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Investigation of the molecular mechanisms of elimination of clinically relevant tumors by killer cells of the immune system.
Libigerová, Martina ; Bosáková, Zuzana (referee) ; Bezouška, Karel (advisor)
Carbohydrates have an essentials role in wide range of biological phenomena. It is well known that most of the eukaryotic proteins are glycosylated and that their glycosylation undergoes dynamic changes, nevertheless the biological imperative for these modifications is still not fully understood. However, one area in which the importace of cell surface glycosylation has recently been the subject of active investigations is the tumor plasma membrane biology, where many changes in glycosylation have been found useful for diagnosis, and mostly recent, even for the therapies of malignant disease. Interestingly cell surface glycoconjugates, namely N-linked and O-linked oligosaccharides have been found therapeutically attractive for treatment of certain tumors. And although our understanding of the participation of these principal glycan classes in tumorigenesis is far from complete, there are already several examples of carbohydrate-based antitumor vaccines. Therefore, we decided to give this issue more attention, especially the molecular mechanisms responsible for identifying changes in glycosylation of the surface of tumor cells of the immune system. Although in the past in our laboratory identified a receptor-type lectin specific lectin receptors on natural killer cells, very little is yet known...
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Characterization of transgenic forms of dipeptidylpeptidase IV expressed in astrocytoma cell line U373MG
Vomelová, Ivana ; Vaníčková, Zdislava (referee) ; Bezouška, Karel (advisor)
Dipeptidyl peptidase IV (DPP-IV) is a serine protease, which executes its proteolytic activity by cleaving X-Pro dipeptides from the N-termini of its substrates. Furthermore, DPP-IV exhibits many biological functions independent of its enzymatic aktivity. Previous studies in our laboratory proved increased expression of DPP-IV in high-grade astrocytic tumours. To evaluate the enzymatic and non-enzymatic functions of DPP-IV in a glioma model, clones of asctrocytic cell line U373MG transfected by enzymatically inactive, mutated DPP-IV (mutDPP-IV) and enzymatically active, wild type DPP-IV (wtDPP-IV), were prepared. Enzymatically inactive mutDPP-IV was prepared using point mutation the active site serine residue. Cells U373MG were transfected using a doxycycline inducible Tet-On® system. For further analysis of the transgenic forms of DPP-IV, methods were used for verification of protein expression, enzymatic activity and subcellular localization. Doxycycline induced U373MG mutDPP-IV and U373MG wtDPP-IV cells, expressing mutated and wild type DPP-IV, respectivelly, exhibited increased expression of transgenic DPP-IV in a concentration and time dependent manner. Doxycycline induced U373MG wtDPP-IV cells exhibited both increased expression and enzymatic activity of DPP-IV. In contrast, DPP-IV enzymatic...
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Barley Proteomic Studies Related to Beer Production
Benkovská, Dagmar ; Márová, Ivana (referee) ; Ehrenbergerová, Jaroslava (referee) ; Zdráhal, Zbyněk (referee) ; Bobáľová, Janette (advisor)
Tato práce se zabývá proteomickými studiemi ječmene v souvislosti s výrobou piva. Ječmen patří mezi nejvýznamnější plodiny na světě a je využíván hlavně pro sladovnické účely, nejčastěji pro pivovarnictví. Studium proteinů ječmene během sladování a výroby piva poskytuje informace o změnách v proteinovém složení nebo jejich posttranslačních modifikacích. Jelikož jsou proteiny v ječmeni a jejich změny zásadní pro kvalitu sladu a piva, proteomické studie ječmene mají potenciál pro zlepšení procesu sladování a pivovarnictví. Hlavním cílem této práce je studium ve vodě rozpustných proteinů ječmene a jejich změn, ke kterým dochází během sladování a výroby piva. Rozdíly v proteinovém složení byly sledovány pomocí gelové elektroforézy, kapalinové chromatografie na reverzní fázi, gelové chromatografie a MALDI-TOF hmotnostní spektrometrie. Během sladování se vlivem klíčení zrna zvyšuje množství některých proteinů a také jsou tvořeny nové proteiny. V průběhu vaření piva se naopak v důsledku vysoké teploty a enzymatické aktivity proteáz mnoho proteinů rozkládá. Těmto drsným podmínkám odolají jen některé proteiny, které přechází až do piva a mohou ovlivnit jeho kvalitu. Dále byly zkoumány různé odrůdy ječmene a jejich rozdíly. Byly porovnány odrůdy povolené pro výrobu certifikovaného Českého piva s jednou osvědčenou sladovnickou odrůdou a jednou nesladovnickou odrůdou ječmene. Kromě toho byly studovány v alkoholu rozpustné proteiny ječmene a jejich změny v průběhu sladování. Zvláštní pozornost byla věnována vybrané skupině posttranslačních modifikací proteinů: glykosylacím. Neenzymaticky glykosylované proteiny ječmene (neboli glykované proteiny) jsou tvořeny v průběhu sladování kvůli přítomnosti velkého množství glukózy uvolněné z rozkladu škrobu. Glykované proteiny ovlivňují stabilitu proteinů a kvalitu piva, obzvlášť pěnotvorný účinek. Enzymatické N-glykosylace představují nejčastěji studované posttranslační modifikace u rostlin, protože glykoproteiny hrají klíčovou roli v různých biologických funkcích. Glykoproteiny jsou často přítomny v malém množství, a proto je pro jejich analýzu potřebné obohacení glykoproteinů z komplexní směsi. Pro studium glykoproteinů byla využita afinitní chromatografie s lektinem concanavalin A. Kromě toho byla také optimalizována analýza sacharidové části glykoproteinů. Tato disertační práce přináší důležité informace o proteinech ječmene, jejich změnách a analýze, které budou užitečné pro další studium.
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Optimization of N-glycopeptides analysis methods and their preliminary application to barley proteins study
Benkovská, Dagmar ; Flodrová, Dana ; Bobálová, Janette ; Laštovičková, Markéta
N-glycosylation is the most frequently studied plant protein post-translational modification. The analysis of Nglycopeptides after protein proteolytic digestion offers information about the structure of both oligosaccharide and peptide moiety. However, this method has so far been less commonly used. Since glycopeptides hardly ionize during MS analysis in the presence of non-glycosylated peptides, they need to be separated from the complex peptide mixture. In this study, the glycopeptides enrichment, purification and analysis methods were successfully optimized on two standard N-glycoproteins. Concanavalin A (ConA) lectin tips were used for glycopeptide capturing, and obtained fractions were purified on carbon tips and analyzed using MALDI-TOF mass spectrometry. The differences in the CID fragmentation of certain types of glycopeptides were found. This technique was then applied to glycopeptide analysis of barley grain and malt proteins. Several barley glycopeptides were found, however, their identification was very difficult. More proteins separation techniques will be required before this enrichment procedure in further studies.
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Flagellin and outer surface proteins from Borrelia burgdorferi are not glycosylated
ŠTĚRBA, Ján
Glycosylation of four proteins from Borrelia burgdorferi s.s. was investigated ? flagellins FlaA, FlaB, and outer surface proteins OspA and OspB. Glycosylation of these four proteins was not proved by any of the used techniques. However, other glycan-staining positive proteins were present in the borrelia samples. These proteins were suggested to originate in the culture medium.
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Identification and biochemical characterization of lectins in the hemolymph of three species of tick in the genus \kur{Rhipicephalus}
FIŠER, Miroslav
Lectins are tissue specific carbohydrate binding proteins with possible functions in invertebrate immunity and pathogen transmission. The main goal of this study was to identify hemolymph lectins in three different tick species. Three proteins with molecular weights of 58 kDa, 75 kDa and 180 kDa were detected in all investigated species using antibodies directed against hemagglutination activity of Rhipicephalus appendiculatus hemolymph. These proteins were characterized by biochemical methods such as Schiff/periodate staining, lectin blotting, enzymatic deglycosylation, hemagglutination analysis, immunoblotting, and mass spectrometry.
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Cultivation of tick-borne encephalitis virus in the presence of inhibitor of glycosylation: its effect on nucleotide sequence of genes encoding viral proteins.
FARKOVÁ, Karolína
In this work I compared nucleotide sequences of virus genes and the resulting aminoacid sequences of viral proteins of TBE virus variant Hypr3TM with the parental virus Hypr50/CB. This comparison serves for identification of nucleotide/aminoacid substitutions resulting for virus replication in an enviroment with inhibited protein glycosylation. I identified four nucleotide substitutions; three of them caused also changes in amino acid sequence. When compared to a low-passaged variant Hypr5, three of the nucleotide substituions were identical with this variant. One amino acid substitution is unique for variant Hypr3TM. This substitution caused changes in electrostatic potencial of receptor domain of protein E and thus can affect the protein-protein interactions within homodimers and homotrimers at the surface of the protein and the interaction of the E protein with host-cell receptors for the TBE virus.
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