se tak umožní výměna s jinými kinázami