Prozessanalyse im Bereich der Gemeindeverwaltung zur Vorbereitung des Elektronischen Akts

Die vorliegende Arbeit beschäftigt sich mit Prozessmanagement in der öffentlichen Verwaltung, das einige Besonderheiten im Vergleich zum privaten Sektor aufweist. Insbesondere wird auf die einzelnen Schritte einer Prozessanalyse (Prozesserfassung, Ist-Prozessmodellierung, Analyse der Ist-Prozesse, Soll-Prozessmodellierung, Implementierung und Kontrolle) eingegangen. Darüber hinaus werden die Herausforderungen in der öffentlichen Verwaltung aufgezeigt. Die Entscheidung, die eigenen Prozesse zu analysieren und verbessern zu wollen, stellt oft den ersten Schritt zu einer Reform der Verwaltung dar. Die Modernisierungen in letzter Zeit konzentrierten sich oft auf die neuen Technologien, wobei eine Prozessanalyse eine Art Vorbedingung darstellt, um E-Government erfolgreich einzuführen. Da es verschiedene Methoden gibt Verwaltungen zu modernisieren, werden diese in der vorliegenden Arbeit näher erläutert. All diese Aspekte werden auch in Hinblick auf den Einsatz und die Entwicklung in Österreich besprochen. Die theoretischen Inhalte werden im Rahmen eines Praxis-Projekts mit der Stadtgemeinde Neulengbach umgesetzt. Die durchgeführte Prozessanalyse (Erfassung der Prozesse, Erhebung des Ist-Standes und Verbesserung zu Soll-Prozessen) in einer Abteilung (Buchhaltung) in dieser Gemeinde, soll in weiterer Folge die Einführung des elektronischen Akts vorbereiten. Aus diesem Grund wurden Anbieter des elektronischen Akts am österreichischen Markt untersucht und auf dessen Eignung für die Stadtgemeinde Neulengbach überprüft

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells. One Sentence Summary While plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair

Vascular surveillance by haptotactic blood platelets in inflammation and infection

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets

Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity

Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz−/− follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity

Isolation of synaptic vesicles from genetically engineered cultured neurons

Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts

Biochemical analyses of the cement float of the goose barnacle <i>Dosima fascicularis</i> – a preliminary study

<div><p>The goose barnacle <i>Dosima fascicularis</i> produces an excessive amount of adhesive (cement), which has a double function, being used for attachment to various substrata and also as a float (buoy). This paper focuses on the chemical composition of the cement, which has a water content of 92%. Scanning electron microscopy with EDX was used to measure the organic elements C, O and N in the foam-like cement. Vibrational spectroscopy (FTIR, Raman) provided further information about the overall secondary structure, which tended towards a β-sheet. Disulphide bonds could not be detected by Raman spectroscopy. The cystine, methionine, histidine and tryptophan contents were each below 1% in the cement. Analyses of the cement revealed a protein content of 84% and a total carbohydrate content of 1.5% in the dry cement. The amino acid composition, 1D/2D-PAGE and MS/MS sequence analysis revealed a <i>de novo</i> set of peptides/proteins with low homologies with other proteins such as the barnacle cement proteins, largely with an acidic pI between 3.5 and 6.0. The biochemical composition of the cement of <i>D</i>. <i>fascicularis</i> is similar to that of other barnacles, but it shows interesting variations<i>.</i></p></div

Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces

WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues

When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes