Optimierung der Aufreinigung und Rekonstitution von humanem P-Glykoprotein in <em>S. cerevisiae </em>zur Charakterisierung neuer Modulatoren

P-Gp ist eine ca. 170 kDa großes Protein, das aus zwei homologen Hälften besteht. Jede homologe Hälfte enthält jeweils sechs a-Helix transmembranäre Domainen und eine Nukleotid-Bindungsstelle im Zytoplasma. Für P-Gp werden mehrere Bindungsstellen postuliert. Es existieren zumindest eine H- und eine R-Bindungsstelle. Unklar ist es, ob sich alle Bindungsstellen innerhalb einer großen Bindungstasche befinden, oder ob sie über den gesamten Bereich des Proteins verstreut sind. Zur Aufklärung werden unter anderem funktionelle Assays (ATPase und Calcein), Mutations- und Photoaffinitäts-Labeling-Studien durchgeführt. Um diese Studien zur Struktur-Wirkungsaufklärung direkt an homogenem Protein durchführen zu können, benötigt man ein rekombinantes Expressionssystem. In dieser Arbeit wurde das aus der Literatur bekannte Expressionssystem für P-Gp in dem Protease-defizienten S. cerevisiae Stamm BJ5457 unter der Kontrolle des Promotors PMA1 etabliert und modifiziert. Die zur Rekonstitution verwendeten Lipide beinflussen die spezifische Aktivität von P-Gp. Eine aus Phosphatidylcholin, Phosphatidylsäure und Cholesterol bestehende Lipidmischung zeigte in den Untersuchungen das höchste Verhältnis zwischen basaler und stimulierter ATPase Aktivität (Messfenster). Daher wurde diese Lipidmischung als neue Standardmischung für alle weitergehenden Untersuchungen verwendet. Desweiteren wurde das P-Gp in extrudierte Liposomen rekonstituiert. Das P-Gp richtet sich hierbei bevorzugt mit der ATP-Bindungsstelle nach außen aus. Weiter wurden zwei bzw. fünf zusätzliche Histidine am C-Terminus des Proteins angefügt, um die Affinität für Nickel-Beads zu erhöhen, damit die Ausbeute an reinem Protein erhöht wird. Die Affinitätserhöhung zu den Nickel-Beads konnte zwar bei dem 15-Histidin markiertem Protein nachgewiesen werden, aber das resultierende homogene P-Gp zeigte nur eine geringe spezifische Aktivität. Die cDNA von P-Gp unter der Kontrolle des Promotors PMA1 in Ergosterol-defiziente Stämme kloniert. Der Stamm JS3 ERG2-4, mit vorwiegendem Zymosterol-Anteil, hat eine Expressionsrate von 8,16% ± 2,09%. Der Stamm JS6 ERG6, mit überwiegend Fecosterol-Anteil, hat eine Expressionsrate von 14,7 ± 1,23%. Das P-Gp ist in der Hefemembran mit veränderter Lipidordnung und –fluidität funktionell exprimiert und kann aus beiden Stämmen funktionell aufgereinigt werden. Die mit diesem System erzielten Expressionsraten sind die bisher höchsten. Das mit Octylglucosid aufgereinigte Protein aus den Ergosterol-defizienten Stämmen ist zu 100% aktiv. Die Vorteile des in dieser Arbeit entwickelten Expressionssystem für P-Gp kann auch für die Expression anderer ABC-Transporter in Hefen genutzt werden. Der zweite Teil dieser Arbeit lieferte Erkenntnisse über die Stimulation der ATPase Aktivität durch bekannte und neue Modulatoren basierend auf der Tariquidar-Struktur. In den Untersuchungen konnte erstmals für den Tyrosinkinase-Inhibitor Imatinib eine Wechselwirkung mit der ATP-Bindungsstelle am aufgereinigten Protein gezeigt werden. Bei geringen Konzentrationen von Imatinib (pEC50-Wert von 4,74 ± 0,30) wird die Affinität von ATP zu der Nukleotid-Bindungsstelle von P-Gp vermindert. Ebenfalls konnte für Hoechst, ein spezifisches Substrat der H-Seite, eine Affinitätserhöhung für Rhodamin 123, ein spezifisches Substrat der R-Seite, gezeigt werden. Daraus lässt sich schließen, dass die beiden Bindungsstellen in positiver Regulation miteinander wechselwirken. Der Vergleich der durch den ATPase-Assay bestimmten Affinitätsdaten mit den Affinitätsdaten aus den Calcein-AM-Assay und Daunorubicin-Influx-Assay mittels einer Hauptkomponentenanalyse zeigte für einen homogenen WK-X Datensatz keine Korrelation. Weiterhin wurden die WK-X-Verbindungen auf Ihre physiko-chemischen Eigenschaften durch pKa und log P-Wert Bestimmung untersucht, da diese Faktoren bei Interaktion von Modulatoren mit der Lipidmembran eine Rolle spielen. Eine Korrelation der P-Gp Affinitätsdaten und der log P-Werte konnte in keinem Fall gezeigt werden. Es bestand allerdings eine Korrelation zwischen den jeweiligen log P und log Pion Werten

Microstructure-Based Lifetime Assessment of Austenitic Steel AISI 347 in View of Fatigue, Environmental Conditions and NDT

The assessment of metallic materials used in power plants’ piping represents a big challenge due to the thermal transients and the environmental conditions to which they are exposed. At present, a lack of information related to degradation mechanisms in structures and materials is covered by safety factors in its design, and in some cases, the replacement of components is prescribed after a determined period of time without knowledge of the true degree of degradation. In the collaborative project “Microstructure-based assessment of maximum service life of nuclear materials and components exposed to corrosion and fatigue (MibaLeb)”, a methodology for the assessment of materials’ degradation is being developed, which combines the use of NDT techniques for materials characterization, an optimized fatigue lifetime analysis using short time evaluation procedures (STEPs) and numerical simulations. In this investigation, the AISI 347 (X6CrNiNb18-10) is being analyzed at different conditions in order to validate the methodology. Besides microstructural analysis, tensile and fatigue tests, all to characterize the material, a pressurized hot water pipe exposed to a series of flow conditions will be evaluated in terms of full-scale testing as well as prognostic evaluation, where the latter will be based on the materials’ data generated, which should prognose changes in the material’s condition, specifically in a pre-cracked stage. This paper provides an overview of the program, while the more material’s related aspects are presented in the subsequent paper

SAFIR2022 Program, KYT2022 Program and SAFER2028 Draft Framework External Evaluation Report

At the request of the Ministry of Economic Affairs and Employment of Finland, an international evaluation team conducted an external review of the SAFIR2022 and KYT2022 research programs. Furthermore, feedback on the proposed framework plan of SAFER2028, a new research program that combines the previous SAFIR and KYT programs, was requested. The team found that, in general, the research programs produce a remarkable level of scientific output for a modest stream of funding. The primary, perceived value added by the research programs is that they provide a pipeline of new talent and expertise necessary for the successful regulation of nuclear power and waste management activities in Finland. The research products had, generally, a high technical quality. In some instances, however, the research aimed towards well-established, or mature fields of study where the value added in terms of increased knowledge or improved safety might be considered marginal or incremental. However, the benefit of these activities vis-à-vis the educational benefit in training new experts in relevant fields could be seen. There are opportunities for key improvements in some of the novel elements of the SAFER2028 framework plan, in particular the doctoral education network

A Short-Time Approach for Fatigue Life Evaluation of AISI 347 Steel for Nuclear Power Energy Applications

AISI 347 austenitic steel is, as an example, used in nuclear energy piping systems. Piping filled with superheated steam or cooled water is particularly exposed to high stresses, whereupon local material properties in the pipes can change significantly, especially in the case of additional corrosive influences, leading to aging of the material. In the absence of appropriate information, such local material property variations are currently covered rather blanketly by safety factors set during the design of those components. An increase in qualified information could improve the assessment of the condition of such aged components. As part of the collaborative project “Microstructure-based assessment of the maximum service life of core materials and components subjected to corrosion and fatigue (MiBaLeB)”, the short-time procedure, StrainLife, was developed and validated by several fatigue tests. With this procedure, a complete S–N curve of a material can be determined on the basis of three fatigue tests only, which reduces the effort compared to a conventional approach significantly and is thus ideal for assessing the condition of aged material, where the material is often rare, and a cost-effective answer is often very needed. The procedure described is not just limited to traditional parameters, such as stress and strain, considered in destructive testing but rather extends into parameters derived from non-destructive testing, which may allow further insight into what may be happening within a material’s microstructure. To evaluate the non-destructive quantities measured within the StrainLife procedure and to correlate them with the aging process in a material, several fatigue tests were performed on unnotched and notched specimens under cyclic loading at room and elevated temperatures, as well as under various media conditions, such as distilled water and reactor pressure vessel boiling water (BWR) conditions

Wissenschaftlicher Abschlussbericht zum mFUND-Projekt: EasyGSH-DB

Weitere Titelangaben: Erstellung anwendungsorientierter synoptischer Referenzdaten zur Geomorphologie, Sedimentologie und Hydrodynamik in der Deutschen Bucht (EasyGSH-DB) Gemeinsamer wissenschaftlicher Abschlussbericht mit Beiträgen aus allen fünf TeilprojektenIllustrationen, Diagramme, Karten Förderkennzeichen BMVI 19F2004A-D Verbundnummer 01178048Siehe auch FuE-Abschlussbericht der BAW https://hdl.handle.net/20.500.11970/107374xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxDatasets Themengebiet - Bathymetrie - https://doi.org/10.48437/02.2020.K2.7000.0002Datasets Themengebiet - Geomorphologie - https://doi.org/10.48437/02.2020.K2.7000.0001Datasets Themengebiet - Hydrodynamik - https://doi.org/10.48437/02.2020.K2.7000.0003Datasets Themengebiet - synoptische Hydrodynamik - https://doi.org/10.48437/02.2020.K2.7000.0004Datasets Themengebiet - Sedimentologie - https://doi.org/10.48437/02.2020.K2.7000.000

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy‑number profle with inactivation of TP53, PTEN, and RB1

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.publishedVersio

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors

Sarcoma classification by DNA methylation profiling

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications

Christian Heckötter Simulation of Impact Tests With Hard, Soft and Liquid Filled Missiles on Reinforced Concrete Structures

Vital parts of nuclear facilities are commonly protected by reinforced concrete (rc-) structures. In order to assess the barrier effectiveness of these structures, different internal and external loads have to be considered. Among others, external missile impacts, for instance due to an airplane crash, are assumed to be relevant loading cases. In this context, impacts of nondeformable (&quot;hard&quot;), deformable (&quot;soft&quot;) as well as liquid filled (&quot;wet&quot;