Entwicklung einer Webapplikation zur Verwaltung von Ergebnissen digitaler Fragebögen

Fragebögen werden häufig in den Bereichen der Psychologie und dem Gesundheitswesen eingesetzt, um gezielt und effizient große Mengen an personenbezogenen Daten zu erheben. Diese werden bis heute größtenteils papierbasiert verwendet, obwohl dies mehrere Nachteile mit sich bringt. Das QuestionSys Framework, das an der Universität Ulm entwickelt wurde, versucht die Nachteile der Erhebung von Daten durch papierbasierte Fragebögen zu eliminieren, indem der gesamte Prozess digitalisiert wird. Das Framework bietet dem Nutzer die Möglichkeit digitale Fragebögen mit einer Konfigurator Anwendung zu erstellen. Die Fragebögen können im Anschluss daran mithilfe von mobilen Endgeräten ausgefüllt und die entstehenden Ergebnisse auf einen Server übertragen werden. Die erfassten Ergebnisdaten können bisher jedoch nicht zentral verwaltet werden. Dies führt dazu, dass ein Nutzer des QuestionSys Frameworks bislang nicht in der Lage ist, mittels eines Softwaresystems sämtliche ihm zugeordneten Fragebögen und deren Ergebnisse verwalten zu können. Die vorliegende Arbeit beschäftigt sich mit der Entwicklung einer Webapplikation, welche die Verwaltung der erfassten Daten digitaler Fragebögen innerhalb des QuestionSys Frameworks ermöglicht. Die Webanwendung zeigt hierfür dem Benutzer sämtliche ihm zugewiesenen Fragebögen und deren Versionen an. Zudem werden in der Anwendung alle bisher erhobenen Resultate einer Fragebogenversion aufgelistet. Einzelne Ergebnisdatensätze können durch die Anwendung editiert und gelöscht werden. In der Arbeit wird hierfür anhand einer Anforderungsanalyse das Konzept der Webanwendung vorgestellt und die resultierende Architektur beschrieben. Des Weiteren werden Implementierungsaspekte im Hinblick auf die verwendeten Vorlagen sowie ausgewählter Bereiche der Anwendung erläutert. Abschließend wird die grafische Benutzeroberfläche der entwickelten Anwendung vorgestellt

The Role of TRP Channels in the Metastatic Cascade

A dysregulated cellular Ca2+ homeostasis is involved in multiple pathologies including cancer. Changes in Ca2+ signaling caused by altered fluxes through ion channels and transporters (the transportome) are involved in all steps of the metastatic cascade. Cancer cells thereby “re-program” and “misuse” the cellular transportome to regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Cancer cells use their transportome to cope with diverse environmental challenges during the metastatic cascade, like hypoxic, acidic and mechanical cues. Hence, ion channels and transporters are key modulators of cancer progression. This review focuses on the role of transient receptor potential (TRP) channels in the metastatic cascade. After briefly introducing the role of the transportome in cancer, we discuss TRP channel functions in cancer cell migration. We highlight the role of TRP channels in sensing and transmitting cues from the tumor microenvironment and discuss their role in cancer cell invasion. We identify open questions concerning the role of TRP channels in circulating tumor cells and in the processes of intra- and extravasation of tumor cells. We emphasize the importance of TRP channels in different steps of cancer metastasis and propose cancer-specific TRP channel blockade as a therapeutic option in cancer treatment

It takes more than two to tango: mechanosignaling of the endothelial surface

The endothelial surface is a highly flexible signaling hub which is able to sense the hemodynamic forces of the streaming blood. The subsequent mechanosignaling is basically mediated by specific structures, like the endothelial glycocalyx building the top surface layer of endothelial cells as well as mechanosensitive ion channels within the endothelial plasma membrane. The mechanical properties of the endothelial cell surface are characterized by the dynamics of cytoskeletal proteins and play a key role in the process of signal transmission from the outside (lumen of the blood vessel) to the interior of the cell. Thus, the cell mechanics directly interact with the function of mechanosensitive structures and ion channels. To precisely maintain the vascular tone, a coordinated functional interdependency between endothelial cells and vascular smooth muscle cells is necessary. This is given by the fact that mechanosensitive ion channels are expressed in both cell types and that signals are transmitted via autocrine/paracrine mechanisms from layer to layer. Thus, the outer layer of the endothelial cells can be seen as important functional mechanosensitive and reactive cellular compartment. This review aims to describe the known mechanosensitive structures of the vessel building a bridge between the important role of physiological mechanosignaling and the proper vascular function. Since mutations and dysfunction of mechanosensitive proteins are linked to vascular pathologies such as hypertension, they play a potent role in the field of channelopathies and mechanomedicine

Simultaneous quantification of selected glycosaminoglycans by butanolysis-based derivatization and LC-SRM/MS analysis for assessing glycocalyx disruption in vitro and in vivo

Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate GAG-specific disaccharide generation and its subsequent retention in LC–reversed-phase mode followed by mass spectrometric detection performed in positive ion-selected reaction monitoring (SRM) mode. GAG contents were measured in media of endothelial cells (EA.hy926) subjected to various GAG-degrading enzymes, as well as in murine plasma and urine in apolipoprotein E/low‐density lipoprotein receptor‐deficient (ApoE/LDLR −/−) mice and age-matched wild-type C57BL/6 mice. Alternatively, GLX disruption was verified by atomic force microscopy (AFM)-based analysis of GLX thickness. The proposed assay to quantify GAG-specific disaccharides presented high sensitivity for each of the analytes (LLOQ: 0.05–0.1 μg/mL) as well as accuracy and precision (86.8–114.9% and 2.0–14.3%, respectively). In medium of EA.hy926 cells subjected to GAG-degrading enzymes various GAG-specific disaccharides indicating the degradation of keratan sulphate (KS), heparan sulphate (HS), chondroitin sulphate (CHS) or hyaluronan (HA) were detected as predicted based on the characteristics of individual enzyme activity. In turn, AFM-based assessment of GLX thickness was reduced to a similar extent by all single enzyme treatments, whereas the most prominent reduction of GLX thickness was detected following the enzyme mixture. Plasma measurements of GAGs revealed age- and hypercholesterolemia-dependent decrease in GAGs concentration. In summary, a novel LC-SRM/MS-based method for GAG profiling was proposed that may inform on GLX status in cell culture for both in vitro and in vivo conditions

Prolonged Door-to-Balloon Time Leads to Endothelial Glycocalyx Damage and Endothelial Dysfunction in Patients with ST-Elevation Myocardial Infarction

Damage to the endothelial glycocalyx (eGC) has been reported during acute ischemic events like ST-elevation myocardial infarction (STEMI). In STEMI, a door-to-balloon time (D2B) of p p 60 min led to significantly higher serum concentrations of eGC components (syndecan-1: p p p 60 min led to the pronounced loss of eGC height/stiffness (both, p p p p < 0.01). An increased D2B led to severe eGC shedding, with endothelial dysfunction in a temporal context. eGC components and pro-inflammatory mediators correlated with a prolonged D2B, indicating a time-dependent immune reaction during STEMI, with a decreased NO concentration. Thus, D2B is a crucial factor for eGC damage during STEMI. Clinical evaluation of the eGC condition might serve as an important predictor for the endothelial function of STEMI patients in the future

Rac1 regulates lipid droplets formation, nanomechanical, and nanostructural changes induced by TNF in vascular endothelium in the isolated murine aorta

Endothelial inflammation is recognized as a critical condition in the development of cardiovascular diseases. TNF-induced inflammation of endothelial cells is linked to the formation of lipid droplets, augmented cortical stiffness, and nanostructural endothelial plasma membrane remodelling, but the insight into the mechanism linking these responses is missing. In the present work, we determined the formation of lipid droplets (LDs), nanomechanical, and nanostructural responses in the model of TNF-activated vascular inflammation in the isolated murine aorta using Raman spectroscopy, fluorescence imaging, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We analysed the possible role of Rac1, a major regulator of cytoskeletal organization, in TNF-induced vascular inflammation. We demonstrated that the formation of LDs, polymerization of F-actin, alterations in cortical stiffness, and nanostructural protuberances in endothelial plasma membrane were mediated by the Rac1. In particular, we revealed a significant role for Rac1 in the regulation of the formation of highly unsaturated LDs formed in response to TNF. Inhibition of Rac1 also downregulated the overexpression of ICAM-1 induced by TNF, supporting the role of Rac1 in vascular inflammation. Altogether, our results demonstrate that LDs formation, an integral component of vascular inflammation, is activated by Rac1 that also regulates nanomechanical and nanostructural alterations linked to vascular inflammation

Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels

Here we report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes. Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signaling pathways is the utilization of C

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor

The contribution of the shear stress-sensitive epithelial Na(+) channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR(-/-) mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR(-/-) vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR(-/-) vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction

Endothelial Glycocalyx and Cardiomyocyte Damage Is Prevented by Recombinant Syndecan-1 in Acute Myocardial Infarction

The outer layer of endothelial cells (ECs), consisting of the endothelial glycocalyx (eGC) and the cortex (CTX), provides a protective barrier against vascular diseases. Structural and functional impairments of their mechanical properties are recognized as hallmarks of endothelial dysfunction and can lead to cardiovascular events, such as acute myocardial infarction (AMI). This study investigated the effects of AMI on endothelial nanomechanics and function and the use of exogenous recombinant syndecan-1 (rSyn-1), a major component of the eGC, as recovering agent. ECs were exposed in vitro to serum samples collected from patients with AMI. In addition, in situ ECs of ex vivo aorta preparations derived from a mouse model for AMI were employed. Effects were quantified by using atomic force microscopy-based nanoindentation measurements, fluorescence staining, and histologic examination of the mouse hearts. AMI serum samples damaged eGC/CTX and augmented monocyte adhesion to the endothelial surface. In particular, the anaphylatoxins C3a and C5a played an important role in these processes. The impairment of endothelial function could be prevented by rSyn-1 treatment. In the mouse model of myocardial infarction, pretreatment with rSyn-1 alleviated eGC/CTX deterioration and reduced cardiomyocyte damage in histologic analyses. However, echocardiographic measurements did not indicate a functional benefit. These results provide new insights into the underlying mechanisms of AMI-induced endothelial dysfunction and perspectives for future studies on the benefit of rSyn-1 in post-AMI treatment

Protonation of Piezo1 Impairs Cell-Matrix Interactions of Pancreatic Stellate Cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells