This master thesis deals with succession and succession mechanisms of beetles (Coleoptera) on carrions of small vertebrates. As a model sample of small carrion a small mouse of weight 20 grams was chosen. In 2014 two experiments occurred. The first one studied the succession of a beetle community in dependence on seasonality and time of death of the sample. The second experiment was aimed to survey the blocking effect of an early insect succession on the final beetle community composition on carrions. It turned out, that during decomposition of the carrion the overall amount of beetles raised equally to the guild of necrophagous beetles. Change of the species spectrum during succession was not statistically confirmed. On the other hand, the effect of seasonality was clear as during the season the abundance and the amount of species increased. The proportion of trophic guilds changed significantly, necrophagous beetles were the most dominant part of the community towards the end of the season. Equally, the taxonomic composition of the community also changed during the season. The seasonal dynamics of carrion beetles was clear; Nicrophorus vespillo (Linnaeus, 1758) and N. investigator Zetterstedt, 1824 showed their peaks in spring and summer while N. humator (Gleditsch, 1767) and N. vespilloides...

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...

Objectives: Hematopoietic stem cell transplantation (HSCT) is a widely used method for treatment of hematological disorders and some other diseases. However, sometimes a suitable donor of hematopoietic stem cells (HSC) is not found for a patient. Because HSC have been described as cells with low proliferative and metabolic activity, their tolerance to the lack of oxygen or metabolic substrates may be assumed. In this study, we explored cadaveric bone marrow as an alternative source of HSC for HSCT, using a mouse experimental model. In addition, the effect of in vitro metabolic inhibition and short-term in vitro storage (1 - 4 days) on functional properties of mouse HSC was investigated. Methods: C57Bl/6 mice (wild-type or p53-/- ) were used in the experiments. To explore cadaveric HSC, bone marrow (BM) was left in intact femurs at 37řC, 20řC and 4řC under the conditions of ischemia. The bone marrow cells were harvested after defined time periods ranging 0 - 48 hours. For metabolic inhibition, the electron transport chain inhibitor potassium cyanide (KCN) and inhibitor of glycolysis 2-deoxy-D-glucose (2-DG) were used in vitro. To determine the impact of ischemia, metabolic inhibition, or in vitro storage on transplantability of HSC, the competitive repopulation assay using Ly5.1/Ly5.2 congenic model...

Bacillus subtilis is a gram positive soil bacterium that is surrounded by many other microorganisms its environment. That is why it is necessary for the bacterium to be able to fight with these microorganisms for the nutrients and living space. B. subtilis contains the modules in its genetic make-up that improve its ability to compete. These modules are called the toxin-antitoxin systems. This Diploma Thesis is trying to identify yet undescribed extracellular toxins produced by the wild type BSB1 strain of B. subtilis. The related microorganism Bacillus megaterium was used as a competing bacterium. The contact-dependent or independent manner of killing the competing bacterium was demonstrated using this model. By deletion analysis and comparisons of the genomes of the various strains of B. subtilis, the SPβ prophage was first identified as a region containing an unknown toxin(s). Analysis of the extracellular proteome of B. subtilis subsequently revealed an unknown toxin (or toxin complex, respectively) of the molecular weight exceeding 100 kDa. Even more fascinating was the finding that such a large protein molecule is resistant to the pancreatic protease, trypsin. Subsequent non-enzymatic cyanogen bromide cleavage of the extracellular proteins and their analysis by mass spectrometry revealed...

N-methyl-D-aspartate (NMDA) receptors are glutamatergic ionotropic receptors involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They are heteromeric complexes of GluN1 combined with GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Many allosteric modulators can influence the activity of these receptors including neurosteroids. Pregnanolone sulfáte (3α5βS) is an endogenous neurosteroid that inhibits NMDA receptors in a use-dependent manner and has neuroprotective effect. Binding site for 3α5βS on the NMDA receptor molecule is still not indentified. The aim of my work was to contribute to the identification of the biding site by kinetic analysis of rate of response return from 3α5βS inhibition. Using the point mutation we also attempted to identify the amino acids residues that could be involved in the neurosteroid binding. In order to study the effect of 3α5βS on NMDA receptors the electropfysiological recordings on human embryonic kidney 293T cells expressing recombinant GluN1/GluN2B receptors was performed. We confirm that the effect of 3α5βS on GluN1/GluN2B receptors is voltage-independent. The results of my work indicate that steroids can reach the binding site on the NMDA receptors through the membrane rather than directly from the aqueous...

IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...

and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...

V práci je popsán princip synchronní detekce (SD), který byl uplatněn při měření s biosenzory. Metoda SD umožňuje dosažení výrazně lepšího poměru signálu k šumu, vyššího limitu detekce a celkové zlepšení robustnosti měření. Uplatnění SD při měření s biosenzory umožní zlepšit analýzu jeho odezvy a umožní odstranění nežádoucích interferencí nebo šumů, které mohou být způsobeny například mícháním roztoku, elektromagnetickými vlivy nebo parazitními proudy. SD také umožňuje rozložit získaný signál na odezvu stimulace a na dlouhodobý signál jiného procesu, a dále také identifikovat jevy druhého řádu. Pro identifikaci stimulačního signálu ve výstupním signálu měření byl na základě lineárního statistického modelu vyvinut specializovaný software. SD byla ověřena na modelovém případu výstupního signálu biosenzoru s aplikovaným komplexem fotosystému II (PSII) a jeho odezvě na stimulaci světlem. Odezva PSII se řídí kinetikou prvního řádu a může být také ovlivněna inhibitory. Kinetické konstanty vazby herbicidu na PSII závisí lineárně na koncentraci herbicidu. To umožňuje jejich měření také při nízkých koncentracích herbicidu.

The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....

Rhododendron tomentosum is an evergreen shrub with a high content of secondary metabolites, particularly essential oils with antimicrobial effects. Diversity of endophytic fungi in this species and their possible adaptation to growth in the essential oil environment is not much explored. Therefore, the first aim of this thesis was to reveal the diversity of endophytic fungi colonising leaves of R. tomentosum on seven localities in the Czech Republic and one in Estonia. I isolated and determined (using comparison of ITS1 and ITS2 rDNA with the sequences from GenBank and morphological signs) 37 species of endophytic fungi. Among them the ubiquitous species colonising the most of the plants as endophytes were dominant. The second aim of my thesis was to explore whether the essential oil from R. tomentosum influences its endophytic fungi. The hypothesis that the strains obtained from R. tomentosum would be adapted to growth in the environment of the essential oil was postulated. I supposed that they would grow better on mediums with different concentrations of these chemical compounds added, in comparison with strains of the same species obtained from different substrates. Within four of seven species tested, the strains obtained from R. tomentosum grew better, but also on the medium without the...