Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation

Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes.

Characterization of calcium oxide in root perforation sealer materials

Root perforation represents an undesirable complication that may lead to an unfavorable prognosis. The aims of this study were to characterize and to compare the presence of calcium oxide (CaO) on the chemical composition of materials used for root perforation therapy: gray and white mineral trioxide aggregate (MTA) and Portland cement (PC), gray MTA+5%CaO and gray MTA+10%CaO. The last two materials were analyzed to evaluate the increase of CaO in the final sample. CaO alone was used as a standard. Eighteen polyethylene tubes with an internal diameter of 3 mm and 3 mm in length were prepared, filled and then transferred to a chamber with 95% relative humidity and a temperature of 37ºC. The chemical compounds (particularly CaO) and the main components were analyzed by energy-dispersive X-ray microanalysis (EDX). EDX revealed the following concentrations of CaO: gray MTA: 59.28%, white MTA: 63.09%; PC: 72.51%; gray MTA+5%CaO: 63.48% and gray MTA+10%CaO: 67.55%. The tested materials presented different concentrations of CaO. Even with an increase of 5 and 10% CaO in gray MTA, the CaO levels found in the MTA samples were lower than those found in PC.

Healing of a tooth with an overinstrumented apex, extensive transportation and periapical lesion using a 5 mm calcium hydroxide apical plug: an 8-year follow-up report

Besides the risk of filling material extrusion throughout the apex, a satisfactory apical seal can be difficult to achieve in canals with open apices or iatrogenic enlargements of the apical constriction. These situations pose a challenge to root canal filling. This paper describes the root canal filling of a maxillary right canine with an overinstrumented apex, complete loss of the apical stop, extensive canal transportation and apical periodontitis. A 5 mm calcium hydroxide apical plug was placed before root canal filling. The plug was made by soaking paper points with saline, dipping the points in calcium hydroxide powder and then applying it to the apex several times, until a consistent apical plug was obtained. The canal was then irrigated with saline in order to remove any residual calcium hydroxide from the root canal walls, dried with paper points and obturated with an inverted #80 gutta-percha cone and zinc oxide-eugenol based sealer by the lateral condensation technique. An 8-year radiographic follow-up showed formation of mineralized tissue sealing the apical foramen, apical remodeling and no signs of apical periodontitis.

In vitro organogenesis of Eucalyptus grandis: effects of boron and calcium

The in vitro organogenesis of woody species plays an essential role in the improvement of forest products by providing saplings with high commercial value. Furthermore, mineral nutrition plays an important role in the induction of organogenic responses. The objective of this study was to evaluate the effects of boron and calcium in the organogenesis of nodal segments from seedlings of Eucalyptus grandis growing under in vitro conditions. The concentration of boron and calcium in MS medium was modified to induce organogenic responses in 45-day-old nodal segments used as explants. After 60 days, the fresh weight, dry weight, ratio of fresh and dry weight, relative water content and relative matter content accumulated by the explants were evaluated. The concentrations of boron and calcium in the culture medium influenced the in vitro organogenic control of Eucalyptus grandis. Reduced combinations of boron and calcium induced callus formation and dry matter accumulation in the explants. A boron concentration of 100% (1.10 mg L-1) combined with 100% (119.950 mg L-1) and 200% (239.900 mg L-1) of calcium, and 200% (2.20 mg L-1) of boron combined with 100% (119.950 mg L-1) of calcium allowed the induction of well-developed buds, which can be used for the regeneration of micro-plants.

The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1

Interactions between calcium and heavy metals in Norway spruce : Accumulation and binding of metals in wood and bark

Waste products from the forest industry are to be spread in forests in Sweden to counteract nutrient depletion due to whole tree harvesting. This may increase the bioavailability of calcium (Ca) and heavy metals, such as cadmium (Cd), copper (Cu) and zinc (Zn) in forest soils. Heavy metals, like Cd, have already been enriched in forest soils in Sweden, due to deposition of air pollutions, and acidification of forest soils has increased the bioavailability of toxic metals for plant uptake. Changes in the bioavailability of metals may be reflected in altered accumulation of Ca and heavy metals in forest trees, changes in tree growth, including wood formation, and altered tree species composition. This thesis aims at examining: A) if inter- or intra- specific differences in sensitivity to Cd occur in the most common tree species of Sweden, and if so, to study if these can be explained by the uptake and distribution of Cd within the plant: B) how elevated levels of Ca, Cd, Cu and Zn affect the accumulation and attachment of metals in bark and wood, and growth of young Norway spruce (Picea abies): C) how waste products from the forest industry, such as wood ash, influence the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce. Sensitivity to Cd, and its uptake and distribution, in seedlings of Picea abies, Pinus sylvestris and Betula pendula from three regions (southern, central and northern parts) of Sweden, treated with varying concentrations of Cd, were compared. Differences in root sensitivity to Cd both among and within woody species were found and the differences could to some extent be explained by differences in uptake and translocation of Cd. The root sensitivity assays revealed that birch was the least, and spruce the most, sensitive species, both to the external and to tissue levels of Cd. The central ecotype of the species tested tended to be most Cd resistant. The radial distribution, accumulation and attachment of, and interactions between Ca and heavy metals in stems of two-year-old Norway spruce trees treated with elevated levels of Cd, Cu, Zn and/or Ca, were investigated. Further, the influence of these metals on growth, and on root metal content, was examined. Accumulation of the metals was enhanced in wood, bark and/or roots at elevated levels of the metal in question. Even at low levels of the metals, similar to after application of wood ash, an enhanced accumulation was apparent in wood and/or bark, except for Cd. The increased accumulation of Zn and Cu in the stem did not affect the growth. However, Cu decreased the accumulation of Ca in wood. Higher levels of Cu and Cd reduced the stem diameter and the toxic effect was associated with a reduced Ca content in wood. Copper and Cd also decreased the accumulation of Zn in the stem. On the other hand, elevated levels of Ca increased the stem diameter and reduced the accumulation of Cd, Cu, Zn and Mn in wood and/or bark. When metals interacted with each other the firmly bound fraction of the metal reduced was in almost all cases not affected. As an exception, Cd decreased the firmly bound fraction of Zn in the stem. The influence of pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD), in the amount of 3000 kg ha-1, on the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce in the field was examined. The effect of the treatments on the metal content of bark and wood was larger after 3 years than after 6 years. Treatment with ash+GLD had less effect on the heavy metal content of bark and wood than treatment with ash alone. The ash treatment increased the Cu and Zn content in bark and wood, respectively, after 3 years, and decreased the Ca content of the wood after 6 years. The ash+GLD treatment increased the Ca content of the bark and decreased the Zn content of bark and wood after 3 years. Both treatments reduced, or tended to decrease, the Cd content in wood and bark at both times. To conclude, small changes in the bioavailability of Ca, Cu, Cd and Zn in forest soils, such as after spreading pellets of wood ash or a mixture of wood ash and green liquor dregs from the forest industry, will be reflected in an altered accumulation of metals in wood and bark of Norway spruce. It will not only be reflected in changed accumulation of those metals in which bioavailability in the soil has been enhanced, but also of other metals, probably partly due to interactions between metals. When metals interact the exchangeable bound fraction of the metal reduced is suggested to be the main fraction affected. The small alterations in accumulation of metals should not affect the growth of Norway spruce, especially since the changes in accumulation of metals are low, and further since these decrease over time. However, as an exception, one positive and maybe persistent effect of the waste products is that these may decrease the accumulation of Cd in Norway spruce, which partly may be explained by competition with Ca for uptake, translocation and binding. A decreased accumulation of Cd in Norway spruce will probably affect the trees positively, since Norway spruce is one of the most sensitive species to Cd of the forest trees in Sweden. Thus, spreading of waste products from the forest industry may be a solution to decrease the accumulation of Cd in Norway spruce. In a longer perspective, this will decrease the risk of Cd altering the tree species composition of the forest ecosystem. An elevated bioavailability of Ca in forest soils will, in addition to Cd, probably also decrease the accumulation of other less competitive heavy metals, like Zn and Mn, in the stem.

Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

Glutamate-induced potentiation of calcium influx in primary hippocampal culture neurons

ABSTRACT This works aim was to test whether LTP-like features can also be measured in cell culture and by methods that allow to analyse a alrger number of cells. A suitable method for this purpose is calcium imaging. The rationale for this approach lies in the fact that LTP/LTD are dependent on changes in intracellular calcium concentrations. Calcium levels have been measured using the calcium sensitive dye fura-2, whose fluorescence spectrum changes upon formation of the [fura-2-Ca2+] complex. Our LTP-inducing protocol comprised of two glutamate stimuli of identical size and duration (50 mM, 30 s) which were separated by 35 min. We could demonstrate that such a stimulation pattern gives rise to approx. 25% larger calcium influx at the second stimulus. It has been shown than such a stimulation pattern gives rise to an average of 25% augmentation (potentiation) of the second response, with 69% of potentiated cells. This experimental paradigm shows the pharmacological properties of LTP, established by previous electrophysiological studies:- blocking of NMDARs and mGluRs eliminates LTP induction;- blocking of AMPARs and L-type VGCCs does not eliminate LTP induction. Having obtained a system for induction and following of LTP-like changes, a preliminary application example was performed. Its purpose was to investigate possible influence of nicotine and galanthamine on our potentiation effect. Nicotine (100 mM) was shown both to increase and to eliminate glutamate-induced potentiation. Galanthamine coapplication (0.5 mM) with nicotine and glutamate exerted no effect on nicotinic modulation. However, galanthamine coapplied with glutamate alone seems to augment glutamate-induced potentiation. An LTP model system presented here could be additionally refined, by variation of glutamate application times, and testing for dependence on various forms of protein kinases. Galanthamine effect would probably be better addressed by cell-to-cell measurements instead of statistical approach, with subsequent identification of the cell type. Alternatively, combined calcium imaging – electrophysiological experiments could be performed. Spatial and temporal properties of intracellular ion dynamics could be utilised as diagnostic tools of the physiological state of the cells, thereby finding its application in functional proteomics.

Nanostructuring by templated synthesis of nanowires and controlled crystallization of calcium phosphate on self-assembled monolayers

The idea was to obtain nanowires in a chemical laboratory under convenient and simple conditions by employing templates. Thus it was possible to produce nanochains by interlinking of gold colloids synthesized by the two-phase-method of M. Brust with by making use of vanadiumoxide nanotubes as template. The length of the resulting nanowires is varying between 1100 nm and 200 nm with a diameter of about 16 nm. Due to a flexible linker the obtained nanowires are not completely rigid. These unique structural features could make them interesting objects for structuring and assembling in the nanoscale range. Another way to produce gold nanowires was realized by a two-step surface metallization procedure, using type I collagen fibres as a template. Gold colloids were used to label the collagen fibres by direct electrostatic interaction, followed by growth steps to enhance the size of the adsorbed colloidal gold crystals, resulting in a complete metallization of the template surface. The length of the resulting gold nanowires reaches several micrometers, with a diameter ~ 100 to 120 nm. To gain a deeper insight into the process of biomineralization the cooperative effect of self-assembled monolayers as substrate and a soluble counterpart on the nucleation and crystal growth of calcium phosphate was studied by diffusion techniques with a pH switch as initiator. As soluble component Perlucin and Nacrein were used. Both are proteins originally extracted from marine organisms, the first one from the Abalone shell and the second one from oyster pearls. Both are supposed to facilitate the calcium carbonate formation in vivo. Studies with Perlucin revealed that this protein shows a clear cooperative effect at a very low concentration with a hydrophobic surface promoting the calcium phosphate precipitation resulting in a sponge like structure of hydroxyapatite. The Perlucin molecule is very flexible and is unfolded by adsorbing to the hydrophobic surface and uncovers its active side. Hydrophilic surfaces did not have a deeper impact. Studies with Nacrein as additive have shown that the protein stabilizes octacalcium phosphate at room temperature on carboxylic self-assembled monolayer and at 34 °C on all other employed surfaces by interaction with the mineral. On the hydroxyl-, alkyl-, and amin-terminated self-assembled monolayers at room temperature the octacalcium phosphate get transformed to hydroxyapatite. Main analytical techniques which are used in this work are transmission electron microscopy, high resolution scanning electron microscopy, surface plasmon resonance spectroscopy, atomic force microscopy, Raman micro-spectroscopy and quartz crystal microbalance.

Charakterisierung der biologischen Funktion der Plasmodium falciparum Calcium-abhängigen Proteinkinase 1

Im Rahmen dieser Arbeit wurden biologische Funktionen einer Proteinkinase des Erregers der Malaria tropica, genauer der „Plasmodium falciparum calcium dependent protein kinase 1“ (PfCDPK1), in parasitären Blutstadien untersucht.rnUm Einblicke in die Funktion der Kinase, die sie in den extrazellulären Kompartimenten des Parasiten übernimmt, zu gewinnen, wurden sechs Proteine untersucht, die dasselbe Translokationsignal wie PfCDPK1 besitzen. Es konnte gezeigt werden, dass fünf der untersuchten Proteine mit der PfCDPK1 im Bereich der parasitophoren Vakuole sowie des tubovesikulären Systems co-lokalisiert sind. Deletionsmutanten, denen das Translokationssignal fehlte, sowie ein Peptid, das lediglich aus diesem bestand, bestätigten, dass die Translokation in die extrazellulären Kompartimente von keinen weiteren Faktoren, außer dem Signalmotiv abhängt. Mit PfCAP und PfRKIP konnten zwei Regulatoren der PfCDPK1 identifiziert werden. PfARM, Pfrab_5b sowie PfGAP45 sind Substrate der PfCDPK1. Mit Hilfe von massenspektrometrischen Messungen wurde der Phosphorylierungsstatus der untersuchten Proteine durch die PfCDPK1 sowie der Autophosphorylierungsstatus der Kinase bestimmt, um Rückschlüsse auf regulatorische Prozesse ziehen zu können.rnDie Phosphorylierung von PfGAP45 durch die PfCDPK1 steht vermutlich mit dem Invasionsprozess des Parasiten in direktem Zusammenhang, da gezeigt wurde, dass eine Hemmung der Kinase mit PP1 einen 90%igen Rückgang an neu infizierten Erythrozyten zur Folge hatte.rnrn

Radiation-induced defects in calcium fluoride and their influence on material properties under 193 nm laser irradiation

Calcium fluoride (CaF2) is one of the key lens materials in deep-ultraviolet microlithography because of its transparency at 193 nm and its nearly perfect optical isotropy. Its physical and chemical properties make it applicable for lens fabrication. The key feature of CaF2 is its extreme laser stability. rnAfter exposing CaF2 to 193 nm laser irradiation at high fluences, a loss in optical performance is observed, which is related to radiation-induced defect structures in the material. The initial rapid damage process is well understood as the formation of radiation-induced point defects, however, after a long irradiation time of up to 2 months, permanent damage of the crystals is observed. Based on experimental results, these permanent radiation-induced defect structures are identified as metallic Ca colloids.rnThe properties of point defects in CaF2 and their stabilization in the crystal bulk are calculated with density functional theory (DFT). Because the stabilization of the point defects and the formation of metallic Ca colloids are diffusion-driven processes, the diffusion coefficients for the vacancy (F center) and the interstitial (H center) in CaF2 are determined with the nudged elastic band method. The optical properties of Ca colloids in CaF2 are obtained from Mie-theory, and their formation energy is determined.rnBased on experimental observations and the theoretical description of radiation-induced point defects and defect structures, a diffusion-based model for laser-induced material damage in CaF2 is proposed, which also includes a mechanism for annealing of laser damage. rn

Development of models and methods to simulate peptide-assisted nucleation and growth of calcium-minerals

In den vergangenen Jahren wurden einige bislang unbekannte Phänomene experimentell beobachtet, wie etwa die Existenz unterschiedlicher Prä-Nukleations-Strukturen. Diese haben zu einem neuen Verständnis von Prozessen, die auf molekularer Ebene während der Nukleation und dem Wachstum von Kristallen auftreten, beigetragen. Die Auswirkungen solcher Prä-Nukleations-Strukturen auf den Prozess der Biomineralisation sind noch nicht hinreichend verstanden. Die Mechanismen, mittels derer biomolekulare Modifikatoren, wie Peptide, mit Prä-Nukleations-Strukturen interagieren und somit den Nukleationsprozess von Mineralen beeinflussen könnten, sind vielfältig. Molekulare Simulationen sind zur Analyse der Formation von Prä-Nukleations-Strukturen in Anwesenheit von Modifikatoren gut geeignet. Die vorliegende Arbeit beschreibt einen Ansatz zur Analyse der Interaktion von Peptiden mit den in Lösung befindlichen Bestandteilen der entstehenden Kristalle mit Hilfe von Molekular-Dynamik Simulationen.rnUm informative Simulationen zu ermöglichen, wurde in einem ersten Schritt die Qualität bestehender Kraftfelder im Hinblick auf die Beschreibung von mit Calciumionen interagierenden Oligoglutamaten in wässrigen Lösungen untersucht. Es zeigte sich, dass große Unstimmigkeiten zwischen etablierten Kraftfeldern bestehen, und dass keines der untersuchten Kraftfelder eine realistische Beschreibung der Ionen-Paarung dieser komplexen Ionen widerspiegelte. Daher wurde eine Strategie zur Optimierung bestehender biomolekularer Kraftfelder in dieser Hinsicht entwickelt. Relativ geringe Veränderungen der auf die Ionen–Peptid van-der-Waals-Wechselwirkungen bezogenen Parameter reichten aus, um ein verlässliches Modell für das untersuchte System zu erzielen. rnDas umfassende Sampling des Phasenraumes der Systeme stellt aufgrund der zahlreichen Freiheitsgrade und der starken Interaktionen zwischen Calciumionen und Glutamat in Lösung eine besondere Herausforderung dar. Daher wurde die Methode der Biasing Potential Replica Exchange Molekular-Dynamik Simulationen im Hinblick auf das Sampling von Oligoglutamaten justiert und es erfolgte die Simulation von Peptiden verschiedener Kettenlängen in Anwesenheit von Calciumionen. Mit Hilfe der Sketch-Map Analyse konnten im Rahmen der Simulationen zahlreiche stabile Ionen-Peptid-Komplexe identifiziert werden, welche die Formation von Prä-Nukleations-Strukturen beeinflussen könnten. Abhängig von der Kettenlänge des Peptids weisen diese Komplexe charakteristische Abstände zwischen den Calciumionen auf. Diese ähneln einigen Abständen zwischen den Calciumionen in jenen Phasen von Calcium-Oxalat Kristallen, die in Anwesenheit von Oligoglutamaten gewachsen sind. Die Analogie der Abstände zwischen Calciumionen in gelösten Ionen-Peptid-Komplexen und in Calcium-Oxalat Kristallen könnte auf die Bedeutung von Ionen-Peptid-Komplexen im Prozess der Nukleation und des Wachstums von Biomineralen hindeuten und stellt einen möglichen Erklärungsansatz für die Fähigkeit von Oligoglutamaten zur Beeinflussung der Phase des sich formierenden Kristalls dar, die experimentell beobachtet wurde.