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Bacterial pathogens exploit eukaryotic pathways for their own end. Upon ingestion, Salmonella enterica serovar Typhimurium passes through the stomach and then catalyzes its uptake across the intestinal epithelium. It survives and replicates in an acidic vacuole through the action of virulence factors secreted by a type three secretion system located on Salmonella pathogenicity island 2 (SPI-2). Two secreted effectors, SifA and SseJ, are sufficient for endosomal tubule formation, which modifies the vacuole and enables Salmonella to replicate within it. Two-color, superresolution imaging of the secreted virulence factor SseJ and tubulin revealed that SseJ formed clusters of conserved size at regular, periodic intervals in the host cytoplasm. Analysis of SseJ clustering indicated the presence of a pearling effect, which is a force-driven, osmotically sensitive process. The pearling transition is an instability driven by membranes under tension; it is induced by hypotonic or hypertonic buffer exchange and leads to the formation of beadlike structures of similar size and regular spacing. Reducing the osmolality of the fixation conditions using glutaraldehyde enabled visualization of continuous and intact tubules. Correlation analysis revealed that SseJ was colocalized with the motor protein kinesin. Tubulation of the endoplasmic reticulum is driven by microtubule motors, and in the present work, we describe how Salmonella has coopted the microtubule motor kinesin to drive the force-dependent process of endosomal tubulation. Thus, endosomal tubule formation is a force-driven process catalyzed by Salmonella virulence factors secreted into the host cytoplasm during infection.
Truffles (Tuber spp.) are the fruiting bodies of symbiotic fungi, which are prized food delicacies. The marked aroma variability observed among truffles of the same species has been attributed to a series of factors that are still debated. This is because factors (i.e. genetics, maturation, geographical location and the microbial community colonizing truffles) often co-vary in truffle orchards. Here, we removed the co-variance effect by investigating truffle flavour in axenic cultures of nine strains of the white truffle Tuber borchii. This allowed us to investigate the influence of genetics on truffle aroma. Specifically, we quantified aroma variability and explored whether strain selection could be used to improve human-sensed truffle flavour. Our results illustrate that aroma variability among strains is predominantly linked to amino acid catabolism through the Ehrlich pathway, as confirmed by 13C labelling experiments. We furthermore exemplified through sensory analysis that the human nose is able to distinguish among strains and that sulfur volatiles derived from the catabolism of methionine have the strongest influence on aroma characteristics. Overall, our results demonstrate that genetics influences truffle aroma much more deeply than previously thought and illustrate the usefulness of strain selection for improving truffle flavour.
Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover.
Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.
In 1957, Craig Mooney published a set of human face stimuli to study perceptual closure: the formation of a coherent percept on the basis of minimal visual information. Images of this type, now known as “Mooney faces”, are widely used in cognitive psychology and neuroscience because they offer a means of inducing variable perception with constant visuo-spatial characteristics (they are often not perceived as faces if viewed upside down). Mooney’s original set of 40 stimuli has been employed in several studies. However, it is often necessary to use a much larger stimulus set. We created a new set of over 500 Mooney faces and tested them on a cohort of human observers. We present the results of our tests here, and make the stimuli freely available via the internet. Our test results can be used to select subsets of the stimuli that are most suited for a given experimental purpose.
Effect of progesterone on Smad signaling and TGF-β/Smad-regulated genes in lung epithelial cells
(2018)
The effect of endogenous progesterone and/or exogenous pre- or postnatal progesterone application on lung function of preterm infants is poorly defined. While prenatal progesterone substitution may prevent preterm birth, in vitro and in vivo data suggest a benefit of postnatal progesterone replacement on the incidence and severity of bronchopulmonary dysplasia (BPD). However, the molecular mechanisms responsible for progesterone’s effects are undefined. Numerous factors are involved in lung development, airway inflammation, and airway remodeling: the transforming growth factor beta (TGF-β)/mothers against decapentaplegic homolog (Smad) signaling pathway and TGF-β-regulated genes, such as connective tissue growth factor (CTGF), transgelin (TAGLN), and plasminogen activator inhibitor-1 (PAI-1). These processes contribute to the development of BPD. The aim of the present study was to clarify whether progesterone could affect TGF-β1-activated Smad signaling and CTGF/transgelin/PAI-1 expression in lung epithelial cells. The pharmacological effect of progesterone on Smad signaling was investigated using a TGF-β1-inducible luciferase reporter and western blotting analysis of phosphorylated Smad2/3 in A549 lung epithelial cells. The regulation of CTGF, transgelin, and PAI-1 expression by progesterone was studied using a promoter-based luciferase reporter, quantitative real-time PCR, and western blotting in the same cell line. While progesterone alone had no direct effect on Smad signaling in lung epithelial cells, it dose-dependently inhibited TGF-β1-induced Smad3 phosphorylation, as shown by luciferase assays and western blotting analysis. Progesterone also antagonized the TGF-β1/Smad-induced upregulation of CTGF, transgelin, and PAI-1 at the promoter, mRNA, and/or protein levels. The present study highlights possible new molecular mechanisms involving progesterone, including inhibition of TGF-β1-activated Smad signaling and TGF-β1-regulated genes involved in BPD pathogenesis, which are likely to attenuate the development of BPD by inhibiting TGF-β1-mediated airway remodeling. Understanding these mechanisms might help to explain the effects of pre- or postnatal application of progesterone on lung diseases of preterm infants.
This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA.
Background and aims: Expression of carbonic anhydrase IX (CA9), an enzyme expressed in response to hypoxia, acidosis and oncogenic alterations, is reported to be a prognostic factor in HCC patients. Here we evaluated serum CA9 levels in HCC and cirrhosis patients.
Methods: HCC and cirrhosis patients were prospectively recruited and CA9 levels were determined. CA9 levels were compared to stages of cirrhosis and HCC stages. The association of the CA9 levels and overall survival (OS) was assessed. Furthermore, immunohistochemical CA9 expression in HCC and cirrhosis was evaluated.
Results: 215 patients with HCC were included. The median serum CA9 concentration in patients with HCC was 370 pg/ml and significantly higher than in a healthy cohort. Patients with advanced cancer stages (BCLC and ALBI score) had hid significant higher levels of CA9 in the serum. HCC patients with high serum CA9 concentrations (>400 pg/ml) had an increased mortality risk (hazard ratio (HR) 1.690, 95% confidence interval (CI) 1.017–2.809, P = 0.043). Serum CA9 concentration in cirrhotic patients did not differ significantly from HCC patients. Higher CA9 levels in cirrhotic patients correlated with portal hypertension and esophageal varices. Patients with ethanol induced cirrhosis had the highest CA9 levels in both cohorts. Levels of CA9 did not correlate with immunohistochemical expression.
Conclusions: We conclude that a high CA9 level is a possible prognostic indicator for a poor outcome in HCC patients. The high CA9 levels are probably mainly associated with portal hypertension. Ductular reactions might be a possible source of serum CA9.
Transmission of temporally correlated spike trains through synapses with short-term depression
(2018)
Short-term synaptic depression, caused by depletion of releasable neurotransmitter, modulates the strength of neuronal connections in a history-dependent manner. Quantifying the statistics of synaptic transmission requires stochastic models that link probabilistic neurotransmitter release with presynaptic spike-train statistics. Common approaches are to model the presynaptic spike train as either regular or a memory-less Poisson process: few analytical results are available that describe depressing synapses when the afferent spike train has more complex, temporally correlated statistics such as bursts. Here we present a series of analytical results—from vesicle release-site occupancy statistics, via neurotransmitter release, to the post-synaptic voltage mean and variance—for depressing synapses driven by correlated presynaptic spike trains. The class of presynaptic drive considered is that fully characterised by the inter-spike-interval distribution and encompasses a broad range of models used for neuronal circuit and network analyses, such as integrate-and-fire models with a complete post-spike reset and receiving sufficiently short-time correlated drive. We further demonstrate that the derived post-synaptic voltage mean and variance allow for a simple and accurate approximation of the firing rate of the post-synaptic neuron, using the exponential integrate-and-fire model as an example. These results extend the level of biological detail included in models of synaptic transmission and will allow for the incorporation of more complex and physiologically relevant firing patterns into future studies of neuronal networks.
Im Zeitraum vom 7. bis zum 9. Dezember 2017 fand an der Universität Breslau (Wrocław) in Kooperation mit der Ruhr-Universität Bochum die Winterschool "Europa: Poetik und Politik" statt. Die gut zwanzig Teilnehmerinnen und Teilnehmer – darunter Professor*innen, wissenschaftliche Mitarbeiter*innen und Studierende beider Universitäten – diskutierten die neuere Geschichte von Europa-Diskursen von der Romantik bis in die Gegenwart. Ausgangspunkt der Tagung war die gegenwärtige Krise der Europäischen Union, die – so zeigte sich im Verlauf der Tagung – als Fortführung der leitmotivischen 'Schwellensituationen' zu verstehen ist, die als das Gemeinsame der diskutierten Texte und somit in gewisser Weise als konstitutiv für den Europa-Diskurs erkannt wurden.
Entwicklung von Immunisierungsstrategien zur Induktion hoher funktionaler Antikörperantworten
(2018)
Neuartige Viren und Erreger, die sich antigenetisch tiefgreifend von bekannten Varianten unterscheiden, können verheerende Epidemien auslösen, da weder gegen diese Erreger eine Immunität in der Bevölkerung besteht, noch prophylaktische oder therapeutische Maßnahmen verfügbar sind. Eine prophylaktisch vermittelte Immunität durch Impfung stellt die bei Weitem effektivste Methode zur Vorbeugung viraler Infektionen dar, jedoch sind die Entwicklungs- und Herstellungszeiten eines neuen Impfstoffs in der Regel mit der Ausbruchsdynamik nicht kompatibel. Inzwischen steht zwar eine überschaubare Anzahl antiviraler Medikamente zur Verfügung, doch ist die Wahrscheinlichkeit gering, dass diese meist hoch spezifischen Wirkstoffe gegen neu auftretende Viren aktiv sind. Das beispiellose Ausmaß der Ebola-Epidemie 2014 führte zum Einsatz experimenteller antikörperbasierter Therapien, welche das Potential der passiven Vermittlung von temporärem Immunschutz naiver Personen verdeutlicht. Für viele neuartige Viren ist die Entwicklung von Therapieansätzen allerdings noch nicht entsprechend weit fortgeschritten. Zudem bedingt eine Verwendung des eigentlichen Erregers oft hohe Sicherheitsmaßnahmen, was die Arbeit erschwert. Aus diesem Grund werden Notfalltherapien benötigt, die schnell in klinisch relevanter Qualität und Quantität unter niedrigen biologischen Sicherheitsmaßnahmen produziert werden können.
Diese Arbeit basiert auf der zentralen Hypothese, dass die Induktion von hohen Titern funktioneller Antikörperantworten die Basis für einen breiteren Schutz gegen antigenetisch entferntere Virusstämme sowie für die schnelle Produktion von therapeutischen Antiseren darstellt.
Um diese Hypothese zu testen und Einblicke in verschiedene Aspekte dieses Prozesses zu bekommen, wurde zunächst die Nutzung von Adjuvanzien als Zusätze für Impfstoffe am Beispiel des pandemischen A(H1N1)pdm09-Impfstoffs untersucht. Neben den alljährlichen Epidemien, die von saisonalen Influenza-A-Viren der Subtypen H1N1 oder H2N3 verursacht werden, können neuartige Subtypen zu weltweiten Pandemien führen. Während die saisonalen Influenza-Impfstoffe in der Regel keine Adjuvanzien enthalten, wurden einige pandemische H1N1-Impfstoffe aus 2009 mit einem reduzierten Antigengehalt formuliert und mit squalenbasierten Adjuvanzien kombiniert, um eine ausreichende Wirksamkeit bei größerer Verfügbarkeit zu gewährleisten. Zur Charakterisierung des Effekts dieser Adjuvanzien auf die Immunantworten wurden Frettchen mit 2 µg des kommerziellen H1N1pmd09-Impfstoffes alleine sowie in Kombination mit verschiedenen Adjuvanzien immunisiert, die Antikörpertiter gegen homologe und heterologe Influenzastämme untersucht und mit dem Schutz vor einer Infektion korreliert. Dabei zeigte sich, dass die Verwendung squalenbasierter Adjuvanzien die funktionalen Antikörperantworten um das 100-fache erhöhte und zu einer signifikant reduzierten Viruslast nach der Infektion mit dem homologen pandemischen Virus führte. Während in keiner Gruppe Antikörper gegen die heterologen Hämagglutinin-(HA-)Proteine H3, H5, H7 und H9 nachweisbar waren, induzierten mit squalenbasierten Adjuvanzien kombinierte Impfstoffe subtypenspezifische Antikörper gegen das N1 Neuraminidase-(NA-)Protein einschließlich H5N1. Darüber hinaus führte die Immunisierung mit squalenbasierten Adjuvanzien zu einer besseren Kontrolle der Influenzavirus-Replikation in den oberen Atemwegen.
Anschließend wurde im zweiten Teil dieser Arbeit unter Einbeziehung der gewonnenen Erkenntnisse eine Immunisierungsstrategie zur schnellen Produktion therapeutischer Hyper-immunseren entwickelt, wobei unterschiedliche Antigenexpressionssysteme miteinander verglichen wurden. Während in den frühen Stadien eines Ausbruchs Rekonvaleszenzseren nicht ohne weiteres verfügbar sind, können Antiseren tierischen Ursprungs innerhalb eines kurzen Zeitraums hergestellt werden. Die Herausforderung liegt in der schnellen Induktion einer schützenden Immunität, wobei die effiziente Produktion und Reinigung von Hyperimmunserum in klinisch relevanten Mengen ebenso essenziell ist wie die Anpassungsfähigkeit der Immunisierungsstrategie an neue oder hinsichtlich ihrer Antigenizität veränderte Viren. Hierzu wurden verschiedene Immunisierungsstrategien in Mäusen und Kaninchen verglichen, die unterschiedliche Expressionssysteme für das Modellantigen Ebolavirus-Glykoprotein (EBOV-GP) verwenden: (i) Ebolavirus-ähnliche Partikel (VLP), (ii) das rekombinante modifizierte Vacciniavirus Ankara (MVA) sowie (iii) das rekombinante Virus der vesikulären Stomatitis (VSV). Im Ergebnis induzierte eine dreimalige Immunisierung mit VLPs in Kombination mit squalenhaltigem Adjuvans neutralisierende Antikörpertiter, die vergleichbar mit der Immunisierung mit replikationskompetentem VSVΔG/EBOV-GP waren. Dies deutet darauf hin, dass nicht die De-novo-Antigenexpression, sondern vielmehr die mehrfache Präsentation des Antigens in nativer Konformation für die Produktion von neutralisierenden Antikörpern essenziell ist. Darüber hinaus waren die funktionalen Antikörpertiter aller Kaninchenseren in der In-vitro-Analyse gegen das Wildtypvirus 10- bis 100-fach höher als der Durchschnitt, der in mit VSVΔG/EBOV-GP geimpften Probanden beobachtet wurde. Die Etablierung eines optimierten mehrstufigen Reinigungsverfahrens unter Verwendung einer zweistufigen Ammoniumsulfat-Präzipitation, gefolgt von einer Protein-A-Affinitätschromatographie, führte zu aufgereinigten IgG-Präparationen mit nahezu unveränderter neutralisierender Aktivität, die über neun Tage im xenogenen In-vivo-Modell stabil waren. Die signifikante Erhöhung von totalen und funktionalen Antikörpertitern in Kombination mit einer größeren Breite der Antikörperantwort im Kontext von squalenbasierten Adjuvanzien stützt die Hypothese dieser Arbeit. Adjuvantierte Immunisierungsstrategien sind damit ein vielversprechender Ansatz nicht nur zur Wirksamkeitssteigerung von Subunit- und Proteinimpfstoffen, sondern auch zur schnellen Herstellung von therapeutischen Antiseren.
Um molekulare Mechanismen in biologischen Prozessen zu verstehen, ist es unerlässlich biologisch aktive Verbindungen zu kontrollieren. Dabei spielt besonders die Aktivierung bzw. Desaktivierung von Genabschnitten eine zentrale Rolle in der gegenwärtigen chemischen, biologischen und medizinischen Forschung. Nukleinsäuren sind dabei offenkundige Zielmoleküle, da sie die Genexpression auf unterster Ebene regulieren und auf vielfältige Art und Weise an biologischen Prozessen beteiligt sind. Um solch eine genaue Steuerung zu erreichen, werden Nukleinsäuren häufig photolabil modifiziert und unter die Kontrolle von Licht gebracht. Da hochentwickelte Technologien es erlauben Photonen bestimmter Energie unter präziser räumlicher und zeitlicher Auflösung zu dosieren, ist Licht als nicht invasives Triggersignal ein besonders geeignetes Werkzeug um molekulare Prozesse zu kontrollieren.
Die Verwendung photolabiler Schutzgruppen („cage“) ermöglicht es, diese lichtaktivierbaren Nukleinsäuren („caged compound“) herzustellen. Üblicherweise werden Oligonukleotide damit an funktionsbestimmenden Stellen versehen, woraufhin die Funktion der Oligonukleotide unterdrückt wird. Die biologische Aktivität kann durch Bestrahlung mit Licht wieder hergestellt werden, da die photolabile Schutzgruppe durch den Lichtimpuls abgespalten wird. Neben der zeitweiligen Maskierung der Nukleinsäureaktivität existiert auch eine Methode, die als „photoaktivierbarer Strangbruch“ (‘‘caged strand break‘‘) bezeichnet wird. Dabei werden mit Hilfe von photolabilen Linkern (‘‘Verknüpfer‘‘) lichtinduzierte Strangbrüche in Oligonukleotiden ausgelöst, um so beispielsweise die Struktur eines Nukleinsäurestrangs zu zerstören. Die Idee der photoaktivierbaren Strangbrüche ist nicht neu, dennoch werden photolabile Schutzgruppen überwiegend nach der erstgenannten Strategie verwendet. Im Rahmen dieses Promotionsvorhabens wurden neue photosensitive Linkerbausteine für Oligonukleotide entwickelt und hergestellt, welche sich vor allem im Hinblick auf die Anwendbarkeit in lebenden biologischen Systemen von den bisherigen photolabilen Linkern unterscheiden.
Im ersten Projekt wurde ein nicht-nukleosidischer, photolabiler Linker, basierend auf dem Cumaringrundgerüst, entwickelt. Das Ziel war hier, vor allem, einen zweiphotonenaktiven Linker für biologische Anwendungen und Zweiphotonen-Fragestellungen nutzbar zu machen. Bisherige Zweiphotonen-Linker konnten hauptsächlich nur für Proteinverknüpfungen bzw. Neurotransmitter verwendet werden oder mussten chemisch umständlich (z.B. Click-Chemie) und postsynthetisch in Oligonukleotide eingeführt werden. Der neu entwickelte Zweiphotonen-Linker wurde als Phosphoramiditbaustein für die Oligonukelotid-Festphasensynthese synthetisiert, was einen problemlosen und automatisierten Einbau garantiert. Mit einem modifizierten Oligonukleotid konnten die photochemischen Eigenschaften des Linkers bestimmt und mit Hilfe eines fluoreszenzbasierten Verdrängungsassays und Lasertechniken der Zweiphotonen-Effekt visualisiert werden. Dazu wurde ein Hairpin-DNA-Strang hergestellt, welcher eine Linkermodifikation im Bereich der Loopregion enthält. Durch eine Thiolmodifikation am 5‘-Ende des Oligonukleotidstranges war es möglich, diesen in einem Maleimid-funktionalisierten Hydrogel zu fixieren. Ein DNA-Duplex mit einem Fluorophor/Quencherpaar und einer korrespondierenden Sequenz zum modifizierten Hairpin-Strang wurde ebenfalls dem System zugegeben, allerdings wurde dieser nicht fixiert, um Diffusion zu ermöglichen. Durch die räumliche Nähe des Fluorophors zum Quencher konnte im unbelichteten Zustand zunächst keine Fluoreszenz gemessen werden. Mit einem (Femtosekunden-)gepulsten Laser und dem damit verbundenen Bindungsbruch im Hairpin-Strang durch Zweiphotonen-Effekte wurde es dem fluoreszierenden Strang des DNA-Duplex ermöglicht, sich vom Quencher-Strang zu lösen und an den fixierten Strang zu hybridisieren. Das Photolyse-Ereignis konnte so in ein lokales Fluoreszenzsignal übersetzt und detektiert werden.
Der eindeutige Beweis, dass es sich tatsächlich um ein Zweiphotonen-induziertes Ereignis handelt, konnte durch die dreidimensional aufgelöste Photolyse und über die quadratische Anhängigkeit des Fluoreszenzsignals von der eingestrahlten Laserleistung erbracht werden.
Die generelle Kompatibilität des Cumarin-Linkers mit biologischen Systemen konnte in Zellkulturexperimenten gezeigt werden. Dazu wurde eine Transkriptionsfaktor-DNA Decoy-Strategie entwickelt, in der Linker-modifizierte DNA Decoys an regulatorische Transkriptionsfaktoren binden und diese aber auch photochemisch wieder freisetzen können („catch and release-Strategie“). Zellkulturexperimente, um mit dieser Methode das Transkriptionsfaktor-gesteuerte und endogene Gen für Cyclooxygenase-2 (COX2) zu regulieren, lieferten keine aussagekräftigen Ergebnisse. Daher wurden die verwendeten Zellen dahingehend manipuliert, sodass sie das Protein GFP (grün fluoreszierendes Protein) in Abhängigkeit von der Anwesenheit eines Transkriptionsfaktors exprimieren. Das so durch die Zellen verursachte Fluoreszenzsignal steht in direkter Abhängigkeit zur Decoy-Aktivität. Mit Hilfe modifizierter GFP-Decoys konnte hierbei eine Regulation auf Transkriptionsebene in biologischen Organismen erreicht werden. Mit dem Electrophoretic Mobility Shift Assay (EMSA), einer molekularbiologischen in vitro-Analysetechnik, wurden die Interaktionen zwischen modifizierten Decoys und dem Transkriptionsfaktor untersucht.
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This thesis is concerned with systematic investigations of electronic noise in novel condensed matter systems. Although fluctuations are frequently considered a nuisance, that is, a disturbance limiting the accuracy of scientific measurements, in many cases they can reveal fundamental information about the inherent system dynamics. During the past decades, the study of electronic fluctuations has evolved into an indispensable tool in condensed matter physics.
The focus of the present work lies both in a further development of the fluctuation spectroscopy technique and in the study of materials of current interest. In particular, a comprehensive study of the charge carrier dynamics in the archetypal diluted magnetic semiconductors (Ga,Mn)As and (Ga,Mn)P was performed. In spite of extensive research work carried out during the last years, there still exists no theoretical consensus on the precise mechanism of ferromagnetic order and the electronic structure in these materials. Moreover, disorder and correlation effects complicate the understanding of these compounds.
Fluctuation spectroscopy experiments presented in this work provide strong evidence that a percolation transition is observed in samples with localized charge carriers, since the normalized resistance noise magnitude displays a significant enhancement around the Curie temperature. In addition, this quantity exhibits a power law scaling behavior as a function of the resistance, which is in good agreement with theoretical models of percolating systems.
By contrast, it was found that the resistance noise in metallic samples is mainly dominated by the physics of defects such as manganese interstitials and arsenic antisites. Furthermore, first noise studies were carried out on hafnia- and yttria-based resistive random access memories. In these memristor devices, the rupture and re-formation of oxygen deficient conducting filaments caused by the electric field and Joule heating driven motion of mobile anions lead to an unusual resistance switching behavior. For the first time, comparative noise measurements on oxygen deficient and stoichiometric hafnium oxide devices, as well as on novel yttrium oxide based devices were performed in this work. Finally, new strategies for noise measurements of highly insulating and extremely low-resistive samples were developed and realized. In detail, an experimental setup for the measurements of dielectric polarization fluctuations in insulating systems was designed and successfully tested. Here, the polarization noise of a sample is measured as current or voltage fluctuations produced within a capacitance cell. The study of dielectric polarization noise allows for conclusions to be drawn regarding equilibrium structural dynamics in insulators such as relaxor ferroelectrics. On the other hand, as successfully demonstrated for a heavy-fermion compound, focused ion beam etching enables to introduce a meander-shaped geometry in single crystal platelets, in order to strongly enhance the sample resistance and thus make resistance noise measurements possible. First results indicate a connection of the noise properties with the Kondo effect in the investigated material.
Infections with the hepatitis B virus (HBV) or the hepatitis C virus (HCV) lead to complications like the development of cirrhosis or hepatocellular carcinoma. These complications end up in 887,000 and 500,000 deaths per year, respectively. Since the development of new direct acting antiviral agents for HCV in the past years a complete cure of an HCV infection can be achieved in the majority of the patients. In contrast, a complete cure of a chronic HBV infection still remains a challenging problem as current treatment regimens mainly suppress the viral replication and cccDNA as well as integrated DNA still persist in these patients. Several viral and host factors were described to impair the efficacy of treatment regimens or influence the course of the infection. Therefore, in this work viral factors as well as host factors were investigated in HBeAg negative chronic HBV infected patients and in chronic HCV infected patients. In the present study, it was demonstrated that mutations and/or deletions in the HBV basal core promoter (BCP), the precore and the preS domain occur in a genotype-specifc pattern in HBeAg negative HBV infected patients. While the BCP double mutation A1762T/G1764A was found with the highest prevalence in genotype E infected patients, the precore mutation G1896A occurred mostly in genotype B infected patients. Variants in the preS domain could be detected with the highest frequency in patients infected with genotype C. In patients, who had to start an antiviral therapy during the course of the disease, mutations in the precore region could be detected with a higher frequency in the samples right before treatment start in comparison to the baseline sample.
While different HBV genotypes and preS mutations were not associated with HBV-DNA serum levels, precore mutations as well as BCP mutations were significantly associated with HBV-DNA levels. Furthermore, precore mutations showed lower and preS mutations higher HBsAg levels. The HBsAg serum levels varied significantly among the different genotypes. Since HBsAg levels < 1000 IU/ml have been described as a prognostic marker in several studies, the prevalence of patients with HBsAg < 1000 IU/ml was analyzed among the genotypes A - E. While most of the patients infected with HBV genotype B had HBsAg < 1000 IU/ml, only a few patients infected HBV genotype E and A had HBsAg < 1000 IU/ml.
Furthermore, HBV genotype A genomes derived from patients harboring a) A1762T/ G1764A (BCP), b) G1896A/G1899A (precore), c) 15 aa deletion in preS1, d) no mutation (reference genome) were cloned and analyzed in vitro. An enhanced expression but reduced secretion of viral genomes was found in the preS-deletion- and the precore-variant. No differences in the HBsAg production and secretion were observed in the cloned precore- or BCP-variant, while the preS-deletion-variant was characterized with an elevated HBsAg release.
Regarding the secretion of viral and subviral particles, a genotype-specifc pattern of the L/M/SHBs ratio was detected in the serum of patients infected with genotypes A - E. This pattern did not change in the serum of patients, who started antiviral treatment. Secreted HBsAg containing particles displayed a higher density as well as a higher filaments/spheres ratio in genotypes B and D compared to genotypes A, C and E. Population-based and deep sequencing revealed large deletions in the preS domain or preS2 start codon mutations in a certain number of the viral genomes. Theoretically, these mutations/deletions should influence the molecular weight of the expressed protein or abolish the expression of the protein at all. In contrast, LHBs/MHBs were detectable and appeared at the same molecular weight in these patient samples in comparison to patient samples without these mutations. Furthermore, in the in vitro analyses comparing the reference genome and the preS1-deletion genome, it was shown that the deletion indeed influenced the molecular weight of LHBs. Therefore, HBsAg might be expressed from a genetically different source than the released viral genomes, meaning the integrated DNA.
Additionally, in the present study the prevalence of resistance associated substitutions (RASs) in the viral genes NS3, NS5A and NS5B of chronic HCV infected patients was analyzed in correlation to single nucleotide polymorphisms (SNPs) in the interferon-λ4 (IFNL4) gene of the infected patients. No significant correlation was found between IFNL4 SNPs and RASs within NS3/NS5B in the present cohort. In contrast, the frequently detected NS5A RAS Y93H could be significantly associated with beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1-infected patients.
Taken together, the present study demonstrated that viral genome mutations as well as the morphology of secreted particles occur in a genotype-dependent pattern in HBeAg negative HBV infected patients with no need of antiviral therapy. As the amount of serum qHBsAg levels varied among the different genotypes, the HBsAg cut-off < 1000 IU/ml should be adapted individually among the various genotypes. Because the composition of the secreted subviral particles varied between the different genotypes, a genotype-specific immune-response might be induced in these patients. Additionally, the results of the present study indicate that in HBeAg negative HBV infected patients with mutations or deletions in the preS domain MHBs and LHBs might be expressed from the integrated DNA and therefore from a genetically different source than the released viral genomes.
Aside from that, the finding of a significant association of the NS5A RAS Y93H with beneficial IFNL4 SNPs in chronic HCV infected patients may explain a lack of a correlation or an inverse correlation of treatment response with the IFNL4 genotype in some NS5A inhibitor-containing IFN-free regimens.
Evoked potentials (EPs) are well established in clinical practice for diagnosis and prognosis in multiple sclerosis (MS). However, their value is limited to the assessment of their respective functional systems. Here, we used transcranial magnetic stimulation (TMS) coupled with electroencephalography (TMS-EEG) to investigate cortical excitability and spatiotemporal dynamics of TMS-evoked neural activity in MS patients. Thirteen patients with early relapsing–remitting MS (RRMS) with a median Expanded Disability Status Scale (EDSS) of 1.0 (range 0–2.5) and 16 age- and gender-matched healthy controls received single-pulse TMS of left and right primary motor cortex (L-M1 and R-M1), respectively. Resting motor threshold for L-M1 and R-M1 was increased in MS patients. Latencies and amplitudes of N45, P70, N100, P180, and N280 TMS-evoked EEG potentials (TEPs) were not different between groups, except a significantly increased amplitude of the N280 TEP in the MS group, both for L-M1 and R-M1 stimulation. Interhemispheric signal propagation (ISP), estimated from the area under the curve of TEPs in the non-stimulated vs. stimulated M1, also did not differ between groups. In summary, findings show that ISP and TEPs were preserved in early-stage RRMS, except for an exaggerated N280 amplitude. Our findings indicate that TMS-EEG is feasible in testing excitability and connectivity in cortical neural networks in MS patients, complementary to conventional EPs. However, relevance and pathophysiological correlates of the enhanced N280 will need further study.
Background: The recurrence rate in lumbar disc herniations (LDH) has been reported between 5 and 25%. There are only few data about this phenomenon that occurs within days of the initial operation. We analyse early recurrent LDH by analysis of data from the German Spine register.
Methods: Data from patients undergoing disc herniation surgery in the lumbar region were extracted from the German Spine Registry between 1st January 2012 and 31st December 2016. Patients with early recurrent LDH within days of initial surgery were separately analysed.
Results: A total of 9310 surgeries for LDH were documented in the German Spine Register. From these patients 115 (1.2%) presented an early recurrent disc surgeries within days of the initial surgery. The mean age was 70 ± 2.50 years. Most affected segment was L4/5 (47 cases, 41%), followed by L3/4 (45 cases, 39%). The most of our patients showed a normal or overweight Body Mass Index. Surgery for early recurrent LDH was associated with a high rate of incidental durotomies (20 cases, 17.6%). In 3 cases (2.6%) therapy with a lumbar drain was necessary.
Conclusions: The rate of early recurrent LDH within days of surgery is 1.2%. Age seems to be an important factor in early recurrent LDH while obesity does not. The data of the German Spine Register seems to have a reliable data collection system that can perform multicentre data analysis. The databases from this Register could be used in the future for various purposes, such as the evaluation of multicentre surgical techniques, results in patients with various surgical procedures and basic research in spine surgery.
Background: Juvenile dermatomyositis (JDM) is the most common inflammatory myopathy in childhood and a major cause of morbidity among children with pediatric rheumatic diseases. The management of JDM is very heterogeneous. The JDM working group of the Society for Pediatric Rheumatology (GKJR) aims to define consensus- and practice-based strategies in order to harmonize diagnosis, treatment and monitoring of JDM.
Methods: The JDM working group was established in 2015 consisting of 23 pediatric rheumatologists, pediatric neurologists and dermatologists with expertise in the management of JDM. Current practice patterns of management in JDM had previously been identified via an online survey among pediatric rheumatologists and neurologists. Using a consensus process consisting of online surveys and a face-to-face consensus conference statements were defined regarding the diagnosis, treatment and monitoring of JDM. During the conference consensus was achieved via nominal group technique. Voting took place using an electronic audience response system, and at least 80% consensus was required for individual statements.
Results: Overall 10 individual statements were developed, finally reaching a consensus of 92 to 100% regarding (1) establishing a diagnosis, (2) case definitions for the application of the strategies (moderate and severe JDM), (3) initial diagnostic testing, (4) monitoring and documentation, (5) treatment targets within the context of a treat-to-target strategy, (6) supportive therapies, (7) explicit definition of a treat-to-target strategy, (8) various glucocorticoid regimens, including intermittent intravenous methylprednisolone pulse and high-dose oral glucocorticoid therapies with tapering, (9) initial glucocorticoid-sparing therapy and (10) management of refractory disease.
Conclusion: Using a consensus process among JDM experts, statements regarding the management of JDM were defined. These statements and the strategies aid in the management of patients with moderate and severe JDM.
The results of this thesis lie in the area of convex algebraic geometry, which is the intersection of real algebraic geometry, convex geometry, and optimization.
We study sums of nonnegative circuit polynomials (SONC) and their related cone, both geometrically and in application to polynomial optimization. SONC polynomials are certain sparse polynomials having a special structure in terms of their Newton polytopes and supports, and serve as a certificate of nonnegativity for real polynomials, which is independent of sums of squares.
The first part of this thesis is dedicated to the convex geometric study of the SONC cone. As main results we show that the SONC cone is full-dimensional in the cone of nonnegative polynomials, we exactly determine the number of zeros of a nonnegative circuit polynomial, and we give a complete and explicit characterization of the number of zeros of SONC polynomials and forms. Moreover, we provide a first approach to the study of the exposed faces of the SONC cone and their dimensions.
In the second part of the thesis we use SONC polynomials to tackle constrained polynomial optimization problems (CPOPs).
As a first step, we derive a lower bound for the optimal value of CPOP based on SONC polynomials by using a single convex optimization program, which is a geometric program (GP) under certain assumptions. GPs are a special type of convex optimization problems and can be solved in polynomial time. We test the new method experimentally and provide examples comparing our new SONC/GP approach with Lasserre's relaxation, a common approach for tackling CPOPs, which approximates nonnegative polynomials via sums of squares and semidefinite programming (SDP). The new approach comes with the benefit that in practice GPs can be solved significantly faster than SDPs. Furthermore, increasing the degree of a given problem has almost no effect on the runtime of the new program, which is in sharp contrast to SDPs.
As a second step, we establish a hierarchy of efficiently computable lower bounds converging to the optimal value of CPOP based on SONC polynomials. For a given degree each bound is computable by a relative entropy program. This program is also a convex optimization program, which is more general than a geometric program, but still efficiently solvable via interior point methods.
Testicular germ cell cancer in a metastatic state is curable with a cisplatin‑based first line chemotherapy. However, 10‑15% of these patients are resistant to first line chemotherapy and are thus left with only palliative options. Immunotherapies and inhibition of angiogenesis used in multiple types of cancer; however, the molecular context of angiogenesis and immune checkpoints in the development and progression of testicular cancers is still unknown. Therefore, the present study performed tissue micro array based analysis of 84 patients with immunohistochemistry of programmed cell death protein 1 (PD‑1), programmed cell death ligand 1 (PD‑L1) and vascular endothelial growth factor receptor 2 (VEGFR2) of testicular cancer and corresponding normal appearing testis tissue, matching the results with clinical data. The results demonstrated that PD‑L1 was significantly upregulated in testicular tumors and that PD‑1 positive cells significantly infiltrated the testicular tumor when compared with normal testicular tissue. VEGFR2 was significantly upregulated in testicular cancer. It was indicated that PD‑1 expressing cytotoxic cells may require pathologic tumor vessels to pass the blood‑testis‑barrier in order to migrate into the tumor. Notably, when matching the clinical data for PD‑1, PD‑L1 and VEGFR2 there were no differences in expression in the different International Germ Cell Cancer Collaborative Group stages of non‑seminoma. These data suggested that the anti‑PD‑1/PD‑L1 immunotherapy and the anti‑angiogenic therapy, sequentially or in combination, may be a promising option in the treatment of testicular cancer.
According to embodied cognition accounts, viewing others’ facial emotion can elicit the respective emotion representation in observers which entails simulations of sensory, motor, and contextual experiences. In line with that, published research found viewing others’ facial emotion to elicit automatic matched facial muscle activation, which was further found to facilitate emotion recognition. Perhaps making congruent facial muscle activity explicit produces an even greater recognition advantage. If there is conflicting sensory information, i.e., incongruent facial muscle activity, this might impede recognition. The effects of actively manipulating facial muscle activity on facial emotion recognition from videos were investigated across three experimental conditions: (a) explicit imitation of viewed facial emotional expressions (stimulus-congruent condition), (b) pen-holding with the lips (stimulus-incongruent condition), and (c) passive viewing (control condition). It was hypothesised that (1) experimental condition (a) and (b) result in greater facial muscle activity than (c), (2) experimental condition (a) increases emotion recognition accuracy from others’ faces compared to (c), (3) experimental condition (b) lowers recognition accuracy for expressions with a salient facial feature in the lower, but not the upper face area, compared to (c). Participants (42 males, 42 females) underwent a facial emotion recognition experiment (ADFES-BIV) while electromyography (EMG) was recorded from five facial muscle sites. The experimental conditions’ order was counter-balanced. Pen-holding caused stimulus-incongruent facial muscle activity for expressions with facial feature saliency in the lower face region, which reduced recognition of lower face region emotions. Explicit imitation caused stimulus-congruent facial muscle activity without modulating recognition. Methodological implications are discussed.
We develop a model that reproduces the average return and volatility spread between sin and non-sin stocks. Our investors do not necessarily boycott sin companies. Rather, they are open to invest in any company while trading off dividends against ethicalness. We show that when dividends and ethicalness are complementary goods and investors are sufficiently risk averse, the model predicts that the dividend share of sin companies exhibits a positive relation with the future return and volatility spreads. Our empirical analysis supports the model's predictions.
Optogenetics offers a unique method to regulate the activity of select neural circuits. However, the electrophysiological consequences of targeted optogenetic manipulation upon the entire circuit remain poorly understood. Analysis of the sensory-CNS-motor circuit in Drosophila larvae expressing eHpHR and ChR2-XXL revealed unexpected patterns of excitability. Optical stimulation of motor neurons targeted to express eNpHR resulted in inhibition followed by excitation of body wall contraction with repetitive stimulation in intact larvae. In situ preparations with direct electrophysiological measures showed an increased responsiveness to excitatory synaptic activity induced by sensory stimulation within a functional neural circuit. To ensure proper function of eNpHR and ChR2-XXL they were expressed in body wall muscle and direct electrophysiological measurements were obtained. Under eNpHR induced hyperpolarization the muscle remained excitable with increased amplitude of excitatory postsynaptic synaptic potentials. Theoretical models to explain the observations are presented. This study aids in increasing the understanding of the varied possible influences with light activated proteins within intact neural circuits.
In the last decade, central bank interventions, flights to safety, and the shift in derivatives clearing resulted in exceptionally high demand for high quality liquid assets, such as German treasuries, in the securities lending market besides the traditional repo market activities. Despite the high demand, the realizable securities lending income has remained economically negligible for most beneficial owners. We provide empirical evidence of pricing inefficiencies in the non-transparent, oligopolistic securities lending market for German treasuries from 2006 to 2015. Consistent with Duffie, Gârleanu and Pedersen (2005)’s theory, we find that the less connected market participants’ interests are underrepresented, evident in the longer maturity segment, where lenders are more likely to be conservative passive investors, such as pension funds and insurance firms. The low price elasticity in this segment hinders these beneficial owners to fully capitalize on the additional income from securities lending, giving rise to important negative welfare implications.
Cervical spine injuries are frequent and often caused by a blunt trauma mechanism. They can have severe consequences, with a high mortality rate and a high rate of neurological lesions.Diagnosis is a three-step process: 1) risk assessment according to the history and clinical features, guided by a clinical decision rule such as the Canadian C-Spine rule; 2) imaging if needed; 3) classification of the injury according to different classification systems in the different regions of the cervical spine.The urgency of treatment is dependent on the presence of a neurological lesion and/or instability. The treatment strategy depends on the morphological criteria as defined by the classification.
In this study we investigate which economic ideas were prevalent in the macroprudential discourse post-crises in order to understand the availability of ideas for reform minded agents. We base our analysis on new findings in the field of ideational shifts and regulatory science, which posit that change-agents engage with new ideas pragmatically and strategically in their effort to have their economic ideas institutionalized. We argue that in these epistemic battles over new regulation, scientific backing by academia is the key resource determining the outcome. We show that the present reforms implemented internationally follow this pattern. In our analysis we contrast the entire discourse on systemic risk and macroprudential regulation with Borio’s initial 2003 proposal for a macroprudential framework. We find that mostly cross-sectional measures targeted towards increasing the resilience of the financial system rather than inter-temporal measures dampening the financial cycle have been implemented. We provide evidence for the lacking support of new macroprudential thinking within academia and argue that this is partially responsible for the lack of anti-cyclical macroprudential regulation. Most worryingly, the financial cycle is largely absent in the academic discourse and is only tacitly assumed instead of fully fledged out in technocratic discourses, pointing to the possibility that no anti-cyclical measures will be forthcoming.
Background: Conversion from calcineurin inhibitor (CNI) therapy to everolimus within 6 months after kidney transplantation improves long-term graft function but can increase the risk of mild biopsy-proven acute cellular rejection (BPAR). We performed a post-hoc analysis of histological data from a randomized trial in order to further analyze histologic information obtained from indication and protocol biopsies up to 5 years after transplantation.
Methods: Biopsy samples obtained up to 5 years post-transplant were analyzed from the randomized ZEUS study, in which kidney transplant patients were randomized at month 4.5 to switch to everolimus (n = 154) or remain on cyclosporine (CsA)-based immunosuppression (n = 146). All patients received mycophenolate and steroids.
Results: At least one investigator-initiated biopsy was undertaken in 53 patients in each group between randomization and year 5, with a mean (SD) of 2.6 (1.7) and 2.2 (1.4) biopsies per patient in the everolimus and CsA groups, respectively. In the everolimus and CsA groups, investigator-initiated biopsies showed (i) BPAR in 12.3 and 7.5% (p = 0.182) of patients, respectively, with episodes graded mild in 22/24 and 18/20 cases (ii) CsA toxicity lesions in 4.5 and 10.3% of patients (p = 0.076) (iii) antibody-mediated rejection in 0.6 and 2.7% of patients (p = 0.204), respectively.
Conclusions: This analysis of histological findings in the ZEUS study to 5 years after kidney transplantation shows no increase in antibody-mediated rejection under everolimus-based therapy with a lower rate of CNI-related toxicity compared to a conventional CsA-based regimen, and confirms the preponderance of mild BPAR seen in the main study after the early switch to CsA-free everolimus therapy.
Trial registration: ClinicalTrials.gov NCT00154310. Date of registration: September 12, 2005.
Background: Subdural hematoma (SDH) is a common disease associated with high morbidity, which is becoming more prominent due to the increasing incidence. Decision for a surgical evacuation is made depending on the clinical appearance and the volume of SDH, wherefore it is important to have a simple ‘bedside’ method to measure and compare the volume of SDH.
Objective: The aim of the study was to verify the accuracy of the simplified ABC/2 volumetric formula to determine a valuable tool for the clinical practice.
Methods: Preoperative CT-scans of 83 patients with SDHs were used for the computer-assisted volumetric measurement via BrainLab® as well as the ABC/2 volumetric measurement. A = largest length (anterior to posterior) of the SDH; B = maximum width (lateral to midline) 90° to A; C = maximum height (coronal plane or multiplication of slices) of the hematoma. These measurements were performed by two independent clinicians in a blinded fashion. Both volumes were compared by linear regression analysis of Pearson and Bland-Altman regression analysis.
Results: Among 100 SDHs, 53% were under an 47% were over 100cm3 showing a well distribution of the hematoma sizes. There was an excellent correlation between computer-assisted volumetric measurement and ABC/2 (R2 = 0.947, p<0.0001) and no undesirable deviation and trend were detected (p = 0.101; p = 0.777). A 95% tolerance region of the ratios of both methods was [0.805–1.201].
Conclusion: The ABC/2 method is a simple and fast bedside formula for the measurement of SDH volume in a timely manner without limited access through simple adaption, which may replace the computer-assisted volumetric measurement in the clinical and research area. Reason for the good accuracy seems to be the spherical form of SDH, which has a similarity to a half ellipsoid.
Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These “molecular vestiges” provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.
Since its founding in 1993 the International Long-term Ecological Research Network (ILTER) has gone through pronounced development phases. The current network comprises 44 active member LTER networks representing 700 LTER Sites and ~ 80 LTSER Platforms across all continents, active in the fields of ecosystem, critical zone and socio-ecological research. The critical challenges and most important achievements of the initial phase have now become state-of-the-art in networking for excellent science. At the same time increasing integration, accelerating technology, networking of resources and a strong pull for more socially relevant scientific information have been modifying the mission and goals of ILTER. This article provides a critical review of ILTER's mission, goals, development and impacts. Major characteristics, tools, services, partnerships and selected examples of relative strengths relevant for advancing ILTER are presented. We elaborate on the tradeoffs between the needs of the scientific community and stakeholder expectations. The embedding of ILTER in an increasingly collaborative landscape of global environmental observation and ecological research networks and infrastructures is also reflected by developments of pioneering regional and national LTER networks such as SAEON in South Africa, CERN/CEOBEX in China, TERN in Australia or eLTER RI in Europe. The primary role of ILTER is currently seen as a mechanism to investigate ecosystem structure, function, and services in response to a wide range of environmental forcings using long-term, place-based research. We suggest four main fields of activities and advancements for the next decade through development/delivery of a: (1) Global multi-disciplinary community of researchers and research institutes; (2) Strategic global framework and strong partnerships in ecosystem observation and research; (3) Global Research Infrastructure (GRI); and (4) a scientific knowledge factory for societally relevant information on sustainable use of natural resources.
The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is well known for its ability to successfully degrade various mixtures of n-alkanes occurring in marine oil spills. For effective growth on these compounds, the bacteria possess the unique capability not only to incorporate but also to modify fatty intermediates derived from the alkane degradation pathway. High efficiency of both these processes provides better competitiveness for a single bacteria species among hydrocarbon degraders. To examine the efficiency of A. borkumensis to cope with different sources of fatty acid intermediates, we studied the growth rates and membrane fatty acid patterns of this bacterium cultivated on diesel, biodiesel and rapeseed oil as carbon and energy source. Obtained results revealed significant differences in both parameters depending on growth substrate. Highest growth rates were observed with biodiesel, while growth rates on rapeseed oil and diesel were lower than on the standard reference compound (hexadecane). The most remarkable observation is that cells grown on rapeseed oil, biodiesel, and diesel showed significant amounts of the two polyunsaturated fatty acids linoleic acid and linolenic acid in their membrane. By direct incorporation of these external fatty acids, the bacteria save energy allowing them to degrade those pollutants in a more efficient way. Such fast adaptation may increase resilience of A. borkumensis and allow them to strive and maintain populations in more complex hydrocarbon degrading microbial communities.
The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.
The photoregulation of nucleic acids by azobenzene photoswitches has recently attracted considerable interest in the context of emerging biotechnological applications. To understand the mechanism of photoinduced isomerisation and conformational control in these complex biological environments, we employ a Quantum Mechanics/Molecular Mechanics (QM/MM) approach in conjunction with nonadiabatic Surface Hopping (SH) dynamics. Two representative RNA–azobenzene complexes are investigated, both of which contain the azobenzene chromophore covalently attached to an RNA double strand via a β-deoxyribose linker. Due to the pronounced constraints of the local RNA environment, it is found that trans-to-cis isomerization is slowed down to a time scale of ∼10–15 picoseconds, in contrast to 500 femtoseconds in vacuo, with a quantum yield reduced by a factor of two. By contrast, cis-to-trans isomerization remains in a sub-picosecond regime. A volume-conserving isomerization mechanism is found, similarly to the pedal-like mechanism previously identified for azobenzene in solution phase. Strikingly, the chiral RNA environment induces opposite right-handed and left-handed helicities of the ground-state cis-azobenzene chromophore in the two RNA–azobenzene complexes, along with an almost completely chirality conserving photochemical pathway for these helical enantiomers.
Drug-induced liver injury (DILI) has become a major problem for patients and for clinicians, academics and the pharmaceutical industry. To date, existing hepatotoxicity test systems are only poorly predictive and the underlying mechanisms are still unclear. One of the factors known to amplify hepatotoxicity is the tumor necrosis factor alpha (TNFα), especially due to its synergy with commonly used drugs such as diclofenac. However, the exact mechanism of how diclofenac in combination with TNFα induces liver injury remains elusive. Here, we combined time-resolved immunoblotting and live-cell imaging data of HepG2 cells and primary human hepatocytes (PHH) with dynamic pathway modeling using ordinary differential equations (ODEs) to describe the complex structure of TNFα-induced NFκB signal transduction and integrated the perturbations of the pathway caused by diclofenac. The resulting mathematical model was used to systematically identify parameters affected by diclofenac. These analyses showed that more than one regulatory module of TNFα-induced NFκB signal transduction is affected by diclofenac, suggesting that hepatotoxicity is the integrated consequence of multiple changes in hepatocytes and that multiple factors define toxicity thresholds. Applying our mathematical modeling approach to other DILI-causing compounds representing different putative DILI mechanism classes enabled us to quantify their impact on pathway activation, highlighting the potential of the dynamic pathway model as a quantitative tool for the analysis of DILI compounds.
Background: Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context.
Methods: CIK cells with EBV-specificity were generated from peripheral blood mononuclear cells (PBMCs), expanded in the presence of interferon-γ, anti-CD3, interleukin (IL)-2 and IL-15 and were pulsed twice with EBV consensus peptide pool. CIK cells with EBV-specificity and conventional CIK cells were phenotypically and functionally analyzed. Additionally, CIK cells with EBV-specificity were applied to a patient with EBV-related PTLD rapidly progressing to highly aggressive B-cell lymphoma on a compassionate use basis after approval and agreement by the regulatory authorities.
Results: Pre-clinical analysis showed that generation of CIK cells with EBV-specificity was feasible. In vitro cytotoxicity analyses showed increased lysis of EBV-positive target cells, enhanced proliferative capacity and increased secretion of cytolytic and proinflammatory cytokines in the presence of EBV peptide-displaying target cells. In addition, 1 week after infusion of CIK cells with EBV-specificity, the patient's highly aggressive B-cell lymphoma persistently disappeared. CIK cells with EBV-specificity remained detectable for up to 32 days after infusion and infusion did not result in acute toxicity.
Discussion: The transfer of both anti-cancer potential and T-cell memory against EBV infection provided by EBV peptide-induced CIK cells might be considered a therapy for EBV-related PTLD.
Im Rahmen dieser Arbeit wurden sRNAs des halophilen Archaeons Haloferax volcanii hinsichtlich ihrer biologischen und ihrer regulatorischen Funktion charakterisiert.
Um einen Überblick über die biologischen Funktionen archaealer sRNAs zu erhalten, wurde eine umfassende phänotypische Charakterisierung von 27 sRNA-Deletionsmutanten im Vergleich zum Wildtyp ausgewertet. Im Zuge dieser phänotypischen Charakterisierungen wurden zehn verschiedene Wachstumsbedingungen, morphologische Unterschiede und Veränderungen in der Zellmotilität untersucht. Hierbei zeigten nahezu alle Deletionsmutanten unter mindestens einer der getesteten Bedingungen phänotypische Unterschiede. Durch den Verlust von sRNAs wurden sowohl sogenannte Gain-of-function als auch Loss-of-function Phänotypen beobachtet. Haloarchaeale sRNAs spielen eine wichtige Rolle beim Wachstum mit verschiedenen Salzkonzentrationen, mit verschiedenen Kohlenstoffquellen und beim Schwärmverhalten, sind jedoch weniger in die Adaptation an diverse Stressbedingungen involviert.
Zur näheren Charakterisierung der regulatorischen Funktion archaealer sRNAs wurden sRNA362, sRNAhtsf468 und sRNA479 mittels molekulargenetischer Methoden wie Northern Blot-Analyse und DNA-Mikroarray sowie bioinformatischer in silico-Analyse untersucht. Das Expressionslevel von sRNA362 konnte bestimmt und potentielle Zielgene für sRNAhtsf468 und sRNA479 identifiziert werden.
Eine vorangegangene Studie zeigte den Einfluss von sRNA30 unter Hitzestress und führte zur Identifikation differentiell produzierter Proteine in Abwesenheit der sRNA. In dieser Arbeit wurde mittels Northern Blot-Analysen die Expression der sRNA30 charakterisiert. Das Wachstum in An- und Abwesenheit von sRNA30 wurde bei 42°C und 51°C phänotypisch charakterisiert und der regulatorische Einfluss der sRNA auf die mRNA differentiell regulierter Proteine durch Northern Blot-Analyse überprüft. Eine Transkriptomanalyse mittels DNA-Mikroarray nach Hitzeschock-Induktion führte zur Identifikation differentiell regulierter Gene involviert in Transportprozesse, Metabolismus, Transkriptionsregulation und die Expression anderer sRNAs. Die differentielle Regulation des Proteoms nach Hitzeschockinduktion in An- und Abwesenheit von sRNA30 konnte bestätigt werden.
Desweiteren wurde in dieser Arbeit sRNA132 und deren phosphatabhängige Regulation der Ziel-mRNA HVO_A0477-80 näher charakterisiert. Eine Induktionskinetik nach Phosphatentzug bestätigte die Bedeutung von sRNA132 für die verstärkte Expression des Operons HVO_A0477-80 unter Phosphatmangel-Bedingungen und verwies auf die Existenz weiterer Regulationsmechanismen. Während vor und nach Phosphatentzug kein Unterschied bezüglich der Zellmorphologie von Wildtyp und Deletionsmutante zu erkennen war, führte das Wachstum mit einem starken Phosphatüberschuss von 5 mM zu einer Zellverlängerung der Deletionsmutante. Die Kompetition der nativen 3‘-UTR des Operons HVO_A0477-80 mit einer Vektor-kodierten artifiziellen 3‘-UTR legt eine Regulation über die Bindung von sRNA132 an die 3‘-UTR nahe. Der Transkriptomvergleich nach Phosphatentzug in An- und Abwesenheit von sRNA132 führte zur Identifikation des Phosphoregulons der sRNA. Zu diesem Phosphoregulon gehören unter anderem zwei Glycerinphosphat-Dehydrogenasen, Transkriptionsregulatoren, eine Polyphosphatkinase und eine Glycerolphosphodiesterase. Zudem waren die Transkriptlevel der beiden ABC-Transporter HVO_A0477-80 und HVO_2375-8 für anorganisches Phosphat und des Transporters HVO_B0292-5 für Glycerinaldehyd-3-Phosphat in Abwesenheit der sRNA verringert. Die beiden ABC-Transportsysteme für anorganisches Phosphat wurden im Rahmen dieser Arbeit deletiert und weiter charakterisiert. Es konnte gezeigt werden, dass das ABC-Transportsystem HVO_2375-8 bei geringen Phosphatkonzentrationen leicht induziert wird und das Transkriptlevel in Anwesenheit von sRNA132 erhöht ist. Wachstumsversuche der jeweiligen Deletionsmutante in direkter Konkurrenz mit dem Wildtyp zeigten, dass keiner der beiden ABC-Transporter den anderen vollständig ersetzen kann und der Wildtyp mit beiden intakten ABC-Transportern unter phosphatlimitierenden Bedingungen einen Wachstumsvorteil besitzt. In silico-Analysen der Promotorbereiche von sRNA und ABC-Transporter legen zudem die Existenz von P-Boxen nahe.
SAFE Newsletter : 2018, Q2
(2018)
Cross-border exchange and comparison of forensic DNA data in the context of the Prüm decision
(2018)
This study, commissioned by the European Parliament’s Policy Department for Citizens’ Rights and Constitutional Affairs at the request of the LIBE Committee, provides an overview of the Prüm regime. It first considers the background of the Prüm Convention and Prüm Decision. The subsequent two chapters summarize the Prüm regime in relation mainly to DNA data looking at value and shortcomings; and ethical, legal and social implications of forensic DNA typing and databasing in relation to the Prüm regime. Finally, based on the analysis, it provides the policy recommendations.
Crystal growth and characterization of cerium- and ytterbium-based quantum critical materials
(2018)
In der Festkörperphysik werden heutzutage Themen wie Supraleitung, Magnetismus und Quantenkritikalität sowohl von experimenteller als auch von theoretischer Seite stark untersucht. Quantenkritikalität und Quantenphasenübergänge können in Systemen erforscht werden, für welche ein Kontroll Parameter existiert, durch den z.B. eine magnetische Ordnung soweit unterdrückt wird, bis der Phasenübergang bei Null Kelvin, bei einem quantenkritischen Punkt (QCP), stattfindet. Vorzugsweise wird quantenkritisches Verhalten an Einkristallen untersucht, da diese in sehr reiner Qualität gezüchtet werden können und da deren gemessenen physikalischen Eigenschaften ausschließlich intrinsisch sind und nicht durch Verunreinigungseffekte überlagert werden. Der Schwerpunkt dieser Arbeit lag auf der Züchtung von Einkristallen und der Charakterisierung von Materialien, die quantenkritische Phänomene aufweisen. Als Ausgangsstoffe dienten dabei Elemente höchstmöglicher Reinheit. Es wurden die Serie YbNi4(P1-xAsx)2 mit einem ferromagnetischen QCP bei x=0,1, die Verbindung YbRh2Si2 mit einem feldinduzierten QCP bei Bcrit = 60mT und die Serie Ce(Ru1-xFex)PO mit einem QCP bei x = 0,86 untersucht. Für alle Verbindungen wurde das Züchtungsverfahren entwickelt, dann wurden Einkristalle gezüchtet und charakterisiert. Die Züchtung wurde zum einen mittels der Bridgman-Methode, zum anderen mit der Czochralski Methode durchgeführt. Neben struktureller und chemischer Charakterisierung der Einkristalle mittels Röntgen-Pulverdiffraktometrie, Laue-Methode und Energie-dispersiver Röntgen-Spektroskopie, wurden auch deren spezifische Wärme, elektrischer Widerstand und Magnetisierung im Temperaturbereich 1,8 – 300 K untersucht. Im weiteren Verlauf wurden die Kristalle in verschiedenen Kooperationen untersucht und bis in den Tieftemperatur- Bereich (20 mK), bei YbRh2Si2 bis in den Submillikelvin-Bereich, charakterisiert. Ausserdem wurden im Rahmen dieser Dissertation Einkristalle weiterer antiferromagnetischer Verbindungen SmRh2Si2, GdRh2Si2, GdIr2Si2, HoRh2Si2 und HoIr2Si2 gezüchtet. Bei diesen Verbindungen stand die Untersuchung elektronischer Oberflächenzustände mittels winkelaufgelöster Photoemissionsspektroskopie im Vordergrund.
Die Forschungsarbeit leistet einen kriminologischen Beitrag zur Systematisierung des Phänomens der sekundären Viktimisierung bei Opfern sexualisierter Gewalt und zeigt gleichzeitig Präventionsansätze auf, die sekundäre Viktimisierung verhindern sollen.
„Sekundäre Viktimisierung“ als die sogenannte „zweite Opferwerdung“ durch soziale Fehlreaktionen einzelner Personen oder gesellschaftlicher Gruppen sowie die Prävention dieses Phänomens ist im Detail noch wenig erforscht. Der Fokus des Forschungs- und Erkenntnisinteresses richtet sich auf die Zielgruppe der Opfer sexualisierter Gewalt, die in ihrer Kindheit sexuell missbraucht und als Erwachsene sekundär viktimisiert wurden. Es wird angenommen, dass Sexualstraftaten, die von den Opfern im Kindesalter erlitten werden, in besonderem Maße die persönliche Unversehrtheit verletzen und dass somit eine zusätzliche sekundäre Viktimisierung als besonders belastend empfunden wird.
Zunächst wird im theoretischen Teil auf wesentliche Begriffe wie sexualisierte Gewalt in Verbindung mit primärer und sekundärer Viktimisierung eingegangen und nimmt deren kriminologische Einordnung vor, stellt das Ausmaß sowie die Spezifika des Phänomens in den Fokus. Dabei wird zunächst der Opferbegriff ausführlich diskutiert, wobei der Opferperspektive viel Raum gegeben wird. Ein weiterer Schwerpunkt liegt auf der Darstellung des aktuellen Forschungsstandes zur sekundären Viktimisierung, insbesondere mit Bezug zu sexualisierter Gewalt.
Im Zentrum des qualitativen Forschungsansatzes stehen folgende Fragen: „Welche Strukturen prägen die Situationen sekundärer Viktimisierung?“ und „Wie ist Prävention von sekundärer Viktimisierung möglich?“. Auf der Grundlage der Forschungsergebnisse wurde ein Präventionskonzept mit praxisorientierten Empfehlungen entwickelt. Zunächst sind typische Strukturen sekundärer Viktimisierung analysiert und in einem Modell verdeutlicht worden. Es ist zu unterscheiden zwischen Strukturen, die sekundäre Viktimisierung begünstigen (Risikofaktoren) sowie Strukturen, die sekundärer Viktimisierung vorbeugen (Schutzfaktoren). Anhand der identifizierten Schutzfaktoren entstand das Modell zur Prävention sekundärer Viktimisierung, aus denen konkrete Präventionsansätze abzuleiten sind.
Hervorgehoben wird die kriminologische Orientierung der Arbeit; wenngleich die Kriminologie interdisziplinär einzuordnen ist, dominiert bei der Analyse die kriminalsoziologische Verortung und speziell die viktimologische Ausrichtung.
Die Tagung rief dazu auf, die Verbindlichkeit von Versprechen als allgemeines Phänomen zu untersuchen, also zu ergründen, inwiefern man unabhängig von ihrer Ausgestaltung in konkreten Rechtsordnungen oder anderen Normensystemen von einer "vor-rechtlichen" Verbindlichkeit sprechen kann. Literarische Texte aller Zeiten bis hin zu den Mythen bezeugen die Bindung an das Wort als Urerfahrung, die von jeder gesellschaftlichen Konvention unabhängig erscheint. Ebenso kennen sie aber Verrat und offenen oder verdeckten Wortbruch, deren Folgen sie thematisieren. Strukturelle Verwandtschaften einerseits und gegenseitige Beeinflussung andererseits der literarischen und der rechtlichen Auseinandersetzung mit dem Versprechen sollten untersucht werden.
This paper examines whether gender features (masculine, feminine, neuter) in German have to be interpreted semantically, along their specific gender, or whether they allow for a gender unrelated interpretation. As to this, two experiments with two different classes of nouns (gender marked and sex marked nouns vs. gender marked and sex neutral nouns) were conducted. The first experiment supports the view that in their function as nominal predicates masculine nouns, contrary to feminine (and neuter) nouns, have the widest extension – which confirms the existence of a ‘Generic Masculine’ (Generisches Maskulinum). On the other hand, the second experiment shows that in their function as subjects masculine nouns, contrary to feminine (and neuter) nouns, are the least flexible agreement controllers – hardly allowing for gender mismatches. Thus, masculine nouns behave differently depending on whether they appear as controllers/sources of agreement or as targets of agreement. The findings are supplemented by corpus data.
"Habt ihr schon mal davon gehört gehabt?" Fällt Ihnen bei diesem Satz etwas auf? Wie würden Sie den Satz interpretieren, insbesondere die Zeitform des Prädikates hören? Weist sie, Ihrer Meinung nach, eher auf Expressivität, seine Abgeschlossenheit, die (Vor-)Vorvergangenheit eines Geschehens oder eine einfache Vergangenheit hin? Im letzteren Fall würde der Satz die gleiche Semantik ausdrücken wie ohne das zweite Partizip II: Habt ihr schon mal davon gehört? Im Fokus dieser Arbeit stehen empirische Evidenzen zum Gebrauch des doppelten Perfekts und Plusquamperfekts in der deutschen Sprache. Im Rahmen dieser Untersuchung wurde ein Fragebogen mit 202 deutschen Muttersprachlern durchgeführt. Die Ergebnisse dieser Studie zeigen, dass das doppelte Plusquamperfekt bei der Interpretation von ca. 86% der untersuchten deutschen Muttersprachler akzeptiert wird. Weiterhin deuten die Ergebnisse dieser Studie auf viele Unterschiede bei der Akzeptanz der doppelten Konstruktionen zwischen Studierenden verschiedener Fachrichtungen hin.
In the paper, German disintegrated verb-final 'obwohl' (‘although’) and 'weil' (‘because’) clauses are compared with constructions in which 'obwohl' and 'weil' precedes clauses with main clause word order. The former constructions constitute independent, yet subsidiary speech acts. Thus, the subordinating connectors and the positioning of the verb do not indicate syntactic but textual dependency. The latter constructions are of a very different kind. Here, 'obwohl' and 'weil' do not form a constituent with the following clause. Instead, they appear as syntactically independent discourse markers connecting two discourse units. As discourse markers, 'obwohl' and 'weil' obtain their special syntactic and semantic properties as elements of the derived, but independent module of Thetic Grammar.
This paper deals with German 'wobei'-clauses and their Italian counterparts. Based on a corpus study of administrative texts, we identify the type and frequency of the Italian constructions that correspond to 'wobei'-clauses. In particular, we will assess to what extent the Italian converb construction gerundio correlates with 'wobei'-clauses. More specifically, we will focus on the thesis put forward by Haspelmath (1995) and Breindl (2014), according to which comitativity is expressed by converb constructions when it applies to state of affairs.
'Je-desto'-Sätze scheinen in struktureller Hinsicht Einzelgänger zu sein. Das Ungewöhnliche ist, dass sie wie eine obligatorische Verb-dritt-Konstruktion daherkommen: An erster Stelle steht scheinbar der durch je eingeleitete Nebensatz im linken Außenfeld bzw. Vor-vor-Feld, dann folgt die desto-Konstituente, die das Vorfeld einnimmt, und dann an dritter Stelle das finite Verb des Matrixsatzes. Angesichts der Semantik der involvierten Konstituenten ist diese Strukturbeschreibung ungewöhnlich und widerspricht plausiblen Erwartungen. Der Aufsatz bietet eine Analyse, nach der der 'je'-Satz und die 'desto'-Konstituente zusammen eine komplexe Konstituente bilden, die eine einzige, ganz reguläre Einheit konstituiert, was bedeutet, dass der Gesamtsatz eine ziemlich reguläre Verb-zweit-Struktur ist.