Techn. University, Diss., 2005--München.

München, Techn. Universiẗat, Diss., 2004.

Abstract: TSG101 is a tumor susceptibility gene exhibits multiple biological function, including the regulation of cell progression, intracellular protein sorting and membrane trafficking, and transcription activity of nuclear recptor such as estrogen recptor. TSG101 contains an UBC domain which is homologous to that in ubiquitin conjugating E2 enzyme. However, it lacks an essential cysteine residue, which is essential for catalytic activity. Cellular protein ubiquitination serves as a signal for protein degradation or sorting into multivesicular body. UBC domain of TSG101 was proved to contain amino acid residues that are important for its interaction with ubquitin (residues V43, N46, D46 and F88) and PTAP sequence found in the late domain of HIV gag protein (residues Y63, M95, V141). SUMO is an ubquitin-like modifier which can modify cellular protein harbors £ZKXE amino acid sequence, thereby change its subcellular localization and biological activities. TSG101 protein contains K98, K243, K264 and K269 residues that localize in potential SUMO modification site. Our preliminary data indicated that TSG101 colocalize with SUMO in nucleus. It is interesting to know whether TSG101 is sumoylated, and its functional significance. In this thesis, a series of site-directed mutageneic mutant HA and GFP-tagged expression plasmids which contain mutation of the above mentioned functional related amino acid residues were constructed for future TSG101 functional studies.

No description available.

Workshop "Netz- und Service-Infrastrukturen" Darstellung wesentlicher technischer und inhaltlicher Aspekte eines einheitlichen Identitätsmanagements an der UB Chemnitz unter konkreter Beachtung des Lokalsystems LIBERO, das an allen wissenschaftlichen Bibliotheken Sachsens betrieben wird

Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...

Ubiquitylation is an important post-translational modification and can be reversed by the action of deubiquitinating (DUB) enzymes. The ubiquitylation and deubiquitylation of target proteins are significant in terms of regulating cellular events such as protein degradation, signal transduction, vesicle trafficking, DNA repair and apoptosis. Chromosomal band 17q23 is frequently amplified in breast cancers and harbors a predicted ubiquitin specific protease gene, USP32 (ubiquitin specific protease 32). Given its potential role in breast cancer, we aimed to characterize USP32 for its potential DUB activity. Bioinformatic analysis of USP32 and known yeast and mouse DUBs suggested presence of Cys-His domains which are common in active DUBs of the USP superfamily. Our in vivo and in vitro DUB activity assays revealed that USP32 was indeed an active deubiquitinating enzyme. To investigate its substrate specificity and kinetic properties, USP32 was expressed in insect cell culture to be isolated and purified. Using isolated USP32 protein, diubiquitin assay was performed with all seven types of diubiquitin (K6, K11, K27, K29, K33, K48 and K63) as well as linear diubiquitin. Results showed that USP32 was able to cleave all seven types of ubiquitin linkages with higher cleavage efficiency for K6, K11, K48 and K63-linked diubiquitin. Moreover, kinetic parameters, Km, kcat and kcat/ Km, suggested that full length protein had lower affinity for potential substrates and lower catalytic activity compared to the catalytic domain alone. These data suggested the importance of USP32 tertiary structure and possible role of other non DUB domains (e.g. EF hand domain) which may be regulated by an as of unknown mechanism in cells. Further investigations are underway to understand the functions of USP32 in cells and how it may contribute to breast tumorigenesis.

Uppsatsen är en studie av fjärrlåneverksamheten av äldre tryck, med digitalisering som en valmöjlighet. Kontexten är dels utvecklingen av digitaliseringsprojekt hos bibliotek, dels det användarcentrerade förslaget hos 2010 års fjärrlåneutredning. Som fallstudier beskrivs två bibliotek i Umeå, universitetsbiblioteket och Sveriges depåbibliotek. Utvärderingens centrala fråga är om digitalisering kan vara ett passande alternativ till fjärrlån av äldre tryck, för att stärka användarvänligheten. Problembilden utgår från en frågeställning över tre områden: Hur ser fjärrlånetrafiken av äldre tryck ut? Vilka problematiska aspekter finns det kring fjärrlån? Vilka konsekvenser får ett alternativ som digitalisering? Fjärrlån utgör en mycket liten del av den totala lånesumman, och det äldre trycket av det samma en nästan obetydlig del. Kostnad, tidsåtgång och ömtålighet är aspekter av verksamheten, och ett tänkt utfall är att effektiviteten skulle öka med digitalisering, vilket samtidigt skulle innebära att kulturarvet synliggörs, vilket är digitaliseringens främsta syfte idag. Dessutom skulle användarnas önskemål leda detta tillgängliggörande, vilket svarar mot både riktlinjer över digitalisering och fjärrlåneutredningens användarbaserade målsättning. Ett försök av implementering av digitalisering i fjärrlåneverksamheten vore intressant att se, och Umeå är en passande stad för ett pilotprojekt.

In Saccharomyces cerevisiae nimmt die membrangebundene RING-Ub-Ligase Doa10 eine bedeutende Rolle in der Proteinqualitätskontrolle (PQC) des Endoplasmatischen Retikulums (ER) und des Nukleus ein. Doa10 katalysiert dabei die Verknüpfung K48- verbundener Ub-Ketten auf Proteine, die entweder in der ER-Membran oder löslich im Cytosol oder dem Nukleoplasma vorliegen. Diese Markierung leitet die Degradation dieser Proteine ein. Interessanterweise kooperiert Doa10, im Gegensatz zu anderen RING-Ub-Ligasen, mit zwei Ub-konjugierenden Enzymen (E2), um ihre Substrate zu prozessieren. In dieser Arbeit wird veranschaulicht, wie die beiden hochspezialisierten E2 Enzyme Ubc6 und Ubc7 sequentiell agieren, um Doa10 Substrate zu modifizieren. Zuerst wird ein einzelnes Ub-Molekül Ubc6-abhängig an ein Substrat konjugiert (Initiation). Von diesem Rest ausgehen katalysiert Ubc7 die Ausbildung einer K48-verbundenen Ub-Kette (Elongation). Die Fähigkeit von Ubc6 nicht nur Lysine, sondern auch hydroxylierten Aminosäuren wie Serin und Threonin mit Ub-Molekülen zu verknüpfen, erweitert das Substratspektrum von Doa10 und ermöglicht die Prozessieren von Proteinen, die keine zugänglichen Lysinreste exponieren. Weiterhin wird gezeigt, dass ein Überangebot von Ubc6 den Doa10-abhängigen Substratabbau beeinträchtigt. Dies weist darauf hin, dass die Generierung eines effizienten Poly-Ub-Signals einer streng kontrollierten Koordination beider E2 Enzyme am Doa10-Ligase-Komplex unterliegt. / In Saccharomyces cerevisiae, the membrane-bound RING-type Ub ligase Doa10 is a key player of Protein Quality Control (PQC) in the endoplasmic reticulum (ER) and the nucleus. Doa10 promotes lysine 48-linked poly-ubiquitylation of proteins that either reside in the ER membrane or are soluble in the cytosol or the nucleus and thereby labels them for degradation. Strikingly, in contrast to other RING Ub ligases, which typically employ a single Ub conjugating enzyme (E2) for substrate ubiquitylation, the Doa10 ligase requires two of such enzymes for client processing. This study demonstrates that the highly specialized E2 enzymes Ubc6 and Ubc7 act in a sequential manner on Doa10 client proteins. In a first step Ubc6 attaches a single Ub molecule to a substrate (priming), which is followed by the elongation of this moiety with K48-linked Ub chains by Ubc7 (elongation). The ability of Ubc6 to conjugate Ub not only to lysine but also to hydroxylated amino acids like serine and threonine broadens the substrate range of Doa10 and allows processing of proteins, which do not expose accessible lysine residues. Overproduction of Ubc6 was shown to impair Doa10 dependent substrate degradation. Apparently, the generation of a productive K48-linked poly-Ub signal requires a tightly coordinated activity of the individual E2 enzymes at the Doa10 ligase complex.

The thesis analyzes the illustrations and author books of Daisy Mrázková directed mainly to the children readers. The thesis puts this part of her artistic work in the context of her work as a whole as well as in the Czechoslovak artistic milieu from sixties till eighties. Detailed attention is paid to Daisy Mrázková's inspiration, her childhood and education, family life and relationship with her friends, especially those from the artistic group UB 12, and their influence on her work. The work also focuses on the influence of English children literature specifically due to the artist's family origin. Her illustrations and author books are compared to the books of the contemporary Czechoslovak fellow artists as well as her teachers and schoolmates. A significant part of the thesis describes and analyzes synesthesia, both in the artist own work as well as in the work of the Czech and foreign artists.