Entwicklung einer iOS Anwendungzur Unterstützung von Mitarbeitern der Qualitätssicherung vor Ort

Mitarbeiter der Qualitätssicherung haben einen umfangreichen Aufgabenbereich, der sich damit beschäftigt, Kosten zu minimieren, die durch fehlerhafte Produkte entstehen. Sie sind dafür zuständig Fehler zu analysieren, um ihre Ursachen herauszufinden. Damit können Verfahren entwickelt werden, die das Auftreten des Fehlers verhindern. Eine detaillierte Analyse der Fehler erfordert ein grundlegendes Fachwissen aller Arbeitsschritte, die ein Produkt durchläuft. Dieses Wissen wird meist über entsprechende Fachliteratur bezogen. Solche Werke sind in der Regel sehr umfangreich und erfordern eine aufwändige Suche, um Informationen zu finden, die für einen speziellen Fehler relevant sind. Aus diesem Grund ist das Ziel dieser Arbeit die Entwicklung einer mobilen Anwendung, welche die Mitarbeiter der Qualitätssicherung bei der Fehleranalyse unterstützt. Dafür werden zunächst einige Grundlagen vorgestellt, die sich mit Normen und der Funktionsweise eines REST Webservices beschäftigen. Außerdem wird die Struktur eines Pakets definiert, das die Fachliteratur der Qualitätssicherung enthält. Anschließend werden die Anforderungen dieser Anwendung analysiert, die den späteren Funktionsumfang beschreiben. Auf diesen Grundlagen wird die Implementierung der Anwendung vorgestellt und mit anderen Anwendungen verglichen, die ähnliche Ziele haben. Abschließend werden mögliche Erweiterungen für die Anwendung vorgestellt

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)

We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology

Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT

Prophylactic titanium elastic nailing (TEN) following femoral lengthening (Lengthening then rodding) with one or two nails reduces the risk for secondary interventions after regenerate fractures: a cohort study in monolateral vs. bilateral lengthening procedures

Background: Femoral fracture rates of up to 30% have been reported following lengthening procedures using fixators. “Lengthening then rodding” uses one or two titanium elastic nails (TENs) for prophylactic intramedullary nailing to reduce this complication. The aim of the study was to decide if usage of only one TEN is safe or has it a higher risk of getting a fracture? And we asked if there is a difference between patients with monolateral or bilateral lengthening procedures according to their fracture rate? Methods: One or two TENs were implanted in two groups of patients (monolateral and bilateral) after femoral lengthening procedures. The regenerate quality was classified using the Li system and fractures were categorized using the Simpson and Kenwright classification. The follow-up period was at least 1 year after removal of the frame. Results: Sixty-seven patients with 101 femoral lengthening procedures were included in 2007–2011. Group A included 34 patients with bilateral lengthening due to congenital short stature. Group B consisted of 33 patients with congenital disorders with leg length discrepancies. Seven fractures in six patients were seen in group A and five fractures in group B. One patient had residual shortening of 1 cm, and 11 fractures healed without relevant deviation (< 5°) or shortening (< 5 mm). A soft-tissue infection in one patient led to early removal of one TEN. Conclusions: Fractures occurred in both groups of patients in total in 12 of the 101 cases (12%). The rate of secondary interventions was markedly reduced. Usage of one or two TENs did not influence the fracture rate.<br

Modulation of microtubule acetylation by the interplay of TPPP/p25, SIRT2 and new anticancer agents with anti-SIRT2 potency

Abstract The microtubule network exerts multifarious functions controlled by its decoration with various proteins and post-translational modifications. The disordered microtubule associated Tubulin Polymerization Promoting Protein (TPPP/p25) and the NAD+-dependent tubulin deacetylase sirtuin-2 (SIRT2) play key roles in oligodendrocyte differentiation by acting as dominant factors in the organization of myelin proteome. Herein, we show that SIRT2 impedes the TPPP/p25-promoted microtubule assembly independently of NAD+; however, the TPPP/p25-assembled tubulin ultrastructures were resistant against SIRT2 activity. TPPP/p25 counteracts the SIRT2-derived tubulin deacetylation producing enhanced microtubule acetylation. The inhibition of the SIRT2 deacetylase activity by TPPP/p25 is evolved by the assembly of these tubulin binding proteins into a ternary complex, the concentration-dependent formation of which was quantified by experimental-based mathematical modelling. Co-localization of the SIRT2-TPPP/p25 complex on the microtubule network was visualized in HeLa cells by immunofluorescence microscopy using Bimolecular Fluorescence Complementation. We also revealed that a new potent SIRT2 inhibitor (MZ242) and its proteolysis targeting chimera (SH1) acting together with TPPP/p25 provoke microtubule hyperacetylation, which is coupled with process elongation only in the case of the degrader SH1. Both the structural and the functional effects manifesting themselves by this deacetylase proteome could lead to the fine-tuning of the regulation of microtubule dynamics and stability

Small Molecule Tools to Study Cellular Target Engagement for the Intracellular Allosteric Binding Site of GPCRs

Abstract A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein‐coupled receptors (GPCRs). Ligands targeting the IABS, so‐called intracellular allosteric antagonists, are highly promising compounds for pharmaceutical intervention and currently evaluated in several clinical trials. Beside co‐crystal structures that laid the foundation for the structure‐based development of intracellular allosteric GPCR antagonists, small molecule tools that enable an unambiguous identification and characterization of intracellular allosteric GPCR ligands are of utmost importance for drug discovery campaigns in this field. Herein, we discuss recent approaches that leverage cellular target engagement studies for the IABS and thus play a critical role in the evaluation of IABS‐targeted ligands as potential therapeutic agents

A Family of Styrylcoumarins: Synthesis, Spectroscopic, Photophysical and Photochemical Properties

A new family of 5-styrylcoumarins exhibit E–Z isomerization around the Cα[DOUBLE BOND]Cβ bond with large extinction coefficients, medium-lived excited states, and moderate fluorescence quantum yields (see picture). The alteration of these photophysical properties is also corroborated by computational studies

Non-Phosphorylatable PEA-15 Sensitises SKOV-3 Ovarian Cancer Cells to Cisplatin

The efficacy of cisplatin-based chemotherapy in ovarian cancer is often limited by the development of drug resistance. In most ovarian cancer cells, cisplatin activates extracellular signal-regulated kinase1/2 (ERK1/2) signalling. Phosphoprotein enriched in astrocytes (PEA-15) is a ubiquitously expressed protein, capable of sequestering ERK1/2 in the cytoplasm and inhibiting cell proliferation. This and other functions of PEA-15 are regulated by its phosphorylation status. In this study, the relevance of PEA-15 phosphorylation state for cisplatin sensitivity of ovarian carcinoma cells was examined. The results of MTT-assays indicated that overexpression of PEA-15AA (a non-phosphorylatable variant) sensitised SKOV-3 cells to cisplatin. Phosphomimetic PEA-15DD did not affect cell sensitivity to the drug. While PEA-15DD facilitates nuclear translocation of activated ERK1/2, PEA-15AA acts to sequester the kinase in the cytoplasm as shown by Western blot. Microarray data indicated deregulation of thirteen genes in PEA-15AA-transfected cells compared to non-transfected or PEA-15DD-transfected variants. Data derived from The Cancer Genome Atlas (TCGA) showed that the expression of seven of these genes including EGR1 (early growth response protein 1) and FLNA (filamin A) significantly correlated with the therapy outcome in cisplatin-treated cancer patients. Further analysis indicated the relevance of nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signalling for the favourable effect of PEA-15AA on cisplatin sensitivity. The results warrant further evaluation of the PEA-15 phosphorylation status as a potential candidate biomarker of response to cisplatin-based chemotherapy. View Full-Tex