Charakterisierung und gezielte Optimierung des primären Energiestoffwechsels von kontinuierlichen Zelllinien zur Produktion rekombinanter pharmazeutischer Proteine

Kontinuierliche Säugetierzelllinien werden zur Produktion rekombinanter, pharmazeutisch nutzbarer Proteine verwendet, da sie in der Lage sind, post-translationale Modifikationen am Protein auszuführen und dieses nativ zu sekretieren. Jedoch weisen diese Zellen einen ineffektiven Energiestoffwechsel auf, da ihnen die enzymatische Verbindung zwischen Glycolyse und Citratzyklus fehlt. Als Folge dessen werden Lactat und Ammonium in den Kulturüberstand sekretiert, welche das Zellwachstum und die Produktivität beeinträchtigen. Zur Optimierung des Energiemetabolismus wurde die cytosolische Pyruvat-Carboxylase 2 (PYC2) aus Saccharomyces cerevisiae in der derzeit wichtigsten industriell genutzten Produktionszelllinie CHO DG44 exprimiert und ihr Einfluss auf den Energiestoffwechsel und die Produktivität untersucht. Durch Nachweis der Enzymaktivität wurde die erfolgreiche Expression rekombinanter PYC2 gezeigt. Entgegen Untersuchungen bei anderen Zelllinien hatte diese Möglichkeit der verbesserten Glucose-Oxidation keinen signifikanten Einfluss auf das vitale Wachstumsverhalten der bereits durch Selektion in der Industrie optimierten Zellen. Um festzustellen, ob dennoch eine Erhöhung der Produktivität der Zelllinie CHO DG44 mit diesem Schritt des Inversen Metabolic Engineering möglich war, wurde ein bicistronisches Plasmid konstruiert, das die Gene für das Modellprotein Human Growth Hormone (HGH) und die PYC2 enthält. CHO DG44 wurde mit diesem Plasmid und dem Dihydrofolat-Reductase-Gen cotransfiziert und die HGH- und PYC2-codierenden Gene wurden coamplifiziert. Jedoch konnten mit dieser Strategie weder der Energiemetabolismus noch die HGH-Produktivität dieser Zelllinie optimiert werden.Continuous mammalian cell lines are used for the production of recombinant pharmaceutical proteins as they are able to carry out post-translational modifications at the native secreted protein. A major drawback of these cell lines is their inefficient energy metabolism because they lack the enzymatic link between the two pathways glycolysis and TCA cycle. Thus, the toxic metabolites lactate and ammonia that impair cell growth and productivity are secreted into the culture supernatant. In order to improve the energy metabolism a cytosolic pyruvate carboxylase (PYC2) from Saccharomyces cerevisiae was introduced into the cell line CHO DG44 that is at present one of the most important industrially used production cell lines. The effect of PYC2 on the energy metabolism and the productivity of CHO DG44 was investigated. Successful expression of PYC2 could be demonstrated by proving enzyme activity. In contrast to other cell lines, the additional possibility to oxidize glucose could not significantly improve the growth behavior of CHO DG44. To achieve optimization of the productivity of CHO DG44 a dicistronic plasmid containing the genes for the model protein Human Growth Hormone (HGH) and PYC2 was constructed. CHO DG44 was cotransfected with this plasmid and the dihydrofolate reductase gene, and the HGH and PYC2 genes were co-amplified. PYC2 activity could not be detected in CHO DG44. With this approach neither the energy metabolism nor the productivity of CHO DG44 could be improved

Laser-Induced-Fluorescence Detection of Nitric Oxide in High-Pressure Flames with A-X(0, 2) Excitation

Laser-induced fluorescence techniques have been used successfully for quantitative two-dimensional measurements of nitric oxide. The commonly applied D–X(0, 1) or A–X(0, 0) schemes are restricted to atmospheric-pressure flames and engines driven with gaseous fuels because of strong attenuation of the exciting laser beam by combustion intermediates. The properties of a detection scheme for which excitation in the nitric oxide A–X(0, 2) band was used were investigated. We discuss the advantages of the A–X(0, 2) system (excited at 247.95 nm) based on measurements in laminar premixed methane/air flames at 1–40 bars.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86764/1/Sick41.pd

Persistence of the biological effect of codling moth granulovirus in the orchard - preliminary field trials

In 2000 and 2001, in a field trial, the persistence of the biological effect of codling moth granulovirus (CpGV) was investigated. With a single treatment at full concentration of CpGV (MADEX 3, 100 ml/ha) a considerable reduction of CM population was achieved over the whole vegetation period. This may indicate that over a considerable period of time after a treatment a biological effect of CpGV sufficient for an increased mortality of the larvae was present in the orchard. However, the onset of mortality was not fast enough to protect the fruit from damage. Further research has to be done to gain more experience in handling this effect. It could be very important for the reduction of the number of treatments in organic apple growing

AI and Literacy for Non-Technical Students: A Hybrid-Augmented Learning Factory

Artificial Intelligence (AI) has made a strong impact on business and private life. Nonetheless, understanding how AI works and which role data specifically plays in this context remain unclear for many people. We argue that especially students with non-technical backgrounds should build up AI & data literacy to understand the key concepts and leverage their potential in their field of study and research. For this purpose, we present the concept of a hybrid-augmented learning factory, where students can explore AI & data concepts with interactive and immersive technologies in a physical and virtual environment. In this workshop paper, we demonstrate our overarching idea of the hybrid-augmented learning factory as well as our current progress on implementing learning applications for the learning factory

Role of Non-Stationary Collisional Dynamics in Determining Nitric Oxide LIF Spectra

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77182/1/AIAA-2004-389-525.pd

Control of the apple sawfly (Hoplocampa testudinea Klug) with extracts from Quassia amara L.: Quality and combination

In the last years the contro I 01 the apple sawfly Hoplocampa testudinea Klug was not efficient in organic Iruit growing. This was due to quality problems 01 the "Quassia wood" used lor the treatments. First orientating criteria based on one 01 the active ingredients, Quassin, were established lor the quality 01 "Quassia" preparations. A lield test on apple sawfly showed, that the quantity 01 Quassin per ha required lor good sawfly control is about 12-18 9 Quassin. In the nex! years, more active ingredients (Le. Neoquassin) have to be included in the quality criteria and evaluated by biotests. Only this way, reliable recommendations can be given to the growers. NeemAzal-T/S was effective only against the secondary inlestation. Thus, it can be recommended only in situations with low inlestation. The combination with Quassia (12 9 Quassin/ ha) did not give better results than Quassia alone. Antileedant effects have to be considered and may provocate antagonistic effects

Antiferromagnetic gap in the Hubbard model

We compute the temperature dependence of the antiferromagnetic order parameter and the gap in the two dimensional Hubbard model at and close to half filling. Our approach is based on truncations of an exact functional renormalization group equation. The explicit use of composite bosonic degrees of freedom permits a direct investigation of the ordered low temperature phase. We show that the Mermin--Wagner theorem is not practically applicable for the spontaneous breaking of the continuous spin symmetry in the antiferromagnetic state. The critical behavior is dominated by the fluctuations of composite Goldstone bosons.Comment: new discussion of critical behavior 4 pages,2 figures, LaTe

Nonstationary Collisional Dynamics in Determining Nitric Oxide Laser-Induced Flourescence Spectra

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77237/1/AIAA-8783-947.pd

Ascorbic Acid/Retinol and/or Inflammatory Stimuli’s Effect on Proliferation/Differentiation Properties and Transcriptomics of Gingival Stem/Progenitor Cells

The present study explored the effects of ascorbic-acid (AA)/retinol and timed inflammation on the stemness, the regenerative potential, and the transcriptomics profile of gingival mesenchymal stem/progenitor cells’ (G-MSCs). STRO-1 (mesenchymal stem cell marker) immuno-magnetically sorted G-MSCs were cultured in basic medium (control group), in basic medium with IL-1β (1 ng/mL), TNF-α (10 ng/mL) and IFN-γ (100 ng/mL, inflammatory-medium), in basic medium with AA (250 µmol/L) and retinol (20 µmol/L) (AA/retinol group) or in inflammatory medium with AA/retinol (inflammatory/AA/retinol group; n = 5/group). The intracellular levels of phosphorylated and total β-Catenin at 1 h, the expression of stemness genes over 7 days, the number of colony-forming units (CFUs) as well as the cellular proliferation aptitude over 14 days, and the G-MSCs’ multilineage differentiation potential were assessed. Next-generation sequencing was undertaken to elaborate on up-/downregulated genes and altered intracellular pathways. G-MSCs demonstrated all mesenchymal stem/progenitor cells characteristics. Controlled inflammation with AA/retinol significantly elevated NANOG (p < 0.05). The AA/retinol-mediated reduction in intracellular phosphorylated β-Catenin was restored through the effect of controlled inflammation (p < 0.05). Cellular proliferation was highest in the AA/retinol group (p < 0.05)