TY - THES A1 - Mambretti, Egle Maria T1 - Opioid receptors as therapeutic targets for nociceptor specific regional analgesia T1 - Opioidrezeptoren als therapeutisches Target einer nozizeptionsspezifischen Regionalanalgesie N2 - Opioids have been, since centuries, the gold standard for pain treatment and relief. They exert their effects after binding to opioid receptors (OP) that are expressed and functional in the central (CNS) and peripheral nervous system (PNS). As their systemic application has many side effects, including sedation and respiratory depression, a peripheral application of opioids and selective targeting of µ-OP (MOP) in nociceptive axons would be extremely beneficial. MOP presence and function has been conclusively demonstrated at nerve terminals; however it is still controversial whether functional MOPs are available on the membrane of peripheral nociceptive axons to mediate opioid-induced antinociception. While under pathologic conditions (i.e. nerve injury) exogenous as well as endogenous MOP agonists applied at the damaged nerve can elicit potent antinociception or anti-allodynia, under physiological conditions no antinociception was seen in rats. This could be caused by either a lack of functional opioid receptors in the axonal membranes or by the inability of injected opioids to cross the intact perineurial barrier and to reach nociceptors. Previous behavioral test results showed an antinociceptive effect (up to 5h) following perisciatic application of the hydrophilic DAMGO (MOP agonist) if coinjected with hypertonic saline solution (HTS; 10% NaCl), a treatment suited to open the perineural barrier. The effect was inhibited by naloxone, a MOP antagonist, documenting its specific action via MOP. Fentanyl, a lipophilic opioid, elicited an effect, which was enhanced by HTS treatment, indicating that HTS may act not only on the barrier but also directly on axonal MOP presence and/or functionality. To provide a basis for testing this hypothesis, the present work was designed to study the axonal localization of MOP in experimental animals under different conditions using molecular and morphological methods. Initially four different commercial antibodies were tested for MOP detection. Immunoreactions with these antibodies specifically detected MOP in the hippocampus and in amygdala, while in the peripheral nervous system the reactions showed varying labeling patterns pointing towards less specificity with low signal-to-noise ratio. Double labelling with calcitonin gene related peptide (CGRP), a neuropeptide expressed in sensory fibers, with the non-compacted myelin marker S100 or with the neuronal marker PGP9.5 documented significant immunoreaction signals outside sensory nerve fibers. Therefore, none of these antibodies appeared suitable. Taking advantage of a new commercial monoclonal rabbit antibody (RabMAb) and of genetically modified mice in which the fluorescent protein mcherry was inserted in the C-tail of MOP (MOP-mcherry knock-in mice), MOP fusion protein expression in rat and mouse CGRP+ sciatic nerve fibers and fiber bundles was confirmed by immunofluorescence labeling. Immunoelectron microscopic analysis indicated MOP/MOP-mcherry-localization in the cytoplasm and the membranes of unmyelinated axons organized in Remak bundles. Both antibodies detected bands of appropriate size in Western Blot in the CNS and additional larger bands in the PNS. Quantitative analyses 60 min after HTS-treatment revealed no change in MOP mRNA in the sciatic nerve and DRG as well as no change in MOP immunoreactivity in the sciatic nerve. Thus, the opioid-induced long lasting antinociception enhanced by perisciatic injection of HTS were not due to a sustained increased MOP expression or content in sensory, putative nociceptive axons. In summary, the current study succeeded to unequivocally document the presence of MOP protein in intact sensory axons of rat and mouse sciatic nerve. Thus, axonal MOPs may indeed mediate antinociceptive opioid effects observed in behavioral studies in naive animals possibly via activation of potassium or calcium channels. As HTS treatment does not lead to a sustained increase in axonal MOP protein or MOP mRNA expression, other mechanisms might enhance MOP function, including inhibition of MOP recycling or changes in functional coupling. Future studies should further explore the axonal mechanisms of antinociception by opioids and enhancing treatments. N2 - Opioide sind seit Jahrhunderten der Goldstandard für die Schmerzbehandlung. Sie entfalten ihre Wirkung nach der Bindung mit Opioidrezeptoren (OP), die im zentralen (ZNS) und peripheren (PNS) Nervensystem exprimiert und funktionell sind. Da die systemische Anwendung viele Nebenwirkungen hat, wie die Beruhigung und Atemdepression, wäre eine Anwendung von Opioiden und die gezielte Targeting von µ-OP (MOP) in nozizeptiven Axone in Rahmen einer Regionalanalgesie besser. Die Anwesenheit und die Funktionalität der MOP wurden zwar schon in Nervenendungen gezeigt, aber es ist noch strittig, ob funktionelle MOP in der Membran von peripheren nozizeptiven Axonen sind, um opioid-induzierte Antinozizeption zu vermitteln. Während bei Erkrankungen der Nerven (z.B. traumatische Nervenbeschädigung) exogene und endogene MOP-Agonisten Antinozizeption und Antiallodynie bewirken, konnte in gesunden Ratten kein Effekt bei perineuraler Injektion am Nerven beobachtet werden. Dies könnte entweder durch einen Mangel an funktionellen OP in axonalen Membranen verursacht sein. Alternativ könnte die mangelde Penetration der injizierten Opioide durch die Barriere des Perineuriums verantwortlich sein, die es verhindert, dass die Opioide die Nozizeptoren erreichen. Vorherige Ergebnisse aus Schmerzverhaltenstests zeigten eine Anhebung von mechanischen nozizeptiven Schwellen (bis 5 h) nach perineuraler Anwendung des hydrophilen MOP-Agonisten DAMGO, wenn dieser mit einer hypertonen Lösung (HTS; 10% NaCl) ko-injiziert war. Denn dies ist eine geeignete Behandlung, die die Barriere des Perineuriums öffnet. Der Effekt wurde von Naloxon, einem MOP-Antagonist, gehemmt, was eine spezifische Wirkung via MOP unterstützt. Die Wirkung von Fentanyl, einem lipophilen Opioid, wurde ebenfalls durch die HTS-Behandlung verbessert. Das führt zu unserer Hypothese, dass HTS nicht nur die Schranke öffnet, sondern auch direkt Expression und/oder Funktionalität von axonalen MOP verbessert. Um eine Grundlage für die Untersuchung dieser Hypothese zu schaffen, war das Ziel dieser Arbeit, die axonale MOP bei Versuchstieren unter verschiedenen Bedingungen mit molekularen und morphologischen Methoden zu charaktiersieren. Am Anfang wurden vier verschiedene kommerzielle Antikörper für die Erkennung der MOP getestet. Immunreaktionen mit diesen Antikörpern wiesen spezifisch MOP in dem Hippocampus und in der Amygdala nach, während im peripheren Nervensystem die Immunreaktion veränderliche Markierungsmuster und weniger Spezifität mit einem ungünstigeren Signal-zu-Hintergund Verhältnis zeigte. Die Doppelmarkierung mit calcitonin gene-related peptide (CGRP), einem Neuropeptid, das in sensorischen Fasern exprimiert ist, mit dem Marker für non-compacted Myelin S100 oder mit dem neuronalen Marker PGP9.5, bestätigte ein reproduzierbares Färbemuster außerhalb sensorischer Nervenfasern. Deshalb war keiner dieser Antikörper geeignet. Mit der Anwendung eines neuen kommerziell erhältlichen monoklonalen Kaninchen Antikörpers (RabMAb) gegen MOP sowie gentechnisch veränderten Mäusen, bei denen das fluoreszierende Protein mCherry in das C-terminale Ende von MOP eingefügt wurde (MOP-mcherry knock-in Mäusen), wurden MOP und das MOP-Fusionprotein im CGRP+ im Ischiasnerv und Fasernbündeln durch Immunfluoreszenzmarkierung von Ratten und Mäuse bestätigt. Die immunelectron-mikroscopische Analyse zeigte MOP/MOP-mcherry im Zytoplasma und der Membran unmyelinizierter Axone, die in Remak Bündlen organisiert sind. Beide Antikörper erkannten Banden in richtige Größe in Western Blot in ZNS und mehrere größere Banden in PNS. Quantitative Analysen 60 min nach HTS-Behandlung zeigten keine Veränderung in MOP mRNA in dem Ischiasnerv und Hinterwurzelganglion sowie keine Veränderung in der MOP-Immunreaktivität in dem Ischiasnerv. Daher müssen noch weitere Ursachen für die verbesserte Wirkung von Opioiden am Nerven nach HTS in Betracht gezogen werden. Zusammenfassend konnte diese Studie die MOP-Proteins in intakten sensorischen Axonen des N. ischiadicus der Ratte und Maus eindeutig nachweisen. Axonale MOPs könnten über Kaliumkanäle oder Calciumkanäle in den Verhaltenstests bei naiven Tiere antinozizeptiv wirken. Da die HTS Behandlung zu keiner deutlichen Steigerung von axonalem MOP-Protein führen kann, sollten anderen Mechanismen wie MOP-Recycling oder Veränderung der intrazellulären Singaltransduktion untersucht werden, die die Funktionalität von MOP erhöhen. Zukünftige Studien ferner den genauen Mechanismus klären, wie axonal Opioide antinozizeptiv wirken, um so die Behandlung von Schmerzen mit Regionalanalgesie weiter zu verbessert. KW - opioid receptors KW - nociceptors KW - regional analgesia KW - Opioide KW - Rezeptor KW - Nozizeptor KW - Lokalanästhesie Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128866 ER - TY - JOUR A1 - Briese, Michael A1 - Saal, Lena A1 - Appenzeller, Silke A1 - Moradi, Mehri A1 - Baluapuri, Apoorva A1 - Sendtner, Michael T1 - Whole transcriptome profiling reveals the RNA content of motor axons JF - Nucleic Acids Research N2 - Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts. KW - RNA KW - motor axons Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126800 ER - TY - THES A1 - Keßler, Martina T1 - Biodegradable solvent cast films and solution electrospun meshes for the prevention of postsurgical adhesions T1 - Bioabbaubare aus der Lösung gegossene Filme und elektrogesponnenen Vliese zur Prävention von Gewebeadhäsionen N2 - Intraperitoneal adhesions are fibrous bands that connect tissues in the peritoneal cavity that are usually separated. These adhesions form as a consequence of trauma, inflammation or surgical interventions and often result in severe consequences such as chronic pain, small bowel obstructions or female infertility. The aim of this thesis was to develop a synthetic barrier device for adhesion prevention made of modified poly(lactide) [PLA]. Solid PLA films (SurgiWrap®) are already successfully in clinical use due to the good biocompatibility and the biodegradability of the material resulting in non-toxic degradation products since lactic acid is naturally part of the metabolic circles of the human body. Considering the brittleness and stiffness of the films, the long degradation time of several months as well as the need for suturing, there is potential for optimization. Through a copolymerization with the hydrophilic poly(ethylene glycol) [PEG], a reduction of the degradation time was intendend. Moreover, the copolymerization should also lead to an improvement of the mechanical properties of the films since PEG acts as plasticizer for PLA. Linear PLA-PEG-PLA triblock copolymers as well as star-shaped PEG-PLA copolymers were synthesized via standard ring opening polymerization to tailor the barrier properties. Besides solid films, solution electrospun meshes from PLA and the synthesized PEG-PLA copolymers were investigated for a potential application as well. Since suturing of a barrier additionally induces adhesion formation, alginate coated membranes were prepared in order to achieve self-adhesiveness. With the intention to reduce infections and consequently inflammation, electrospun meshes and solvent cast films were loaded with the antibacterial drug triclosan and drug release as well as antibacterial efficacy was investigated. Mechanical tests confirmed that through the variation of the PEG content and branching the mechanical properties can be tailored and are in good accordance with the glass transition temperatures [Tg] of the polymers. Consequently, potentially adequate mechanical properties for surgical handling as well as for the performance within the patient’s body were successfully achieved. Degradation studies revealed that the degradation time was significantly shorter for PEG-PLA membranes than for PLA films and with an appropriate PEG content could be adjusted to the intended time frame. Cell adhesion and viability tests confirmed the non-toxicity of the clinically used PLA films as well as of PEG-PLA films and meshes. With a bioadhesion test the benefit of an alginate coated side towards the pure PLA film concerning self-adhesiveness was successfully demonstrated. Moreover, optical evaluations and a T-peel test of different alginate coated PLA films showed that the cohesion between the chemically different layers was distinctly enhanced by the use of an appropriate PEG-PLA mesh as intermediate cohesion promoting layer. In in vitro release studies with triclosan loaded films a higher release was determined for PEG-PLA than for PLA films. In agar diffusion tests a higher and longer inhibition of staphylococcus aureus growth was observed confirming the release results. Moreover, drug loaded meshes (especially drug loaded after electrospinning) showed enhanced and elongated bacterial inhibition in comparison to films. N2 - Intraperitoneale Adhäsionen sind fibröse Bänder, die Gewebe in der Peritonealhöhle miteinander verbinden, die normalerweise voneinander getrennt sind. Diese Adhäsionen entstehen als Folge von Trauma, Entzündung oder chirurgischen Eingriffen und bringen oft schwerwiegende Folgen mit sich wie chronische Schmerzen, Dünndarmobstruktionen oder Unfruchtbarkeit bei Frauen. Ziel dieser Arbeit war es, synthetische Barrieren aus modifiziertem Poly(laktid) [PLA] für die Adhäsionsprävention zu entwickeln. PLA-Filme (SurgiWrap®) werden bereits erfolgreich klinisch eingesetzt aufgrund der guten Biokompatibilität und der Bioabbaubarkeit des Materials mit seinen ungiftigen Abbauprodukten, da Milchsäure natürlicher Bestandteil der metabolischen Zyklen im menschlichen Körper ist. Betrachtet man jedoch die Sprödigkeit und Steifigkeit der Filme, die lange Abbauzeit von mehreren Monaten sowie die Notwendigkeit des Annähens, erkennt man noch Verbesserungspotential. Durch eine Copolymerisation mit hydrophilem Poly(ethylen glykol) [PEG] wurde eine Reduktion der Abbauzeit angestrebt. Zusätzlich sollte die Copolymerisation zu einer Verbesserung der mechanischen Eigenschaften der Filme führen, da PEG als Weichmacher von PLA dient. Lineare PLA-PEG-PLA-Triblock-Copolymere sowie sternförmige PEG-PLA-Copolymere wurden über eine Standard-Ringöffnungspolymerisation synthetisiert um die Barriereeigenschaften maßzuschneidern. Neben massiven Filmen wurden aus der Lösung elektrogesponnene Vliese aus PLA und den synthetisierten PEG-PLA-Copolymeren für eine potentielle Anwendung untersucht. Da das Annähen einer Barriere zusätzlich Adhäsionsbildung hervorruft, wurden mit Alginat beschichtete Membranen hergestellt um eine Selbsthaftung zu erreichen. Mit der Intention, Infektionen und damit Entzündungen zu reduzieren, wurden elektrogesponnene Vliese und aus der Lösung gegossene Filme mit dem antibakteriellen Arzneistoff Triclosan beladen und die Freisetzung sowie die antibakterielle Wirksamkeit untersucht. Mechanische Tests bestätigten, dass mit der Variation von PEG-Gehalt und Verzweigung die mechanischen Eigenschaften zugeschnitten werden können und gut mit den Glasübergangstemperaturen der Polymere in Einklang stehen. Folglich konnten potentiell passende mechanische Eigenschaften für die chirurgische Handhabung sowie für das Verhalten im Körper des Patienten erfolgreich erreicht werden. Abbaustudien zeigten, dass die Abbauzeit von PEG-PLA-Membranen signifikant kürzer war als von PLA-Filmen und mit geeignetem PEG-Gehalt erfolgreich auf den erzielten Zeitraum hin angepasst werden konnte. Zelladhäsion und Zellviabilitätstests bestätigten die Ungiftigkeit der klinisch eingesetzten PLA-Filme sowie der PEG-PLA-Filme und –Vliese. Mit einem Bioadhäsionstest konnte der Nutzen einer Alginatbeschichtung gegenüber einem reinen PLA-Film bezüglich einer Selbsthaftung erfolgreich gezeigt werden. Außerdem ergaben visuelle Begutachtungen und ein T-Peeltest von verschiedenen mit Alginat beschichteten PLA-Filmen, dass die Kohäsion zwischen den chemisch unterschiedlichen Schichten durch den Einsatz eines geeigneten PEG-PLA-Vlieses als kohäsionsfördernde Zwischenschicht deutlich verstärkt wurde. In in vitro Freisetzungsstudien mit Triclosan-beladenen Filmen wurde eine höhere Freisetzung für PEG-PLA- als für PLA-Filme bestimmt. In Agardiffusionstests wurde eine höhere und längere Wachstumshemmung von Staphylococcus aureus beobachtet, was die Freisetzungsergebnisse bestätigt. Weiterhin zeigten Arzneistoff-beladene Vliese (insbesondere mit Arzneistoffbeladung nach dem Elektrospinning) eine verstärkte und verlängerte bakterielle Inhibition im Vergleich zu Filmen. KW - Polymere KW - Biologischer Abbau KW - Polyethylenglykole KW - postsurgical adhesion KW - biodegradable polymer KW - Polymilchsäure KW - Adhäsion KW - polylactid KW - polyethylenglykole KW - Blockcopolymere Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129358 ER - TY - THES A1 - Glaser, Jan T1 - Antileishmanial compounds from Nature - Elucidation of the active principles of an extract from Valeriana wallichii rhizomes T1 - Antileishmaniale Wirkstoffe aus der Natur - Aufklärung des Wirkprinzips eines Wurzelextraktes von Valeriana wallichii N2 - This study is dealing with the bioactivity-guided fractionation of a chloroform extract from pulverized rhizomes of Valeriana wallichii with focus on isolation and structure elucidation of the antileishmanial active principles. N2 - Die vorliegende Studie beschäftigt sich mit der bioaktivitätsgeleiteten Fraktionierung eines Chloroformextraktes aus pulverisierten Rhizomen von Valeriana wallichii mit Schwerpunkt auf der Isolierung und Strukturaufklärung des antileishmanialen Wirkprinzips. KW - Leishmaniose KW - Valeriana wallichii KW - Extrakt KW - Rhizom KW - Naturstoff KW - natural products KW - antileishmanial KW - Valeriana wallichii Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129140 ER - TY - THES A1 - Schmitt, Alexandra T1 - Role of Peroxiredoxin 6 in human melanoma T1 - Die Funktion von Peroxiredoxin 6 im humanen Melanom N2 - Peroxiredoxin 6 (PRDX6) is a bifunctional enzyme comprising a peroxidase and a Ca2+-independent phospholipase (iPLA2) activity. This renders the enzyme capable of detoxifying reactive oxygen species (ROS) and of catalyzing the liberation of arachidonic acid (AA) from cellular membranes. Released AA can be further metabolized to bioactive lipids including eicosanoids, which are involved in inflammation, cell growth, differentiation, invasion and proliferation. Human melanoma cells are often characterized by imbalances in both ROS and lipid levels, which can be generated by oncogenic signaling, altered metabolism or UV irradiation. In previous studies, a comparative proteome analysis of the Xiphophorus fish melanoma model revealed a strong upregulation of Prdx6 in benign and malignant lesions compared to healthy skin. As the Xiphophorus melanoma model displays in many respects molecular characteristics that are similar to human melanoma, I investigated the functional role of PRDX6 in human melanoma cells. The first part of the study deals with the regulation of PRDX6 in melanocytes and human melanoma cells. I could demonstrate that the protein level of PRDX6 was strongly enhanced by the induction of the EGFR orthologue Xmrk from the Xiphophorus fish as well as the human EGFR. The upregulation of PRDX6 was further shown to be mediated in a PI3K-dependent and ROS-independent manner. The main part of the thesis comprises the investigation of the functional role of PRDX6 in human melanoma cells as well as the analysis of the underlying mechanism. I could show that knockdown of PRDX6 enhanced the oxidative stress response and led to decreased proliferation of melanoma cells. This cell growth effect was mainly mediated by the iPLA2 activity of PRDX6. Under conditions of strongly enhanced oxidative stress, the peroxidase activity became also important for cellular proliferation. Furthermore, the anti-proliferative effect in cells with lowered PRDX6 levels was the result of reduced cellular AA content and the decrease in the activation of SRC family proteins. Similarly, supplementation with AA led to regeneration of SRC family kinase activity and to an improvement in the reduced proliferation after knockdown of PRDX6. Since AA can be further processed into the prostaglandin PGE2, which has a pro-tumorigenic function in some cancer types, I further examined whether this eicosanoid is involved in the proliferative function of PRDX6. In contrast to AA, PGE2 was not consistently required for melanoma proliferation. In summary, I could demonstrate that PRDX6 plays a major role in AA-dependent lipid signaling in melanoma cells and thereby regulates proliferation. Interestingly, the proliferation relevant iPLA2 activity can be pharmacologically targeted, and melanoma cell growth was clearly blocked by the inhibitor BEL. Thus, I could identify the phospholipase activity of PRDX6 as a new therapeutically interesting target for melanoma treatment. N2 - Peroxiredoxin 6 (PRDX6) ist ein bifunktionales Enzym, welches neben seiner Peroxidase-Aktivität auch eine Ca2+-unabhängige Phospholipase-Aktivität besitzt. Aufgrund dieser beiden Aktivitäten ist das Enzym in der Lage, sowohl oxidativen Stress zu bekämpfen als auch die Freisetzung von Arachidonsäure aus zellulären Membranen zu katalysieren. Freie Arachidonsäure (AA) dient der Generierung von bioaktiven Lipiden wie zum Beispiel Eicosanoiden, welche an Entzündungsreaktionen, Zellwachstum, Differenzierung, Invasion und Proliferation beteiligt sind. Humane Melanomzellen zeichnen sich oft durch ein gestörtes Gleichgewicht reaktiver Sauerstoffspezies und zellulärer Lipide aus. Dieses Ungleichgewicht kann durch onkogene Signalgebung, einen veränderten Metabolismus oder UV-Bestrahlung hervorgerufen werden. Eine vorangegangene Proteomanalyse des Xiphophorus-Fisch-Melanommodells zeigte, dass im Vergleich zur gesunden Haut die Menge an PRDX6 in benignen und malignen Läsionen stark erhöht ist. Da das Xiphophorus-Melanommodell in vielerlei Hinsicht die molekulare Situation des humanen Melanoms wiederspiegelt, habe ich die funktionale Rolle von PRDX6 in humanen Melanomzellen untersucht. Der erste Teil der Studie beschäftigt sich mit der Regulierung von PRDX6 in Melanozyten und humanen Melanomzellen. Ich konnte nachweisen, dass die Menge an PRDX6 Protein durch die Induktion des EGFR Orthologs Xmrk aus Xiphophorus Fischen, sowie des humanen EGFR stark erhöht wurde. Auch konnte ich zeigen, dass die Heraufregulierung von PRDX6 von der Signalgebung der PI3 Kinase, aber nicht von reaktiven Sauerstoffspezies abhängig war. Der Hauptteil der vorliegenden Forschungsarbeit befasst sich mit der Ermittlung der funktionalen Rolle von PRDX6 in humanen Melanomzellen und der Analyse des zugrundeliegenden Mechanismus. Ich konnte nachweisen, dass ein Knockdown von PRDX6 die oxidative Stress-Antwort verstärkte und die Proliferation von Melanomzellen reduzierte. Der Effekt auf das zelluläre Wachstum wurde hierbei hauptsächlich durch die iPLA2-Aktivität von PRDX6 verursacht. Bei stark erhöhtem oxidativem Stress konnte auch eine Relevanz der Peroxidase-Aktivität für die zelluläre Proliferation nachgewiesen werden. Auch ging der anti-proliferative Effekt mit einer Abnahme zellulärer AA und der Reduktion aktiver Kinasen der SRC-Familie einher. Die Zugabe von AA zu Zellen mit PRDX6-Knockdown führte zur Regeneration der SRC-Kinase-Aktivität und konnte die Proliferation wieder verbessern. Da AA zum Prostaglandin PGE2 prozessiert werden kann, welches in einigen Krebsarten pro-tumorigene Funktionen erfüllt, untersuchte ich, ob dieses Eicosanoid auch für die proliferative Funktion von PRDX6 relevant ist. Im Gegensatz zu AA wies PGE2 jedoch keine kontinuierliche pro-proliferative Funktion auf. Zusammenfassend konnte ich zeigen, dass PRDX6 eine entscheidende Rolle im AA- Stoffwechsel von Melanomzellen spielt und hierdurch die Proliferation reguliert. Interessanterweise ist die proliferationsrelevante iPLA2-Aktivität pharmakologisch hemmbar, und auch das Wachstum der Melanomzellen wurde durch den Inhibitor BEL deutlich inhibiert. Mit der Phospholipase-Aktivität von PRDX6 konnte ich somit einen neuen therapeutisch nutzbaren Angriffspunkt für das Melanom identifizieren. KW - Melanom KW - Peroxiredoxin 6 KW - peroxiredoxin 6 KW - Melanom KW - melanoma KW - Arachidonsäure KW - arachidonic acid KW - Prostaglandin E2 KW - prostaglandin E2 KW - Melanomzellen KW - melanoma cells KW - Peroxiredoxin KW - Arachidonsäure KW - Prostaglandin E2 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111465 ER - TY - THES A1 - Mülek, Melanie T1 - Distribution and metabolism of constituents and metabolites of a standardized maritime pine bark extract (Pycnogenol®) in human serum, blood cells and synovial fluid of patients with severe osteoarthritis T1 - Verteilung und Metabolismus von Bestandteilen und Metaboliten eines standardisierten Kiefernrindenextraktes (Pycnogenol®) in humanem Serum, Blutzellen und Synovialflüssigkeit von Patienten mit schwerer Osteoarthritis N2 - Dietary polyphenols have been related to beneficial effects on humans’ health. Pycnogenol®, a dietary polyphenol-rich food supplement complies with the monograph “Maritime pine extract” in the United States Pharmacopeia (USP) and has demonstrated effects in different diseases. Several human trials concerning knee osteoarthritis have shown significant improvement of the symptoms like reducing the pain and the stiffness of the joint(s) upon intake of Pycnogenol®. After oral intake of multiple doses of Pycnogenol® previously low concentrations in the nanomolar range of monomeric extract constituents have been found in human plasma as well as a bioactive metabolite, δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1), which is formed by the human intestinal flora from the procyanidins’ catechin units. It is not clear yet which compound(s) of the complex extract is (are) mainly responsible for the described clinical effects of Pycnogenol®. To gain deeper insights into the in vivo fate of the pine bark extract the distribution of its constitutents and metabolites was closer investigated in the present thesis. Initial in vitro experiments suggested a facilitated cellular uptake of M1 into human erythrocytes, possibly via GLUT-1 transporter. For elucidating further the in vitro and in vivo metabolism of M1 in human blood cells, a metabolomic approach was performed using UPLC-ESI-qTOF-MSE analysis, which revealed a comprehensive and rapid metabolism of M1 to a variety of biotransformation products in human blood cells. Predominant metabolites were found to be conjugates of glutathione (GSH) isomers, namely M1-S-GSH and M1-N-GSH. Further sulfur-containing biotransformation products of M1 were conjugates with oxidized glutathione (M1-GSSG) and cysteine (M1-CYS) and the sulfated derivative of M1 (M1-sulfated). Other in vitro biotransformation products constituted the open-chained ester form of M1 (M1-COOH), hydroxybenzoic acid and the methylated (M1-methylated), acetylated (M1-acetylated), hydroxylated (M1-hydroxylated) and ethylated (M1-ethylated) derivatives of M1. Indeed, six of these in vitro metabolites, respectively M1-COOH, M1-sulfated, hydroxybenzoic acid, M1-S-GSH, M1-methylated and M1-acetylated, were also identified in vivo in blood cells of human volunteers after ingestion of Pycnogenol®. Related reference material was synthesized for reliable confirmation of the metabolites M1-GSH, M1-GSSG, M1-CYS and M1-COOH. In the course of a randomized controlled clinical trial patients suffering from severe osteoarthritis ingested multiple doses of 200 mg/day Pycnogenol® for three weeks before they were scheduled for an elective knee replacement surgery. Various biological specimen, respectively blood cells, synovial fluid and serum samples, were to be analyzed to investigate the distribution and disposition of possibly bioactive constituents and metabolites. Therefore, highly sensitive methods were developed using liquid chromatography tandem mass spectrometry (LC-MS/MS)- technology because of the expected low concentrations of the analytes in the related matrices. Initially, for each matrix different sample preparation techniques (protein precipitation, liquid-liquid extraction, solid phase extraction and useful combinations thereof) were compared to achieve maximum detection sensitivity of the analytes that were of highest interest, namely M1, ferulic acid and taxifolin. By comparing 32 various sample clean-up procedures in human serum, the highest recovery of the metabolite M1 was achieved using a liquid-liquid extraction with ethyl acetate and tert-butyl methyl ether at a serum pH-value of 3.2. A similar extraction method was also chosen for analyte detection in human synovial fluid after comparing 31 different sample preparation techniques. Whole blood or blood cells are difficult to handle because of their high viscosity and strong coloration. The QuEChERS (quick, easy, cheap, effective, rugged and safe) approach which was originally developed for the food safety and thus for the determination of pesticide residues in fruits and vegetables yielded the highest total recovery rate of M1 in human blood cells when assessing 18 different sample clean-up techniques. By applying the QuEChERS method for the first time for the simultaneous and highly sensitive quantification of selected polyphenols in human blood cells it was demonstrated that this fast and inexpensive technique can be applied in clinical fields for cleaning-up highly complex and thus challenging biological matrices. All developed methods for the different biological specimen were optimized to achieve maximum sensitivity of the target analytes. The determined lower limits of quantification (LLOQs) were sufficient for the quantification of the study samples. The LLOQs ranged from 113 pg/mL for taxifolin to 48 ng/mL for caffeic acid in blood cells and from 80 pg/mL for taxifolin to 3 ng/mL for caffeic acid in synovial fluid. In human serum the LLOQs even ranged down to 35 pg/mL for taxifolin and up to 8 ng/mL for caffeic acid. All analytical methods were subjected to a full validation according to current EMA and FDA guidelines and fulfilled those criteria, showing excellent performance and reliability of the developed and optimized methods. Serum, blood cells and synovial fluid samples of the osteoarthritis patients were all processed with an enzymatic incubation with ß-glucuronidase/sulfatase to hydrolyse conjugates (phase-II-metabolism) prior the actual sample preparation. Additionally, serum samples of the osteoarthritis patients were prepared without enzymatic hydrolysis to determine the individual degree of conjugation with sulfate and glucuronic acid of the analytes. All determined concentrations in the patients’ samples were in the lower ng/mL range. Notably, highest total concentrations of the polyphenols were not detected in serum, in which the degree of analyte conjugation with sulfate and glucuronic acid ranged from 54.29 ± 26.77% for catechin to 98.34 ± 4.40% for M1. The flavonoids catechin and taxifolin mainly partitioned into blood cells, whereas the metabolite M1, ferulic and caffeic acid primarily resided in the synovial fluid. The concentration of M1 in the blood cells was low, however, this could be explained by the previously observed extensive and rapid intracellular metabolism in vitro. This was now supported by the in vivo evidence in samples of patients who received Pycnogenol® in which the open-chained ester form of M1 (M1-COOH) as well as the glutathione conjugate of M1 (M1-GSH) were identified, indicating that M1 does not accumulate in its original form in vivo. Possibly, a variety of bioactive metabolites exist which might play an important role for the clinical effects of Pycnogenol®. Although the study participants were requested to avoid polyphenol-rich food and beverages within the last two days before the blood samplings this was obviously difficult for most of the patients. Hence, no statistically significantly difference was observed in the mean polyphenol concentrations in serum, blood cells and synovial fluid between the intervention and the control group. Nevertheless, it was possible to identify marker compounds for Pycnogenol® intake under real life conditions with occasional or regular consumption of polyphenol-rich foods and beverages. Thereby, ferulic acid was found in serum samples exclusively after intake of Pycnogenol®, confirming that ferulic acid is a suitable marker of consumption of French maritime pine bark extract. Taxifolin was present in serum and synovial fluid exclusively in the intervention group indicating a role as further marker of Pycnogenol® intake. Taxifolin, ferulic acid and caffeic acid were detected in both serum and synovial fluid only in the intervention group. Moreover, the metabolite M1, taxifolin and ferulic acid were only detected simultaneously in all matrices (serum, blood cells and synovial fluid) after ingestion of Pycnogenol®. Thus, deeper insights into the distribution of bioactive constituents and metabolites of Pycnogenol® into serum, blood cells and synovial fluid after oral administration to patients with severe osteoarthritis were gained. The present study provides the first evidence that polyphenols indeed distribute into the synovial fluid of patients with osteoarthritis where they might contribute to clinical effects. N2 - Polyphenole in Nahrungsmitteln werden mit positiven Wirkungen auf die menschliche Gesundheit in Verbindung gebracht. Pycnogenol®, ein polyphenolreiches Nahrungs-ergänzungsmittel, welches der Monographie "Maritime Pine Extract" im US-Amerikanischen Arzneibuch (United States Pharmacopeia, USP) entspricht, wurde bereits Effekte bei verschiedenen Krankheiten zugeschrieben. Eine orale Einnahme von Pycnogenol® hat in mehreren Humanstudien, welche sich mit Arthrose am Knie beschäftigt haben, eine signifikante Verbesserung der Symptome wie die Reduzierung von Schmerzen und der Steifheit des Gelenks gezeigt. Nach Mehrfacheinnahmen von Pycnogenol® wurden im menschlichen Plasma bereits niedrige Konzentrationen (im nanomolaren Bereich) von monomeren Extraktbestandteilen gefunden sowie ein bioaktiver Metabolit, δ-(3,4-Dihydroxy-phenyl)-γ-Valerolacton (M1), welcher durch die menschliche Darmflora aus den Catechin-Einheiten der Procyanidine gebildet wird. Bis jetzt ist noch unklar, welche Verbindung(en) des komplexen Extraktes für die beschriebenen klinischen Wirkungen von Pycnogenol® hauptsächlich verantwortlich ist (sind). Um einen tieferen Einblick in das in vivo Verhalten des Kiefernrindenextraktes zu gewinnen, wurde in der vorliegenden Arbeit die Verteilung von Bestandteilen und Metaboliten des Extraktes näher untersucht. Erste in vitro Experimente wiesen auf eine erleichterte zelluläre Aufnahme von M1 in menschliche Erythrozyten hin, möglicherweise vermittelt über den GLUT-1-Transporter. Um den in vitro und in vivo Metabolismus von M1 in menschlichen Blutzellen weiter aufzuklären, wurden metabolomische Untersuchungen mittels UPLC-ESI-qTOF-MS-Analyse durchgeführt, welche eine umfassende und schnelle Metabolisierung von M1 in menschlichen Blutzellen zu einer Vielzahl von Biotransformationsprodukten zeigten. Die Hauptmetabolite waren Konjugate von Glutathion(GSH)-Isomeren, nämlich M1-S-GSH und M1-N-GSH. Daneben entstanden schwefelhaltige Biotransformationsprodukte von M1, nämlich Konjugate mit oxidiertem Glutathion (M1-GSSG) und Cystein (M1-CYS) sowie ein Derivat von M1 mit Sulfat (M1-sulfatiert). Andere in vitro Biotransformationsprodukte waren die offenkettige Esterform von M1 (M1-COOH), Hydroxybenzoesäure, die methylierte (M1-methyliert), acetylierte (M1-acetyliert), hydroxylierte (M1-hydroxyliert) und ethylierte (M1-ethyliert) Form von M1. Sechs dieser in vitro Metabolite, nämlich M1-COOH, M1-sulfatiert, Hydroxybenzoesäure, M1-S-GSH, M1-methyliert und M1-acetyliert, wurden tatsächlich auch in vivo in humanen Blutzellen von freiwilligen Spendern identifiziert, welche zuvor Pycnogenol® oral eingenommen hatten. Für eine zuverlässige Bestätigung der Metaboliten M1-GSH, M1-GSSG, M1-CYS und M1-COOH wurde entsprechendes Referenzmaterial synthetisiert. Im Rahmen einer randomisiert-kontrollierten Studie wurden Patienten, welche an einer schweren Arthrose litten, eine orale Mehrfachdosis von 200 mg Pycnogenol® pro Tag über drei Wochen hinweg verabreicht, bevor sich diese anschließend einer notwendigen Kniegelenksersatz-Operation unterzogen. Um das Auftreten und die Verteilung von möglichen bioaktiven Bestandteilen und Metaboliten zu untersuchen, wurden verschiedene biologische Flüssigkeiten, nämlich Serum, Blutzellen und die Gelenkflüssigkeit analysiert. Da sehr geringe Konzentrationen der Analyten in den einzelnen Matrizes erwartet wurden, waren hochempfindliche Methoden erforderlich. Daher wurde Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) eingesetzt. Zunächst wurden unterschiedliche Probenvorbereitungstechniken (Proteinfällung, Flüssig-Flüssig-Extraktion, Festphasenextraktion und sinnvolle Kombinationen davon) für jede Matrix verglichen, um eine maximal empfindliche Detektion der wichtigsten Analyten, nämlich M1, Ferulasäure und Taxifolin, zu erzielen. Durch den Vergleich von 32 verschiedenen Probenaufarbeitungen in humanem Serum wurde die höchste Wiederfindung des Metaboliten M1 unter Verwendung einer Flüssig-Flüssig-Extraktion mit Essigsäureethylester und Methyl-tert-butylether bei einem pH-Wert im Serum von 3,2 erreicht. Zum Nachweis der Analyten in der humanen Gelenkflüssigkeit wurde nach einem Vergleich von 31 verschiedenen Probenaufarbeitungen eine ähnliche Extraktion angewandt. Aufgrund der hohen Viskosität und der starken Färbung ist die Aufarbeitung von Vollblut oder Blutzellen sehr anspruchsvoll. Das QuEChERS (quick, easy, cheap, effective, rugged and safe) Verfahren, welches ursprünglich für die Lebensmittelüberwachung zur Bestimmung von Pestizidrückständen in Obst und Gemüse entwickelt wurde, ergab bei der Bewertung von 18 Probenaufarbeitungs-techniken die höchste Gesamtwiederfindungsrate von M1 in menschlichen Blutzellen. Durch die erstmalige Anwendung von QuEChERS zur hochempfindlichen und simultanen Quantifizierung von ausgewählten Polyphenolen in menschlichen Blutzellen wurde gezeigt, dass diese schnelle und kostengünstige Methode durchaus auch in klinischen Bereichen zur Aufreinigung von sehr komplexen und anspruchsvollen biologischen Matrizes angewendet werden kann. Alle entwickelten Methoden wurden umfassend optimiert um eine maximal empfindliche Quantifizierung der Analyten zu erhalten. Die ermittelten unteren Bestimmungs-grenzen (lower limit of quantification, LLOQ) waren ausreichend für die Quantifizierung der Studienproben. Die LLOQs reichten in humanen Blutzellen von 113 pg/mL für Taxifolin bis 48 ng/mL für Kaffeesäure und in der menschlichen Gelenkflüssigkeit von 80 pg/mL für Taxifolin bis hin zu 3 ng/mL für Kaffeesäure. In humanem Serum bewegten sich die Bestimmungsgrenzen sogar bis zu 35 pg/mL für Taxifolin und bis zu 8 ng/mL für Kaffeesäure. Alle analytischen Methoden wurden einer „Full Validation“ nach den gegenwärtigen EMA- und FDA-Richtlinien unterzogen und erfüllten deren Kriterien, was eine hervorragende Leistungsfähigkeit und Zuverlässigkeit der entwickelten und optimierten Methoden bewies. Serum-, Blutzell- und Gelenkflüssigkeitsproben der Arthrose-Patienten wurden einer enzymatischen Inkubation mit ß-Glucuronidase/Sulfatase unterworfen, um die konjugierten (Phase-II-Metabolismus) Verbindungen vor der eigentlichen Probenvorbereitung zu hydrolysieren. Die Serumproben der Studienteilnehmer wurden zusätzlich noch ohne enzymatische Hydrolyse aufgearbeitet, um den individuellen Grad der Analytkonjugation mit Sulfat und Glucuronsäure zu bestimmen. Alle ermittelten Konzentrationen in den Patientenproben lagen im unteren ng/mL-Bereich. Bemerkenswerterweise wurden die höchsten Gesamtkonzentrationen der Polyphenole nicht in Serum, in welchem der Grad der Analytkonjugation mit Sulfat und Glucuronsäure von 54,29 ± 26,77 % für Catechin bis 98,34 ± 4,40 % für M1 reichte, bestimmt. Die beiden Flavonoide Catechin und Taxifolin verteilten sich vor allem in die Blutzellen, während der Metabolit M1, Ferulasäure und Kaffeesäure in erster Linie in Gelenkflüssigkeit zu finden war. Die Konzentration von M1 in den Blutzellen war gering, was durch den zuvor beobachteten umfangreichen und schnellen intrazellulären in vitro Metabolismus erklärt werden konnte. Durch den in vivo Nachweis der offenkettigen Esterform von M1 (M1-COOH) als auch des Glutathion-Konjugats von M1 (M1-GSH) in Proben von Patienten, welche zuvor Pycnogenol® eingenommen hatten, konnte dies bestätigt werden. Dies deutet darauf hin, dass M1 in vivo nicht in der ursprünglichen Form akkumuliert und möglicherweise eine Vielzahl von biologisch aktiven Metaboliten vorliegt, was für die klinische Wirkung von Pycnogenol® eine wichtige Rolle spielen könnte. Obwohl die Studienteilnehmer darum gebeten wurden in den letzten zwei Tagen vor den Blut-entnahmen weitestgehend auf polyphenolreiche Nahrungsmittel und Getränke zu verzichten, war die Umsetzung für die meisten der Patienten doch sehr schwierig. Daher wurden keine statistisch signifikanten Unterschiede zwischen der Interventions- und der Kontrollgruppe in den mittleren Polyphenolkonzentrationen im Serum, Blutzellen und in der Gelenkflüssigkeit beobachtet. Dennoch war es möglich, einige Markerverbindungen für eine Aufnahme von Pycnogenol® unter Alltagsbedingungen mit gelegentlichem oder regelmäßigem Konsum von polyphenolreichen Lebensmitteln und Getränken zu identifizieren. So wurde Ferulasäure nur in Serumproben nach der Einnahme von Pycnogenol® gefunden, was bestätigt, dass Ferulasäure ein geeigneter Marker für die Einnahme des Kiefernrindenextraktes ist. Taxifolin wurde ausschließlich im Serum und Gelenkflüssigkeit der Interventionsgruppe nachgewiesen, was auf einen weiteren Marker der Pycnogenol®-Einnahme hindeutet. Taxifolin, Ferulasäure und Kaffeesäure wurden nur in der Interventionsgruppe in den beiden Matrizes Serum und Gelenkflüssigkeit nachgewiesen. Darüber hinaus wurde das gleichzeitige Vorhandensein des Metaboliten M1, Taxifolin und Ferulasäure in allen Körperflüssigkeiten (Serum, Blutzellen und Gelenkflüssigkeit) nur nach einer Aufnahme von Pycnogenol® festgestellt. Somit konnten tiefere Einblicke in die Verteilung von bioaktiven Inhaltsstoffen und Metaboliten von Pycnogenol® in Serum, Blutzellen und Gelenkflüssigkeit nach oraler Verabreichung an Patienten mit schwerer Arthrose gewonnen werden. Die vorliegende Studie liefert den ersten Beweis dafür, dass sich Polyphenole durchaus in die Gelenkflüssigkeit von Patienten mit Osteoarthritis verteilen, in welcher diese möglicherweise zu klinischen Effekten beitragen können. KW - Pycnogenol KW - Pharmakokinetik KW - Metabolismus KW - Arthrose KW - LC-MS/MS KW - Kiefernrindenextrakt KW - Matrix Effekte KW - biologische Körperflüssigkeiten KW - Probenaufarbeitung KW - Osteoarthritis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128085 ER - TY - THES A1 - Schmidt, Thomas Christian T1 - Theoretical Investigations on the Interactions of Small Compounds with their Molecular Environments T1 - Theoretische Untersuchungen der Wechselwirkungen Kleiner Moleküle mit deren Molekularen Umgebungen N2 - Im ersten Teil dieser Arbeit wird eine Kombination theoretischer Methoden für die strukturbasierte Entwicklung neuer Wirkstoffe präsentiert. Ausgehend von der Kristallstruktur eines kovalenten Komplexes einer Modellverbindung mit dem Zielprotein wurde mit Hilfe von quantenmechanischen und QM/MM Rechnungen die genaue Geometrie des vorausgehenden nicht-kovalenten Komplexes betimmt. Letztere ist der bestimmende Faktor für die Reaktivität des Inhibitors gegenüber der katalytisch aktiven Aminosäure und damit für die Ausbildung einer kovalenten Bindung. Aus diesem Grund wurde diese Geometrie auch für die Optimierung der Substitutionsmusters des Ihnibitors verwendet, um dessen Affinität zum Zielenzyme zu verbessern ohne dass dieser seine Fähigkeit kovalent an das aktive Zentrum zu binden verliert. Die Optimierung des Substitutionsmuster wurde doch Methode des Molekularen Dockings unterstützt, das diese optimal dazu geeignet sind, Bindungsaffinitäten vorherzusagen, die durch eine Modifikation der chemischen Struktur entstehen. Eine Auswahl der besten Strukturen wurde anschließend verwendet, um zu überprüfen, ob die veränderten Moleküle noch genügen Reaktivität gegenüber dem Zielprotein aufweisen. Moleküldynamik Simulationen der neuen Verbindungen haben jedoch gezeigt, dass die veränderten Verbindungen nur so and das Protein binden, dass die Bilung eine kovalenten Bindung zum Enzym nicht mehr möglich ist. Daher wurden in einem weiteren Schritt die Modellverbindungen weiter modifiziert. Neben Änderungen im Substitutionsmuster wurde auch die chemische Struktur im Kern verändert. Die Bindungsaffinitäten wurde wieder mittels Docking überprüft. Für die besten Bindungsposen wurden wieder Simulationen zur Moleküldynamik durchgeführt, wobei diesmal die Ausbildung einer kovalenten Bindung zum Enzyme möglich erscheint. In einer abschließenden Serie von QM/MM Rechnungen unter Berücksichtigung verschiedener Protonierungszustände des Inhibitors und des Proteins konnten Reaktionspfade und zugehörige Reaktionsenergien bestimmt werden. Die Ergebnisse lassen darauf schließen, dass eines der neu entwickelten Moleküle sowohl eine stark verbesserte Bindungsaffinität wie auch die Möglichkeit der kovalenten Bindung an Enzyme aufweist. Der zweite Teil der Arbeit konzentriert sich auf die Umgebungseinflüsse auf die Elektronenverteilung eines Inhibitormodells. Als Grundlage dient ein vinylsulfon-basiertes Moekül, für das eine experimentell bestimmte Kristallstruktur sowie ein theoretisch berechneter Protein Komplex verfügbar sind. Ein Referendatensatz für diese Systeme wurde erstellt, indem der Konformationsraum des Inhibitors nach möglichen Minimumsstrukturen abgesucht wurde, welche später mit den Geometrien des Moleküls im Kristall und im Protein verglichen werden konnten. The Geometrie in der Kristallumgebung konnte direkt aus den experimentellen Daten übernommen werden. Rechnungen zum nicht-kovalenten Protein Komplex hingegen haben gezeigt, dass für das Modellsystem mehrere Geometrien des Inhibiors sowie zwei Protonierungszustände für die katalytisch aktiven Aminosäuren möglich sind. Für die Analyse wurden daher alle möglichen Proteinkomplexe mit der Kristallstruktur verglichen. Ebenso wurden Vergleiche mit der Geometrie des isolierten Moleküls im Vakuum sowie der Geometrie in wässriger Lösung angestellt. Für die Geometrie des Moleküls an sich ergab sich eine gute Übereinstimmung für alle Modellsysteme, für die Wechselwirkungen mit der Umgebung jedoch nicht. Die Ausbildung von Dimeren in der Kristallumgebung hat einen stark stablisierenden Effekt und ist einer der Gründe, warum dieser Kristall so gut wie keine Fehlordungen aufweist. In den Proteinkomplexen hingegen ergibt sich eine Abstoßung zwischen dem Inhibitor und einer der katalytisch aktiven Aminosäuren. Als Ursache für diese Abstoßung konnte die Einführung der Methylaminfunktion ausgemacht werden. Vermutlicherweise führt diese strukturelle Änderung auch dazu, dass der Modellinhibitor nicht in der Lage ist, so wie die Leitstruktur K11777 an das aktive Zentrum des Enzyms zu binden. N2 - In the first part of this work, a combination of theoretical methods for the rational design of covalent inhibitor is presented. Starting from the crystal structure of the covalent complex of a lead compound, quantum mechanical and QM/MM calculations were used to derive the exact geometry of the preceeding non-covalent enzyme inhibitor complex. The geometry of the latter mainly determines the reactivity of the inhibitor against its target enzyme concerning the formation of the covalent bond towards an active site residue. Therefore, this geometry was used as starting point for the optimization of the substitution pattern of the inhibitor such as to increase its binding affinity without loosing its ability to covalently bind to the target protein. The optimization of the chemical structure was supported by using docking procedures, which are best suited to estimate binding affinities that arise from the introduced changes. A screening of the novel substitution patterns resulted in a first generation of model compounds which were further tested for their reactivity against the target. Dynamic simulations on the novel compounds revealed that the orientation that compounds adopt within the active site are such that a covalent interaction with the enzyme is no longer possible. Hence, the chemical structure was further modified, including not only changes in the substituents but also within the core of the molecule. Docking experiments have been conducted to assure sufficiently high binding affinities and to obtain the most favored binding poses. Those have then again been used for dynamic simulations which resulted in structures, for which the bond formation process appeared feasible. A final series of QM/MM calculations considering various protonation states was computed to estimate the reaction energies for the covalent attachment of the inhibitor to the enzyme. The theoretical results indicate a reasonable high inhibition potency of the novel compounds. The second part concentrates on the environmental influences on the electron density of an inhibitor molecule. Therefore, a vinylsulfone-based model compound was selected for which an experimental crystal structure for the pure compound as well as a theoretically determined enzyme-inhibitor complex have been available. To provide reference data for the larger systems, the conformational space of the isolated molecule was screened for favorable geometries which were later compared to those within the crystal and protein surrounding. The geometry of the crystal structure could readily be taken from the experimental data whereas calculations on the protein complex revealed four potential non-covalent complexes exhibiting different arrangements of the molecule within the active site of the protein as well as two possible protonation states of the catalytic dyad. Hence, all four protein complexes have been compared to the crystal structure of the molecule as well as against the more favorable geometries of the isolated molecule being determined within vacuum or aqueous surrounding. Whereas the molecule itself was found to adopt comparable geometries within all investigated environments, the interactions pattern between the crystal surrounding and the protein differed largely from each other. The favorable formation of dimers within the crystal has a strong stabilizing effect and explains the extraordinarily good quality of the crystal. Within the protein however, repulsive forces have been found between the protein and the inhibitor. The origin of the repulsion could be traced back to effect of on of the substituents to the vinyl scaffold. The difference in the chemical structure in comparison to a well known inhibitor might also explain the experimentally found loss of activity for the model compound in comparison to K11777. KW - Theoretische Chemie KW - theoretical chemistry KW - electron density KW - inhibition KW - Elektronendichte KW - Inhibitor Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127860 ER - TY - THES A1 - Puhl, Sebastian T1 - Methods for protein crystal delivery: Exploring new techniques for encapsulation and controlled release T1 - Methoden der Proteinkristallverabreichung: Erforschung neuer Techniken der Verkapselung und kontrollierter Freisetzung N2 - More and more newly registered drugs are proteins. Although many of them suffer from instabilities in aqueous media, the most common way of protein drug administration still is the injection of a solution. Numerous protein drugs require frequent administration, but suitable controlled release systems for proteins are rare. Chapter 1 presents current advances in the field of controlled delivery of particulate protein formulations. While the main focus lies on batch crystallized proteins, amorphous particulate proteins are also discussed in this work. The reason is that, on the one hand precipitated protein particles hold some of the advantages of crystalline proteins and on the other hand the physical state of the protein may simply be unknown for many drug delivery systems or semi-crystalline particles have been used. Crystallization and precipitations methods as well as controlled delivery methods with and without encapsulation in a polymeric delivery system are summarized and critically discussed. In chapter 2 a novel way of protein crystal encapsulation by electrospinning is introduced. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals with a diameter of 0.7 or 2.1 μm and a PCL fiber diameter between 1.6 and 10 μm, release within the first 24 h could be varied between approximately 10 and 100%. Lysozyme loading of PCL microfibers between 0.5 and 5% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved. Chapter 3 takes on the findings made in chapter 2 and further modifies the properties of the nonwovens as protein crystal delivery system. Nonwoven scaffolds consisting of poly-ε-caprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and polidocanol (PD), and loaded with lysozyme crystals were prepared by electrospinning. The composition of the matrix was varied and the effect of PD content in binary mixtures, and of PD and PLGA content in ternary mixtures regarding processability, fiber morphology, water sorption, swelling and drug release was studied. Binary PCL/PD blend nonwovens showed a PD-dependent increase in swelling of up to 30% and of lysozyme burst release of up to 45% associated with changes of the fiber morphology. Furthermore, addition of free PD to the release medium resulted in a significant increase of lysozyme burst release from pure PCL nonwovens from approximately 2% to 35%. Using ternary PCL/PD/PLGA blends, matrix degradation could be significantly improved over PCL/PD blends, resulting in a biphasic release of lysozyme with constant release over 9 weeks, followed by constant release with a reduced rate over additional 4 weeks. Based on these results, protein release from PCL scaffolds is improved by blending with PD due to improved lysozyme desorption from the polymer surface and PD-dependent matrix swelling. Chapter 4 gives deeper insight on lysozyme batch crystallization and shows the influences of the temperature on the precipitation excipients. Yet up to now protein crystallization in a pharmaceutical useful scale displays a challenge with crystal size and purity being important but difficult to control parameters. Some of these influences are being discussed here and a detailed description of crystallization methods and the achieved crystals are demonstrated. Therapeutic use of such protein crystals may require further modification of the protein release rate through encapsulation. Silk fibroin (SF) harvested from the cocoons of Bombyx mori is a well-established protein suitable for encapsulation of small molecules as well as proteins for controlled drug delivery. This novel polymer was deployed for as carrier for the model drug crystals. Lysozyme again was used as a crystallizable protein and the effect of process- as well as formulation parameters of batch crystallization on crystal size were investigated using statistical design of experiments. Lysozyme crystal size depended on temperature and sodium chloride and poly(ethylenglycol) concentration of precipitant solution. Under optimized conditions, lysozyme crystals in a size range of approximately 0.3 to 10 µm were obtained. Furthermore, a solid-in-oil-in-water process for encapsulation of lysozyme crystals into SF was developed. Using this process, coating of protein crystals with another protein was achieved for the first time. Encapsulation resulted in a significant reduction of dissolution rate of lysozyme crystals, leading to prolonged release over up to 24 hours. N2 - Immer mehr neu zugelassene Arzneimittel sind Proteine. Obwohl viele von ihnen in wässrigen Milieus sehr instabil sind, werden die meisten Proteine immer noch als Parenteralia formuliert. Kontrollierte Freisetzungssysteme sind selten, und das obwohl zahlreiche Proteine regelmäßig verabreicht werden müssen. Kapitel 1 befasst sich mit den aktuellen Fortschritten im Bereich der kontrollierten Darreichungsformen für Proteinpartikel. Das Hauptaugenmerk liegt klar auf den „batch“ kristallisierten Proteinen, aber auch allgemeine ‚amorphe‘ Proteinpartikel werden diskutiert. Das liegt daran, dass zum einen präzipitierte Proteinpartikel ebenfalls einige Vorteile der Proteinkristalle haben und zum anderen wurde in einigen Fällen der physikalische Zustand der Proteine nicht bestimmt oder es wurden zumindest semi-kristalline Proteinpartikel verwendet. In diesem Kapitel werden Kristallisations- und Präzipitationsmethoden sowie kontrollierte Freisetzungssysteme mit und ohne vorherige Einbettung in ein Polymersystem zusammengefasst und kritisch diskutiert. Kapitel 2 stellt eine neue Methode der Proteinkristalleinbettung durch Electrospinning vor. Electrospinning von Proteinen hat sich aufgrund des häufigen Einsatzes von organischen Lösemitteln als schwierig heraus gestellt und führt häufig zum Entfalten oder Denaturieren des Proteins. Aufgrund höherer thermodynamischer Stabilität und verbesserter Kompatibilität mit organischen Lösemitteln haben sich Proteinkristalle hier als attraktive wenngleich bisher ziemlich unbeachtete Alternative heraus gestellt. In diesem Kapitel wird das das Electrospinning von Proteinkristallsuspensionen erforscht und grundlegende Bedingungen für diese neuartige Art der Proteinverkapselung etabliert. Poly-ε-caprolacton (PCL) eignet sich hervorragend für das Electrospinning und zeichnet sich durch matrixkontrollierte Freisetzung, einfache Handhabbarkeit sowie gute Biokompatibilität aus. PCL wurde als Matrix und Lysozym als Modelprotein eingesetzt. Mittels Kombination zweier Lysozymkristalldurchmesser, 0,7 oder 2,1 µm, und der Variation des PCL-Fadendurchmessers zwischen 1,6 und 10 µm konnte die Initialfreisetzung innerhalb der ersten 24 Stunden zwischen ca. 10 und 100 % modifiziert werden. Die Beladung der PCL Mikrofäden mit Lysozym zwischen 0,5 und 5 % konnte ohne Beeinträchtigung des Herstellungsprozesses erreicht werden. Hierdurch konnte die gesamt freigesetzte Menge an Lysozym variiert werden, während die relative freigesetzte Menge konstant blieb. Um weitere Modifikationen an der Freisetzungsrate vorzunehmen, wurden Poly(ethylen glycol) und Poly(lactic-co-glycolic acid) (PLGA) dem PCL beigemischt. Dadurch konnte unter optimierten Bedingungen eine Freisetzungsdauer von über 11 Wochen erreicht werden. Kapitel 3 greift die Entdeckungen des zweiten Kapitels auf und nimmt weitere Modifikationen an den Fasermatten vor. Die mit Proteinkristallen beladenen Fasermatten wurden wieder durch Electrospinning hergestellt und bestehen aus PCL, PLGA und Polidocanol (PD). Die Zusammensetzung der Matrix wurde variiert und die Auswirkungen des PD-Gehalts in binären Mischung, sowie des PD- und PLGA-Gehalts in ternären Mischsystemen auf die Verarbeitbarkeit, Fadenmorphologie, Wassersorption, Quellung und Wirkstofffreisetzung untersucht. Binäre Fasermatten aus einer Mischung aus PCL/PD zeigten PD-abhängige Zunahme der Quellung von bis zu 30 % und eine Zunahme der Initialfreisetzung von bis 45 %. Dieser Anstiege werden einer Veränderung der Fasermorphologie zugeschrieben. Das Hinzufügen von freiem PD zum Freisetzungsmedium von reinen PCL Fasermatten führte zu einer Zunahme der Initialfreisetzung von ca. 2 % auf etwa 35 %. Durch den Einsatz von ternären Mischsystem, bestehend aus PCL/PD/PLGA, kam es zu einem signifikant erhöhten Matrixabbau gegenüber den PCL/PD Mischungen. Das führte wiederrum zu einem zweiphasigen Freisetzungsverhalten, das durch eine konstante Freisetzungsrate innerhalb von 9 Wochen, sowie eine anschließende langsamere, ebenfalls konstante Freisetzung über weitere 4 Wochen gekennzeichnet war. Aufgrund dieser Ergebnisse kann gesagt werden, dass die Proteinfreisetzung aus PCL Matrices durch das Hinzufügen von PD aufgrund von verbesserter Lysozymdesorption vom Polymer sowie PD-abhängiger Matrixquellung verbessert werden konnte. Kapitel 4 bringt tiefere Erkenntnisse bezüglich der Lysozym Batchkristallisation und zeigt den Einfluss der Temperatur auf die verwendeten Fällungsreagenzien. Bis heute stellt die Kristallisation von Proteinen in einem pharmazeutisch sinnvollen Maßstab eine Herausforderung dar, wobei die Kristallgröße und die Reinheit wichtige und dennoch schwer kontrollierbare Parameter sind. Einige dieser Einflüsse werden hier näher beleuchtet und Kristallisationsmethoden sowie die erhaltenen Kristalle näher beschrieben. Lysozym wurde erneut als kristallisierbares Protein eingesetzt und die Einflüsse der Prozess- sowie Formulierungsparameter der Batch-Kristallisation mit Hilfe von statistischen Experimentendesigns (Design of Experiment, DoE) untersucht. Die Kristallgröße wurde durch die Temperatur sowie die Natriumchlorid- und Poly(ethylenglycol)konzentration der Fällungslösung bestimmt. Unter kontrollierten Bedingungen konnten Kristallgrößen in einem Bereich zwischen ca. 0.3 bis 10 µm erreicht werden. Darüber hinaus wurde ein „solid-in-oil-in-water“ (Feststoff-in-Öl-in-Wasser) Verfahren entwickelt mit dem Lysozymkristalle in Seidenfibroin eingebettet werden konnten. Mit Hilfe dieses Verfahrens wurden erstmals Proteinkristalle mit einem anderen Protein überzogen. Diese Einbettung brachte eine signifikante Auflösungsverzögerung mit einer verlängerten Freisetzung über 24 Stunden. KW - Kontrollierte Wirkstofffreisetzung KW - Verkapselung KW - Proteine KW - Electrospinning KW - Silk Fibroin KW - Kristall KW - Protein crystal KW - Encapsulation KW - Controlled release Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126371 ER - TY - THES A1 - Holzmeier, Fabian T1 - Photoionization of Nitrogen-Containing Reactive Molecules with Synchrotron Radiation T1 - Photoionisation von stickstoffhaltigen reaktiven Molekülen mit Synchrotronstrahlung N2 - The photoionization of several nitrogen-containing reactive intermediates relevant in combustion processes was investigated in the gas phase employing VUV synchrotron radiation. The intermediates were either freshly prepared and stored under cryogenic temperatures during the experiment or generated in situ by vacuum flash pyrolysis of suitable precursor molecules. The iPEPICO (imaging photoelectron photoion coincidence) setups of the VUV beamlines at the Swiss Light Source and Synchrotron SOLEIL were then used to record mass-selected threshold photoelectron (TPE) spectra. TPE spectra reveal the ionization energy and vibrational structure in the cationic states can often be resolved, which enables to distinguish different isomers. Accurate ionization energies for the radicals carbonyl amidogen, pyrrolyl, and 3-picolyl, and for the closed shell molecules isocyanic acid and cyanovinylacetylene were obtained. The analysis of the dissociative photoionization of the pyrolysis precursors enables in some cases to retrieve thermochemical data. Beyond, the absolute photoionization cross section of the cyclic carbene cyclopropenylidene was determined, NEXAFS and normal Auger spectra of isocyanic acid were recorded and analyzed at the O1s, N1s, and C1s edges, and the dissociative photoionization and pyrolysis of 1,4-di-tert-butyl-1,4-azaborinine was studied. N2 - Die Photoionisiation von stickstoffhaltigen reaktiven Intermediaten, die in Verbrennungsprozessen vorkommen, wurde in der Gasphase mit VUV Synchrotronstrahlung untersucht. Die Intermediate wurden entweder unmittelbar vor dem Experiment hergestellt und während des Experiments bei sehr niedrigen Temperaturen gehalten oder in situ durch Vakuum Flash Pyrolyse eines geeigneten Vorläufermoleküls erzeugt. Massenselektive Schwellenphotoelektronen(TPE)-Spektren wurden an den iPEPICO (imaging photoion photoelectron coincidence) Setups der VUV Strahllinien der Swiss Light Source und des Synchrotrons SOLEIL aufgenommen. Die Ionisierungsenergie kann in TPE-Spektren bestimmt werden und eine Auflösung von Schwingungsstruktur im Kation ist in vielen Fällen möglich, wodurch verschiedene Isomere unterschieden werden können. Verlässliche Ionisierungsenergien konnten für die Radikale Carbonylamidogen, Pyrrolyl und 3-Picolyl sowie für die geschlossenschaligen Moleküle Isocyansäure und Cyanovinylacetylen erhalten werden. Die Analyse der dissoziativen Photoionisation der Pyrolysevorläufer eröffnet in manchen Fällen Zugang zu thermochemischen Daten. Darüber hinaus wurde der absolute Photoionisationsquerschnitt des cyclischen Carbens Cyclopropenyliden bestimmt, wurden die NEXAFS und nicht-resonanten Auger Spektren von Isocyansäure an der O1s, N1s und C1s Kante aufgenommen und analysiert und die dissoziative Photoionisation und Pyrolyse von 1,4-di-tert-butyl-1,4-azaborinin untersucht. KW - Dissoziative Photoionisation KW - Synchrotronstrahlung KW - Ultraviolett-Photoelektronenspektroskopie KW - Pyrolyse KW - Photoelektron-Photoion-Koinzidenz KW - Fotoionisation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127763 ER - TY - THES A1 - Schneider, Johannes T1 - Functional diversification of membrane microdomains in Bacillus subtilis T1 - Funktionale Diversifizierung von Membran-Mikrodomänen in Bacillus subtilis N2 - Eukaryotic cells are considered as evolutionary complex organisms because they possess organelles that enable them to regulate the spatio-temporal organization of cellular processes. Spatio-temporal organization of signal transduction cascades occurs in eukaryotic cells via organization of membrane-associated microdomains or lipid rafts. Lipid rafts are nanoscale-sized domains in the plasma membrane that are constituted by a specific set of lipids and proteins and harbor a number of proteins related to signal transduction and trafficking. The integrity of lipid rafts is important for the assembly and functional coordination of a plethora of signaling networks and associated processes. This integrity is partially mediated by a chaperone protein called flotillin. Disruption of lipid raft integrity, for example via depletion or overproduction of flotillin, alters raft-associated signal transduction cascades and causes severe diseases like Alzheimer’s, Parkinson’s disease or cardiovascular disease. It was traditionally assumed that a sophisticated compartmentalization of cellular processes like the one exhibited in lipid rafts was exclusive to eukaryotic cells and therefore, lipid rafts have been considered as a hallmark in the evolution of cellular complexity, suggesting that prokaryotic cells were too simple organisms to organize such sophisticated membrane platforms. However, it was recently discovered that bacteria are also able to organize Functional Membrane Microdomains (FMMs) in their cellular membrane that are able to organize and catalyze the functionality of many diverse cellular processes. These FMMs of bacterial membranes contain flotillin-like proteins which play important roles in the organization of FMM-associated cellular processes. In this dissertation I describe the structural and biological significance of the existence of two distinct flotillin proteins, FloA and FloT, in the FMMs of the bacterial model Bacillus subtilis. Localization studies, proteomic data and transcriptomic analyses show that FloA and FloT are individual scaffold proteins that activate different regulatory programs during bacterial growth. Using the tractable bacterial model system, I show that the functionality of important regulatory proteins, like the protease FtsH or the signaling kinases KinC, PhoR and ResE, is linked to the activity of FMMs and that this is a direct consequence of the scaffold activity of the bacterial flotillins. FloA and FloT distribute heterogeneously along the FMMs of B. subtilis thereby generating a heterogeneous population of FMMs that compartmentalize different signal transduction cascades. Interestingly, diversification of FMMs does not occur randomly, but rather in a controlled spatio-temporal program to ensure the activation of given signaling networks at the right place and time during cell growth. N2 - Eukaryotische Zellen werden als evolutionär komplexe Organismen betrachtet, weil sie Organellen besitzen, mit denen sie die raum-zeitliche Organisation von zellulären Prozessen steuern können. Die räumliche und zeitliche Organisation von Signalwegen in eukaryotischen Zellen erfolgt durch die Abgrenzung von membran-assoziierten Mikrodomänen oder Lipid Rafts. Lipid Rafts sind wenige Nanometer große Felder in der Plasmamembran, die aus einem spezifischen Set von Lipiden und Proteinen zusammengesetzt sind und eine Reihe von für die Signaltransduktion und den Proteintransfer erforderlichen Proteine enthalten. Die Integrität der Lipid Rafts ist wichtig um zahlreiche Signalwege und damit assoziierte Prozesse zu verbinden und funktional zu koordinieren. Diese Integrität wird zum Teil von einem Chaperon-Protein namens Flotillin vermittelt. Eine Beeinträchtigung der Integrität der Lipid Rafts, z.B. aufgrund eines Mangels an Flotillin oder einer Überproduktion von Flotillin, verändert Raft-assoziierte Signalwege und verursacht schwere Erkrankungen wie Alzheimer, Parkinson oder kardiovaskuläre Erkrankungen. Bislang wurde angenommen, dass eine so anspruchsvolle Kompartimentierung zellulärer Prozesse wie im Falle der Lipid Rafts ausschließlich in eukaryotischen Zellen vorkommt. Lipid Rafts galten daher als Meilenstein in der Evolution der zellulären Komplexität und prokaryotische Zellen als zu einfache Organismen, um solch komplexe Plattformen in der Membran einzurichten. Vor kurzem wurde jedoch herausgefunden, dass Bakterien ebenfalls in der Lage sind, Funktionale Mikrodomänen in der Membran (FMMs) zu formen, die viele verschiedene zelluläre Prozesse organisieren und katalysieren können. Diese FMMs in bakteriellen Membranen enthalten Flotillin-ähnliche Proteine, die wichtige Aufgaben bei der Organisation von FMM-assoziierten Prozessen übernehmen. In dieser Dissertation beschreibe ich die strukturelle und biologische Signifikanz des Vorkommens der beiden verschiedenen Flotillin-Proteine FloA und FloT in den FMMs des bakteriellen Modellorganismus Bacillus subtilis. Lokalisationsstudien, proteomische Daten und transkriptomische Analysen demonstrieren, dass FloA und FloT individuelle Gerüstproteine sind, die während des Bakterienwachstums verschiedene regulatorische Programme aktivieren. Mit Hilfe des zugänglichen bakteriellen Modellorganismus zeige ich, dass die Funktionsweise von wichtigen regulatorischen Proteinen, wie z.B. der Protease FtsH oder der Signalwegskinasen KinC, PhoR und ResE, an die Aktivität der FMMs gebunden ist, und dass dies eine direkte Folge der stützenden Tätigkeit der bakteriellen Flotilline ist. FloA und FloT sind unterschiedlich in den FMMs von B. subtilis verteilt, wodurch sie eine heterogene Population von FMMs erzeugen, die verschiedene Signalwege abgrenzen kann. Interessanterweise erfolgt die Diversifizierung der FMMs nicht zufällig, sondern durch ein räumlich und zeitlich kontrolliertes Programm, um die Aktivierung von bestimmten Signalwegen am richtigen Ort und zur richtigen Zeit während des Zellwachstums sicherzustellen. KW - Heubacillus KW - Plasmamembran KW - Diversifikation KW - FMMs KW - Bacillus subtilis KW - Flotillin KW - Lipid Rafts Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127569 ER - TY - JOUR A1 - Neumann, Yvonne A1 - Ohlsen, Knut A1 - Donat, Stefanie A1 - Engelmann, Susanne A1 - Kusch, Harald A1 - Albrecht, Dirk A1 - Cartron, Michael A1 - Hurd, Alexander A1 - Foster, Simon J. T1 - The effect of skin fatty acids on Staphylococcus aureus JF - Archives of Microbiology N2 - Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS KW - S. aureus KW - skin fatty acid KW - C-6-H KW - resistance Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121657 VL - 197 ER - TY - JOUR A1 - Planes, Maria D. A1 - Niñoles, Regina A1 - Rubio, Lourdes A1 - Bissoli, Gaetano A1 - Bueso, Eduardo A1 - García-Sánchez, María J. A1 - Alejandro, Santiago A1 - Gonzalez-Guzmán, Miguel A1 - Hedrich, Rainer A1 - Rodriguez, Pedro L. A1 - Fernández, José A. A1 - Serrano, Ramón T1 - A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane \(H^+\)-ATPase and decreased cytosolic pH, \(K^+\), and anions JF - Journal of Experimental Botany N2 - The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for \(H^+\) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit \(H^+\) efflux (\(H^+\)-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (\(H^+\) efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to \(K^+\) efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the \(H^+\)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of (\(H^+\)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of (\(H^+\)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not. KW - ABA receptors KW - cytosolic pH KW - ion channels KW - microelectrodes KW - protein kinase KW - proton efflux Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121221 VL - 66 IS - 3 ER - TY - JOUR A1 - Galluzzi, L. A1 - Bravo-San Pedro, J. M. A1 - Vitale, I. A1 - Aaronson, S. A. A1 - Abrams, J. M. A1 - Adam, D. A1 - Alnemri, E. S. A1 - Altucci, L. A1 - Andrews, D. A1 - Annicchiarico-Petruzelli, M. A1 - Baehrecke, E. H. A1 - Bazan, N. G. A1 - Bertrand, M. J. A1 - Bianchi, K. A1 - Blagosklonny, M. V. A1 - Blomgren, K. A1 - Borner, C. A1 - Bredesen, D. E. A1 - Brenner, C. A1 - Campanella, M. A1 - Candi, E. A1 - Cecconi, F. A1 - Chan, F. K. A1 - Chandel, N. S. A1 - Cheng, E. H. A1 - Chipuk, J. E. A1 - Cidlowski, J. A. A1 - Ciechanover, A. A1 - Dawson, T. M. A1 - Dawson, V. L. A1 - De Laurenzi, V. A1 - De Maria, R. A1 - Debatin, K. M. A1 - Di Daniele, N. A1 - Dixit, V. M. A1 - Dynlacht, B. D. A1 - El-Deiry, W. S. A1 - Fimia, G. M. A1 - Flavell, R. A. A1 - Fulda, S. A1 - Garrido, C. A1 - Gougeon, M. L. A1 - Green, D. R. A1 - Gronemeyer, H. A1 - Hajnoczky, G. A1 - Hardwick, J. M. A1 - Hengartner, M. O. A1 - Ichijo, H. A1 - Joseph, B. A1 - Jost, P. J. A1 - Kaufmann, T. A1 - Kepp, O. A1 - Klionsky, D. J. A1 - Knight, R. A. A1 - Kumar, S. A1 - Lemasters, J. J. A1 - Levine, B. A1 - Linkermann, A. A1 - Lipton, S. A. A1 - Lockshin, R. A. A1 - López-Otín, C. A1 - Lugli, E. A1 - Madeo, F. A1 - Malorni, W. A1 - Marine, J. C. A1 - Martin, S. J. A1 - Martinou, J. C. A1 - Medema, J. P. A1 - Meier, P. A1 - Melino, S. A1 - Mizushima, N. A1 - Moll, U. A1 - Muñoz-Pinedo, C. A1 - Nuñez, G. A1 - Oberst, A. A1 - Panaretakis, T. A1 - Penninger, J. M. A1 - Peter, M. E. A1 - Piacentini, M. A1 - Pinton, P. A1 - Prehn, J. H. A1 - Puthalakath, H. A1 - Rabinovich, G. A. A1 - Ravichandran, K. S. A1 - Rizzuto, R. A1 - Rodrigues, C. M. A1 - Rubinsztein, D. C. A1 - Rudel, T. A1 - Shi, Y. A1 - Simon, H. U. A1 - Stockwell, B. R. A1 - Szabadkai, G. A1 - Tait, S. W. A1 - Tang, H. L. A1 - Tavernarakis, N. A1 - Tsujimoto, Y. A1 - Vanden Berghe, T. A1 - Vandenabeele, P. A1 - Villunger, A. A1 - Wagner, E. F. A1 - Walczak, H. A1 - White, E. A1 - Wood, W. G. A1 - Yuan, J. A1 - Zakeri, Z. A1 - Zhivotovsky, B. A1 - Melino, G. A1 - Kroemer, G. T1 - Essential versus accessory aspects of cell death: recommendations of the NCCD 2015 JF - Cell Death and Differentiation N2 - Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121207 VL - 22 ER - TY - JOUR A1 - Wehrle, Esther A1 - Liedert, Astrid A1 - Heilmann, Aline A1 - Wehner, Tim A1 - Bindl, Ronny A1 - Fischer, Lena A1 - Haffner-Luntzer, Melanie A1 - Jakob, Franz A1 - Schinke, Thorsten A1 - Amling, Michael A1 - Ignatius, Anita T1 - The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice JF - Disease Models & Mechanisms N2 - Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations. KW - fracture healing KW - LMHFV KW - oestrogen receptor signalling KW - whole-body vibration KW - Wnt-signalling Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121109 VL - 8 ER - TY - THES A1 - Bruttel, Valentin Stefan T1 - Soluble HLA-G binds to dendritic cells which likely suppresses anti-tumour immune responses in regional lymph nodes in ovarian carcinoma T1 - Lösliches HLA-G wird von dendritischen Zellen gebunden, was beim Ovarialkarzinom zur Hemmung von Immunraktionen in regionalen Lymphknoten führen kann N2 - Zusammenfassung Einleitung HLA-G, ein nicht-klassisches HLA bzw. MHC Klasse Ib Molekül, kann sowohl als membrangebundenes als auch als lösliches Molekül verschiedenste Immunzellpopulationen effektiv inhibieren. Unter physiologischen Bedingungen wird HLA-G vor allem in der Plazenta exprimiert, wo es dazu beiträgt den semiallogenen Embryo vor einer Abstoßung durch das mütterliche Immunsystem zu beschützen. Außerdem wird HLA-G in einer Vielzahl von Tumoren wie zum Beispiel in Ovarialkarzinomen überexprimiert. Ziel dieser Arbeit war es besonders die Rolle von löslichem HLA-G im Ovarialkarzinom und die Expression von HLA-G in verschiedenen Subtypen des Ovarialkarzinoms genauer zu untersuchen. Ergebnisse Anhand eines Tissue Microarrays wurde bestätigt dass HLA-G unter physiologischen Bedingungen nur in sehr wenigen Geweben wie Plazenta oder Testes exprimiert wird. Außerdem wurden erstmals auch im Nebennierenmark hohe Expressionslevel detektiert. Im Gegensatz zur physiologischen Expression wurde HLA-G in serösen, muzinösen, endometrioiden und Klarzellkarzinomen und somit in Tumoren aller untersuchten Subtypen des Ovarialkarzinoms detektiert. Am häufigsten war HLA-G in hochgradigen serösen Karzinomen überexprimiert. Hier konnte gezeigt werden dass auf Genexpressionslevel in Ovarialkarzinomen die Expression des immunsuppressiven HLA-G mit der Expression von klassischen MHC Molekülen wie HLA-A, -B oder -C hochsignifikant korreliert. Außerdem konnte in Aszitesproben von Patientinnen mit Ovarialkarzinomen hohe Konzentrationen von löslichem HLA-G nachgewiesen werden. Auch auf metastasierten Tumorzellen in regionalen Lymphknoten war HLA-G nachweisbar. Überraschenderweise wurde aber besonders viel HLA-G auf Dendritischen Zellen in Lymphknoten detektiert. Da in Monozyten und Dendritischen Zellen von gesunden Spendern durch IL-4 oder IL-10 im Gegensatz zu Literatur keine Expression von HLA-G induzierbar war, untersuchten wir ob Dendritische Zellen lösliches HLA-G binden. Es konnte gezeigt werden, dass besonders Dendritische Zellen die in Gegenwart von IL-4, IL-10 und GM-CSF aus Monozyten generiert wurden (DC-10) effektiv lösliches HLA-G über ILT Rezeptoren binden. In Abhängigkeit von ihrer Beladung mit HLA-G hemmen auch fixierte DC-10 Zellen noch die Proliferation von zytotoxischen CD8+ T Zellen. Zudem wurden regulatorische T Zellen induziert. Schlussfolgerungen Besonders in den am häufigsten diagnostizierten hochgradigen serösen Ovarialkarzinomen ist HLA-G in den meisten Fällen überexprimiert. Durch die Expression immunsuppressiver MHC Klasse Ib Moleküle wie HLA-G können wahrscheinlich auch Tumore wachsen, die noch klassische MHC Moleküle exprimieren und aufgrund ihrer Mutationslast eigentlich vom Immunsystem erkannt und eliminiert werden müssten. Lösliches HLA-G könnte zudem lokal Immunantworten gegen Tumorantigene unterdrücken indem es an Dendritische Zellen in regionalen Lymphknoten bindet. Diese Zellen präsentieren nomalerweise zytotoxischen T Zellen Tumorantigene und spielen daher eine entscheidende Rolle in der Entstehung von protektiven Immunantworten. Mit löslichem HLA-G beladene Dendritische Zellen hemmen jedoch die Proliferation von CD8+ T Zellen und induzieren regulatorische T Zellen. Dadurch könnten Ovarialkarzinome “aus der Ferne” auch in metastasenfreien Lymphknoten die Entstehung von gegen den Tumor gerichteten Immunantworten unterdrücken. Dieser erstmals beschriebene Mechanismus könnte auch in anderen malignen Erkrankungen eine Rolle spielen, da lösliches HLA-G in einer Vielzahl von Tumorindikationen nachgewiesen wurde. N2 - Abstract Background HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases. Results As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde. Conclusion The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G. KW - HLA-G KW - HLA-G KW - Dendritische Zelle KW - Eierstocktumor KW - ovarian cancer KW - ovarian carcinoma KW - transfer KW - dendritic cells KW - immune escape Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127252 ER - TY - JOUR A1 - Hefner, Jochen A1 - Csef, Herbert A1 - Frantz, Stefan A1 - Glatter, Nina A1 - Warrings, Bodo T1 - Recurrent Tako-Tsubo cardiomyopathy (TTC) in a pre-menopausal woman: late sequelae of a traumatic event? JF - BMC Cardiovascular Disorders N2 - Background “Tako-Tsubo cardiomyopathy” (TTC) is a syndrome characterized by left ventricular (LV) wall motion abnormalities, usually without coronary artery disease, mimicking the diagnosis of acute coronary syndrome. It most often affects post-menopausal women and TTC tends to run a benign course with very low rates of recurrence, complications or mortality. The condition is also called “stress-induced cardiomyopathy” because acute physical or emotional stress appears to be frequently related to its onset. The pathogenic role of premorbid or comorbid psychiatric illnesses has been discussed controversially. For the first time, we present a case of fourfold recurrent TTC with severe complications in a pre-menopausal woman. Furthermore, a long history of flaring posttraumatic stress symptoms anteceded the first event. Case presentation A 43-year old, pre-menopausal Caucasian woman was hospitalized with symptoms of acute coronary syndrome. Clinical examination revealed hypokinetic wall motion in the apical ventricular region with no signs of coronary artery disease and diagnosis of TTC was established. She experienced recurrence three times within the following ten months, which led to thrombembolism and myocardial scarring among others. The circumstances of chronic distress were striking. 16 years ago she miscarried after having removed a myoma according to her doctor’s suggestion. Since then, she has suffered from symptoms of posttraumatic distress which peaked annually at the day of abortion. Chronic distress became even more pronounced after the premature birth of a daughter some years later. The first event of TTC occurred after a family dispute about parenting. Conclusion This is the first case report of fourfold TTC in a pre-menopausal woman. From somatic perspectives, the course of the disease with recurrences and complications underlines the fact that TTC is not entirely benign. Furthermore, it is the first case report of long lasting symptoms of traumatic stress anteceding TTC. Close connections between adrenergic signaling and late onset of clinical stress symptoms are well known in the psychopathology of traumatization. Although larger clinical trials are needed to elucidate possible interactions of premorbid psychiatric illnesses and TTC, cardiologists should be vigilant especially in cases of recurrent TTC. KW - recurrent Tako-Tsubo cardiomyopathy KW - chronic distress KW - gene-environment interaction KW - comprehensive psychosomatic assessment Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124949 VL - 15 IS - 3 ER - TY - JOUR A1 - Gassenmaier, Tobias A1 - Petritsch, Bernhard A1 - Kunz, Andreas S. A1 - Gkaniatsas, Spyridon A1 - Gaudron, Philipp D. A1 - Weidemann, Frank A1 - Nordbeck, Peter A1 - Beer, Meinrad T1 - Long term evolution of MRI characteristics in a case of atypical left lateral wall hypertrophic cardiomyopathy JF - World Journal of Cardiology N2 - We are reporting a long-time magnetic resonance imaging (MRI) follow-up in a rare case of cardiac left lateral wall hypertrophy. Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disorder and a significant cause of sudden cardiac death. Cardiac magnetic resonance (CMR) imaging can be a valuable tool for assessment of detailed information on size, localization, and tissue characteristics of hypertrophied myocardium. However, there is still little knowledge of long-term evolution of HCM as visualized by magnetic resonance imaging. Recently, our group reported a case of left lateral wall HCM as a rare variant of the more common forms, such as septal HCM, or apical HCM. As we now retrieved an old cardiac MRI acquired in this patient more than 20 years ago, we are able to provide the thrilling experience of an ultra-long MRI follow-up presentation in this rare case of left lateral wall hypertrophy. Furthermore, this case outlines the tremendous improvements in imaging quality within the last two decades of CMR imaging. KW - cardiac magnetic resonance imaging KW - hypertrophic cardiomyopathy KW - follow-up KW - atypical KW - left lateral wall Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124934 VL - 7 IS - 6 ER - TY - JOUR A1 - Fluri, Felix A1 - Fleischer, Michael A1 - Kleinschnitz, Christoph T1 - Accidental Thrombolysis in a Stroke Patient Receiving Apixaban JF - Cerebrovascular Diseases Extra N2 - No abstract available. KW - acute management of stroke KW - acute ischemic stroke KW - acute neurology KW - acute stroke imaging KW - acute stroke management KW - acute stroke outcome Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126326 VL - 5 ER - TY - JOUR A1 - Kraft, Peter A1 - Drechsler, Christiane A1 - Schuhmann, Michael K. A1 - Gunreben, Ignaz A1 - Kleinschnitz, Christoph T1 - Characterization of Peripheral Immune Cell Subsets in Patients with Acute and Chronic Cerebrovascular Disease: A Case-Control Study JF - International Journal of Molecular Science N2 - Immune cells (IC) play a crucial role in murine stroke pathophysiology. However, data are limited on the role of these cells in ischemic stroke in humans. We therefore aimed to characterize and compare peripheral IC subsets in patients with acute ischemic stroke/transient ischemic attack (AIS/TIA), chronic cerebrovascular disease (CCD) and healthy volunteers (HV). We conducted a case-control study of patients with AIS/TIA (n = 116) or CCD (n = 117), and HV (n = 104) who were enrolled at the University Hospital Würzburg from 2010 to 2013. We determined the expression and quantity of IC subsets in the three study groups and performed correlation analyses with demographic and clinical parameters. The quantity of several IC subsets differed between the AIS/TIA, CCD, and HV groups. Several clinical and demographic variables independently predicted the quantity of IC subsets in patients with AIS/TIA. No significant changes in the quantity of IC subsets occurred within the first three days after AIS/TIA. Overall, these findings strengthen the evidence for a pathophysiologic role of IC in human ischemic stroke and the potential use of IC-based biomarkers for the prediction of stroke risk. A comprehensive description of IC kinetics is crucial to enable the design of targeted treatment strategies. KW - chronic cerebrovascular disease KW - lymphocytes KW - leukocytes KW - immune cells KW - biomarker KW - monocytes KW - regulatory T cells KW - ischemic stroke KW - thromboinflammation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126319 VL - 16 IS - 10 ER - TY - JOUR A1 - Boelch, Sebastian Philipp A1 - Barthel, Thomas A1 - Goebel, Sascha A1 - Rudert, Maximilian A1 - Plumhoff, Piet T1 - Calcinosis universalis - a rare case with classical presentation JF - Case Reports in Orthopedics N2 - Juvenile Dermatomyositis (JDM) is a rare autoimmune disease in children and adolescents. In these patients calcinosis might be the most characteristic symptom. However there are only few reported cases of intramuscular calcinosis in Dermatomyositis. We report a case of calcinosis universalis (CU) of the elbow in JDM successfully treated with broaching. The patient, a 24-year-old woman, suffered from a long history of JDM. On examination she presented with a fistula lateral to the olecranon and pain of the right elbow joint. Plain X-rays displayed a diffuse pattern of multiple periarticular, subcutaneous, and intramuscular calcifications. The patient underwent surgery for histological and microbiological sampling as well as broaching. Intraoperatively sinus formation and subfascial hard calcium deposition were found. Due to the risk of collateral tissue damage, incomplete broaching was performed. A local infection with Staphylococcus was diagnosed and treated with antibiotics. On six-week and 30-month follow-up the patient was free of pain and had very good function. Calcifications on standard radiographs had almost resolved entirely. This case report gives a summary on calcinosis in Dermatomyositis and adds a new case of recalcitrant CU to the literature. Broaching surgery proved to be a reliable treatment option in symptomatic calcinosis. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126300 VL - 2015 ER - TY - JOUR A1 - Zinner, Christoph A1 - Hauser, Anna A1 - Born, Dennis-Peter A1 - Wehrlin, Jon P. A1 - Holmberg, Hans-Christer A1 - Sperlich, Billy T1 - Influence of Hypoxic Interval Training and Hyperoxic Recovery on Muscle Activation and Oxygenation in Connection with Double-Poling Exercise JF - PLoS One N2 - Here, we evaluated the influence of breathing oxygen at different partial pressures during recovery from exercise on performance at sea-level and a simulated altitude of 1800 m, as reflected in activation of different upper body muscles, and oxygenation of the m. triceps brachii. Ten well-trained, male endurance athletes (25.3±4.1 yrs; 179.2±4.5 cm; 74.2±3.4 kg) performed four test trials, each involving three 3-min sessions on a double-poling ergometer with 3-min intervals of recovery. One trial was conducted entirely under normoxic (No) and another under hypoxic conditions \((Ho; F_iO_2 = 0.165)\). In the third and fourth trials, the exercise was performed in normoxia and hypoxia, respectively, with hyperoxic recovery \((HOX; F_iO_2 = 1.00)\) in both cases. Arterial hemoglobin saturation was higher under the two HOX conditions than without HOX (p<0.05). Integrated muscle electrical activity was not influenced by the oxygen content (best d = 0.51). Furthermore, the only difference in tissue saturation index measured via near-infrared spectroscopy observed was between the recovery periods during the NoNo and HoHOX interventions (P<0.05, d = 0.93). In the case of HoHo the athletes’ \(P_{mean}\) declined from the first to the third interval (P < 0.05), whereas Pmean was unaltered under the HoHOX, NoHOX and NoNo conditions. We conclude that the less pronounced decline in \(P_{mean}\) during 3 x 3-min double-poling sprints in normoxia and hypoxia with hyperoxic recovery is not related to changes in muscle activity or oxygenation. Moreover, we conclude that hyperoxia \((F_iO_2 = 1.00)\) used in conjunction with hypoxic or normoxic work intervals may serve as an effective aid when inhaled during the subsequent recovery intervals. KW - triceps KW - bood KW - medical hypoxia KW - blood KW - arms KW - hyperoxia KW - breathing KW - near-infrared spectroscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126299 VL - 10 IS - 10 ER - TY - JOUR A1 - Zinner, Christoph A1 - Sperlich, Billy A1 - Wahl, Patrick A1 - Mester, Joachim T1 - Classification of selected cardiopulmonary variables of elite athletes of different age, gender, and disciplines during incremental exercise testing JF - SpringerPlus N2 - Incremental exercise testing is frequently used as a tool for evaluating determinants of endurance performance. The available reference values for the peak oxygen uptake \((VO_{2peak})\), % of \(VO_{2peak}\) , running speed at the lactate threshold \((v_{LT})\), running economy (RE), and maximal running speed \((v_{peak})\) for different age, gender, and disciplines are not sufficient for the elite athletic population. The key variables of 491 young athletes (age range 12–21 years; 250 males, 241 females) assessed during a running step test protocol \((2.4 m s^{−1} ; increase 0.4 m s^{−1} 5 min^{−1})\) were analysed in five subgroups, which were related to combat-, team-, endurance-, sprint- and power-, and racquet-related disciplines. Compared with female athletes, male athletes achieved a higher \(v_{peak}\) (P = 0.004). The body mass, lean body mass, height, abs. \(VO_{2peak} (ml min^{−1})\), rel. \(VO_{2peak} (ml kg^{−1} min^{−1})\), rel. \(VO_{2peak} (ml min^{−1} kg^{−0.75})\), and RE were higher in the male participants compared with the females (P < 0.01). The % of \(VO_2\) at \(v_{LT}\) was lower in the males compared with the females (P < 0.01). No differences between gender were detected for the \(v_{LT}\) (P = 0.17) and % of \(VO_2\) at \(v_{LT}\) (P = 0.42). This study is one of the first to provide a broad spectrum of data to classify nearly 500 elite athletes aged 12–21 years of both gender and different disciplines. KW - performance KW - data KW - competition KW - reference values Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126275 VL - 4 IS - 544 ER - TY - JOUR A1 - Jordan, Martin C. A1 - Hoelscher-Doht, Stefanie A1 - Fehske, Kai A1 - Gilbert, Fabian A1 - Jansen, Hendrik A1 - Meffert, Rainer H. T1 - Bunnell or cross-lock Bunnell suture for tendon repair? Defining the biomechanical role of suture pretension JF - Journal of Orthopaedic Surgery and Research N2 - Background Suture pretension during tendon repair is supposed to increase the resistance to gap formation. However, its effects on the Bunnell suture technique are unknown. The purpose of this study was to determine the biomechanical effects of suture pretension on the Bunnell and cross-lock Bunnell techniques for tendon repair. Methods Eighty porcine hindlimb tendons were randomly assigned to four different tendon repair groups: those repaired with or without suture pretension using either a simple Bunnell or cross-lock Bunnell technique. Pretension was applied as a 10 % shortening of the sutured tendon. After measuring the cross-sectional diameter at the repair site, static and cyclic biomechanical tests were conducted to evaluate the initial and 5-mm gap formation forces, elongation during cyclic loading, maximum tensile strength, and mode of failure. The suture failure mechanism was also separately assessed fluoroscopically in two tendons that were repaired with steel wire. Results Suture pretension was accompanied by a 10 to 15 % increase in the tendon diameter at the repair site. Therefore, suture pretension with the Bunnell and cross-lock Bunnell repair techniques noticeably increased the resistance to initial gap formation and 5-mm gap formation. The tension-free cross-lock Bunnell repair demonstrated more resistance to initial and 5-mm gap formation, less elongation, and higher maximum tensile strength than the tension-free Bunnell repair technique. The only difference between the tensioned cross-lock Bunnell and tensioned Bunnell techniques was a larger resistance to 5-mm gap formation with the cross-lock Bunnell technique. Use of the simple instead of cross-lock suture configuration led to failure by suture cut out, as demonstrated fluoroscopically. Conclusion Based on these results, suture pretension decreases gapping and elongation after tendon repair, and those effects are stronger when using a cross-lock, rather than a regular Bunnell suture. However, pretension causes an unfavorable increase in the tendon diameter at the repair site, which may adversely affect wound healing. KW - suture KW - cross-lock KW - Bunnell KW - achilles KW - pretension KW - tendon Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126262 VL - 10 IS - 192 ER - TY - JOUR A1 - Lauterbach, Helge A. A1 - Borrmann, Dorit A1 - Heß, Robin A1 - Eck, Daniel A1 - Schilling, Klaus A1 - Nüchter, Andreas T1 - Evaluation of a Backpack-Mounted 3D Mobile Scanning System JF - Remote Sensing N2 - Recently, several backpack-mounted systems, also known as personal laser scanning systems, have been developed. They consist of laser scanners or cameras that are carried by a human operator to acquire measurements of the environment while walking. These systems were first designed to overcome the challenges of mapping indoor environments with doors and stairs. While the human operator inherently has the ability to open doors and to climb stairs, the flexible movements introduce irregularities of the trajectory to the system. To compete with other mapping systems, the accuracy of these systems has to be evaluated. In this paper, we present an extensive evaluation of our backpack mobile mapping system in indoor environments. It is shown that the system can deal with the normal human walking motion, but has problems with irregular jittering. Moreover, we demonstrate the applicability of the backpack in a suitable urban scenario. KW - man-portable mapping KW - backpack mobile mapping KW - SLAM KW - mobile laser scanning KW - personal laser scanning Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126247 VL - 7 IS - 10 ER - TY - JOUR A1 - Boelch, S. P. A1 - Jansen, H. A1 - Meffert, R. H. A1 - Frey, S. P. T1 - Six Sesamoid Bones on Both Feet: Report of a Rare Case JF - Journal of Clinical and Diagnostic Research N2 - There is a variation of the total number of distinct bones in the human in the literature. This difference is mainly caused by the variable existence of sesamoid bones. Sesamoid bones at the first MTP are seen regularly. In contrast additional sesamoid bones at the divond to fifth MTP are rare. We report a case of additional sesamoid bones at every metatarsophalangeal joint (MTP) of both feet. A 22-year-old female Caucasian presented with weight-dependent pain of the divond MTP of the left foot. In the radiographs of both feet additional sesamoid bones at every MTP could be seen. This case reports a very rare variation in human anatomy. A similar case has not been displayed to the academic society and therefore should be acknowledged. KW - anatomy KW - genetics KW - variation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126073 VL - 9 IS - 8 ER - TY - JOUR A1 - Schick, Martin Alexander A1 - Baar, Wolfgang A1 - Bruno, Raphael Romano A1 - Wollborn, Jakob A1 - Held, Christopher A1 - Schneider, Reinhard A1 - Flemming, Sven A1 - Schlegel, Nicolas A1 - Roewer, Norbert A1 - Neuhaus, Winfried A1 - Wunder, Christian T1 - Balanced hydroxyethylstarch (HES 130/0.4) impairs kidney function in-vivo without inflammation JF - PLoS One N2 - Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES). In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI). Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP). sCASP-group as well as control group (C) remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL) and control+HES (C+VOL) received 50 ml/KG balanced 6% HES (VOL) 130/0.4 over 6h. After 24h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1–4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion KW - colloids KW - kidneys KW - histopathology KW - blood KW - creatinine KW - sepsis KW - urine KW - inflammation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126068 VL - 10 IS - 9 ER - TY - JOUR A1 - Sollfrank, Teresa A1 - Hart, Daniel A1 - Goodsell, Rachel A1 - Foster, Jonathan A1 - Tan, Tele T1 - 3D visualization of movements can amplify motor cortex activation during subsequent motor imagery JF - Frontiers in Human Neuroscience N2 - A repetitive movement practice by motor imagery (MI) can influence motor cortical excitability in the electroencephalogram (EEG). This study investigated if a realistic visualization in 3D of upper and lower limb movements can amplify motor related potentials during subsequent MI. We hypothesized that a richer sensory visualization might be more effective during instrumental conditioning, resulting in a more pronounced event related desynchronization (ERD) of the upper alpha band (10–12 Hz) over the sensorimotor cortices thereby potentially improving MI based brain-computer interface (BCI) protocols for motor rehabilitation. The results show a strong increase of the characteristic patterns of ERD of the upper alpha band components for left and right limb MI present over the sensorimotor areas in both visualization conditions. Overall, significant differences were observed as a function of visualization modality (VM; 2D vs. 3D). The largest upper alpha band power decrease was obtained during MI after a 3-dimensional visualization. In total in 12 out of 20 tasks the end-user of the 3D visualization group showed an enhanced upper alpha ERD relative to 2D VM group, with statistical significance in nine tasks.With a realistic visualization of the limb movements, we tried to increase motor cortex activation during subsequent MI. The feedback and the feedback environment should be inherently motivating and relevant for the learner and should have an appeal of novelty, real-world relevance or aesthetic value (Ryan and Deci, 2000; Merrill, 2007). Realistic visual feedback, consistent with the participant’s MI, might be helpful for accomplishing successful MI and the use of such feedback may assist in making BCI a more natural interface for MI based BCI rehabilitation. KW - 3-dimensional visualization KW - motor cortex activation KW - EEG KW - brain-computer interfaces Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126058 VL - 9 IS - 463 ER - TY - JOUR A1 - Danhof, Sophia A1 - Schreder, Martin A1 - Strifler, Susanne A1 - Einsele, Hermann A1 - Knop, Stefan T1 - Long-Term Disease Control by Pomalidomide-/Dexamethasone-Based Therapy in a Patient with Advanced Multiple Myeloma: A Case Report and Review of the Literature JF - Case Reports in Oncology N2 - Background: Therapy for multiple myeloma (MM) has substantially improved in the era of immunomodulatory drugs and bortezomib. However, the prognosis of patients with progressive disease despite treatment with these ‘novel agents' remains poor. Recently, pomalidomide was approved in this setting, but a median progression-free survival of <4 months still leaves room for improvement. Pomalidomide-based combination therapies are currently under investigation, but data on long-term treatment are lacking. Case Report: We present the case of a 68-year-old woman with refractory MM who received pomalidomide in combination with various drugs including anthracyclines, alkylators and proteasome inhibitors. Initially, major hematological toxicities and infectious complications including a hepatitis B virus reactivation were encountered. With careful dose adjustments and selection of combination partners, pomalidomide treatment was maintained for over 4 years and led to a sustained partial remission. In particular, the well-tolerated regimen of bortezomib, cyclophosphamide and dexamethasone together with pomalidomide was administered for >30 cycles. Conclusion: This case illustrates the value of an individualized approach to myeloma care given an increasing availability of ‘novel agents'. Tailored treatment using these drugs as a backbone is essential to achieve long-lasting responses and minimize side effects. KW - Hepatitis B virus reactivation KW - pomalidomide KW - combination therapy KW - multiple myeloma Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126093 VL - 8 IS - 1 ER - TY - JOUR A1 - Kuger, Sebastian A1 - Flentje, Michael A1 - Djuzenova, Cholpon S. T1 - Simultaneous perturbation of the MAPK and the PI3K/mTOR pathways does not lead to increased radiosensitization JF - Radiation Oncology N2 - Background The mitogen-activated protein kinases (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are intertwined on various levels and simultaneous inhibition reduces tumorsize and prolonges survival synergistically. Furthermore, inhibiting these pathways radiosensitized cancer cells in various studies. To assess, if phenotypic changes after perturbations of this signaling network depend on the genetic background, we integrated a time series of the signaling data with phenotypic data after simultaneous MAPK/ERK kinase (MEK) and PI3K/mTOR inhibition and ionizing radiation (IR). Methods The MEK inhibitor AZD6244 and the dual PI3K/mTOR inhibitor NVP-BEZ235 were tested in glioblastoma and lung carcinoma cells, which differ in their mutational status in the MAPK and the PI3K/mTOR pathways. Effects of AZD6244 and NVP-BEZ235 on the proliferation were assessed using an ATP assay. Drug treatment and IR effects on the signaling network were analyzed in a time-dependent manner along with measurements of phenotypic changes in the colony forming ability, apoptosis, autophagy or cell cycle. Results Both inhibitors reduced the tumor cell proliferation in a dose-dependent manner, with NVP-BEZ235 revealing the higher anti-proliferative potential. Our Western blot data indicated that AZD6244 and NVP-BEZ235 perturbed the MAPK and PI3K/mTOR signaling cascades, respectively. Additionally, we confirmed crosstalks and feedback loops in the pathways. As shown by colony forming assay, the AZD6244 moderately radiosensitized cancer cells, whereas NVP-BEZ235 caused a stronger radiosensitization. Combining both drugs did not enhance the NVP-BEZ235-mediated radiosensitization. Both inhibitors caused a cell cycle arrest in the G1-phase, whereas concomitant IR and treatment with the inhibitors resulted in cell line- and drug-specific cell cycle alterations. Furthermore, combining both inhibitors synergistically enhanced a G1-phase arrest in sham-irradiated glioblastoma cells and induced apoptosis and autophagy in both cell lines. Conclusion Perturbations of the MEK and the PI3K pathway radiosensitized tumor cells of different origins and the combination of AZD6244 and NVP-BEZ235 yielded cytostatic effects in several tumor entities. However, this is the first study assessing, if the combination of both drugs also results in synergistic effects in terms of radiosensitivity. Our study demonstrates that simultaneous treatment with both pathway inhibitors does not lead to synergistic radiosensitization but causes cell line-specific effects. KW - autophagy KW - radiosensitivity KW - NVP-BEZ235 KW - AZD6244 KW - cell cycle arrest KW - apoptosis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126104 VL - 10 IS - 214 ER - TY - JOUR A1 - Baur, Johannes A1 - Schedelbeck, Ulla A1 - Pulzer, Alina A1 - Bluemel, Christina A1 - Wild, Vanessa A1 - Fassnacht, Martin A1 - Steger, U. T1 - A case report of a solitary pancreatic metastasis of an adrenocortical carcinoma JF - BMC Surgery N2 - Background Solitary metastases to the pancreas are rare. Therefore the value of resection in curative intention remains unclear. In the literature there are several promising reports about resection of solitary metastasis to the pancreas mainly of renal origin. Case presentation Here we report for the first time on the surgical therapy of a 1.5 cm solitary pancreatic metastasis of an adrenocortical carcinoma. The metastasis occurred almost 6 years after resection of the primary tumor. A partial pancreatoduodenectomy was performed and postoperatively adjuvant mitotane treatment was initiated. During the follow-up of 3 years after surgery no evidence of tumor recurrence occurred. Conclusion Resection of pancreatic tumors should be considered, even if the mass is suspicious for metastatic disease including recurrence of adrenocortical cancer. KW - surgical treatment KW - adrenocortical KW - carcinoma metastases to pancreas Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126130 VL - 15 IS - 93 ER - TY - THES A1 - Hauschild, Dirk T1 - Electron and soft x-ray spectroscopy of indium sulfide buffer layers and the interfaces in Cu(In,Ga)(S,Se)2-based thin-film solar cells T1 - Elektronen- und Weichröntgenemissionsspektroskopie von Indiumsulfid-Pufferschichten und Grenzflächen in Cu(In,Ga)(S,Se)2-basierten Dünnschichtsolarzellen N2 - In this thesis, thin-film solar cells on the basis of Cu(In,Ga)(S,Se)2 (CIGSSe) were investigated. Until today, most high efficient CIGSSe-based solar cells use a toxic and wetchemical deposited CdS buffer layer, which doesn’t allow a dry inline production. However, a promising and well-performing alternative buffer layer, namely indium sulfide, has been found which doesn’t comprise these disadvantages. In order to shed light on these well-performing devices, the surfaces and in particular the interfaces which play a major role for the charge carrier transport are investigated in the framework of this thesis. Both, the chemical and electronic properties of the solar cells’ interfaces were characterized. In case of the physical vapor deposition of an InxSy-based buffer layer, the cleaning step of the CdS chemical-bath deposition is not present and thus changes of the absorber surface have to be taken into account. Therefore, adsorbate formation, oxidation, and segregation of absorber elements in dependence of the storing temperature and the humidity are investigated in the first part of this thesis. The efficiencies of CIGSSe-based solar cells with an InxSy buffer layer depend on the nominal indium concentration x and display a maximum for x = 42 %. In this thesis, InxSy samples with a nominal indium concentration of 40.2% ≤ x ≤ 43.2% were investigated by surface-sensitive and surface-near bulk-sensitive techniques, namely with photoemission spectroscopy (PES) and x-ray emission spectroscopy (XES). The surfaces of the films were found to be sulfur-poor and indium-rich in comparison with stoichiometric In2S3. Moreover, a direct determination of the band alignment at the InxSy/CISSe interface in dependence of the nominal indium concentration x was conducted with the help of PES and inverse PES (IPES) and a flat band alignment was found for x = 42 %. In order to study the impact of a heat treatment as it occurs during subsequent cell process steps, the indium sulfide-buffered absorbers were annealed for 30 minutes under UHV conditions at 200 °C after the initial data set was taken. Besides a reported enhanced solar cell performance, a significant copper diffusion from the absorber into the buffer layer takes place due to the thermal treatment. Accordingly, the impact of the copper diffusion on the hidden InxSy/CISSe interface was discussed and for x = 40.2% a significant cliff (downwards step in the conduction band) is observed. For increasing x, the alignment in the conduction band turns into a small upwards step (spike) for the region 41% ≤ x ≤ 43.2%. This explains the optimal solar cell performance for this indium contents. In a further step, the sodium-doped indium sulfide buffer which leads to significantly higher efficient solar cells was investigated. It was demonstrated by PES/IPES that the enhanced performance can be ascribed to a significant larger surface band gap in comparison with undoped InxSy. The occurring spike in the Na:InxSy/CISSe band alignment gets reduced due to a Se diffusion induced by the thermal treatment. Furthermore, after the thermal treatment the sodium doped indium sulfide layer experiences a copper diffusion which is reduced by more than a factor of two compared to pure InxSy. Next, the interface between the Na:InxSy buffer layer and the i-ZnO (i = intrinsic, non-deliberately doped), as a part of the transparent front contact was analyzed. The i-ZnO/Na:InxSy interface shows significant interdiffusion, leading to the formation of, e.g., ZnS and hence to a reduction of the nominal cliff in the conduction band alignment. In the last part of this thesis, the well-established surface-sensitive reflective electron energy loss spectroscopy (REELS) was utilized to study the CIGSSe absorber, the InxSy buffer, and annealed InxSy buffer surfaces. By fitting the characteristic inelastic scattering cross sections λK(E) with Drude-Lindhard oscillators the dielectric function was identified. The determined dielectric functions are in good agreement with values from bulk-sensitive optical measurements on indium sulfide layers. In contrast, for the chalcopyrite-based absorber significant differences appear. In particular, a substantial larger surface band gap of the CIGSSe surface of E^Ex_Gap = (1.4±0.2) eV in comparison with bulk values is determined. This provides for the first time an independent verification of earlier PES/IPES results. Finally, the electrons’ inelastic mean free paths l for the three investigated surfaces are compared for different primary energies with theoretical values and the universal curve. N2 - Die vorliegende Arbeit untersucht Dünnschichtsolarzellen auf Basis von Cu(In,Ga)(S,Se)2 (CIGSSe). Um hohe Effizienzen bei CIGSSe-basierten Solarzellen zu erreichen, wurde bisher meist eine toxische und schlecht in einen Vakuumprozess integrierbare nasschemische CdS Pufferschicht verwendet. Mit Indiumsulfid konnte stattdessen eine vielversprechende alternative Pufferschicht gefunden werden, die diese nachteiligen Eigenschaften von CdS nicht aufweist und Solarzellen mit diesem Puffermaterial zeigen gute bis sehr gute Wirkungsgrade. Um die Ursachen der guten Leistungen herauszufinden, wurden die in der Solarzelle vorkommenden Oberflächen und Grenzflächen, die für den Ladungstransport eine zentrale Rolle spielen, Schritt für Schritt als Modellsysteme charakterisiert. Für einen InxSy-basierten Puffer, der durch die physikalische Gasphasenabscheidung aufgebracht wird, fehlt der Reinigungsprozess der Absorberoberflächen durch die nasschemische CdS Abscheidetechnik. Deshalb müssen Adsorbatbildung, Oxidation und Segregation von Absorberelementen die innerhalb der ersten Tage nach der Herstellung auftreten (je nach Feuchtigkeitsgehalt und Temperatur der Umgebung) berücksichtigt werden. Im ersten Teil der Arbeit werden solche Einflüsse auf die Oberfläche des Absorbers untersucht. Zellen mit einem Indiumsulfidpuffer zeigen Wirkungsgrade, die von der nominellen Indiumkonzentration x abhängen und bei x = 42% ein Optimum aufweisen. Eine stöchiometrische Analyse der InxSy Oberflächen ergab für 40.2% ≤ x ≤ 43.2% eine schwefelarme bzw. indiumreiche Oberfläche im Vergleich zu stöchiometrischem In2S3 (40% In und 60% S). Allerdings zeigen die untersuchten Proben für verschiedene Indiumkonzentrationen im Rahmen der oberflächensensitiven Photoemission (PES) und volumensensitiven Röntgenemission (XES) keine quantitativen Unterschiede. Mit Hilfe der PES und inversen PES (IPES) wurde der Bandverlauf an der InxSy/CISSe Grenzfläche in Abhängigkeit von der Indiumkonzentration untersucht und für x = 42% konnte ein flacher Bandverlauf ermittelt werden. Um den Einfluss des im Herstellungsprozess vorkommenden Temperaturschritts zu untersuchen, wurden die Proben für 30 Minuten auf 200 °C geheizt. Dabei konnte eine signifikante Diffusion von Kupfer aus dem Absorber in den Puffer beobachtet werden. Der Temperaturschritt führt neben der bereits bekannten Effizienzerhöhung vor allem zu einer Verringerung der Bandlücke des Puffers. Der Einfluss der Kupferdiffusion auf die verborgene InxSy/CISSe Grenzfläche wurde analysiert und für x = 40:2% wurde ein deutlicher "Cliff" (Stufe im Leitungsband nach unten) gefunden. Für Indiumkonzentrationen 41% ≤ x ≤ 43.2% wurde ein kleiner "Spike" (Stufe im Leitungsband nach oben) identifiziert, was dabei im Einklang mit den optimalen Wirkungsgraden ist. In einem weiteren Schritt wurde ein mit Natrium dotierter Indiumsulfidpuffer Na:InxSy, der verbesserte Wirkungsgrade zeigt, untersucht. Diese konnte zum einen auf eine deutlich vergrößerte Oberflächenbandlücke des Puffers zurückgeführt werden. Zum anderen wurde nach dem Temperaturschritt im Vergleich zu dem InxSy Puffer eine um den Faktor zwei verringerte Kupferdiffusion an der Oberfläche festgestellt. Des Weiteren konnte bei dem Temperaturschritt eine Diffusion von Selen festgestellt werden, die den vor dem Temperaturschritt vorhandenen "Spike" im Leitungsbandverlauf verringert. Nach dem Aufbringen der i-ZnO Schicht (i = intrinsisch, nicht absichtlich dotiert) als Teil des Frontkontakts auf den Na:InxSy Puffer, wurden Durchmischungseffekte an der i-ZnO/Na:InxSy Grenzfläche gefunden. Im weiteren Verlauf zeigte sich, dass der nominell auftretende "Cliff" zwischen i-ZnO und Na:InxSy durch die Bildung von ZnS reduziert bzw. vernachlässigt werden kann. Im letzten Teil der Arbeit wurde die etablierte oberflächensensitive reflektive Elektronenenergieverlustspektroskopie auf die Absorber- sowie Indiumsulfidoberflächen angewandt. Die ermittelten inelastisch gestreuten Verlustspektren λK(E) wurden mit dem Drude-Lindhard Modell simuliert und somit die dielektrische Funktion der jeweiligen Oberflächen bestimmt. Ein Vergleich mit volumensensitiven optischen Werten zeigt für die InxSy Schichten eine gute Übereinstimmung. Bei der CIGSSe Oberfläche konnten hingegen signifikante Unterschiede festgestellt werden. Dabei wurde erstmals die Oberflächenbandlücke eines Cu(In,Ga)(S,Se)2 Absorbers unabhängig von PES/IPES zu E^Ex_Gap = (1.4 ±0.2) eV verifiziert. Abschließend wurden die mittleren freien Weglängen der Elektronen l für die drei untersuchten Oberflächen für unterschiedliche Energien mit theoretischen Werten und der universellen Kurve verglichen. KW - Photoelektronenspektroskopie KW - Dünnschichtsolarzelle KW - Elektronische Eigenschaften KW - semiconductor interfaces KW - inverse photoemission KW - photoelectron spectroscopy KW - x-ray emission KW - Halbleitergrenzflächen KW - Photoelektronenspektroskopie KW - Röntgenemission KW - Solarzellen KW - Grenzfläche KW - Oberfläche Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126766 ER - TY - JOUR A1 - Walz, Yvonne A1 - Wegmann, Martin A1 - Dech, Stefan A1 - Vounastou, Penelope A1 - Poda, Jean-Noel A1 - N'Goran, Eliézer K. A1 - Raso, Giovanna A1 - Utzinger, Jürg T1 - Modeling and Validation of Environmental Suitability for Schistosomiasis Transmission Using Remote Sensing JF - PLoS Neglected Tropical Diseases N2 - Background Schistosomiasis is the most widespread water-based disease in sub-Saharan Africa. Transmission is governed by the spatial distribution of specific freshwater snails that act as intermediate hosts and human water contact patterns. Remote sensing data have been utilized for spatially explicit risk profiling of schistosomiasis. We investigated the potential of remote sensing to characterize habitat conditions of parasite and intermediate host snails and discuss the relevance for public health. Methodology We employed high-resolution remote sensing data, environmental field measurements, and ecological data to model environmental suitability for schistosomiasis-related parasite and snail species. The model was developed for Burkina Faso using a habitat suitability index (HSI). The plausibility of remote sensing habitat variables was validated using field measurements. The established model was transferred to different ecological settings in Côte d’Ivoire and validated against readily available survey data from school-aged children. Principal Findings Environmental suitability for schistosomiasis transmission was spatially delineated and quantified by seven habitat variables derived from remote sensing data. The strengths and weaknesses highlighted by the plausibility analysis showed that temporal dynamic water and vegetation measures were particularly useful to model parasite and snail habitat suitability, whereas the measurement of water surface temperature and topographic variables did not perform appropriately. The transferability of the model showed significant relations between the HSI and infection prevalence in study sites of Côte d’Ivoire. Conclusions/Significance A predictive map of environmental suitability for schistosomiasis transmission can support measures to gain and sustain control. This is particularly relevant as emphasis is shifting from morbidity control to interrupting transmission. Further validation of our mechanistic model needs to be complemented by field data of parasite- and snail-related fitness. Our model provides a useful tool to monitor the development of new hotspots of potential schistosomiasis transmission based on regularly updated remote sensing data. KW - schistosomiasis KW - Burkina Faso KW - remote sensing KW - surface water KW - habitats KW - agricultural irrigation KW - rivers KW - snails Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125845 VL - 9 IS - 11 ER - TY - JOUR A1 - Doppler, Kathrin A1 - Appeltshauser, Luise A1 - Krämer, Heidrun H. A1 - King Man Ng, Judy A1 - Meinl, Edgar A1 - Villmann, Carmen A1 - Brophy, Peter A1 - Dib-Hajj, Sulayman D. A1 - Waxman, Stephen G. A1 - Weishaupt, Andreas A1 - Sommer, Claudia T1 - Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy JF - PLoS One N2 - Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients’ sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy. KW - motor proteins KW - enzyme-linked immunoassays KW - binding analysis KW - neuropathy KW - nerve fibers KW - cell binding assay KW - antibodies KW - enzyme assays Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126156 VL - 10 IS - 7 ER - TY - JOUR A1 - Strube-Bloss, Martin F. A1 - Brown, Austin A1 - Spaethe, Johannes A1 - Schmitt, Thomas A1 - Rössler, Wolfgang T1 - Extracting the Behaviorally Relevant Stimulus: Unique Neural Representation of Farnesol, a Component of the Recruitment Pheromone of Bombus terrestris JF - PLoS One N2 - To trigger innate behavior, sensory neural networks are pre-tuned to extract biologically relevant stimuli. Many male-female or insect-plant interactions depend on this phenomenon. Especially communication among individuals within social groups depends on innate behaviors. One example is the efficient recruitment of nest mates by successful bumblebee foragers. Returning foragers release a recruitment pheromone in the nest while they perform a ‘dance’ behavior to activate unemployed nest mates. A major component of this pheromone is the sesquiterpenoid farnesol. How farnesol is processed and perceived by the olfactory system, has not yet been identified. It is much likely that processing farnesol involves an innate mechanism for the extraction of relevant information to trigger a fast and reliable behavioral response. To test this hypothesis, we used population response analyses of 100 antennal lobe (AL) neurons recorded in alive bumblebee workers under repeated stimulation with four behaviorally different, but chemically related odorants (geraniol, citronellol, citronellal and farnesol). The analysis identified a unique neural representation of the recruitment pheromone component compared to the other odorants that are predominantly emitted by flowers. The farnesol induced population activity in the AL allowed a reliable separation of farnesol from all other chemically related odor stimuli we tested. We conclude that the farnesol induced population activity may reflect a predetermined representation within the AL-neural network allowing efficient and fast extraction of a behaviorally relevant stimulus. Furthermore, the results show that population response analyses of multiple single AL-units may provide a powerful tool to identify distinct representations of behaviorally relevant odors. KW - instinct KW - plant-insect interactions KW - pheromones KW - bumblebees KW - odorants KW - principal component analysis KW - neurons KW - action potentials Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125875 VL - 10 IS - 9 ER - TY - JOUR A1 - Zoungrana, Benewinde Jean-Bosco A1 - Conrad, Christopher A1 - Amekudzi, Leonard K. A1 - Thiel, Michael A1 - Dapola Da, Evariste A1 - Forkuor, Gerald A1 - Löw, Fabian T1 - Multi-Temporal Landsat Images and Ancillary Data for Land Use/Cover Change (LULCC) Detection in the Southwest of Burkina Faso, West Africa JF - Remote Sensing N2 - Accurate quantification of land use/cover change (LULCC) is important for efficient environmental management, especially in regions that are extremely affected by climate variability and continuous population growth such as West Africa. In this context, accurate LULC classification and statistically sound change area estimates are essential for a better understanding of LULCC processes. This study aimed at comparing mono-temporal and multi-temporal LULC classifications as well as their combination with ancillary data and to determine LULCC across the heterogeneous landscape of southwest Burkina Faso using accurate classification results. Landsat data (1999, 2006 and 2011) and ancillary data served as input features for the random forest classifier algorithm. Five LULC classes were identified: woodland, mixed vegetation, bare surface, water and agricultural area. A reference database was established using different sources including high-resolution images, aerial photo and field data. LULCC and LULC classification accuracies, area and area uncertainty were computed based on the method of adjusted error matrices. The results revealed that multi-temporal classification significantly outperformed those solely based on mono-temporal data in the study area. However, combining mono-temporal imagery and ancillary data for LULC classification had the same accuracy level as multi-temporal classification which is an indication that this combination is an efficient alternative to multi-temporal classification in the study region, where cloud free images are rare. The LULCC map obtained had an overall accuracy of 92%. Natural vegetation loss was estimated to be 17.9% ± 2.5% between 1999 and 2011. The study area experienced an increase in agricultural area and bare surface at the expense of woodland and mixed vegetation, which attests to the ongoing deforestation. These results can serve as means of regional and global land cover products validation, as they provide a new validated data set with uncertainty estimates in heterogeneous ecosystems prone to classification errors. KW - Burkina Faso KW - West Africa KW - multi-temporal images KW - mono-temporal image KW - ancillary data KW - LULCC Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125866 VL - 7 IS - 9 ER - TY - JOUR A1 - Körner, Anita A1 - Topolinski, Sascha A1 - Strack, Fritz T1 - Routes to Embodiment JF - Frontiers of Psychology N2 - Research on embodiment is rich in impressive demonstrations but somewhat poor in comprehensive explanations. Although some moderators and driving mechanisms have been identified, a comprehensive conceptual account of how bodily states or dynamics influence behavior is still missing. Here, we attempt to integrate current knowledge by describing three basic psychological mechanisms: direct state induction, which influences how humans feel or process information, unmediated by any other cognitive mechanism; modal priming, which changes the accessibility of concepts associated with a bodily state; sensorimotor simulation, which affects the ease with which congruent and incongruent actions are performed. We argue that the joint impact of these mechanisms can account for most existing embodiment effects. Additionally, we summarize empirical tests for distinguishing these mechanisms and suggest a guideline for future research about the mechanisms underlying embodiment effects. KW - priming KW - simulation KW - grounded cognition KW - embodied cognition KW - metaphors Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125960 VL - 9 IS - 940 ER - TY - JOUR A1 - Walz, Yvonne A1 - Wegmann, Martin A1 - Leutner, Benjamin A1 - Dech, Stefan A1 - Vounatsou, Penelope A1 - N'Goran, Eliézer K. A1 - Raso, Giovanna A1 - Utzinger, Jürg T1 - Use of an ecologically relevant modelling approach to improve remote sensing-based schistosomiasis risk profiling JF - Geospatial Health N2 - Schistosomiasis is a widespread water-based disease that puts close to 800 million people at risk of infection with more than 250 million infected, mainly in sub-Saharan Africa. Transmission is governed by the spatial distribution of specific freshwater snails that act as intermediate hosts and the frequency, duration and extent of human bodies exposed to infested water sources during human water contact. Remote sensing data have been utilized for spatially explicit risk profiling of schistosomiasis. Since schistosomiasis risk profiling based on remote sensing data inherits a conceptual drawback if school-based disease prevalence data are directly related to the remote sensing measurements extracted at the location of the school, because the disease transmission usually does not exactly occur at the school, we took the local environment around the schools into account by explicitly linking ecologically relevant environmental information of potential disease transmission sites to survey measurements of disease prevalence. Our models were validated at two sites with different landscapes in Côte d’Ivoire using high- and moderateresolution remote sensing data based on random forest and partial least squares regression. We found that the ecologically relevant modelling approach explained up to 70% of the variation in Schistosoma infection prevalence and performed better compared to a purely pixelbased modelling approach. Furthermore, our study showed that model performance increased as a function of enlarging the school catchment area, confirming the hypothesis that suitable environments for schistosomiasis transmission rarely occur at the location of survey measurements. KW - Côte d’Ivoire KW - schistosomiasis KW - spatial risk profiling KW - remote sensing KW - ecological relevant model Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126148 VL - 10 IS - 2 ER - TY - THES A1 - Holz, Elisa Mira T1 - Systematic evaluation of non-invasive brain-computer interfaces as assistive devices for persons with severe motor impairment based on a user-centred approach – in controlled settings and independent use T1 - Systematische Evaluation nicht-invasiver Gehirn-Computer Schnittstellen als Hilfsmittel für Personen mit schweren motorischen Einschränkungen auf Basis eines nutzerzentrierten Ansatzes – im kontrollierten Setting und experten-unabhängigen Gebrauch N2 - Brain-computer interfaces (BCIs) are devices that translate signals from the brain into control commands for applications. Within the last twenty years, BCI applications have been developed for communication, environmental control, entertainment, and substitution of motor functions. Since BCIs provide muscle independent communication and control of the environment by circumventing motor pathways, they are considered as assistive technologies for persons with neurological and neurodegenerative diseases leading to motor paralysis, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal muscular atrophy and stroke (Kübler, Kotchoubey, Kaiser, Wolpaw, & Birbaumer, 2001). Although most researcher mention persons with severe motor impairment as target group for their BCI systems, most studies include healthy participants and studies including potential BCI end-users are sparse. Thus, there is a substantial lack of studies that investigate whether results obtained in healthy participants can be transferred to patients with neurodegenerative diseases. This clearly shows that BCI research faces a translational gap between intense BCI research and bringing BCI applications to end-users outside the lab (Kübler, Mattia, Rupp, & Tangermann, 2013). Translational studies are needed that investigate whether BCIs can be successfully used by severely disabled end-users and whether those end-users would accept BCIs as assistive devices. Another obvious discrepancy exists between a plethora of short-term studies and a sparse number of long-term studies. BCI research thus also faces a reliability gap (Kübler, Mattia, et al., 2013). Most studies present only one BCI session, however the few studies that include several testing sessions indicate high inter- and intra-individual variance in the end-users’ performance due to non-stationarity of signals. Long-term studies, however, are needed to demonstrate whether a BCI can be reliably used as assistive device over a longer period of time in the daily-life of a person. Therefore there is also a great need for reliability studies. The purpose of the present thesis was to address these research gaps and to bring BCIs closer to end-users in need, especially into their daily-lives, following a user-centred design (UCD). The UCD was suggested as theoretical framework for bringing BCIs to end-users by Kübler and colleagues (Kübler et al., 2014; Zickler et al., 2011). This approach aims at the close and iterative interaction between BCI developers and end-users with the final goal to develop BCI systems that are accepted as assistive devices by end-users. The UCD focuses on usability, that is, how well a BCI technology matches the purpose and meets the needs and requirements of the targeted end-users and was standardized with the ISO 9241-210. Within the UCD framework, usability of a device can be defined with regard to its effectiveness, efficiency and satisfaction. These aspects were operationalized by Kübler and colleagues to evaluate BCI-controlled applications. As suggested by Vaughan and colleagues, the number of BCI sessions, the total usage duration and the impact of the BCI on the life of the person can be considered as indicators of usefulness of the BCI in long-term daily-life use (Vaughan, Sellers, & Wolpaw, 2012). These definitions and metrics for usability and usefulness were applied for evaluating BCI applications as assistive devices in controlled settings and independent use. Three different BCI applications were tested and evaluated by in total N=10 end-users: In study 1 a motor-imagery (MI) based BCI for gaming was tested by four end-users with severe motor impairment. In study 2, a hybrid P300 event-related (ERP) based BCI for communication was tested by four severely motor restricted end-users with severe motor impairment. Study 1 and 2 are short-term studies conducted in a controlled-setting. In study 3 a P300-ERP BCI for creative expression was installed for long-term independent use at the homes of two end-users in the locked-in state. Both end-users are artists who had gradually lost the ability to paint after being diagnosed with ALS. Results reveal that BCI controlled devices are accepted as assistive devices. Main obstacles for daily-life use were the not very aesthetic design of the EEG-cap and electrodes (cap is eye-catching and looks medical), low comfort (cables disturb, immobility, electrodes press against head if lying on a head cushion), complicated and time-consuming adjustment, low efficiency and low effectiveness, and not very high reliability (many influencing factors). While effectiveness and efficiency in the MI based BCI were lower compared to applications using the P300-ERP as input channel, the MI controlled gaming application was nevertheless better accepted by the end-users and end-users would rather like to use it compared to the communication applications. Thus, malfunctioning and errors, low speed, and the EEG cap are rather tolerated in gaming applications, compared to communication devices. Since communication is essential for daily-life, it has to be fast and reliable. BCIs for communication, at the current state of the art, are not considered competitive with other assistive devices, if other devices, such as eye-gaze, are still an option. However BCIs might be an option when controlling an application for entertainment in daily-life, if communication is still available. Results demonstrate that BCI is adopted in daily-life if it matches the end-users needs and requirements. Brain Painting serves as best representative, as it matches the artists’ need for creative expression. Caveats such as uncomfortable cap, dependence on others for set-up, and experienced low control are tolerated and do not prevent BCI use on a daily basis. Also end-users in real need of means for communication, such as persons in the locked-in state with unreliable eye-movement or no means for independent communication, do accept obstacles of the BCI, as it is the last or only solution to communicate or control devices. Thus, these aspects are “no real obstacles” but rather “challenges” that do not prevent end-users to use the BCI in their daily-lives. For instance, one end-user, who uses a BCI in her daily-life, stated: “I don’t care about aesthetic design of EEG cap and electrodes nor amplifier”. Thus, the question is not which system is superior to the other, but which system is best for an individual user with specific symptoms, needs, requirements, existing assistive solutions, support by caregivers/family etc.; it is thereby a question of indication. These factors seem to be better “predictors” for adoption of a BCI in daily-life, than common usability criterions such as effectiveness or efficiency. The face valid measures of daily-life demonstrate that BCI-controlled applications can be used in daily-life for more than 3 years, with high satisfaction for the end-users, without experts being present and despite a decrease in the amplitude of the P300 signal. Brain Painting re-enabled both artists to be creatively active in their home environment and thus improved their feelings of happiness, usefulness, self-esteem, well-being, and consequently quality of life and supports social inclusion. This thesis suggests that BCIs are valuable tools for people in the locked-in state. N2 - Gehirn-Computer Schnittstellen (engl. Brain-computer interfaces, Abk.: BCIs) sind technische Systeme, die Gehirnsignale in Kontrollbefehle für Computeranwendungen übersetzen. In den vergangenen zwanzig Jahren wurden verschiedenste BCI Anwendungen entwickelt, beispielsweise zur Kommunikation, Umweltsteuerung, Unterhaltung und Ersatz von Motorfunktionen. Da BCIs muskelunabhängige Kommunikation und Kontrolle ermöglichen, werden sie als mögliche Hilfsmittel für Personen mit neurologischen und neurodegenerativen Krankheiten, die zu motorischen Lähmungen führen, wie beispielsweise bei Amyotrophe Lateralsklerose (ALS), Muskeldystrophie, Spinale Muskelatrophie und Schlaganfall, in Betracht gezogen (Kübler, Kotchoubey, et al., 2001). Auch wenn die meisten BCI Forscher Personen mit starken motorischen Einschränkungen als Zielgruppe für ihre BCI Systeme angeben, so testen sie ihre Systeme nur in Stichproben von gesunden Probanden. BCI Studien, die Patienten einschließen, sind dagegen selten. Daher gibt es einen beträchtlichen Mangel an Studien, die untersuchen, ob die Forschungsergebnisse, die basierend auf einer gesunden Stichprobe verzeichnet wurden, auch auf Patienten mit neurodegenerativen Erkrankungen übertragen werden können. Das macht deutlich, dass es in der BCI Forschung eine erhebliche Translationslücke zwischen der intensiven BCI Grundlagenforschung und dem Transfer von BCI Anwendungen aus dem Labor zu den Patienten, den sogenannten BCI End-Nutzern, gibt (Kübler, Mattia, et al., 2013). Es werden deshalb Translationsstudien benötigt, die untersuchen, ob BCIs von stark motorisch eingeschränkten Patienten verwendet werden können und ob diese sogenannten End-Nutzer BCIs als Hilfsmittel akzeptieren. Zusätzlich ist eine deutliche Diskrepanz zwischen der Vielzahl an Kurzzeitstudien und der geringen Anzahl an Langzeitstudien zu verzeichnen. BCI Forschung ist daher auch mit einer Reliabilitätslücke konfrontiert (Kübler, Mattia, et al., 2013). Die meisten Studien basieren nur auf einer BCI Sitzung, jedoch zeigen die wenigen Studien, die auf mehreren Sitzungen beruhen, hohe inter- und intraindividuelle Varianz in der Performanz der Patienten. Langzeitzeitstudien werden daher benötigt, um aufzuzeigen, ob ein BCI reliabel als Hilfsmittel über einen längeren Zeitraum im Alltagsleben eines Patienten verwendet werden kann. Demzufolge gibt es einen starken Bedarf an Translations- und Reliabilitätsstudien. Das Anliegen der vorliegenden Dissertation war es, diesen Forschungslücken zu begegnen und, basierend auf einem nutzerzentrierten Vorgehen, BCIs näher zu den BCI End-Nutzern zu bringen, besonders in ihr Alltagsleben. Der nutzerzentrierte Ansatz wurde von Kübler und Kollegen (Kübler et al., 2014; Zickler et al., 2011) als theoretisches Gerüst nahegelegt, um BCIs näher zu Patienten zu bringen. Dieser Ansatz beabsichtigt eine enge und iterative Interaktion zwischen BCI Entwicklern und den End-Nutzern mit dem finalen Ziel BCI Systeme zu entwickeln, die von den End-Nutzern als Hilfsmittel akzeptiert werden. Der nutzerzentrierte Ansatz fokussiert auf die Benutzbarkeit, das heißt, wie gut eine BCI Technologie den Bedürfnissen und den Ansprüchen der Zielgruppe entspricht. Dieser Ansatz wurde standardisiert mit dem ISO 9241-210. Demnach ist die Benutzbarkeit eines Gerätes definiert hinsichtlich der Effektivität, Effizienz, und Zufriedenheit. Um BCI Systeme zu evaluieren, wurden diese Aspekte von Kübler und Kollegen operationalisiert. Nach Vaughan und Kollegen können die Anzahl der BCI Sitzungen, die Gesamtnutzungsdauer und der Einfluss eines BCIs auf die Lebensqualität einer Person als Indikatoren der Nützlichkeit eines BCI betrachtet werden (Vaughan et al., 2012). Diese Definitionen und entsprechenden Operationalisierungen wurden in dieser Dissertation verwendet, um BCI Anwendungen hinsichtlich ihrer Benutzbarkeit und Nützlichkeit als Hilfsmittel zu evaluieren. N=10 End-Nutzer testeten und evaluierten drei BCI Anwendungen: In Studie 1 wurde ein auf Bewegungsvorstellung basiertes BCI (engl. motor imagery, Abk: MI) zur Steuerung einer Spielanwendung von vier potentiellen End-Nutzern mit unterschiedlichen neurologischen Erkrankungen getestet. In Studie 2 wurde ein Hybrid BCI, das das P300 ereignis-korrelierte Potential (EKP) und Muskelaktivität als Inputkanäle zur Steuerung eines Kommunikationsprogramms verwendet, von vier potentiellen End-Nutzern getestet. Studie 1 und 2 sind Kurzzeitstudien, welche in einem kontrollierten Design durchgeführt wurden. In Studie 3 wurde ein P300-EKP basiertes BCI zum künstlerischen Ausdruck bei zwei End-Nutzern zuhause für einen experten-unabhängigen und längerfristigen Gebrauch implementiert. Beide End-Nutzer sind Künstler, die, aufgrund der Diagnose ALS und der damit verbundenen fast kompletten körperlichen Lähmung (Locked-in Zustand), nicht mehr in der Lage waren zu malen (Studie 3). Die Ergebnisse zeigen, dass BCI Systeme als Hilfsmittel akzeptiert werden. Das nicht sehr ästhetische Design der EEG-Kappe und der Elektroden (Kappe zu auffällig, sieht medizinisch aus), der geringe Komfort (Kabel stören, Immobilität, Elektroden drücken gegen den Kopf), die komplizierte und zeitaufwändige Einstellung und Anpassung, die geringe Effizienz und geringe Effektivität und die nicht sehr hohe Reliabilität (viele Einflussfaktoren), wurden jedoch für einen Alltagsgebrauch als problematisch angesehen. Obwohl die Effektivität und Effizienz beim MI BCI geringer, verglichen mit beiden P300-EKP BCI Systemen, waren, wurde das MI basierte BCI-Spiel von den End-Nutzern besser akzeptiert und die End-Nutzer konnten sich eher vorstellen es im Alltag zu verwenden, als das Kommunikationsprogramm. Das zeigt, dass Störungen und Fehler, eine geringe Geschwindigkeit, und die EEG Kappe bei BCI Systemen zur Unterhaltung eher toleriert werden, als bei Systemen zur Kommunikation. Da Kommunikation im Leben essentiell ist, muss sie schnell und zuverlässig sein. BCI Systeme zur Kommunikation sind daher zum aktuellen Stand der Technik nicht konkurrenzfähig mit anderen Hilfsmitteln, wenn andere Hilfsmittel zur Kommunikation, wie Augensteuerung, verwendet werden können. BCI Systeme sind aber eine Option im Bereich Unterhaltung, sofern Möglichkeiten zur Kommunikation (noch) bestehen. Die Ergebnisse zeigen, dass ein BCI für einen Alltagsgebrauch übernommen wird, wenn es den Bedürfnissen und Anforderungen des End-Nutzers entspricht. Brain Painting zeigt hierbei beispielhaft, wie negative Facetten, wie die wenig komfortable EEG Kappe, die Abhängigkeit von anderen aufgrund der komplexen Einstellung, und eine subjektiv empfundene geringe Kontrolle toleriert werden, da es genau den Bedürfnissen der Künstler sich kreativ auszudrücken entspricht. Ebenso Patienten, die Bedarf an Kommunikation haben, wie Patienten im Locked-in Zustand, die keine zuverlässigen Augen-Bewegungen aufweisen, oder Patienten, die keine Hilfsmittel zur unabhängigen Kommunikation haben, akzeptieren diese Umstände beim BCI Gebrauch. Das zeigt, dass diese Umstände keine richtigen „Hindernisse“, sondern vielmehr Herausforderungen sind, die eine Übernahme eines BCI im Alltag eines Patienten nicht verhindern. Es ist daher nicht die Frage, welches BCI System überlegen ist, sondern welches BCI System das Beste für ein Individuum mit spezifischen Symptomen, Bedürfnissen, Ansprüchen, vorhandenen Hilfsmitteln, Unterstützung durch Familie und Pflegern, ist; es ist deshalb eine Frage der Indikation. Eine End-Nutzerin, die ein BCI im Alltag verwendet, sagte beispielsweise: „Mir ist das ästhetische Design der EEG-Kappe und der Elektroden, oder des EEG-Verstärkers völlig egal“. Diese Faktoren können als die besten “Prädiktoren” für eine Übernahme eines BCI Systems im Alltag eines Patienten angesehen werden, weniger hingegen die üblichen Kriterien zur Bewertung der Benutzbarkeit, wie Effektivität und Effizienz. Die Ergebnisse hinsichtlich des Alltagsgebrauches belegen ferner, dass ein P300-EKP basiertes BCI mit hoher Zufriedenheit über einen Zeitraum von 3 Jahren, ohne die Hilfe eines BCI Experten, und trotz einer Abnahme der Amplitude des P300-Signales verwendet werden kann. Brain Painting ermöglichte beiden Künstlern sich wieder kreativ auszudrücken und beeinflusste somit positiv das Empfinden von Freude, das Gefühl von Nützlichkeit, das Selbstwertgefühl, das Wohlbefinden und folglich die Lebensqualität der Künstler und förderte ihre soziale Inklusion. Die vorliegende Dissertation zeigt, dass BCIs wertvolle Hilfsmittel für Personen im Locked-in Zustand sein können. KW - Gehirn-Computer-Schnittstelle KW - Benutzerfreundlichkeit KW - brain-computer interface KW - evaluation KW - Evaluation KW - usability KW - Benutzerfreundlichkeit/ Benutzbarkeit KW - assistive device KW - Hilfsmittel KW - user-centred design KW - nutzerzentrierter Ansatz Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126334 ER - TY - JOUR A1 - Lichtenstein, Leonie A1 - Sommerlandt, Frank M. J. A1 - Spaethe, Johannes T1 - Dumb and Lazy? A Comparison of Color Learning and Memory Retrieval in Drones and Workers of the Buff-Tailed Bumblebee, Bombus terrestris, by Means of PER Conditioning JF - PLoS One N2 - More than 100 years ago, Karl von Frisch showed that honeybee workers learn and discriminate colors. Since then, many studies confirmed the color learning capabilities of females from various hymenopteran species. Yet, little is known about visual learning and memory in males despite the fact that in most bee species males must take care of their own needs and must find rewarding flowers to obtain food. Here we used the proboscis extension response (PER) paradigm to study the color learning capacities of workers and drones of the bumblebee, Bombus terrestris. Light stimuli were paired with sucrose reward delivered to the insects’ antennae and inducing a reflexive extension of the proboscis. We evaluated color learning (i.e. conditioned PER to color stimuli) in absolute and differential conditioning protocols and mid-term memory retention was measured two hours after conditioning. Different monochromatic light stimuli in combination with neutral density filters were used to ensure that the bumblebees could only use chromatic and not achromatic (e.g. brightness) information. Furthermore, we tested if bees were able to transfer the learned information from the PER conditioning to a novel discrimination task in a Y-maze. Both workers and drones were capable of learning and discriminating between monochromatic light stimuli and retrieved the learned stimulus after two hours. Drones performed as well as workers during conditioning and in the memory test, but failed in the transfer test in contrast to workers. Our data clearly show that bumblebees can learn to associate a color stimulus with a sugar reward in PER conditioning and that both workers and drones reach similar acquisition and mid-term retention performances. Additionally, we provide evidence that only workers transfer the learned information from a Pavlovian to an operant situation. KW - memory KW - bumblebees KW - behavioral conditioning KW - honey bees KW - flowers KW - sucrose KW - bees KW - learning Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125832 VL - 10 IS - 7 ER - TY - JOUR A1 - Westermaier, Thomas A1 - Koehler, Stefan A1 - Linsenmann, Thomas A1 - Kinderlen, Michael A1 - Pakos, Paul A1 - Ernestus, Ralf-Ingo T1 - Intraoperative Myelography in Cervical Multilevel Stenosis Using 3D Rotational Fluoroscopy: Assessment of Feasibility and Image Quality JF - Radiology Research and Practice N2 - Background. Intraoperative myelography has been reported for decompression control in multilevel lumbar disease. Cervical myelography is technically more challenging. Modern 3D fluoroscopy may provide a new opportunity supplying multiplanar images. This study was performed to determine the feasibility and image quality of intraoperative cervical myelography using a 3D fluoroscope. Methods. The series included 9 patients with multilevel cervical stenosis. After decompression, 10 mL of water-soluble contrast agent was administered via a lumbar drainage and the operating table was tilted. Thereafter, a 3D fluoroscopy scan (O-Arm) was performed and visually evaluated. Findings. The quality of multiplanar images was sufficient to supply information about the presence of residual stenosis. After instrumentation, metal artifacts lowered image quality. In 3 cases, decompression was continued because myelography depicted residual stenosis. In one case, anterior corpectomy was not completed because myelography showed sufficient decompression after 2-level discectomy. Interpretation. Intraoperative myelography using 3D rotational fluoroscopy is useful for the control of surgical decompression in multilevel spinal stenosis providing images comparable to postmyelographic CT. The long duration of contrast delivery into the cervical spine may be solved by preoperative contrast administration. The method is susceptible to metal artifacts and, therefore, should be applied before metal implants are placed. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125779 VL - 2015 ER - TY - JOUR A1 - Salat, Daniela A1 - Winkler, Anja A1 - Urlaub, Henning A1 - Gessler, Manfred T1 - Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus JF - PLoS One N2 - The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors. KW - ubiquitination KW - glycerol KW - transcription factors KW - DNA-binding proteins KW - immunoprecipitation KW - protein interactions KW - protein domains Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125769 VL - 10 IS - 6 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Vollmer, Tina A1 - Sefrin, Peter A1 - Kraus, Martin A1 - Happel, Oliver A1 - Wunder, Christian A1 - Steinisch, Andrias A1 - Roewer, Norbert A1 - Maier, Sebastian T1 - Monitoring of in-hospital cardiac arrest events with the focus on Automated External Defibrillators – a retrospective observational study JF - Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine N2 - Background Patients with cardiac arrest have lower survival rates, when resuscitation performance is low. In In-hospital settings the first responders on scene are usually nursing staff without rhythm analysing skills. In such cases Automated External Defibrillators (AED) might help guiding resuscitation performance. At the Wuerzburg University Hospital (Germany) an AED-program was initiated in 2007. Aim of the presented study was to monitor the impact of Automated External Defibrillators on the management of in-hospital cardiac arrest events. Methods The data acquisition was part of a continuous quality improvement process of the Wuerzburg University Hospital. For analysing the CPR performance, the chest compression rate (CCR), compression depth (CCD), the no flow fraction (NFF), time interval from AED-activation to the first compression (TtC), the time interval from AED-activation to the first shock (TtS) and the post schock pause (TtCS) were determined by AED captured data. A questionnaire was completed by the first responders. Results From 2010 to 2012 there were 359 emergency calls. From these 53 were cardiac arrests with an AED-application. Complete data were available in 46 cases. The TtC was 34 (32–52) seconds (median and IQR).The TtS was 30 (28–32) seconds (median and IQR) . The TtCS was 4 (3–6) seconds (median and IQR) . The CCD was 5.5 ± 1 cm while the CCR was 107 ± 11/min. The NFF was calculated as 41 %. ROSC was achieved in 21 patients (45 %), 8 patients (17 %) died on scene and 17 patients (37 %) were transferred under ongoing CPR to an Intensive Care Unit (ICU). Conclusion The TtS and TtC indicate that there is an AED-user dependent time loss. These time intervals can be markedly reduced, when the user is trained to interrupt the AED’s “chain of advices” by placing the electrode-paddles immediately on the patient’s thorax. At this time the AED switches directly to the analysing mode. Intensive training and adaption of the training contents is needed to optimize the handling of the AED in order to maximize its advantages and to minimize its disadvantages. KW - cardio-pulmonary resuscitation KW - team-training KW - chest-compression rate KW - automated external defibrillators KW - in-hospital cardiac arrest Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125756 VL - 23 IS - 87 ER - TY - JOUR A1 - Wohlleben, Gisela A1 - Scherzad, Agmal A1 - Güttler, Antje A1 - Vordermark, Dirk A1 - Kuger, Sebastian A1 - Flentje, Michael A1 - Polat, Buelent T1 - Influence of hypoxia and irradiation on osteopontin expression in head and neck cancer and glioblastoma cell lines JF - Radiation Oncology N2 - Background Tumor hypoxia is a known risk factor for reduced response to radiotherapy. The evaluation of noninvasive methods for the detection of hypoxia is therefore of interest. Osteopontin (OPN) has been discussed as an endogenous hypoxia biomarker. It is overexpressed in many cancers and is involved in tumor progression and metastasis. Methods To examine the influence of hypoxia and irradiation on osteopontin expression we used different cell lines (head and neck cancer (Cal27 and FaDu) and glioblastoma multiforme (U251 and U87)). Cells were treated with hypoxia for 24 h and were then irradiated with doses of 2 and 8 Gy. Osteopontin expression was analyzed on mRNA level by quantitative real-time RT-PCR (qPCR) and on protein level by western blot. Cell culture supernatants were evaluated for secreted OPN by ELISA. Results Hypoxia caused an increase in osteopontin protein expression in all cell lines. In Cal27 a corresponding increase in OPN mRNA expression was observed. In contrast the other cell lines showed a reduced mRNA expression under hypoxic conditions. After irradiation OPN mRNA expression raised slightly in FaDu and U87 cells while it was reduced in U251 and stable in Cal27 cells under normoxia. The combined treatment (hypoxia and irradiation) led to a slight increase of OPN mRNA after 2 Gy in U251 (24 h) and in U87 (24 and 48 h) cell lines falling back to base line after 8 Gy. This effect was not seen in Cal27 or in FaDu cells. Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions. Again the combined treatment resulted in a minor increase in OPN secretion 48 hours after irradiation with 8 Gy. Conclusion Osteopontin expression is strongly modulated by hypoxia and only to a minor extent by irradiation. Intracellular OPN homeostasis seems to vary considerably between cell lines. This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting. KW - glioblastoma multiforme KW - head and neck cancer KW - irridation KW - hypoxia KW - osteopontin Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125746 VL - 10 IS - 167 ER - TY - JOUR A1 - Sickel, Wiebke A1 - Ankenbrand, Markus J. A1 - Grimmer, Gudrun A1 - Holzschuh, Andrea A1 - Härtel, Stephan A1 - Lanzen, Jonathan A1 - Steffan-Dewenter, Ingolf A1 - Keller, Alexander T1 - Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach JF - BMC Ecology N2 - Background Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing. Results We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase). Conclusions This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples. KW - pollination ecology KW - next generation sequencing KW - ITS2 KW - illumina MiSeq platform KW - high throughput sequencing KW - DNA barcoding KW - NGS KW - osmia KW - palynolog Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125730 VL - 15 IS - 20 ER - TY - JOUR A1 - Popp, Michael A1 - Thielman, Ina A1 - Nieswandt, Bernhard A1 - Stegner, David T1 - Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5) JF - PLoS One N2 - Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice. KW - platelet activation KW - fibrinogen KW - integrins KW - platelets KW - thrombin KW - flow cytometry KW - platelet aggregation KW - blood Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125724 VL - 10 IS - 7 ER - TY - JOUR A1 - Glaser, Jan A1 - Schurigt, Uta A1 - Suzuki, Brian M. A1 - Caffrey, Connor R. A1 - Holzgrabe, Ulrike T1 - Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl JF - Molecules N2 - Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1–30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy. KW - thymyl cinnamate KW - vacuoles KW - autophagy KW - anti-schistosomal activity KW - schistosoma KW - schistosomula KW - parasite KW - eugenyl cinnamate Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125712 VL - 20 ER - TY - JOUR A1 - Adelfinger, Marion A1 - Bessler, Simon A1 - Cecil, Alexander A1 - Langbein-Laugwitz, Johanna A1 - Frentzen, Alexa A1 - Gentschev, Ivaylo A1 - Szalay, Aladar A. T1 - Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy JF - Viruses N2 - Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS. KW - canine cancer therapy KW - canine soft tissue sarcoma (CSTS) KW - oncolytic virus KW - cancer KW - canine cancer cell lines KW - antibody production KW - angiogenesis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125705 VL - 7 ER - TY - JOUR A1 - Merget, Benjamin A1 - Sotriffer, Christoph A. T1 - Slow-Onset Inhibition of Mycobacterium tuberculosis InhA: Revealing Molecular Determinants of Residence Time by MD Simulations JF - PLoS One N2 - An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring. KW - crystal structure KW - ethers KW - oxygen KW - cofactors (biochemistry) KW - binding analysis KW - biochemical simulations KW - hydrogen bonding mycobacterium tuberculosis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125607 VL - 10 IS - 5 ER - TY - JOUR A1 - Loeffler, Claudia A1 - Loeffler, Jürgen A1 - Kobsar, Anna A1 - Speer, Christian P. A1 - Eigenthaler, Martin T1 - Septic Vs Colonizing Group B Streptococci Differentially Regulate Inflammation and Apoptosis in Human Coronary Artery Endothelial Cells - a Pilot Study JF - Journal of Pediatrics and Neonatal Care N2 - In this pilot study, we exemplify differences between a septic and a colonizing GBS strain during their interaction with Endothelial Cells by evaluating cytokine levels, surface and apoptosis-related molecules. These preliminary results indicate that in vitro infection using an exemplary septic GBS strain results in diminished activation of the innate immune response. KW - streptococci KW - apoptosis KW - inflammation KW - endothelial cells KW - innate immunity KW - early onset sepsis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125596 VL - 2 IS - 2 ER - TY - JOUR A1 - Meierjohann, Svenja T1 - Hypoxia independent drivers of melanoma angiogenesis JF - Frontiers in Oncology N2 - Tumor angiogenesis is a process which is traditionally regarded as the tumor’s response to low nutrient supply occurring under hypoxic conditions. However, hypoxia is not a pre-requisite for angiogenesis. The fact that even single tumor cells or small tumor cell aggregates are capable of attracting blood vessels reveals the early metastatic capability of tumor cells. This review sheds light on the hypoxia-independent mechanisms of tumor angiogenesis in melanoma. KW - melanoma KW - angiogenesis KW - hypoxia-independent KW - reactive oxygen species KW - NF-κB Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125586 VL - 5 IS - 120 ER - TY - JOUR A1 - Subbarayal, Prema A1 - Karunakaran, Karthika A1 - Winkler, Ann-Cathrin A1 - Rother, Marion A1 - Gonzalez, Erik A1 - Meyer, Thomas F. A1 - Rudel, Thomas T1 - EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis JF - PLoS Pathogens N2 - The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance. KW - membrane proteins KW - chlamydia infection KW - chlamydia trachomatis KW - chlamydia KW - HeLa cells KW - apoptosis KW - host cells KW - membrane receptor signaling Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125566 VL - 11 IS - 4 ER - TY - JOUR A1 - Schneider, Johannes A1 - Klein, Teresa A1 - Mielich-Süss, Benjamin A1 - Koch, Gudrun A1 - Franke, Christian A1 - Kuipers, Oskar P. A1 - Kovács, Ákos T. A1 - Sauer, Markus A1 - Lopez, Daniel T1 - Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium JF - PLoS Genetics N2 - Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. KW - membrane proteins KW - gene expression KW - bacillus subtilis KW - fluorescence microscopy KW - cell fusion KW - signal transduction KW - gene regulation KW - lipids Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125577 VL - 11 IS - 4 ER - TY - JOUR A1 - Masic, Anita A1 - Valencia Hernandez, Ana Maria A1 - Hazra, Sudipta A1 - Glaser, Jan A1 - Holzgrabe, Ulrike A1 - Hazra, Banasri A1 - Schurigt, Uta T1 - Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice JF - PLoS One N2 - Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches. KW - leishmania major KW - promastigotes KW - apoptosis KW - mitochondria KW - parasitic diseases KW - leishmania KW - leishmaniasis KW - mouse models Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125354 VL - 10 IS - 11 ER - TY - JOUR A1 - Winkler, Karol A1 - Fischer, Julian A1 - Schade, Anne A1 - Amthor, Matthias A1 - Dall, Robert A1 - Geßler, Jonas A1 - Emmerling, Monika A1 - Ostrovskaya, Elena A. A1 - Kamp, Martin A1 - Schneider, Christian A1 - Höfling, Sven T1 - A polariton condensate in a photonic crystal potential landscape JF - New Journal of Physics N2 - The possibility of investigating macroscopic coherent quantum states in polariton condensates and of engineering polariton landscapes in semiconductors has triggered interest in using polaritonic systems to simulate complex many-body phenomena. However, advanced experiments require superior trapping techniques that allow for the engineering of periodic and arbitrary potentials with strong on-site localization, clean condensate formation, and nearest-neighbor coupling. Here we establish a technology that meets these demands and enables strong, potentially tunable trapping without affecting the favorable polariton characteristics. The traps are based on a locally elongated microcavity which can be formed by standard lithography. We observe polariton condensation with non-resonant pumping in single traps and photonic crystal square lattice arrays. In the latter structures, we observe pronounced energy bands, complete band gaps, and spontaneous condensation at the M-point of the Brillouin zone. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125050 VL - 17 ER - TY - JOUR A1 - Kleinschnitz, Christoph A1 - Linker, Ralf A. A1 - Magnus, Tim A1 - Korn, Thomas A1 - Meuth, Sven G. T1 - Report on the 6th scientific meeting of the “Verein zur Förderung des Wissenschaftlichen Nachwuchses in der Neurologie” (NEUROWIND e.V.) held in Motzen, Germany, Oct. 31th – Nov. 2nd, 2014 JF - Experimental & Translational Stroke Medicine N2 - From October 31th – November 2nd, 2014, the 6th NEUROWIND e.V. meeting was held in Motzen, Brandenburg, Germany. 70 doctoral students and postdocs from over 25 different groups working in German and Swiss university hospitals or research institutes attended the meeting to discuss their latest experiments and findings in the fields of neuroimmunology, neurodegeneration and neurovascular research. The meeting was regarded as a very well organized platform to support research of young investigators in Germany and all participants enjoyed the stimulating environment for lively in depth discussions. According to the major aim of NEUROWIND e.V. to support younger researchers in Germany the 4th NEUROWIND YOUNG SCIENTIST AWARD for experimental neurology was awarded to Michael Breckwoldt on his work in the group of Thomas Misgeld (Institute of Neuronal Cell Biology, Technische Universität München, Germany). The successful project was published in Nature Medicine entitled “Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo”. This outstanding paper deals with a molecular imaging approach in living mice to optically analyze the role of mitochondrial redox signals in axons in health and disease. The award is endowed with 20.000 Euro sponsored by Merck Serono GmbH, Darmstadt, Germany (unrestricted educational grant). This year’s keynote lecture was given by Bernhard Hemmer, Head of the Department of Neurology at the Klinikum rechts der Isar, Technische Universität München. Dr. Hemmer highlighted the particular role of B cells and (auto)antibodies in multiple sclerosis (MS). As a new highlight Dr. Urbahns, head of global discovery technologies at Merck research laboratories, gave insights from research practice in the pharmaceutical industry and introduced a shift in the view on present-day drug discovery paradigms. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125049 VL - 7 IS - 1 ER - TY - JOUR A1 - Klein, Dennis A1 - Groh, Janos A1 - Weishaupt, Andreas A1 - Martini, Rudolf T1 - Endogenous antibodies contribute to macrophage-mediated demyelination in a mouse model for CMT1B JF - Journal of Neuroinflammation N2 - Background We could previously identify components of both the innate and the adaptive immune system as disease modifiers in the pathogenesis of models for Charcot-Marie-Tooth (CMT) neuropathies type 1B and 1X. As part of the adaptive immune system, here we investigated the role of antibodies in a model for CMT1B. Methods Antibodies were localized and characterized in peripheral nerves of the CMT1B model by immunohistochemistry and Western blot analysis. Experimental ablation of antibodies was performed by cross breeding the CMT1B models with mutants deficient in B-lymphocytes (JHD−/− mutants). Ameliorated demyelination by antibody deficiency was reverted by intravenous injection of mouse IgG fractions. Histopathological analysis was performed by immunocytochemistry and light and quantitative electron microscopy. Results We demonstrate that in peripheral nerves of a mouse model for CMT1B, endogenous antibodies strongly decorate endoneurial tubes of peripheral nerves. These antibodies comprise IgG and IgM subtypes and are preferentially, but not exclusively, associated with nerve fiber aspects nearby the nodes of Ranvier. In the absence of antibodies, the early demyelinating phenotype is substantially ameliorated. Reverting the neuropathy by reconstitution with murine IgG fractions identified accumulating antibodies as potentially pathogenic at this early stage of disease. Conclusions Our study demonstrates that in a mouse model for CMT1B, endogenous antibodies contribute to early macrophage-mediated demyelination and disease progression. Thus, both the innate and adaptive immune system are mutually interconnected in a genetic model for demyelination. Since in Wallerian degeneration antibodies have also been shown to be involved in myelin phagocytosis, our study supports our view that inherited demyelination and Wallerian degeneration share common mechanisms, which are detrimental when activated under nonlesion conditions. KW - adaptive immune system KW - macrophages KW - antibodies KW - demyelination KW - Charcot-Marie-Tooth KW - B-lymphocytes KW - Fc-receptor KW - complement Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125036 VL - 12 IS - 49 ER - TY - JOUR A1 - Zusan, Andreas A1 - Gieseking, Björn A1 - Zerson, Mario A1 - Dyakonov, Vladimir A1 - Magerle, Robert A1 - Deibel, Carsten T1 - The Effect of Diiodooctane on the Charge Carrier Generation in Organic Solar Cells Based on the Copolymer PBDTTT-C JF - Scientific Reports N2 - Microstructural changes and the understanding of their effect on photocurrent generation are key aspects for improving the efficiency of organic photovoltaic devices. We analyze the impact of a systematically increased amount of the solvent additive diiodooctane (DIO) on the morphology of PBDTTT-C:PC71BM blends and related changes in free carrier formation and recombination by combining surface imaging, photophysical and charge extraction techniques. We identify agglomerates visible in AFM images of the 0% DIO blend as PC71BM domains embedded in an intermixed matrix phase. With the addition of DIO, a decrease in the size of fullerene domains along with a demixing of the matrix phase appears for 0.6% and 1% DIO. Surprisingly, transient absorption spectroscopy reveals an efficient photogeneration already for the smallest amount of DIO, although the largest efficiency is found for 3% DIO. It is ascribed to a fine-tuning of the blend morphology in terms of the formation of interpenetrating donor and acceptor phases minimizing geminate and nongeminate recombination as indicated by charge extraction experiments. An increase in the DIO content to 10% adversely affects the photovoltaic performance, most probably due to an inefficient free carrier formation and trapping in a less interconnected donor-acceptor network. KW - electronic properties and materials KW - photonic devices Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125022 VL - 5 ER - TY - JOUR A1 - Seyfried, Florian A1 - von Rahden, Burkhard H. A1 - Miras, Alexander D. A1 - Gasser, Martin A1 - Maeder, Uwe A1 - Kunzmann, Volker A1 - Germer, Christoph-Thomas A1 - Pelz, Jörg O. W. A1 - Kerscher, Alexander G. T1 - Incidence, time course and independent risk factors for metachronous peritoneal carcinomatosis of gastric origin – a longitudinal experience from a prospectively collected database of 1108 patients JF - BMC Cancer N2 - Background Comprehensive evidence on the incidence, time course and independent risk factors of metachronous peritoneal carcinomatosis (metaPC) in gastric cancer patients treated with curative intent in the context of available systemic combination chemotherapies is lacking. Methods Data from a prospectively collected single-institutional Center Cancer Registry with 1108 consecutive patients with gastric adenocarcinoma (GC), clinical, histological and survival data were analyzed for independent risk factors and prognosis with focus on the development of metaPC. Findings were then stratified to the time periods of treatment with surgery alone, 5-Fluorouracil-only and contemporary combined systemic perioperative chemotherapy strategies, respectively. Results Despite R0 D2 gastrectomy (n = 560), 49.6% (±5.4%) of the patients were diagnosed with tumour recurrence and 15.5% (±1.8%) developed metaPC after a median time of 17.7 (15.1-20.3) months after surgery resulting in a tumour related mortality of 100% with a median survival of 3.0 months (2.1 – 4.0). Independent risk factors for the development of metaPC were serosa positive T-category, nodal positive-status, signet cell and undifferentiated gradings (G3/G4). Contemporary systemic combination chemotherapy did not improve the incidence and prognosis of metaPC (p = 0.54). Conclusions Despite significant improvements in the overall survival for the complete cohort with gastric cancer over time, those patients with metaPC did not experience the same benefits. The lack of change in the incidence, and persistent poor prognosis of metaPC after curative surgery expose the need for further prevention and/or improved treatment options for this devastating condition. KW - recurrence survival KW - metachronous KW - peritoneal carcinomatosis KW - gastric cancer KW - risk factors KW - perioperative chemotherapy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125014 VL - 15 IS - 73 ER - TY - JOUR A1 - Liu, Dan A1 - Hu, Kai A1 - Pelzer, Heinz-Theo A1 - Störk, Stefan A1 - Weidemann, Frank T1 - Journey of a patient with chronic thromboembolic pulmonary hypertension JF - European Journal of Medical Research N2 - Right ventricle (RV) dysfunction is a key outcome determinant and a leading cause of death for patients with chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we followed the 5-year clinical journey of a patient with CTEPH. The tricuspid pressure gradient was significantly increased in the early phase of CTEPH and “normalized” at the late phase of this patient’s clinical journey, but this “normalized” gradient is not a positive treatment response but rather an ominous sign of advancing right heart failure owing to an exhaustion of RV contractile function. Thus, appropriate interpretation of the tricuspid pressure gradient change is of importance for assessing RV dysfunction and treatment outcome during follow-up in patients with CTEPH. Besides systolic pulmonary artery pressure (SPAP), other RV functional parameters such as tricuspid annular plane systolic excursion, RV fractional area change, and RV longitudinal strain, together with clinical markers, may provide additional guidance regarding functional improvement or progression in patients with CTEPH. KW - tricuspid pressure gradient KW - chronic thromboembolic pulmonary hypertension Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125009 VL - 20 IS - 20 ER - TY - JOUR A1 - Frank, Benjamin A1 - Marcu, Ana A1 - de Oliveira Almeida Petersen, Antonio Luis A1 - Weber, Heike A1 - Stigloher, Christian A1 - Mottram, Jeremy C. A1 - Scholz, Claus Jürgen A1 - Schurigt, Uta T1 - Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210 JF - Parasites & Vectors N2 - Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients. KW - autophagy KW - BNIP3 KW - CTSE KW - electron tomography KW - leishmania major KW - macrophages KW - miRNAs KW - MTOR KW - siRNAs KW - transmission electron microscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124997 VL - 8 IS - 404 ER - TY - JOUR A1 - Kirscher, Lorenz A1 - Deán-Ben, Xosé Luis A1 - Scadeng, Miriam A1 - Zaremba, Angelika A1 - Zhang, Qian A1 - Kober, Christina A1 - Fehm, Thomas Felix A1 - Razansky, Daniel A1 - Ntziachristos, Vasilis A1 - Stritzker, Jochen A1 - Szalay, Aladar A. T1 - Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent JF - Theranostics N2 - We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system. KW - reporter gene KW - oncolysis KW - molecular imaging KW - virotherapy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124987 VL - 5 IS - 10 ER - TY - JOUR A1 - Hamouda, Khaled A1 - Oezkur, Mehmet A1 - Sinha, Bhanu A1 - Hain, Johannes A1 - Menkel, Hannah A1 - Leistner, Marcus A1 - Leyh, Rainer A1 - Schimmer, Christoph T1 - Different duration strategies of perioperative antibiotic prophylaxis in adult patients undergoing cardiac surgery: an observational study JF - Journal of Cardiothoracic Surgery N2 - Background All international guidelines recommend perioperative antibiotic prophylaxis (PAB) should be routinely administered to patients undergoing cardiac surgery. However, the duration of PAB is heterogeneous and controversial. Methods Between 01.01.2011 and 31.12.2011, 1096 consecutive cardiac surgery patients were assigned to one of two groups receiving PAB with a second-generation cephalosporin for either 56 h (group I) or 32 h (group II). Patients’ characteristics, intraoperative data, and the in-hospital follow-up were analysed. Primary endpoint was the incidence of surgical site infection (deep and superficial sternal wound-, and vein harvesting site infection; DSWI/SSWI/VHSI). Secondary endpoints were the incidence of respiratory-, and urinary tract infection, as well as the mortality rate. Results 615/1096 patients (56,1%) were enrolled (group I: n = 283 versus group II: n = 332). There were no significant differences with regard to patient characteristics, comorbidities, and procedure-related variables. No statistically significant differences were demonstrated concerning primary and secondary endpoints. The incidence of DSWI/SSWI/VHSI were 4/283 (1,4%), 5/283 (1,7%), and 1/283 (0,3%) in group I versus 6/332 (1,8%), 9/332 (2,7%), and 3/332 (0,9%) in group II (p = 0,76/0,59/0,63). In univariate analyses female gender, age, peripheral arterial obstructive disease, operating-time, ICU-duration, transfusion, and respiratory insufficiency were determinants for nosocomial infections (all ≤ 0,05). Subgroup analyses of these high-risk patients did not show any differences between the two regimes (all ≥ 0,05). Conclusions Reducing the duration of PAB from 56 h to 32 h in adult cardiac surgery patients was not associated with an increase of nosocomial infection rate, but contributes to reduce antibiotic resistance and health care costs. KW - nosocomial infection KW - cardiac surgery KW - antibiotic prophylaxis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124977 VL - 10 IS - 25 ER - TY - JOUR A1 - Herter, Eva K. A1 - Stauch, Maria A1 - Gallant, Maria A1 - Wolf, Elmar A1 - Raabe, Thomas A1 - Gallant, Peter T1 - snoRNAs are a novel class of biologically relevant Myc targets JF - BMC Biology N2 - Background Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation. Results We combine chromatin immunoprecipitation-sequencing (ChIPseq) and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes we find the non-coding snoRNA genes as a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors. Conclusions This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc’s transforming effects are partially mediated by this class of non-coding transcripts. KW - Drosophila KW - ribosome KW - snoRNA KW - Myc Transcription KW - growth Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124956 VL - 13 IS - 25 ER - TY - JOUR A1 - Kober, Christina A1 - Rohn, Susanne A1 - Weibel, Stephanie A1 - Geissinger, Ulrike A1 - Chen, Nanhai G. A1 - Szalay, Aladar A. T1 - Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps JF - Journal of Translational Medicine N2 - Background Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM. Methods VACV LIVP 1.1.1 replication in C57BL/6 and \(Foxn1^{nu/nu}\) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system. Results We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of \(Iba1^+\) microglia and \(GFAP^+\) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected. Conclusion Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development. KW - GBM KW - tumor microenvironment KW - microglia KW - polarization KW - VACV KW - OSC KW - IMA2.1 KW - BV-2 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126517 VL - 13 IS - 216 ER - TY - JOUR A1 - Mahmood, Zafar A1 - Muhammad, Khalid A1 - Schmalzing, Marc A1 - Roll, Petra A1 - Dörner, Thomas A1 - Tony, Hans-Peter T1 - CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis JF - Arthritis Research & Therapy N2 - Introduction Enhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ). Methods DN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VH gene rearrangements and specific isotypes. Results Phenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06). Conclusions In RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126506 VL - 17 IS - 61 ER - TY - JOUR A1 - Adolfi, Mateus C. A1 - Carreira, Ana C. O. A1 - Jesus, Lázaro W. O. A1 - Bogerd, Jan A1 - Funes, Rejane M. A1 - Schartl, Manfred A1 - Sogayar, Mari C. A1 - Borella, Maria I. T1 - Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae JF - Reproductive Biology and Endocrinology N2 - Background The dmrt1 and sox9 genes have a well conserved function related to testis formation in vertebrates, and the group of fish presents a great diversity of species and reproductive mechanisms. The lambari fish (Astyanax altiparanae) is an important Neotropical species, where studies on molecular level of sex determination and gonad maturation are scarce. Methods Here, we employed molecular cloning techniques to analyze the cDNA sequences of the dmrt1 and sox9 genes, and describe the expression pattern of those genes during development and the male reproductive cycle by qRT-PCR, and related to histology of the gonad. Results Phylogenetic analyses of predicted amino acid sequences of dmrt1 and sox9 clustered A. altiparanae in the Ostariophysi group, which is consistent with the morphological phylogeny of this species. Studies of the gonad development revealed that ovary formation occurred at 58 days after hatching (dah), 2 weeks earlier than testis formation. Expression studies of sox9 and dmrt1 in different tissues of adult males and females and during development revealed specific expression in the testis, indicating that both genes also have a male-specific role in the adult. During the period of gonad sex differentiation, dmrt1 seems to have a more significant role than sox9. During the male reproductive cycle dmrt1 and sox9 are down-regulated after spermiation, indicating a role of these genes in spermatogenesis. Conclusions For the first time the dmrt1 and sox9 were cloned in a Characiformes species. We show that both genes have a conserved structure and expression, evidencing their role in sex determination, sex differentiation and the male reproductive cycle in A. altiparanae. These findings contribute to a better understanding of the molecular mechanisms of sex determination and differentiation in fish. KW - spermatogenesis KW - SOX9 KW - DMRT1 KW - sex differentiation KW - teleostei Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126486 VL - 13 IS - 2 ER - TY - JOUR A1 - Lakovic, Milica A1 - Poethke, Hans-Joachim A1 - Hovestadt, Thomas T1 - Dispersal timing: Emigration of insects living in patchy environments JF - PLoS One N2 - Dispersal is a life-history trait affecting dynamics and persistence of populations; it evolves under various known selective pressures. Theoretical studies on dispersal typically assume 'natal dispersal', where individuals emigrate right after birth. But emigration may also occur during a later moment within a reproductive season ('breeding dispersal'). For example, some female butterflies first deposit eggs in their natal patch before migrating to other site(s) to continue egg-laying there. How breeding compared to natal dispersal influences the evolution of dispersal has not been explored. To close this gap we used an individual-based simulation approach to analyze (i) the evolution of timing of breeding dispersal in annual organisms, (ii) its influence on dispersal (compared to natal dispersal). Furthermore, we tested (iii) its performance in direct evolutionary contest with individuals following a natal dispersal strategy. Our results show that evolution should typically result in lower dispersal under breeding dispersal, especially when costs of dispersal are low and population size is small. By distributing offspring evenly across two patches, breeding dispersal allows reducing direct sibling competition in the next generation whereas natal dispersal can only reduce trans-generational kin competition by producing highly dispersive offspring in each generation. The added benefit of breeding dispersal is most prominent in patches with small population sizes. Finally, the evolutionary contests show that a breeding dispersal strategy would universally out-compete natal dispersal. KW - animal migration KW - statistical disperison KW - organismal evolution KW - animal sexual behavior KW - habitats KW - insects KW - carrying capacity KW - moths and butterflies Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126466 VL - 10 IS - 7 ER - TY - JOUR A1 - Simon, Nadine A1 - Käthner, Ivo A1 - Ruf, Carolin A. A1 - Pasqualotto, Emanuele A1 - Kübler, Andrea A1 - Halder, Sebastian T1 - An auditory multiclass brain-computer interface with natural stimuli: Usability evaluation with healthy participants and a motor impaired end user JF - Frontiers in Human Neuroscience N2 - Brain-computer interfaces (BCIs) can serve as muscle independent communication aids. Persons, who are unable to control their eye muscles (e.g., in the completely locked-in state) or have severe visual impairments for other reasons, need BCI systems that do not rely on the visual modality. For this reason, BCIs that employ auditory stimuli were suggested. In this study, a multiclass BCI spelling system was implemented that uses animal voices with directional cues to code rows and columns of a letter matrix. To reveal possible training effects with the system, 11 healthy participants performed spelling tasks on 2 consecutive days. In a second step, the system was tested by a participant with amyotrophic lateral sclerosis (ALS) in two sessions. In the first session, healthy participants spelled with an average accuracy of 76% (3.29 bits/min) that increased to 90% (4.23 bits/min) on the second day. Spelling accuracy by the participant with ALS was 20% in the first and 47% in the second session. The results indicate a strong training effect for both the healthy participants and the participant with ALS. While healthy participants reached high accuracies in the first session and second session, accuracies for the participant with ALS were not sufficient for satisfactory communication in both sessions. More training sessions might be needed to improve spelling accuracies. The study demonstrated the feasibility of the auditory BCI with healthy users and stresses the importance of training with auditory multiclass BCIs, especially for potential end-users of BCI with disease. KW - P300 KW - EEG KW - auditory BCI KW - brain-computer interface KW - communication KW - ALS Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126450 VL - 8 IS - 1039 ER - TY - JOUR A1 - Steinert, Andre F. A1 - Kunz, Manuela A1 - Prager, Patrick A1 - Göbel, Sascha A1 - Klein-Hitpass, Ludger A1 - Ebert, Regina A1 - Nöth, Ulrich A1 - Jakob, Franz A1 - Gohlke, Frank T1 - Characterization of bursa subacromialis-derived mesenchymal stem cells JF - Stem Cell Research & Therapy N2 - Introduction The bursa subacromialis (BS) provides the gliding mechanism of the shoulder and regenerates itself after surgical removal. Therefore, we explored the presence of mesenchymal stem cells (MSCs) within the human adult BS tissue and characterized the BS cells compared to MSCs from bone marrow (BMSCs) on a molecular level. Methods BS cells were isolated by collagenase digest from BS tissues derived from patients with degenerative rotator cuff tears, and BMSCs were recovered by adherent culture from bone-marrow of patients with osteoarthritis of the hip. BS cells and BMSCs were compared upon their potential to proliferate and differentiate along chondrogenic, osteogenic and adipogenic lineages under specific culture conditions. Expression profiles of markers associated with mesenchymal phenotypes were comparatively evaluated by flow cytometry, immunohistochemistry, and whole genome array analyses. Results BS cells and BMSCs appeared mainly fibroblastic and revealed almost similar surface antigen expression profiles, which was \(CD44^+, CD73^+, CD90^+, CD105^+, CD106^+\),\(STRO-1^+, CD14^−, CD31^−, CD34^− , CD45^−, CD144^−\). Array analyses revealed 1969 genes upregulated and 1184 genes downregulated in BS cells vs. BMSCs, indicating a high level of transcriptome similarity. After 3 weeks of differentiation culture, BS cells and BMSCs showed a similar strong chondrogenic, adipogenic and osteogenic potential, as shown by histological, immunohistochemical and RT-PCR analyses in contrast to the respective negative controls. Conclusions Our in vitro characterizations show that BS cells fulfill all characteristics of mesenchymal stem cells, and therefore merit further attention for the development of improved therapies for various shoulder pathologies. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126446 VL - 6 IS - 114 ER - TY - JOUR A1 - Tran-Gia, Johannes A1 - Wech, Tobias A1 - Bley, Thorsten A1 - Köstler, Herbert T1 - Model-Based Acceleration of Look-Locker T1 Mapping JF - PLoS One N2 - Mapping the longitudinal relaxation time \(T_1\) has widespread applications in clinical MRI as it promises a quantitative comparison of tissue properties across subjects and scanners. Due to the long scan times of conventional methods, however, the use of quantitative MRI in clinical routine is still very limited. In this work, an acceleration of Inversion-Recovery Look-Locker (IR-LL) \(T_1\) mapping is presented. A model-based algorithm is used to iteratively enforce an exponential relaxation model to a highly undersampled radially acquired IR-LL dataset obtained after the application of a single global inversion pulse. Using the proposed technique, a \(T_1\) map of a single slice with 1.6mm in-plane resolution and 4mm slice thickness can be reconstructed from data acquired in only 6s. A time-consuming segmented IR experiment was used as gold standard for \(T_1\) mapping in this work. In the subsequent validation study, the model-based reconstruction of a single-inversion IR-LL dataset exhibited a \(T_1\) difference of less than 2.6% compared to the segmented IR-LL reference in a phantom consisting of vials with \(T_1\) values between 200ms and 3000ms. In vivo, the \(T_1\) difference was smaller than 5.5% in WM and GM of seven healthy volunteers. Additionally, the \(T_1\) values are comparable to standard literature values. Despite the high acceleration, all model-based reconstructions were of a visual quality comparable to fully sampled references. Finally, the reproducibility of the \(T_1\) mapping method was demonstrated in repeated acquisitions. In conclusion, the presented approach represents a promising way for fast and accurate \(T_1\) mapping using radial IR-LL acquisitions without the need of any segmentation. KW - algorithms KW - cerebrospinal fluid KW - NMR relaxation KW - data acquisition KW - relaxation (physics) KW - relaxation time KW - central nervous system KW - magnetic resonance imaging Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126436 VL - 10 IS - 4 ER - TY - JOUR A1 - Weiß, Clemens Leonard A1 - Schultz, Jörg T1 - Identification of divergent WH2 motifs by HMM-HMM alignments JF - BMC Research Notes N2 - Background The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved. Findings To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of ‘proximal to raf’ (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified. Conclusion In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families. KW - WH2 domain KW - spire KW - shootin-1 KW - actin nucleation KW - HHblits Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126413 VL - 8 IS - 18 ER - TY - JOUR A1 - Andronic, Joseph A1 - Shirakashi, Ryo A1 - Pickel, Simone U. A1 - Westerling, Katherine M. A1 - Klein, Teresa A1 - Holm, Thorge A1 - Sauer, Markus A1 - Sukhorukov, Vladimir L. T1 - Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy JF - PLoS One N2 - Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. KW - electrolytes KW - isotonic KW - membrane proteins KW - cell membranes KW - hypotonic KW - hypotonic solutions KW - tonicity KW - permeability Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126408 VL - 10 IS - 3 ER - TY - JOUR A1 - Kämmerer, Ulrike A1 - Gires, Olivier A1 - Pfetzer, Nadja A1 - Wiegering, Armin A1 - Klement, Rainer Johannes A1 - Otto, Christoph T1 - TKTL1 expression in human malign and benign cell lines JF - BMC Cancer N2 - Background Overexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression. Methods 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the “Warburg effect”. Results Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively. Conclusion Using RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy. KW - RT-qPCT KW - immunohistochemistry KW - TKTL1 KW - cancer cell lines Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126397 VL - 15 IS - 2 ER - TY - THES A1 - Rothe, Dietrich Gernot T1 - Spin Transport in Topological Insulators and Geometrical Spin Control T1 - Spintransport in topologischen Isolatoren und geometrische Spinkontrolle N2 - In the field of spintronics, spin manipulation and spin transport are the main principles that need to be implemented. The main focus of this thesis is to analyse semiconductor systems where high fidelity in these principles can be achieved. To this end, we use numerical methods for precise results, supplemented by simpler analytical models for interpretation. The material system of 2D topological insulators, HgTe/CdTe quantum wells, is interesting not only because it provides a topologically distinct phase of matter, physically manifested in its protected transport properties, but also since within this system, ballistic transport of high quality can be realized, with Rashba spin-orbit coupling and electron densities that are tunable by electrical gating. Extending the Bernvevig-Hughes-Zhang model for 2D topological insulators, we derive an effective four-band model including Rashba spin-orbit terms due to an applied potential that breaks the spatial inversion symmetry of the quantum well. Spin transport in this system shows interesting physics because the effects of Rashba spin-orbit terms and the intrinsic Dirac-like spin-orbit terms compete. We show that the resulting spin Hall signal can be dominated by the effect of Rashba spin-orbit coupling. Based on spin splitting due to the latter, we propose a beam splitter setup for all-electrical generation and detection of spin currents. Its working principle is similar to optical birefringence. In this setup, we analyse spin current and spin polarization signals of different spin vector components and show that large in-plane spin polarization of the current can be obtained. Since spin is not a conserved quantity of the model, we first analyse the transport of helicity, a conserved quantity even in presence of Rashba spin-orbit terms. The polarization defined in terms of helicity is related to in-plane polarization of the physical spin. Further, we analyse thermoelectric transport in a setup showing the spin Hall effect. Due to spin-orbit coupling, an applied temperature gradient generates a transverse spin current, i.e. a spin Nernst effect, which is related to the spin Hall effect by a Mott-like relation. In the metallic energy regimes, the signals are qualitatively explained by simple analytic models. In the insulating regime, we observe a spin Nernst signal that originates from the finite-size induced overlap of edge states. In the part on methods, we discuss two complementary methods for construction of effective semiconductor models, the envelope function theory and the method of invariants. Further, we present elements of transport theory, with some emphasis on spin-dependent signals. We show the connections of the adiabatic theorem of quantum mechanics to the semiclassical theory of electronic transport and to the characterization of topological phases. Further, as application of the adiabatic theorem to a control problem, we show that universal control of a single spin in a heavy-hole quantum dot is experimentally realizable without breaking time reversal invariance, but using a quadrupole field which is adiabatically changed as control knob. For experimental realization, we propose a GaAs/GaAlAs quantum well system. N2 - Manipulation und Transport von elektronischen Spins sind die wesentlichen Elemente, die für das Funktionieren einer zukünftigen Spin-basierten Elektronik implementiert werden müssen. Diese Arbeit befasst sich schwerpunktmäßig mit Halbleitersystemen, in denen diese Prinzipien mit hoher Zuverlässigkeit möglich sind. Dazu wurden sowohl numerische als auch analytische Berechnungsmethoden genutzt, letztere oft in der Form einfacher Modelle zur Interpretation der numerischen Ergebnisse. Das Halbleitersystem von HgTe/CdTe Quantentrögen, auch bekannt als zweidimensionaler topologischer Isolator, ist sowohl von fundamentalem wissenschaftlichen Interesse, da die topologisch nichttriviale Energiestruktur zu einem Schutz von Transporteigenschaften führt, als auch von angewandterem Interesse, da aus diesem Materialsystem Proben gefertigt werden können, die ballistischen Transport hoher Qualität zeigen, und da zudem die Rashba Spin-Bahn-Kopplung sowie die elektronische Dichte durch elektrische Steuerelektroden einstellbar sind. Wir erweitern das Bernevig-Hughes-Zhang Modell für zweidimensionale topologische Isolatoren, indem wir ein Vierbandmodell herleiten, das Rashba Spin-Bahn-Kopplungsterme enthält, die durch ein äußeres elektrisches Feld hervorgerufen werden, wenn dieses die Inversionssymmetrie des Quantentroges bricht. Der Transport von Spins in diesem System zeigt ein interessantes Wechselspiel zwischen Effekten der Rashba Spin-Bahn-Kopplung und Effekten der intrinsischen Dirac-artigen Spin-Bahn-Kopplung. Dabei dominiert die Rashba Spin-Bahn-Kopplung das Verhalten des Spin-Hall-Signals. Basierend auf der einstellbaren Rashba Spin-Bahn-Kopplung, schlagen wir einen spinselektiven Polarisator zur rein elektrischen Erzeugung und Detektion von Spinströmen vor. Das Funktionsprinzip ist vergleichbar mit demjenigen eines doppelbrechenden Kristalls. In der vorgeschlagenen Anordnung untersuchen wir die Spinpolarisation in verschieden Spinvektorkomponenten und zeigen die Realisierbarkeit von hoher Spinpolarisation in der Ebene. Da der Spin keine Erhaltungsgröße des Halbleitermodells ist, analysieren wir in einem ersten Schritt den Transport von der Erhaltungsgröße Helizität, und setzen die erzeugte Polarisation dann in Bezug zur Spinpolarisation. Des Weiteren analysieren wir thermoelektrischen Transport in einem System, das auch den Spin-Hall-Effekt zeigt. Aufgrund von Spin-Bahn-Kopplung kommt es beim Anlegen eines Temperaturgradienten zu einem transversalen Spinstrom, genannt Spin-Nernst-Effekt. Dieser ist über eine Mott-artige Beziehung mit dem Spin-Hall-Effekt verknüpft. Im metallischen Energiebereich können wir die Signale qualitativ anhand von einfachen analytischen Modellen verstehen. Im Energiebereich der elektronischen Bandlücke finden wir ein Spin-Nernst-Signal, das vom räumlichen Überlapp der Randzustände herrührt, die an gegenüberliegenden Kanten des Halbleitersystems lokalisiert sind. Im methodischen ersten Teil dieser Arbeit diskutieren wir zwei komplementäre Methoden zur Konstruktion von effektiven Halbleitermodellen, nämlich die Methode der Envelopefunktionen und die Methode der Invarianten. Außerdem präsentieren wir Elemente der elektronischen Transporttheorie, unter besonderer Beachtung von Spintransport. Wir diskutieren die Zusammenhänge zwischen dem adiabatischen Theorem in der Quantenmechanik einerseits, und semiklassischer Transporttheorie sowie der topologischen Klassifizierung von Phasen andererseits. Als weitere Anwendung des adiabatischen Theorems zeigen wir, wie universelle Kontrolle eines einzelnen Spins in einem Quantenpunkt aus Schwerlochzuständen experimentell realisiert werden kann, ohne dabei die Zeitumkehrsymmetrie zu brechen. Zu diesem Zweck führen wir ein elektrisches Quadrupolfeld ein, dessen Konfiguration als adiabatischer Kontrollparameter dient. Wir schlagen die experimentelle Realisierung des Quantenpunktes in einem QaAs/GaAlAs Quantentrogsystem vor. KW - Elektronischer Transport KW - Topologischer Isolator KW - Spintronik KW - topological insulators KW - topologische Isolatoren KW - mesoskopische Physik KW - mesoscopic physics KW - Halbleiterphysik KW - Thermoelektrizität KW - Quanteninformation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125628 ER - TY - THES A1 - Wiemer, Julian T1 - Maintaining factors of fear-relevant illusory correlations T1 - Aufrechterhaltende Faktoren von angstbezogenen Zusammenhangsverschätzungen N2 - Biased cognitive processes are very likely involved in the maintenance of fears and anxiety. One of such cognitive processes is the perceived relationship between fear-relevant stimuli and aversive consequences. If this relationship is perceived although objective contingencies have been random, it is called an (a posteriori) illusory correlation. If this relationship is overestimated before objective contingencies are experienced, it is called an (a priori) expectancy bias. Previous investigations showed that fear-relevant illusory correlations exist, but very few is known about how and why this cognitive bias develops. In the present dissertation thesis, a model is proposed based on a review of the literature on fear-relevant illusory correlations. This model describes how psychological factors might have an influence on fear and illusory correlations. Several critical implications of the model were tested in four experiments. Experiment 1 tested the hypothesis that people do not only overestimate the proportion of aversive consequences (startle sounds) following emotionally negative stimuli (pictures of mutilations) relative to neutral stimuli (pictures of household objects), but also following highly arousing positive stimuli (pictures of erotic scenes), because arousal might be an important determinant of illusory correlations. The result was a significant expectancy bias for negative stimuli and a much smaller expectancy bias for positive stimuli. Unexpectedly, expectancy bias was restricted to women. An a posteriori illusory correlation was not found overall, but only in those participants who perceived the aversive consequences following negative stimuli as particularly aversive. Experiment 2 tested the same hypothesis as experiment 1 using a paradigm that evoked distinct basic emotions (pictures inducing fear, anger, disgust or happiness). Only negative emotions resulted in illusory correlations with aversive outcomes (startle sounds), especially the emotions of fear and disgust. As in experiment 1, the extent of these illusory correlations was correlated with the perceived aversiveness of aversive outcomes. Moreover, only women overestimated the proportion of aversive outcomes during pictures that evoked fear, anger or disgust. Experiment 3 used functional Magnetic Resonance Imaging (fMRI) to measure biased brain activity in female spider phobics during an illusory correlation paradigm. Both spider phobics and healthy controls expected more aversive outcomes (painful electrical shocks) following pictures of spiders than following neutral control stimuli (pictures of mushrooms). Spider phobics but not healthy controls overestimated the proportion of aversive outcomes following pictures of spiders in a trial-by-trial memory task. This a posteriori illusory correlation was correlated with enhanced shock aversiveness and activity in primary sensory-motor cortex in phobic participants. Moreover, spider phobics’ brain activity in the left dorsolateral prefrontal cortex was elevated in response to spider images. This activity also predicted the extent of the illusory correlation, which supports the theory that executive and attentional resources play an important role in the maintenance of illusory correlations. Experiment 4 tested the hypothesis that the enhanced aversiveness of some outcomes would be sufficient to causally induce an illusory correlation. Neutral images (colored geometric figures) were paired with differently aversive outcomes (three startle sounds varying in intensity). Participants developed an illusory correlation between those images, which predicted the most aversive sound and this sound, which means that this association was overestimated relative to the other associations. The extent of the illusory correlation was positively correlated with participants’ self-reported anxiety. The results imply that the previously found relationship between illusory correlations and outcome aversiveness might reflect a causal impact of outcome aversiveness or salience on illusory correlations. In sum, the conducted experiments indicate that illusory correlations between fear-relevant stimuli and aversive consequences might persist – among other factors - because of an enhanced aversiveness or salience of aversive consequences following feared stimuli. This assumption is based on correlational findings, a neural measure of outcome perception and a causal influence of outcome aversiveness on illusory correlations. Implications of these findings were integrated into a model of fear-relevant illusory correlations and potential implications are discussed. Future investigations should further elucidate the role of executive functions and gender effects. Moreover, the trial-by-trial assessment of illusory correlations is recommended to increase reliability of the concept. From a clinical perspective, the down-regulation of aversive experiences and the allocation of attention to non-aversive experiences might help to cure anxiety and cognitive bias. N2 - Verzerrte kognitive Prozesse sind sehr wahrscheinlich an der Aufrechterhaltung von Furcht und Angst beteiligt. Ein solcher kognitiver Prozess ist der wahrgenommene Zusammenhang zwischen Reizen, vor denen bereits Angst besteht und unangenehmen Konsequenzen. Wenn so ein Zusammenhang wahrgenommen wird, obwohl die objektiven Kontingenzen zufällig sind, spricht man von einer illusorischen Korrelation (a posteriori). Wenn so ein Zusammenhang überschätzt wird, bevor objektive Kontingenzen erlebt werden, spricht man von einer Erwartungsverzerrung (a priori). Frühere Untersuchungen zeigten, dass angstrelevante illusorische Korrelationen existieren, aber bisher ist nur wenig darüber bekannt, wie und warum diese entstehen. In der vorliegenden Dissertation wird ein Modell vorgeschlagen, dass auf bisherigen Erkenntnissen zu angstrelevanten illusorischen Korrelationen beruht. Das Modell beschreibt, welche psychologischen Faktoren die Entstehung von Angst und illusorischen Korrelationen begünstigen könnten. Mehrere Implikationen dieses Modells wurden in vier Experimenten getestet. Experiment 1 überprüfte die Hypothese, dass Menschen nicht nur die Häufigkeit unangenehmer Konsequenzen (Schreckgeräusche) nach emotional negativen Reizen (Bilder von Verletzungen) überschätzen, verglichen mit neutralen Reizen (Bilder von Haushaltsgegenständen), sondern auch nach sehr aufregenden positiven Reizen (Bilder von erotischen Szenen), weil die allgemeine Erregung einen Einfluss auf illusorische Korrelationen haben sollte. Das Ergebnis war eine signifikante Erwartungsverzerrung bei negativen Reizen und eine sehr viel kleinere Erwartungsverzerrung bei positiven Reizen. Unerwarteter Weise waren Erwartungsverzerrungen auf Frauen beschränkt. Eine illusorische Korrelation (a posteriori) konnte im Allgemeinen nicht festgestellt werden, sondern lediglich bei solchen Probanden, die die unangenehmen Konsequenzen nach negativen Reizen als besonders unangenehm empfanden. Experiment 2 überprüfte die gleiche Hypothese wie Experiment 1 anhand einer Versuchsanordnung, die verschiedene Basisemotionen hervorrufen sollte (durch Bilder, die Angst, Ärger, Ekel oder Freude induzierten). Nur negative Emotionen führten zu illusorischen Korrelationen (a posteriori) mit unangenehmen Ereignissen (Schreckgeräusche), insbesondere die Emotionen Angst und Ekel. Wie auch in Experiment 1 korrelierte das Ausmaß der illusorischen Korrelation mit der wahrgenommenen Unangenehmheit der unangenehmen Ereignisse bei der entsprechenden Bildkategorie. Darüber hinaus überschätzten nur Frauen den Anteil negativer Ereignisse bei Bildern, die Angst, Ekel, oder Ärger hervorriefen. Experiment 3 verwendete funktionelle Magnetresonanztomografie (fMRT), um verzerrte Gehirnaktivität bei Spinnenphobikerinnen während eines Versuchs zu illusorischen Korrelationen zu messen. Sowohl Spinnenphobikerinnen als auch gesunde Kontrollprobandinnen erwarteten mehr unangenehme Konsequenzen (schmerzhafte elektrische Reize) bei Bildern von Spinnen als bei neutralen Kontrollreizen (Bilder von Pilzen). Spinnenphobikerinnen, aber nicht gesunde Kontrollprobandinnen überschätzten jedoch im Nachhinein den Anteil unangenehmer Konsequenzen bei Bildern von Spinnen in einer Trial-by-Trial Gedächtnisaufgabe. Diese illusorische Korrelation (a posteriori) korrelierte mit der erhöhten Unangenehmheit der elektrischen Reize und mit Aktivierung im primären senso-motorischen Kortex der phobischen Teilnehmerinnen. Darüber hinaus wiesen Spinnenphobikerinnen in Reaktion auf die Bilder von Spinnen eine erhöhte Aktivierung im linken dorsolateralen präfrontalen Kortex auf. Diese Aktivität sagte auch das Ausmaß der illusorischen Korrelation vorher, was die These unterstützt, dass exekutive und Aufmerksamkeitsressourcen eine wichtige Rolle in der Aufrechterhaltung illusorischer Korrelationen spielen. Experiment 4 überprüfte die Hypothese, dass die erhöhte Unangenehmheit mancher Konsequenzen hinreichend sein würde, um kausal eine illusorische Korrelation hervorzurufen. Neutrale Bilder (geometrische Figuren in drei verschiedenen Farben) wurden mit unterschiedlich unangenehmen Konsequenzen gepaart (Schreckgeräusche in drei verschiedenen Intensitäten). Bei den Probanden entwickelte sich eine illusorische Korrelation mit der Farbe, die das unangenehmste Geräusch voraussagte und diesem Geräusch, das heißt, der Zusammenhang wurde im Vergleich zu den anderen Zusammenhängen überschätzt. Das Ausmaß der illusorischen Korrelation korrelierte positiv mit der Ängstlichkeit der Teilnehmer. Die Ergebnisse legen nahe, dass der bisher gefundene Zusammenhang zwischen illusorischen Korrelationen und der Unangenehmheit der unangenehmen Konsequenzen auf einen kausalen Einfluss der Unangenehmheit oder Salienz der Konsequenzen auf illusorische Korrelationen zurückgehen könnte. Zusammengefasst zeigten die durchgeführten Experimente, dass illusorische Korrelationen zwischen angstrelevanten Reizen und unangenehmen Konsequenzen – neben anderen Einflussfaktoren – aufgrund einer erhöhten Unangenehmheit oder Salienz unangenehmer Konsequenzen bei gefürchteten Reizen bestehen könnten. Diese Annahme basiert auf korrelativen Ergebnissen, einem neuralen Maß der Konsequenzverarbeitung und dem gefundenen kausalen Einfluss der Unangenehmheit unangenehmer Konsequenzen auf illusorische Korrelationen. Implikationen dieser Befunde werden in ein Modell zu angstrelevanten illusorischen Korrelationen integriert und diskutiert. Zukünftige Studien sollten die Rolle exekutiver Funktionen und Geschlechtsunterschiede genauer untersuchen. Es empfiehlt sich dabei, illusorische Korrelationen Trial-by-Trial zu erfassen, um die Reliabilität des Konzepts zu erhöhen. Aus klinischer Sicht könnten die Beruhigung unangenehmer Erfahrungen und die Aufmerksamkeitsallokation auf nicht-unangenehme Erfahrungen helfen, Ängste und kognitive Verzerrungen zu vermindern. KW - Verzerrte Kognition KW - Angststörung KW - Verzerrte Kognition KW - Illusorische Korrelation KW - Gefahrenlernen KW - Funktionelle Kernspintomografie KW - cognitive bias KW - illusory correlation KW - threat learning KW - functional magnetic resonance imaging KW - Assoziatives Gedächtnis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116960 ER - TY - THES A1 - Stumpf, Anette D. T1 - Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors T1 - Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren N2 - Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements. For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future. N2 - Adenosin Rezeptoren, die zur Gruppe der Rhodopsin-ähnlichen G Protein-gekoppelten Rezeptoren (GPCRs) gehören, sind in eine Vielzahl regulatorischer Prozesse eingebunden und weit im Körper verbreitet. Das macht sie zu einer interessanten Zielstruktur für Arzneistoffe. Das Wissen über die Struktur der Adenosin Rezeptoren ist jedoch noch begrenzt. Ein großer Fortschritt zu mehr strukturellem Wissen war die Entwicklung der ersten Kristallstruktur des Adenosin A2A Rezeptors im Jahr 2008. Mit der Kristallstruktur wurden die Aminosäuren bekannt, die die Ligandenbindetasche dieses Rezeptors formen. Zudem gab die Kristallstruktur Einblick in den Endpunkt der dynamischen Rezeptorbewegung nach Ligandenbindung. Im Rahmen der hier vorgestellten Arbeit wurden zwei Mitglieder der Adenosin Rezeptor Familie, der Adenosin A1 Rezeptor und der Adenosin A2A Rezeptor (A1R, A2AR), genauer untersucht. Der A1R wurde auf Basis des vor kurzem veröffentlichten A2AR entwickelt. Die Rezeptoren wurden mit Fluorophoren versehen, zum einen mit dem cyan fluoreszierenden Protein (CFP) am C-Terminus des Rezeptors und zum anderen mit der Bindesequenz des kleinen Fluorophors "Fluorescein Arsenical Hairpin binder" (FlAsH) in der dritten intrazellulären Schleife des Rezeptors. Die daraus resultierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP wurden mit Hilfe des Fluoreszenz Resonanz Energie Transfers (FRET) in lebenden Zellen erforscht. FRET Messungen ermöglichen es, eine Änderung der Distanz zwischen den beiden Fluorophoren und damit Rezeptorbewegungen zu untersuchen. Um A1R und A2AR bezüglich dynamischer Rezeptorbewegungen nach Ligandenbindung vergleichen zu können, wurden die fluoreszierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP mit verschiedenen Liganden umspült. Die Ergebnisse der FRET Messungen bezüglich ihrer Höhe des FRET Ratio wurden verglichen, welche mit der Konformationsänderung des Rezeptors nach Ligandenbindung zusammenhängt. Neben der unterschiedlichen Richtung des FRET Ratio nach Ligandenbindung am A1R und A2AR Sensor waren Unterschiede bezüglich der Signalhöhe und der Bindungs- und Dissoziationskinetiken feststellbar, wenn die verschiedenen Liganden miteinander verglichen wurden. Unterschiede zwischen den Adenosin Rezeptor Subtypen waren speziell für den A1R subtypselektiven Agonist CPA und für den A2AR subtypselektiven Agonist CGS 21680 feststellbar. Einen weiteren Punkt in diesem Projekt stellte die Erforschung des Einflusses, den einzelne Aminosäuren im fluoreszierenden A1R Sensor auf den Prozess der Ligandenbindung haben, dar. Die Position der Aminosäuren wurde der Kristallstruktur des A2AR entnommen und entsprechende Aminosäuren im A1R mutiert. Die konfokalmikroskopische Analyse des A1R Sensors und seiner Mutanten ergab, dass einige Aminosäuren direkt an der zellulären Expression des Rezeptors beteiligt waren. Wurden diese Aminosäuren mutiert, wurde der Rezeptor nicht in der Plasmamembran der Zellen exprimiert. Einige Aminosäuren die untersucht wurden,hatten einen generellen Einfluss auf die Bindung der Liganden, andere Aminosäuren hatten mehr Einfluss auf die Bindung bestimmter struktureller Gruppen der untersuchten Liganden. In einem weiteren Schritt wurden A1R und A2AR am N-terminalen Rezeptorende mit SNAP oder CLIP versehen, was eine Markierung der Rezeptoren mit einer Vielzahl an Fluorophoren erlaubt. Mit Hilfe dieser Technik konnte die Verteilung der Rezeptoren in der Zelle mit konfokaler Mikroskopie untersucht werden. Des Weiteren wurde die Bindung von fluoreszierenden Adenosin Rezeptor Liganden und deren Verdrängung mit einem nicht-fluoreszierenden Adenosin Rezeptor Antagonist erforscht. Am Ende des Projekts wurden die zuvor beschriebenen fluoreszierenden Rezeptorsensoren zu dreifach fluorophormarkierten Rezeptorsensoren kombiniert, was zu den Sensoren SNAP A1 Fl3 CFP und SNAP A2A Fl3 CFP führte. Beide Rezeptorsensoren waren funktionell bezüglich FRET Experimenten und der Expression in der Plasmamembran der Zellen. In Zukunft können mit dieser Methode gleichzeitig die Bindung von fluoreszierenden Liganden am SNAP-markierten Rezeptor, so wie die Rezeptorbewegung beobachtet werden, die durch eine Distanzänderung zwischen CFP und FlAsH angezeigt wird. Das kann zu einem besseren Verständnis der Rezeptorfunktion und der dynamischen Rezeptorbewegung nach Ligandenbindung führen, die in Zukunft zur Entwicklung spezifischerer Wirkstoffe am A1R und A2AR beitragen könnte. KW - Adenosinrezeptor KW - Fluoreszenz-Resonanz-Energie-Transfer KW - G-Protein gekoppelte Rezeptoren KW - Adenosine receptors Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125469 ER - TY - JOUR A1 - Brill, Martin F. A1 - Meyer, Anneke A1 - Roessler, Wolfgang T1 - It takes two—coincidence coding within the dual olfactory pathway of the honeybee JF - Frontiers in Physiology N2 - To rapidly process biologically relevant stimuli, sensory systems have developed a broad variety of coding mechanisms like parallel processing and coincidence detection. Parallel processing (e.g., in the visual system), increases both computational capacity and processing speed by simultaneously coding different aspects of the same stimulus. Coincidence detection is an efficient way to integrate information from different sources. Coincidence has been shown to promote associative learning and memory or stimulus feature detection (e.g., in auditory delay lines). Within the dual olfactory pathway of the honeybee both of these mechanisms might be implemented by uniglomerular projection neurons (PNs) that transfer information from the primary olfactory centers, the antennal lobe (AL), to a multimodal integration center, the mushroom body (MB). PNs from anatomically distinct tracts respond to the same stimulus space, but have different physiological properties, characteristics that are prerequisites for parallel processing of different stimulus aspects. However, the PN pathways also display mirror-imaged like anatomical trajectories that resemble neuronal coincidence detectors as known from auditory delay lines. To investigate temporal processing of olfactory information, we recorded PN odor responses simultaneously from both tracts and measured coincident activity of PNs within and between tracts. Our results show that coincidence levels are different within each of the two tracts. Coincidence also occurs between tracts, but to a minor extent compared to coincidence within tracts. Taken together our findings support the relevance of spike timing in coding of olfactory information (temporal code). KW - olfaction KW - mushroom body KW - insect KW - coincidence KW - multi-electrode-recording KW - antennal lobe Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126179 VL - 6 IS - 208 ER - TY - JOUR A1 - Fritz, Melanie A1 - Vanselow, Jens A1 - Sauer, Nadja A1 - Lamer, Stephanie A1 - Goos, Carina A1 - Siegel, T. Nicolai A1 - Subota, Ines A1 - Schlosser, Andreas A1 - Carrington, Mark A1 - Kramer, Susanne T1 - Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve JF - Nucleic Acids Research N2 - RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126180 ER - TY - JOUR A1 - Klammert, Uwe A1 - Müller, Thomas D. A1 - Hellmann, Tina V. A1 - Wuerzler, Kristian K. A1 - Kotzsch, Alexander A1 - Schliermann, Anna A1 - Schmitz, Werner A1 - Kuebler, Alexander C. A1 - Sebald, Walter A1 - Nickel, Joachim T1 - GDF-5 can act as a context-dependent BMP-2 antagonist JF - BMC Biology N2 - Background Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. Results Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites. Conclusions Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs. KW - growth and differentiation factor 5 KW - ligand-receptor complex KW - crystal structure KW - antagonist Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125550 VL - 13 IS - 77 ER - TY - JOUR A1 - Reuss, Heiko A1 - Pohl, Carsten A1 - Kiesel, Andrea A1 - Kunde, Wilfried T1 - Instructed illiteracy reveals expertise-effects on unconscious processing JF - Frontiers in Psychology N2 - We used a new methodological approach to investigate whether top-down influences like expertise determine the extent of unconscious processing. This approach does not rely on preexisting differences between experts and novices, but instructs essentially the same task in a way that either addresses a domain of expertise or not. Participants either were instructed to perform a lexical decision task (expert task) or to respond to a combination of single features of word and non-word stimuli (novel task). The stimuli and importantly also the mapping of responses to those stimuli, however, were exactly the same in both groups. We analyzed congruency effects of masked primes depending on the instructed task. Participants performing the expert task responded faster and less error prone when the prime was response congruent rather than incongruent. This effect was significantly reduced in the novel task, and even reversed when excluding identical prime-target pairs. This indicates that the primes in the novel task had an effect on a perceptual level, but were not able to impact on response activation. Overall, these results demonstrate an expertise-based top-down modulation of unconscious processing that cannot be explained by confounds that are otherwise inherent in comparisons between novices and experts. KW - top-down control KW - expertise KW - masked priming KW - unconscious processing KW - lexical decision task Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125332 VL - 6 IS - 239 ER - TY - JOUR A1 - Boivin, Valérie A1 - Beyersdorf, Niklas A1 - Palm, Dieter A1 - Nikolaev, Viacheslav O. A1 - Schlipp, Angela A1 - Müller, Justus A1 - Schmidt, Doris A1 - Kocoski, Vladimir A1 - Kerkau, Thomas A1 - Hünig, Thomas A1 - Ertl, Georg A1 - Lohse, Martin J. A1 - Jahns, Roland T1 - Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model JF - PLoS One N2 - Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195–225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure. KW - memory B cells KW - antibodies KW - T cells KW - B cells KW - heart KW - heart failure KW - kidneys KW - enzyme-linked immunoassays Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126028 VL - 10 IS - 2 ER - TY - JOUR A1 - Vaman V. S., Anjana A1 - Poppe, Heiko A1 - Houben, Roland A1 - Grunewald, Thomas G. P. A1 - Goebeler, Matthias A1 - Butt, Elke T1 - LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release, but Not in Melanoma Progression JF - PLoS One N2 - The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release. KW - cell membranes KW - vesicles KW - melanoma cells KW - melanomas KW - melanocytes KW - membrane proteins KW - melanin KW - cell staining Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125994 VL - 10 IS - 6 ER - TY - JOUR A1 - Eder, Andreas B. A1 - Deutsch, Roland T1 - Watch the target! Effects in the affective misattribution procedure become weaker (but not eliminated) when participants are motivated to provide accurate responses to the target JF - Frontiers in Psychology N2 - Previous research showed that priming effects in the affective misattribution procedure (AMP) are unaffected by direct warnings to avoid an influence of the primes. The present research examined whether a priming influence is diminished by task procedures that encourage accurate judgments of the targets. Participants were motivated to categorize the affective meaning of nonsense targets accurately by being made to believe that a true word was presented in each trial and by providing feedback on (allegedly) incorrect responses. This condition produced robust priming effects. Priming was however reduced and less reliable relative to more typical AMP conditions in which participants guessed the meaning of openly presented nonsense targets. Affective judgments of nonsense targets were not affected by advance knowledge of the response mapping during the priming phase, which argues against a response-priming explanation of AMP effects. These findings show that affective primes influence evaluative judgments even in conditions in which the motivation to provide accurate responses is high and a priming of motor responses is not possible. Priming effects were however weaker with high accuracy motivation, suggesting that a focus on accurate judgments is an effective strategy to control for an unwanted priming influence in the AMP. KW - implicit attitude measurement KW - accuracy motivation KW - affect misattribution procedure KW - response priming Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125982 VL - 6 ER - TY - JOUR A1 - Kleih, Sonja C. A1 - Herweg, Andreas A1 - Kaufmann, Tobias A1 - Staiger-Sälzer, Pit A1 - Gerstner, Natascha A1 - Kübler, Andrea T1 - The WIN-speller: a new intuitive auditory brain-computer interface spelling application JF - Frontiers in Neuroscience N2 - The objective of this study was to test the usability of a new auditory Brain-Computer Interface (BCI) application for communication. We introduce a word based, intuitive auditory spelling paradigm the WIN-speller. In the WIN-speller letters are grouped by words, such as the word KLANG representing the letters A, G, K, L, and N. Thereby, the decoding step between perceiving a code and translating it to the stimuli it represents becomes superfluous. We tested 11 healthy volunteers and four end-users with motor impairment in the copy spelling mode. Spelling was successful with an average accuracy of 84% in the healthy sample. Three of the end-users communicated with average accuracies of 80% or higher while one user was not able to communicate reliably. Even though further evaluation is required, the WIN-speller represents a potential alternative for BCI based communication in end-users. KW - Brain-Computer Interface (BCI) KW - communication KW - P300 KW - motor-impaired end-user KW - auditory Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125972 VL - 9 ER - TY - JOUR A1 - Güder, Gülmisal A1 - Brenner, Susanne A1 - Störk, Stefan A1 - Held, Matthias A1 - Broekhuizen, Berna D. L. A1 - Lammers, Jan-Willem J. A1 - Hoes, Arno W. A1 - Rutten, Frans H. T1 - Diagnostic and prognostic utility of mid-expiratory flow rate in older community-dwelling persons with respiratory symptoms, but without chronic obstructive pulmonary disease JF - BMC Pulmonary Medicine N2 - Background The maximal expiratory flow at 50 % of the forced vital capacity (MEF50) is the flow where half of forced vital capacity (FVC) remains to be exhaled. A reduced MEF50 has been suggested as a surrogate marker of small airways disease. The diagnostic and prognostic utility of this easy to assess spirometric variable in persons with respiratory symptoms, but without COPD is unclear. Methods We used data from the UHFO-COPD cohort in which 405 community-dwelling persons aged 65 years or over, and a general practitioner’s diagnosis of chronic obstructive pulmonary disease (COPD) underwent pulmonary function testing and echocardiography. In total 161 patients had no COPD according to the spirometric GOLD criteria. We considered MEF50 as reduced if < 60 % of predicted. Results Of the 161 patients without COPD (mean age 72 ± 5.7 years; 35 % male; follow-up 4.5 ± 1.1 years), 61 (37.9 %) had a reduced MEF50. They were older, had more pack-years of smoking, more respiratory symptoms, and used more frequently inhaled medication than the remaining 100 subjects. A reduced MEF50 was nearly twice as often associated with newly detected heart failure (HF) at assessment (29.5 % vs. 15.6 %, p = 0.045). In age-and sex-adjusted Cox regression analysis, a reduced MEF50 was significantly associated with episodes of acute bronchitis (hazard ratio 2.54 95 % confidence interval (1.26; 5.13) P = 0.009), and in trend with pneumonia (2.14 (0.98; 4.69) P = 0.06) and hospitalizations for pulmonary reasons (2.28 (0.93; 5.62) P = 0.07). Conclusions In older community-dwelling persons with pulmonary symptoms but without COPD, a reduced MEF50 may help to uncover unrecognized HF, and identify those at a higher risk for episodes of acute bronchitis, pneumonia and hospitalizations for pulmonary reasons. Echocardiography and close follow-up should be considered in these patients. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125547 VL - 15 IS - 83 ER - TY - JOUR A1 - Wiegering, V. A1 - Schlegel, P. G. A1 - Winkler, B. A1 - Lazarus, M. A1 - Wirth, C. A1 - Ernestus, K. A1 - Walles, T. A1 - Liese, J. T1 - Persisting Cough as the Single Presenting Symptom of an Intrathoracic Tumor in a Nine-Month-Old Child with Adenovirus Airway Infection JF - Journal of Case Reports and Studies N2 - We report on a nine-month-old girl who presented with persisting cough, and diminished ventilation of the left hemithorax. Viral pneumonia was suspected after Adenovirus detection by PCR, but chest X-rays showed a persistent shadowing of the left hemithorax and persistent coughing despite clinical improvement. Because of the discrepancy between clinical and radiological signs further investigations by ultrasound and CT scan were performed, which visualized an intrathroracic tumor. Histopathology confirmed diagnosis of a teratoma. This case highlights the need for careful evaluation by the treating physicians. If the chest X-ray provides a discrepancy to the clinical findings or persistent pathologies exist, differential diagnosis should be discussed and further diagnostics be performed. KW - tumor KW - children Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125536 VL - 3 IS - 2 ER - TY - JOUR A1 - Falibene, Agustina A1 - Roces, Flavio A1 - Rössler, Wolfgang T1 - Long-term avoidance memory formation is associated with a transient increase in mushroom body synaptic complexes in leaf-cutting ants JF - Frontiers in Behavioral Neuroscience N2 - Long-term behavioral changes related to learning and experience have been shown to be associated with structural remodeling in the brain. Leaf-cutting ants learn to avoid previously preferred plants after they have proved harmful for their symbiotic fungus, a process that involves long-term olfactory memory. We studied the dynamics of brain microarchitectural changes after long-term olfactory memory formation following avoidance learning in Acromyrmex ambiguus. After performing experiments to control for possible neuronal changes related to age and body size, we quantified synaptic complexes (microglomeruli, MG) in olfactory regions of the mushroom bodies (MBs) at different times after learning. Long-term avoidance memory formation was associated with a transient change in MG densities. Two days after learning, MG density was higher than before learning. At days 4 and 15 after learning—when ants still showed plant avoidance—MG densities had decreased to the initial state. The structural reorganization of MG triggered by long-term avoidance memory formation clearly differed from changes promoted by pure exposure to and collection of novel plants with distinct odors. Sensory exposure by the simultaneous collection of several, instead of one, non-harmful plant species resulted in a decrease in MG densities in the olfactory lip. We hypothesize that while sensory exposure leads to MG pruning in the MB olfactory lip, the formation of long-term avoidance memory involves an initial growth of new MG followed by subsequent pruning. KW - microglomeruli KW - olfaction KW - avoidance learning KW - leaf-cutting ants KW - acromyrmex ambiguus KW - synaptic plasticity KW - mushroom body Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125522 VL - 9 IS - 84 ER - TY - JOUR A1 - Wagner, Martin A1 - Ashby, Damien R. A1 - Kurtz, Caroline A1 - Alam, Ahsan A1 - Busbridge, Mark A1 - Raff, Ulrike A1 - Zimmermann, Josef A1 - Heuschmann, Peter U. A1 - Wanner, Christoph A1 - Schramm, Lothar T1 - Hepcidin-25 in diabetic chronic kidney disease is predictive for mortality and progression to end stage renal disease JF - PLoS One N2 - Background Anemia is common and is associated with impaired clinical outcomes in diabetic chronic kidney disease (CKD). It may be explained by reduced erythropoietin (EPO) synthesis, but recent data suggest that EPO-resistance and diminished iron availability due to inflammation contribute significantly. In this cohort study, we evaluated the impact of hepcidin-25—the key hormone of iron-metabolism—on clinical outcomes in diabetic patients with CKD along with endogenous EPO levels. Methods 249 diabetic patients with CKD of any stage, excluding end-stage renal disease (ESRD), were enrolled (2003–2005), if they were not on EPO-stimulating agent and iron therapy. Hepcidin-25 levels were measured by radioimmunoassay. The association of hepcidin-25 at baseline with clinical variables was investigated using linear regression models. All-cause mortality and a composite endpoint of CKD progression (ESRD or doubling of serum creatinine) were analyzed by Cox proportional hazards models. Results Patients (age 67 yrs, 53% male, GFR 51 ml/min, hemoglobin 131 g/L, EPO 13.5 U/L, hepcidin-25 62.0 ng/ml) were followed for a median time of 4.2 yrs. Forty-nine patients died (19.7%) and forty (16.1%) patients reached the composite endpoint. Elevated hepcidin levels were independently associated with higher ferritin-levels, lower EPO-levels and impaired kidney function (all p<0.05). Hepcidin was related to mortality, along with its interaction with EPO, older age, greater proteinuria and elevated CRP (all p<0.05). Hepcidin was also predictive for progression of CKD, aside from baseline GFR, proteinuria, low albumin- and hemoglobin-levels and a history of CVD (all p<0.05). Conclusions We found hepcidin-25 to be associated with EPO and impaired kidney function in diabetic CKD. Elevated hepcidin-25 and EPO-levels were independent predictors of mortality, while hepcidin-25 was also predictive for progression of CKD. Both hepcidin-25 and EPO may represent important prognostic factors of clinical outcome and have the potential to further define “high risk” populations in CKD. KW - diabetes mellitus KW - inflammation KW - type 2 diabetes KW - hemoglobin KW - chronic kidney disease KW - anemia KW - ferritin KW - proteinuria Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125514 VL - 10 IS - 4 ER - TY - JOUR A1 - Toepfer, Martin A1 - Corovic, Hamo A1 - Fette, Georg A1 - Klügl, Peter A1 - Störk, Stefan A1 - Puppe, Frank T1 - Fine-grained information extraction from German transthoracic echocardiography reports JF - BMC Medical Informatics and Decision Making N2 - Background Information extraction techniques that get structured representations out of unstructured data make a large amount of clinically relevant information about patients accessible for semantic applications. These methods typically rely on standardized terminologies that guide this process. Many languages and clinical domains, however, lack appropriate resources and tools, as well as evaluations of their applications, especially if detailed conceptualizations of the domain are required. For instance, German transthoracic echocardiography reports have not been targeted sufficiently before, despite of their importance for clinical trials. This work therefore aimed at development and evaluation of an information extraction component with a fine-grained terminology that enables to recognize almost all relevant information stated in German transthoracic echocardiography reports at the University Hospital of Würzburg. Methods A domain expert validated and iteratively refined an automatically inferred base terminology. The terminology was used by an ontology-driven information extraction system that outputs attribute value pairs. The final component has been mapped to the central elements of a standardized terminology, and it has been evaluated according to documents with different layouts. Results The final system achieved state-of-the-art precision (micro average.996) and recall (micro average.961) on 100 test documents that represent more than 90 % of all reports. In particular, principal aspects as defined in a standardized external terminology were recognized with f 1=.989 (micro average) and f 1=.963 (macro average). As a result of keyword matching and restraint concept extraction, the system obtained high precision also on unstructured or exceptionally short documents, and documents with uncommon layout. Conclusions The developed terminology and the proposed information extraction system allow to extract fine-grained information from German semi-structured transthoracic echocardiography reports with very high precision and high recall on the majority of documents at the University Hospital of Würzburg. Extracted results populate a clinical data warehouse which supports clinical research. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125509 VL - 15 IS - 91 ER - TY - THES A1 - Wegner, Julia T1 - Restoring tissue-like functionality in circulating CD8 T-cells: mechanistic studies and application in immunomonitoring of cancer patients T1 - Wiederherstellen einer gewebeartigen Funktionalität in humanen CD8 T-Zellen des Blutes: mechanistische Studien und Anwendung beim Immunomonitoring von Krebspatienten N2 - Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunologic research and for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However the large majority of human T-cells resides in tissues, especially in lymphatic organs, while only 1 % of the body’s T-cells circulate in the blood stream. Previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by leaving lymphoid organs such as lymph nodes (LNs) and entering the circulation. In this study, these findings were extended to CD8 T-cells. Thus, CD8 T-cell responses of the human tonsil show a significant drop in sensitivity to viral antigens if tissue-exit was simulated by keeping cells in dispersed culture at body temperature for two hours. Conversely, tissue-like functionality in blood-derived CD8 T-cells was restored by applying the simple and robust RESTORE protocol. Indeed, application of the RESTORE protocol, i.e. pre-culturing PBMCs for two days at a high cell density before initiation of antigenic stimulation, demonstrated that CD8 T-cell responses to a broad range of viral and to tumor-associated antigens are greatly underestimated, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 (WT1)-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. My studies on the mechanism of the RESTORE protocol show that HD preculture of PBMCs does not involve antigen-or cytokine-driven clonal expansion of T-cells. Moreover, the gain in antigen sensitivity cannot be explained by a decreased activity of regulatory T-cells during the preculture step. The increased antigen sensitivity of CD8 T-cells from HD precultures of PBMCs is associated with tonic T-cell receptor signaling as indicated by enhanced tyrosine phosphorylation of the CD3 ζ chains and the tyrosine kinase Lck, thereby preparing T-cells for full responses. The upregulation of genes involved in aerobic glycolysis in “restored” CD8 memory T-cells relative to fresh cells might be an essential requirement for increased T-cell functionality including the regulation of IFN-γ production. Taken together, the RESTORE protocol, which was initially described for the CD4 T-cell response to the antibody TGN1412 permits a more meaningful monitoring of CD8 T-cell responses to viral infections and tumors. Furthermore, when generating T-cell lines for adoptive T-cell therapy, the RESTORE protocol allows the generation of CD8 T-cell lines with an improved representation of clones responding to low antigen concentrations. N2 - Mononukleäre Zellen des peripheren Blutes (PBMCs: peripheral blood mononuclear cells) stellen die einzige routinemäßig zugängliche Quelle für humane Lymphozyten dar, welche für die immunologische Forschung und das „Immunomonitoring“ von T-Zellantworten gegen mikrobielle und Tumor-assoziierte Antigene verwendet werden. Jedoch befindet sich der Großteil der T-Zellen des Menschen in Geweben, insbesondere den lymphatischen Organen, wohingegen sich nur 1 % der T-Zellen im Blut aufhalten. Frühere Studien, die sowohl mit murinen als auch mit humanen Zellen durchgeführt wurden, zeigten, dass CD4 T-Zellen ihre Sensitivität gegenüber Antigenen zeitweise verlieren sobald zelluläre Kontakte unterbrochen werden. Dies erfolgt beispielsweise beim Verlassen der T-Zellen von Geweben und dem Eintreten in die Blutzirkulation. In dieser Arbeit wurden diese Beobachtungen auf CD8 T-Zellen ausgeweitet. So weisen humane tonsilläre CD8 T-Zellen eine signifikant niedrigere Sensitivität gegenüber viralen Antigenen auf, wenn diese in Dispersion bei Körpertemperatur für zwei Stunden gehalten werden, um das Verlassen von Geweben und somit den Verlust von zellulären Kontakten zu simulieren. Im Gegenzug konnte eine gewebeähnliche T-Zellfunktionalität bei Blutzellen durch Anwendung des RESTORE Protokolls wiederhergestellt werden. In der Tat zeigte die Anwendung des RESTORE Protokolls, welches eine Vorkultur von PBMCs für zwei Tage bei hoher Zelldichte vor antigenspezifischer T-Zellstimulation einschließt, dass CD8 T-Zellantworten gegen eine Vielzahl viraler und Tumor-assoziierter Antigene deutlich unterschätzt werden, wenn herkömmliche Stimulationsansätze verwendet werden. Teilweise können diese so gemessenen T-Zellantworten bei Verwendung herkömmliche Stimulationsansätze auch gar nicht nachgewiesen werden. Dieser Effekt war bei der Detektion von Wilms Tumor 1 (WT1)-spezifischen CD8 T-Zellantworten bei Leukämiepatienten nach allogener Stammzelltransplantation besonders deutlich zu beobachten. Meine mechanistischen Studien zeigten, dass die Vorkultur von PBMCs bei hoher Zelldichte selbst nicht zu einer Antigen- oder Zytokin-getriebenen T-Zell Expansion führt. Des Weiteren wurde gezeigt, dass der RESTORE Effekt durch den Zugewinn an CD8 T-Zellsensitivität erklärt werden kann und nicht auf eine verringerte CD8 T-Zellsuppression durch regulatorische T-Zellen während der Vorkultur zurückzuführen ist. Die erhöhte Antigensensitivität von vorkultivierten CD8 T-Zellen steht im Zusammenhang mit tonischer T-Zell Signalweiterleitung, welche anhand von erhöhter Tyrosin Phosphorylierung der CD3 ζ Ketten des T-Zell-Rezeptors und der Tyrosinkinase Lck nachgewiesen werden kann. Diese tonischen T-Zellsignale bereiten CD8 T-Zellen darauf vor, bereits auf kleine Mengen Antigen effektiv zu reagieren. Auch die Hochregulierung von Genen, welche der aeroben Glykolyse zuzuordnen sind in vorkultivierten CD8 Gedächtniszellen im Vergleich zu CD8 Gedächtniszellen, welche direkt aus dem Blut isoliert wurden, trägt zu einer erhöhten T-Zellfunktionalität bei, welche die Regulation der IFN-γ Produktion einschließt. Zusammenfassend lässt sich sagen, dass die Anwendung des RESTORE Protokolls, welches ursprünglich zum Nachweis von CD4 T-Zellantworten gegen den Antikörper TGN1412 entwickelt wurde, eine verlässliche Methode zum Nachweis von CD8 T-Zellantworten gegen virale Infektionen und Tumore darstellt. Des Weiteren kann das RESTORE Protokoll zur Generierung von T-Zelllinien in der adoptive T-Zelltherapie eingesetzt werden. Die Anwendung des Protokolls erlaubt das Generieren von Zelllinien, welche auch T-Zellklone beinhalten, die durch Immunantworten auf geringe Antigenkonzentrationen entstanden sind. KW - Antigen CD8 KW - CD8 T cell KW - antigen KW - CD8 T-Zelle KW - RESTORE protocol KW - sensitivity KW - T-Lymphozyt KW - Gewebe Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124177 ER - TY - JOUR A1 - Lapa, Constantin A1 - Linsenmann, Thomas A1 - Lückerath, Katharina A1 - Samnick, Samuel A1 - Herrmann, Ken A1 - Stoffer, Carolin A1 - Ernestus, Ralf-Ingo A1 - Buck, Andreas K. A1 - Löhr, Mario A1 - Monoranu, Camelia-Maria T1 - Tumor-Associated Macrophages in Glioblastoma Multiforme—A Suitable Target for Somatostatin Receptor-Based Imaging and Therapy? JF - PLoS One N2 - Background Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Tumor-associated macrophages (TAM) have been shown to promote malignant growth and to correlate with poor prognosis. [1,4,7,10-tetraazacyclododecane-NN′,N″,N′″-tetraacetic acid]-d-Phe1,Tyr3-octreotate (DOTATATE) labeled with Gallium-68 selectively binds to somatostatin receptor 2A (SSTR2A) which is specifically expressed and up-regulated in activated macrophages. On the other hand, the role of SSTR2A expression on the cell surface of glioma cells has not been fully elucidated yet. The aim of this study was to non-invasively assess SSTR2A expression of both glioma cells as well as macrophages in GBM. Methods 15 samples of patient-derived GBM were stained immunohistochemically for macrophage infiltration (CD68), proliferative activity (Ki67) as well as expression of SSTR2A. Anti-CD45 staining was performed to distinguish between resident microglia and tumor-infiltrating macrophages. In a subcohort, positron emission tomography (PET) imaging using \(^{68}Ga-DOTATATE\) was performed and the semiquantitatively evaluated tracer uptake was compared to the results of immunohistochemistry. Results The amount of microglia/macrophages ranged from <10% to >50% in the tumor samples with the vast majority being resident microglial cells. A strong SSTR2A immunostaining was observed in endothelial cells of proliferating vessels, in neurons and neuropile. Only faint immunostaining was identified on isolated microglial and tumor cells. Somatostatin receptor imaging revealed areas of increased tracer accumulation in every patient. However, retention of the tracer did not correlate with immunohistochemical staining patterns. Conclusion SSTR2A seems not to be overexpressed in GBM samples tested, neither on the cell surface of resident microglia or infiltrating macrophages, nor on the surface of tumor cells. These data suggest that somatostatin receptor directed imaging and treatment strategies are less promising in GBM. KW - glioma KW - positron emission tomography KW - glioblastoma multiforme KW - macrophages KW - somatostatin KW - microglial cells KW - immunostaining KW - magnetic resonance imaging Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125498 VL - 10 IS - 3 ER - TY - JOUR A1 - Glaser, Jan A1 - Schultheis, Martina A1 - Moll, Heidrun A1 - Hazra, Banasri A1 - Holzgrabe, Ulrike T1 - Antileishmanial and Cytotoxic Compounds from Valeriana wallichii and Identification of a Novel Nepetolactone Derivative JF - Molecules N2 - The chloroform extract of Valeriana wallichii (V. wallichii) rhizomes was investigated to elucidate the structures responsible for reported antileishmanial activity. Besides bornyl caffeate (1, already been reported by us previously), bioassay-guided fractionation resulted in two additional cinnamic acid derivatives 2–3 with moderate leishmanicidal activity. The structure of a novel nepetolactone derivative 4 having a cinnamic acid moiety was elucidated by means of spectral analysis. To the best of our knowledge villoside aglycone (5) was isolated from this plant for the first time. The bioassay-guided fractionation yielded two new (compounds 6–7) and two known valtrates (compounds 8–9) with leishmanicidal potential against Leishmania major (L. major) promastigotes. In addition, β-bisabolol (10), α-kessyl alcohol (11), valeranone (12), bornyl isovalerate (13) and linarin-2-O-methylbutyrate (14) were identified. This is the first report on the isolation of 4'-demethylpodophyllotoxin (15), podophyllotoxin (16) and pinoresinol (17) in V. wallichii. In total thirteen known and four new compounds were identified from the extract and their cytotoxic and antileishmanial properties were evaluated. KW - novel nepetolactone derivative KW - cytotoxicity KW - antileishmanial KW - V. wallichii KW - podophyllotoxin KW - valtrates Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125320 VL - 20 IS - 4 ER - TY - JOUR A1 - Lukasczik, Matthias A1 - Gerlich, Christian A1 - Schuler, Michael A1 - Neuderth, Silke A1 - Dlugosch, Gabriele A1 - Faller, Hermann T1 - Stress and resources in women attending an inpatient prevention/rehabilitation measure for parents: Secondary analysis of quality assurance data JF - Open Journal of Medical Psychology N2 - Questionnaire data from two projects on the development of quality assurance instruments for an inpatient rehabilitation/prevention program for parents were used for a secondary analysis. In this analysis, the associations of gains in a psychosocial resource (parenting self-efficacy) and two types of stressors experienced by mothers at the start of treatment (parenting hassles, depressive symptoms) with general life satisfaction and satisfaction with health at the end of treatment were explored. Structural equation modeling was applied to data from N = 1724 female patients. Potential resource-stressor interactions were tested using the Latent Moderated Structural Equations approach. Results showed that parenting hassles were negatively associated with general life satisfaction and satisfaction with health while self-efficacy gains were weakly positively correlated with both variables. No interaction of parenting hassles and self-efficacy gains was found. Depressive symptoms were negatively associated with both satisfaction measures. In these models, self-efficacy gains were not substantially correlated with life satisfaction, but showed a small association with satisfaction with health. There was no significant interaction of depressive symptoms and self-efficacy gains. The findings imply that interventions for distressed mothers—as exemplarily illustrated by this inpatient setting—should focus on identifying and reducing initial stressors as these may continue to impair mothers’ subjective health despite gains in parenting-related resources. KW - parenting stress KW - resource KW - self-efficacy KW - depression KW - mothers Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125316 VL - 4 ER - TY - THES A1 - Wurst, Jan-Eric T1 - Hp-Finite Elements for PDE-Constrained Optimization N2 - Diese Arbeit behandelt die hp-Finite Elemente Methode (FEM) für linear quadratische Optimal-steuerungsprobleme. Dabei soll ein Zielfunktional, welches die Entfernung zu einem angestrebten Zustand und hohe Steuerungskosten (als Regularisierung) bestraft, unter der Nebenbedingung einer elliptischen partiellen Differentialgleichung minimiert werden. Bei der Anwesenheit von Steuerungsbeschränkungen können die notwendigen Bedingungen erster Ordnung, die typischerweise für numerische Lösungsverfahren genutzt werden, als halbglatte Projektionsformel formuliert werden. Folglich sind optimale Lösungen oftmals auch nicht-glatt. Die Technik der hp-Diskretisierung berücksichtigt diese Tatsache und approximiert raue Funktionen auf feinen Gittern, während Elemente höherer Ordnung auf Gebieten verwendet werden, auf denen die Lösung glatt ist. Die erste Leistung dieser Arbeit ist die erfolgreiche Anwendung der hp-FEM auf zwei verwandte Problemklassen: Neumann- und Interface-Steuerungsprobleme. Diese werden zunächst mit entsprechenden a-priori Verfeinerungsstrategien gelöst, mit der randkonzentrierten (bc) FEM oder interface konzentrierten (ic) FEM. Diese Strategien generieren Gitter, die stark in Richtung des Randes beziehungsweise des Interfaces verfeinert werden. Um für beide Techniken eine algebraische Reduktion des Approximationsfehlers zu beweisen, wird eine elementweise interpolierende Funktion konstruiert. Außerdem werden die lokale und globale Regularität von Lösungen behandelt, weil sie entscheidend für die Konvergenzgeschwindigkeit ist. Da die bc- und ic- FEM kleine Polynomgrade für Elemente verwenden, die den Rand beziehungsweise das Interface berühren, können eine neue L2- und L∞-Fehlerabschätzung hergeleitet werden. Letztere bildet die Grundlage für eine a-priori Strategie zum Aufdatieren des Regularisierungsparameters im Zielfunktional, um Probleme mit bang-bang Charakter zu lösen. Zudem wird die herkömmliche hp-Idee, die daraus besteht das Gitter geometrisch in Richtung der Ecken des Gebiets abzustufen, auf die Lösung von Optimalsteuerungsproblemen übertragen (vc-FEM). Es gelingt, Regularität in abzählbar normierten Räumen für die Variablen des gekoppelten Optimalitätssystems zu zeigen. Hieraus resultiert die exponentielle Konvergenz im Bezug auf die Anzahl der Freiheitsgrade. Die zweite Leistung dieser Arbeit ist die Entwicklung einer völlig adaptiven hp-Innere-Punkte-Methode, die Probleme mit verteilter oder Neumann Steuerung lösen kann. Das zugrundeliegende Barriereproblem besitzt ein nichtlineares Optimilitätssystem, das eine numerische Herausforderung beinhaltet: die stabile Berechnung von Integralen über Funktionen mit möglichen Singularitäten in Elementen höherer Ordnung. Dieses Problem wird dadurch gelöst, dass die Steuerung an den Integrationspunkten überwacht wird. Die Zulässigkeit an diesen Punkten wird durch einen Glättungsschritt garantiert. In dieser Arbeit werden sowohl die Konvergenz eines Innere-Punkte-Verfahrens mit Glättungsschritt als auch a-posteriori Schranken für den Diskretisierungsfehler gezeigt. Dies führt zu einem adaptiven Lösungsalgorithmus, dessen Gitterverfeinerung auf der Entwicklung der Lösung in eine Legendre Reihe basiert. Hierbei dient das Abklingverhalten der Koeffizienten als Glattheitsindikator und wird für die Entscheidung zwischen h- und p-Verfeinerung herangezogen. N2 - This thesis deals with the hp-finite element method (FEM) for linear quadratic optimal control problems. Here, a tracking type functional with control costs as regularization shall be minimized subject to an elliptic partial differential equation. In the presence of control constraints, the first order necessary conditions, which are typically used to find optimal solutions numerically, can be formulated as a semi-smooth projection formula. Consequently, optimal solutions may be non-smooth as well. The hp-discretization technique considers this fact and approximates rough functions on fine meshes while using higher order finite elements on domains where the solution is smooth. The first main achievement of this thesis is the successful application of hp-FEM to two related problem classes: Neumann boundary and interface control problems. They are solved with an a-priori refinement strategy called boundary concentrated (bc) FEM and interface concentrated (ic) FEM, respectively. These strategies generate grids that are heavily refined towards the boundary or interface. We construct an elementwise interpolant that allows to prove algebraic decay of the approximation error for both techniques. Additionally, a detailed analysis of global and local regularity of solutions, which is critical for the speed of convergence, is included. Since the bc- and ic-FEM retain small polynomial degrees for elements touching the boundary and interface, respectively, we are able to deduce novel error estimates in the L2- and L∞-norm. The latter allows an a-priori strategy for updating the regularization parameter in the objective functional to solve bang-bang problems. Furthermore, we apply the traditional idea of the hp-FEM, i.e., grading the mesh geometrically towards vertices of the domain, for solving optimal control problems (vc-FEM). In doing so, we obtain exponential convergence with respect to the number of unknowns. This is proved with a regularity result in countably normed spaces for the variables of the coupled optimality system. The second main achievement of this thesis is the development of a fully adaptive hp-interior point method that can solve problems with distributed or Neumann control. The underlying barrier problem yields a non-linear optimality system, which poses a numerical challenge: the numerically stable evaluation of integrals over possibly singular functions in higher order elements. We successfully overcome this difficulty by monitoring the control variable at the integration points and enforcing feasibility in an additional smoothing step. In this work, we prove convergence of an interior point method with smoothing step and derive a-posteriori error estimators. The adaptive mesh refinement is based on the expansion of the solution in a Legendre series. The decay of the coefficients serves as an indicator for smoothness that guides between h- and p-refinement. KW - Finite-Elemente-Methode KW - Optimale Kontrolle KW - Elliptische Differentialgleichung KW - finite elements KW - optimal control KW - higher order methods KW - partial differetial equations Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115027 SN - 978-3-95826-024-5 (print) SN - 978-3-95826-025-2 (online) PB - Würzburg University Press CY - Würzburg ER - TY - JOUR A1 - Ott, Christine A1 - Dorsch, Eva A1 - Fraunholz, Martin A1 - Straub, Sebastian A1 - Kozjak-Pavlovic, Vera T1 - Detailed Analysis of the Human Mitochondrial Contact Site Complex Indicate a Hierarchy of Subunits JF - PLoS One N2 - Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components. KW - co-immunoprecipitation KW - motor proteins KW - mitochondria KW - membrane potential KW - membrane proteins KW - protein complexes KW - mitochondrial membrane KW - outer membrane proteins Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125347 VL - 10 IS - 3 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Zimmermann, Marcus A1 - Katzer, Astrid A1 - Fiedler, Vanessa A1 - Distel, Luitpold V. A1 - Gasser, Martin A1 - Waaga-Gasser, Anna-Maria A1 - Flentje, Michael A1 - Polat, Bülent T1 - A prospective study on histone γ-H2AX and 53BP1 foci expression in rectal carcinoma patients: correlation with radiation therapy-induced outcome JF - BMC Cancer N2 - Background The prognostic value of histone γ-H2AX and 53BP1 proteins to predict the radiotherapy (RT) outcome of patients with rectal carcinoma (RC) was evaluated in a prospective study. High expression of the constitutive histone γ-H2AX is indicative of defective DNA repair pathway and/or genomic instability, whereas 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. Methods Using fluorescence microscopy, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53BP1 in peripheral blood mononuclear cells derived from unselected RC patients (n = 53) undergoing neoadjuvant chemo- and RT. Cells from apparently healthy donors (n = 12) served as references. Results The γ-H2AX assay of in vitro irradiated lymphocytes revealed significantly higher degree of DNA damage in the group of unselected RC patients with respect to the background, initial (0.5 Gy, 30 min) and residual (0.5 Gy and 2 Gy, 24 h post-radiation) damage compared to the control group. Likewise, the numbers of 53BP1 foci analyzed in the samples from 46 RC patients were significantly higher than in controls except for the background DNA damage. However, both markers were not able to predict tumor stage, gastrointestinal toxicity or tumor regression after curative RT. Interestingly, the mean baseline and induced DNA damage was found to be lower in the group of RC patients with tumor stage IV (n = 7) as compared with the stage III (n = 35). The difference, however, did not reach statistical significance, apparently, because of the limited number of patients. Conclusions The study shows higher expression of γ-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome. KW - radiosensitivity KW - peripheral blood lymphocytes KW - DNA repair KW - DNA damage Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125303 VL - 15 IS - 856 ER - TY - JOUR A1 - Bratengeier, Klaus A1 - Holubyev, Kostyantyn T1 - Characteristics of non-coplanar IMRT in the presence of target-embedded organs at risk JF - Radiation Oncology N2 - Background The aim is to analyze characteristics and to study the potentials of non-coplanar intensity modulated radiation therapy (IMRT) techniques. The planning study applies to generalized organ at risk (OAR) – planning target volume (PTV) geometries. Methods The authors focus on OARs embedded in the PTV. The OAR shapes are spherically symmetric (A), cylindrical (B), and bended (C). Several IMRT techniques are used for the planning study: a) non-coplanar quasi-isotropic; b) two sets of equidistant coplanar beams, half of beams incident in a plane perpendicular to the principal plane; c) coplanar equidistant (reference); d) coplanar plus one orthogonal beam. The number of beam directions varies from 9 to 16. The orientation of the beam sets is systematically changed; dose distributions resulting from optimal fluence are explored. A selection of plans is optimized with direct machine parameter optimization (DMPO) allowing 120 and 64 segments. The overall plan quality, PTV coverage, and OAR sparing are evaluated. Results For all fluence based techniques in cases A and C, plan quality increased considerably if more irradiation directions were used. For the cylindrically symmetric case B, however, only a weak beam number dependence was observed for the best beam set orientation, for which non-coplanar directions could be found where OAR- and PTV-projections did not overlap. IMRT plans using quasi-isotropical distributed non-coplanar beams showed stable results for all topologies A, B, C, as long as 16 beams were chosen; also the most unfavorable beam arrangement created results of similar quality as the optimally oriented coplanar configuration. For smaller number of beams or application in the trunk, a coplanar technique with additional orthogonal beam could be recommended. Techniques using 120 segments created by DMPO could qualitatively reproduce the fluence based results. However, for a reduced number of segments the beam number dependence declined or even reversed for the used planning system and the plan quality degraded substantially. Conclusions Topologies with targets encompassing sensitive OAR require sufficient number of beams of 15 or more. For the subgroup of topologies where beam incidences are possible which cover the whole PTV without direct OAR irradiation, the quality dependence on the number of beams is much less pronounced above 9 beams. However, these special non-coplanar beam directions have to be found. On the basis of this work the non-coplanar IMRT techniques can be chosen for further clinical planning studies. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125292 VL - 10 IS - 207 ER - TY - JOUR A1 - Streng, Andrea A1 - Prifert, Christiane A1 - Weissbrich, Benedikt A1 - Liese, Johannes G. T1 - Continued high incidence of children with severe influenza A(H1N1)pdm09 admitted to paediatric intensive care units in Germany during the first three post-pandemic influenza seasons, 2010/11–2012/13 JF - BMC Infectious Diseases N2 - Background Previous influenza surveillance at paediatric intensive care units (PICUs) in Germany indicated increased incidence of PICU admissions for the pandemic influenza subtype A(H1N1)pdm09. We investigated incidence and clinical characteristics of influenza in children admitted to PICUs during the first three post-pandemic influenza seasons, using active screening. Methods We conducted a prospective surveillance study in 24 PICUs in Bavaria (Germany) from October 2010 to September 2013. Influenza cases among children between 1 month and 16 years of age admitted to these PICUs with acute respiratory infection were confirmed by PCR analysis of respiratory secretions. Results A total of 24/7/20 influenza-associated PICU admissions were recorded in the post-pandemic seasons 1/2/3; incidence estimates per 100,000 children were 1.72/0.76/1.80, respectively. Of all 51 patients, 80 % had influenza A, including 65 % with A(H1N1)pdm09. Influenza A(H1N1)pdm09 was almost absent in season 2 (incidence 0.11), but dominated PICU admissions in seasons 1 (incidence 1.35) and 3 (incidence 1.17). Clinical data was available for 47 influenza patients; median age was 4.8 years (IQR 1.6–11.0). The most frequent diagnoses were influenza-associated pneumonia (62 %), bronchitis/bronchiolitis (32 %), secondary bacterial pneumonia (26 %), and ARDS (21 %). Thirty-six patients (77 %) had underlying medical conditions. Median duration of PICU stay was 3 days (IQR 1–11). Forty-seven per cent of patients received mechanical ventilation, and one patient (2 %) extracorporeal membrane oxygenation; 19 % were treated with oseltamivir. Five children (11 %) had pulmonary sequelae. Five children (11 %) died; all had underlying chronic conditions and were infected with A(H1N1)pdm09. In season 3, patients with A(H1N1)pdm09 were younger than in season 1 (p = 0.020), were diagnosed more often with bronchitis/bronchiolitis (p = 0.004), and were admitted to a PICU later after the onset of influenza symptoms (p = 0.041). Conclusions Active screening showed a continued high incidence of A(H1N1)pdm09-associated PICU admissions in the post-pandemic seasons 1 and 3, and indicated possible underestimation of incidence in previous German studies. The age shift of severe A(H1N1)pdm09 towards younger children may be explained by increasing immunity in the older paediatric population. The high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in Germany. KW - post-pandemic KW - intensive care KW - children KW - influenza Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125280 VL - 15 IS - 573 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Kupper, Maria A1 - Ratzka, Carolin A1 - Feldhaar, Heike A1 - Vilcinskas, Andreas A1 - Gross, Roy A1 - Dandekar, Thomas A1 - Förster, Frank T1 - Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing JF - BMC Genomics N2 - Background Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. Carpenter ants are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, carpenter ants carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus. Results The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8 % of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of carpenter ants comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults. Conclusions The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle. KW - immune system KW - transcriptome KW - carpenter ant KW - camponotus floridanus KW - re-annotation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125279 VL - 16 IS - 540 ER - TY - JOUR A1 - Neuhaus, Winfried A1 - Schlundt, Marian A1 - Fehrholz, Markus A1 - Ehrke, Alexander A1 - Kunzmann, Steffen A1 - Liebner, Stefan A1 - Speer, Christian P. A1 - Förster, Carola Y. T1 - Multiple Antenatal Dexamethasone Treatment Alters Brain Vessel Differentiation in Newborn Mouse Pups JF - PLoS One N2 - Antenatal steroid treatment decreases morbidity and mortality in premature infants through the maturation of lung tissue, which enables sufficient breathing performance. However, clinical and animal studies have shown that repeated doses of glucocorticoids such as dexamethasone and betamethasone lead to long-term adverse effects on brain development. Therefore, we established a mouse model for antenatal dexamethasone treatment to investigate the effects of dexamethasone on brain vessel differentiation towards the blood-brain barrier (BBB) phenotype, focusing on molecular marker analysis. The major findings were that in total brains on postnatal day (PN) 4 triple antenatal dexamethasone treatment significantly downregulated the tight junction protein claudin-5, the endothelial marker Pecam-1/CD31, the glucocorticoid receptor, the NR1 subunit of the N-methyl-D-aspartate receptor, and Abc transporters (Abcb1a, Abcg2 Abcc4). Less pronounced effects were found after single antenatal dexamethasone treatment and in PN10 samples. Comparisons of total brain samples with isolated brain endothelial cells together with the stainings for Pecam-1/CD31 and claudin-5 led to the assumption that the morphology of brain vessels is affected by antenatal dexamethasone treatment at PN4. On the mRNA level markers for angiogenesis, the sonic hedgehog and the Wnt pathway were downregulated in PN4 samples, suggesting fundamental changes in brain vascularization and/or differentiation. In conclusion, we provided a first comprehensive molecular basis for the adverse effects of multiple antenatal dexamethasone treatment on brain vessel differentiation. KW - endothelial cells KW - protein expression KW - central nervous system KW - mouse models KW - pregnancy KW - tight junctions KW - sheep KW - angiogenesis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125471 VL - 10 IS - 8 ER -