Composite manufacturing technology selection and strategic implementation timing

CEMSEmbraer Compósitos has problems keeping up with the aerospace market requirements for the production of carbon fiber parts. This work project will investigate what manufacturing capabilities Embraer Compósitos has to implement in the next 8 years and if first-mover advantages justify an early start of development. Criteria based on market requirements, competition and the company characteristics lead to three recommended technologies: Liquid Composite Molding, Laser Ablation and Carbon Nanotubes. The assessment of first-mover advantages specifically related to the aerospace composite manufacturing industry demonstrates the benefits of a prompt start of development and the necessity of a development partnership

Der Einfluss von SRC-Kinasen auf die Signaltransduktion des Interleukin-33-Rezeptors in Mastzellen

Interaktionen zwischen dem Zytokin Interleukin-33 und seinem Rezeptor IL-33R sind bis dato vor allem an Mastzellen und Typ-2-T-Helferzellen (Th2-Lymphozyten, Th2­Zellen) untersucht worden. Durch ihre pathogenetische Rolle u.a. in Tiermodellen des Asthma bronchiale, der rheumatoiden Arthritis sowie der Atherosklerose deuteten sie bereits auf eine hohe klinische Relevanz hin. Dennoch ist die Signaltransduktion des IL-33R aktuell nur unzureichend erforscht. Die Bindung von IL-33 an seinen Rezeptor bewirkt über die Assoziation intrazellulärer Adapterproteine eine Aktivierung diverser MAP-Kinasen (engl. mitogen-activated protein kinases) wie ERK1/2, JNK1/2 und p38 sowie des Transkriptionsfaktors NF-.B. Diese Signalprozesse führen letztendlich zur Initiierung zelltyp-spezifischer Effektorfunktionen wie z.B. der Sekretion von Entzündungsmediatoren und proinflammatorischer Zytokine durch Mastzellen. Drube et al. konnten 2010 in ihrer Studie zeigen, dass die biologische Funktion des IL-33R von der Aktivität der Rezeptortyrosinkinase c-Kit, welche auf Mastzellen konstitutiv exprimiert wird, abhängig ist: Die Kostimulation mit dem c-Kit-Liganden stem cell factor (SCF) ist für die vollständige Aktivierung der IL-33R-vermittelten Signaltransduktion und Zytokinsekretion notwendig. Als strukturelle Basis für diese Interaktion konnte die Formation eines Rezeptorkomplexes, bestehend aus c-Kit, IL-33R und dem IL-1­receptor-accesory protein (IL-1RAcP), identifiziert werden. Der genaue Mechanismus dieser Rezeptorinteraktion ist jedoch noch nicht aufgeklärt worden. Src-Kinasen aus der Gruppe der Non-Rezeptor-Tyrosinkinasen spielen eine bedeutende Rolle in der Signaltransduktion diverser Mastzell-Rezeptoren wie c-Kit und dem hochaffinen Immunglobulin-E-Rezeptor FceRI. Aus diesem Grund ist im Rahmen dieser Arbeit der hypothetische Einfluss von Src-Kinasen auf das von Drube et al. beschriebene, gemeinsam vom IL-33R und c-Kit ausgehende Signalnetzwerk in Mastzellen untersucht worden. Zur Bearbeitung der Fragestellung dieser Arbeit sind zunächst Voruntersuchungen mit Hilfe der Mastzellline HMC-1, welche u.a. eine konstitutiv aktive c-Kit-Mutante exprimieren, durchgeführt worden. Die Erkenntnisse dieser Voruntersuchungen konnten auf primäre, in vitro ausdifferenzierte murine Mastzellen (engl. bone marrow derived mast cells, BMMCs) übertragen werden. Zur Analyse der c-Kit-Aktivierung als Voraussetzung für die Beteiligung von Src-Kinasen an den untersuchten Signalprozessen erfolgten äquivalente Experimente mit c-Kit-defizienten kitW-sh/w-sh-BMMCs sowie mit kitD814V-transgenen BMMCs, welche die konstitutiv aktive c-Kit-Mutante D814V exprimieren. Der Spezifitätsnachweis erfolgte mittels IL-33R­defizienten il-33r-/--BMMCs. Bezug nehmend auf frühere Publikationen wurden die verwendeten Zellen mit IL-33 und SCF (jeweils als Einzel-bzw. Kostimulation) stimuliert und anschließend die Phosphorylierung diverser intrazellulärer Signalproteine mittels Western Blot detektiert sowie die Sekretion von IL-6 bzw. IL-8 mit Hilfe eines spezifischen ELISA gemessen. Durch die jeweilige Präinkubation mit dem spezifischen Src-Kinase-Inhibitor SU6656 konnte der zu untersuchende Einfluss von Src-Kinasen auf die IL-33R-/c-Kit-Signaltransduktion charakterisiert werden. Die Ergebnisse der Untersuchungen dieser Arbeit zeigen, dass wichtige Bestandteile der IL-33R-Signaltransduktion in Mastzellen wie ERK1/2, JNK1/2, NF-.B sowie der Proteinkinase-B-/Akt-Signalweg durch Src-Kinasen aktiviert werden. Ebenso wird eine IL-33-induzierte Phosphorylierung der c-Kit-Tyrosinreste 719 (murin) bzw. 721 (human) durch Src-Kinasen vermittelt. Als Voraussetzung für die Beteiligung von Src-Kinasen an der IL-33R-Signaltransduktion konnte die Aktivierung von c-Kit durch SCF identifiziert werden. Src-Kinasen stellen somit erst nach Prästimulation von c-Kit mit SCF einen Bestandteil des IL-33R-Signalings dar. Auch die beispielhaft für IL-33R-vermittelte Mastzell-Effektorfunktionen untersuchte Sekretion von IL-6 erfolgt lediglich nach Kostimulation mit SCF unter Beteiligung von Src-Kinasen. Src-Kinasen spielen somit eine wesentliche Rolle nicht nur in der Initiierung und Regulierung eines von dem Zytokinrezeptor IL-33R sowie der Rezeptortyrosinkinase c-Kit gemeinsam ausgehenden Signalnetzwerkes, sondern auf diesem Weg auch in der Induzierung von IL-33R-vermittelten Mastzell-Effektorfunktionen. Dysregulationen derartiger komplexer Signalkaskaden spielen somit möglicherweise eine Rolle in der Entstehung verschiedener Krankheitsprozesse und lassen Src-Kinasen aus diesem Grund als einen hypothetischen pharmakologischen Angriffspunkt erscheinen. Notwendig sind hierzu neben der Identifizierung der exakt beteiligten Src-Kinase und ihrer genauen Position innerhalb des beschriebenen Signalnetzwerkes auch entsprechende in-vivo-Experimente am Tiermodell

Inhibition of diacylglycerol–sensitive TRPC channels by synthetic and natural steroids

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca2+ signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC50s of 3–5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC50s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy

Activation of Rac-1 and RhoA contributes to podocyte injury in chronic kidney disease

Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD

Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca2+ sparks in airway myocytes

Short-lived, localized Ca2+ events mediate Ca2+ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca2+-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca2+ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca2+ signaling system composed of Ca2+ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca2+-activated K+ (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca2+ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca2+ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca2+] resulting from Ca2+ sparks, and the kinetic model of BK channels, we estimate that an average Ca2+ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca2+ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca2+ source) and BK channels (the Ca2+ target)

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account

Fatal Cardiac Arrhythmia and Long-QT Syndrome in a New Form of Congenital Generalized Lipodystrophy with Muscle Rippling (CGL4) Due to PTRF-CAVIN Mutations

We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae