Subcelulární lokalizace a úloha komplexu exocyst v savčích buňkách během cytokineze

Cytokinesis is the last step of cell cycle when two individual daughter cells separate in process called abscission. This process involves various cellular membrane structures such as endoplasmic reticulum or trans-Golgi network. Moreover, recent investigation has also highlighted an important role of recycling endosomes. The membrane dynamics appear to be important during cell division especially for the formation of new plasma membrane between two daughter cells. Numerous studies suggest that cytokinesis is tightly linked with highly sophisticated transmembrane shuttle that is controlled by Ras-superfamily members such as Rab and Ral proteins. Moreover, during last years has also been revealed the involvement of tethering factors which mediate the fusion of intracellular vesicles with the target plasma membrane. The best known tethering factor is the evolutionary conserved exocyst complex found in all eukaryotic cells. This protein complex is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) and was found to interact with members of Ras- superfamily suggesting its involvement in the regulation of cytokinesis. Although the exact mechanism remains shrouded in fog this work suppose the possible interactions among Ras- like proteins and exocyst members which may...Posledním krokem buněčného cyklu je cytokineze. Během ní dojde k oddělení dvou dceřiných buněk. Tohoto procesu se účastní množství membránových struktur jako jsou endoplazmatické retikulum a trans-Golgi komplex. Kromě toho je v posledních letech stále více zdůrazňována úloha recyklačních endozomů. Všechny tyto organely spolu vzájemně komunikují pomocí vnitrobuněčného transportu, který je velice důležitý pro úspěšný průběh cytokineze zejména z pohledu nově vznikající plazmatické membrány oddělujících se dceřinných buněk. Nedávné studie odhalily, že tato membránová dynamika je regulována tzv. malými GTPázami z proteinové nadrodiny Ras. Další proteiny s významnou úlohou během membránového transportu jsou takzvané poutací komplexy, které směrují příslušné vnitrobuněčné komponenty k cílové plazmatické membráně, kde usnadňují vzájemnou fúzi. Nejlépe prozkoumaným poutacím komplexem je exocyst komplex. Jeho přítomnost byla prokázána ve všech typech eukaryotních buněk. Tento proteinový komplex je složený z osmi podjednotek Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 a Exo84. Ačkoliv přesný mechanizmus zůstává neznámý, zdá se, že dochází k interakcím mezi podjednotkami exocyst komplexu a zástupci Ras rodiny proteinů. Je proto možné, že se tyto proteiny podílejí na regulaci transportu vnitrobuněčných váčků...Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/significanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy

Application of set-covering problem to fire district station Ovčáry

This thesis deals with the set-covering problem which belongs to distribution problems of the linear programming and in a practice can be found in many forms. The main goal of the standard set-covering problem is the construction of a new operating station which contains a degree of danger, but this thesis is focused on one operating station and examines the decision about its construction like a station of type JPO I, the problem contains so-called degrees of danger. Thesis is based on the real data provided by Fire Brigade of the Central region (Hasičský záchranný sbor Středočeského kraje). The theory of the distribution problems and optimization system MPL (which is the problem solved by) is presented in the first part. There a reader can also find a little information from the history of The Fire Brigade of the Czech Republic (HZS ČR). In the next part there is the description of modification for examining area coverage of the territory, where fire units are included instead of operating stations

Subcelulární lokalizace a úloha komplexu exocyst v savčích buňkách během cytokineze

Cytokinesis is the last step of cell cycle when two individual daughter cells separate in process called abscission. This process involves various cellular membrane structures such as endoplasmic reticulum or trans-Golgi network. Moreover, recent investigation has also highlighted an important role of recycling endosomes. The membrane dynamics appear to be important during cell division especially for the formation of new plasma membrane between two daughter cells. Numerous studies suggest that cytokinesis is tightly linked with highly sophisticated transmembrane shuttle that is controlled by Ras-superfamily members such as Rab and Ral proteins. Moreover, during last years has also been revealed the involvement of tethering factors which mediate the fusion of intracellular vesicles with the target plasma membrane. The best known tethering factor is the evolutionary conserved exocyst complex found in all eukaryotic cells. This protein complex is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) and was found to interact with members of Ras- superfamily suggesting its involvement in the regulation of cytokinesis. Although the exact mechanism remains shrouded in fog this work suppose the possible interactions among Ras- like proteins and exocyst members which may..

Journey through the beauty of the Czech Republic – application of the travelling salesman problem using the goal programming

Tato diplomová práce se zabývá úlohou obchodního cestujícího, která patří do kategorie distribučních úloh lineárního programování a kterou je možné v praxi nalézt v mnoha podobách. V této aplikaci je úloha rozšířena na úlohu vícekriteriální a k jejímu řešení je použit jak klasický model lineárního programování, tak cílové programování, které je součástí teorie rozhodování. Úloha je aplikována v oblasti cestovního ruchu. Cílem je nalézt nejvhodnější okruh po vybraných místech České republiky. V první části práce je popsána úloha obchodního cestujícího, druhá kapitola je zaměřena na teorii cílového programování, které je též stěžejním postupem této práce. Další kapitola pojednává o postupech, které jsou důležité pro praktickou část. Součástí práce je též kapitola zaměřená na optimalizační systém MPL, pomocí kterého je úloha řešena. V přílohách je dále popis jednotlivých míst.This diploma thesis deals with the travelling salesman problem which belongs to distribution problems of the linear programming and in practice can be found in many forms. This application in tourism is the multi-criteria decision problem solved by the standard linear model and also by the goal programming which is the part of the decision making theory. The aim of this thesis is to find the best route contains the part of the selected interesting places in the Czech Republic. The first part comprises description of the travelling salesman problem, the second chapter focus on goal programming which is the key approach in this thesis. The next chapter deals with approaches which are relevant for the practical part. The thesis included also the chapter focuses on optimization system MPL which is the problem solved by. Description of the selected interesting places is in the annex

Determination of functional activity of trimeric G-proteins in brain cortex tissue

Katedra biochemieDepartment of BiochemistryFaculty of SciencePřírodovědecká fakult

Characterization of serine proteases of Schistosoma mansoni involved in interaction with host

Schistosomiasis is a serious parasitic disease, caused by blood flukes of the genus Schistosoma. It remains a global health problem in the 21st century, with more than 250 million people infected in 78 countries. Current therapy relies on the drugs praziquantel and oxamniquine, for which there are concerns of emerging drug resistance. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising targets for the development of new therapeutic strategies against schistosomiasis. This work focuses on the characterization of Schistosoma mansoni serine proteases (SmSPs) and the determination of their role in the interaction with the human host using a variety of genomic, bioinformatic, RNA- and protein-based techniques. First, the major types of proteolytic activities secreted by the blood-dwelling developmental stages of S. mansoni were classified using functional proteomics. The analysis revealed the complexity of proteolytic activities secreted by the schistosome life stages parasitizing the human host. All stages secreted significant serine protease activities, and consequently their genes were retrieved from the genome database and annotated. Localization in adult worms determined by fluorescence in situ RNA hybridization revealed complex expression..

Charakterizace serinových proteáz u motolice Schistosoma mansoni participujících na interakci s hostitelem

Schistosomiasis is a serious parasitic disease, caused by blood flukes of the genus Schistosoma. It remains a global health problem in the 21st century, with more than 250 million people infected in 78 countries. Current therapy relies on the drugs praziquantel and oxamniquine, for which there are concerns of emerging drug resistance. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising targets for the development of new therapeutic strategies against schistosomiasis. This work focuses on the characterization of Schistosoma mansoni serine proteases (SmSPs) and the determination of their role in the interaction with the human host using a variety of genomic, bioinformatic, RNA- and protein-based techniques. First, the major types of proteolytic activities secreted by the blood-dwelling developmental stages of S. mansoni were classified using functional proteomics. The analysis revealed the complexity of proteolytic activities secreted by the schistosome life stages parasitizing the human host. All stages secreted significant serine protease activities, and consequently their genes were retrieved from the genome database and annotated. Localization in adult worms determined by fluorescence in situ RNA hybridization revealed complex expression...Schistosomóza je závažné parazitární onemocnění způsobené motolicemi rodu Schistosoma. I ve 21. století zůstává celosvětovým zdravotním problémem, neboť je jí nakaženo více než 250 milionů lidí v 78 zemích světa. Současná terapie je založena především na dvou lécích, praziquantelu a oxamniquinu. Vzrůstající riziko vzniku rezistence vyvolává potřebu nových alternativních chemoterapeutik nebo vakcín. Proteázy schistosom se podílejí na mnoha kritických interakcích parazita s hostitelem, a proto představují slibné cíle pro vývoj nových léčebných strategií proti schistosomóze. Tato dizertační práce se zaměřuje na charakterizaci serinových proteáz krevničky Schistosoma mansoni a na určení jejich role v interakci s lidským hostitelem za použití genomických a bioinformatických přístupů, včetně metod molekulární biologie RNA a proteinové biochemie. Nejprve byly pomocí funkční proteomiky klasifikovány hlavní typy proteolytických aktivit v exkrečně sekrečních produktech vylučovaných vývojovými stadii S. mansoni žijícími v krvi hostitele. Analýza odhalila komplexnost těchto proteolytických aktivit, které jsou jedinečné pro každé stádium parazitující v lidském hostiteli. U všech studovaných stádií byla v sekrečních produktech detekován významný podíl aktivit serinových proteáz. Následně byla v genomové databázi...Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

ProlyL oligopeptidase from the blood fluke schistosoma mansoni: from functional analysis to anti-schistosomal inhibitors

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs