The influence of test specimens preparation and instaling of grid on low temperature characteristics of bituminous mixture

Bakalářská práce byla věnována zkoumání chování asfaltové směsi s asfaltem modifikovaným pryžovým granulátem za nízkých teplot. Práce je rozdělena na praktickou a teoretickou část. Praktická část pojednává o výrobě zkušebních těles a provedení zkoušky pro zjištění nízkoteplotních vlastností s rovnoměrným řízeným poklesem teploty dle ČSN EN 12697-46. Teoretická část byla věnována rešerši literatury, popisu postupu výroby a analýze naměřených hodnot. Byly vyrobeny tři druhy těles z jedné směsi.This thesis was focused on studying of asphalt mixture behaviour with Crumb Rubber modified Bitumen at low temperatures. The work is divided into practical and theoretical part. The practical part is focused on manufacturing of test specimens and using of thermal stress restrained specimen test according to ČSN EN 12697-46. The literature review, description of the specimens manufacturing process and analysis of the measured values are introduced in the theoretical part of the work. Three kinds of test specimens were manufactured from one asphalt mixture.

Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme

<p>Abstract</p> <p>Background</p> <p>Penicillin G acylase of <it>Escherichia coli </it>(PGA_Ec) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in <it>Pichia</it>, we have cloned the leader-less gene encoding PGA_Ecin this host and studied yeast production capacity and enzyme authenticity.</p> <p>Results</p> <p>Leader-less <it>pga </it>gene encoding PGA_Ecunder the control of AOX1 promoter was cloned in <it>Pichia pastoris </it>X-33. The intracellular overproduction of heterologous PGA_Ec(hPGA_Ec) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l^-1was reached. The hPGA_Ecwas purified, characterized and compared with the wild-type PGA_Ec. The α-subunit of the hPGA_Ecformed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin.</p> <p>Conclusions</p> <p>Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGA_Ecin <it>P. pastoris </it>X-33. Aberrant processing of the precursor of PGA_Ecin the cytosol yielded the mature enzyme with modified traits.</p

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸with Cys⁴⁸³stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p

I/18 Příbram - South-east Bypass

katedra silničních stave

Brachiopoda and polychaeta in Taphocenoses of the Bohemian Cretaceous Basis

The sesile suspension feeders of Brachiopoda and Polychaeta phyla are relatively well represented in the Bohemian Cretaceous Basin (BCB), and show significant diversity. All three subphyla of Brachiopoda (Linguliformea, Craniiformea and Rhynchonelliformea) are present in the fossil record of the sedimentary basin infill; from Polychaeta, there has hith- erto been evidence of only the Canalipalpata subclass members that created solid, usually carbonatic tubes. In both abundancy and diversity, the most richest associations of fossil brachiopods and polychaets occure in near-shore facies of the Cenomanian to the Lower Turonian, and hemipelagic facies of the Late Turonian age. Prior research, as shows the literature survey given by the author, has been focused mainly on representatives of both groups of near-shore facies origin. The aim of this thesis is to fill in the existing gaps in our understanding of these groups within the BCB. This objective is accomplished in the form of a series of studies (Sklenář & Simon 2009, Vodrážka & al. 2009, Sklenář & al., in press) elaborating the following topics in detail: (A) taxonomy, distribution, morphology, variabil- ity, evolutionary relationships and stratigraphic significance of cancellothyrid brachiopod Gyrosoria abundant in the Middle and particularly in..

Cyklický peptid sekretovaný Pseudallescheria boydii – potenciální marker v diagnostice fugálních infekcí

The work consisted in isolation of fungal secondary metabolite, its purification by means of liquid chromatography, and structure determination by mass spectrometry and nulear magnetic resonance. Due to the stability of the cycliv peptide the molekule will be tested as marker of fungal infection in anímal mode

Padesát deklamovánek pro mládež

monografi

Taxonomy And Stratigraphic Distribution Of The Ammonite Schloenbachia Neumayr, 1875 From The Bohemian Cretaceous Basin

Košťák, Martin, Sklenář, Jan, Mazuch, Martin, Čech, Stanislav (2019): Taxonomy And Stratigraphic Distribution Of The Ammonite Schloenbachia Neumayr, 1875 From The Bohemian Cretaceous Basin. Fossil Imprint 75 (1): 64-69, DOI: 10.2478/if-2019-0005, URL: http://dx.doi.org/10.2478/if-2019-000