Eine Studie über die Kontrolle der Zellform in Epithelgewebe

Epithelial tissues line out all inner and outer cavities of the body, and serve as a barrier between the organism and the external world, or between different organs and their compartments. Epithelia have important sensory and signalling roles, while responding to changes in external conditions. The epithelial cell layers are constantly exposed to deformations, exerted on the level of the moving and developing organism, as well as due to internal flows and stresses that act on the cellular level. Nevertheless, the tissue has to maintain its shape and architecture over all scales – from the internal structure of comprising cells, to the shapes of the cells themselves, their connectivity, and ultimately, the shape and macroscopic structure of the tissue. Compromised morphology of the tissue is associated with numerous pathological conditions although it is not clear if the disease is the cause or the result of the altered morphology. The reason for this ambiguity is that the coupling between tissue architecture, the response to stress and the tissue function is still not understood. This relation is investigated in this thesis using epithelial model tissues. The first part of the thesis focuses on the effect of uniaxial stretch and shear deformations on the maintenance of the tissue structure. To this end, MDCK II tissues are grown on elastic substrates, while the deformation is imposed using mechanical manipulators developed with collaborators. In agreement with previous reports in literature, our experiments show a linear elastic behaviour on short time scales, and on length scales of several cells. This linear response can be understood from the observations that on short time scales very little topological restructuring occurs, and the proliferation rate is decreased. However, if the deformation is maintained for more than 30 minutes, the active response sets in targeting the reconstitution of tissue architecture. Cell proliferation increases significantly and, concomitantly, changes of the cell neighbourhood become more common. Depending on the amplitude of the deformation, the tissue needs 48 to 72 hours to restore its initial structure, with most of the recovery taking place in the first 6 hours. On these long time scales, small stretching amplitudes were found to speed up the overall tissue growth, compared to the control, while fast changes of area of more than 30% slow down the development, presumably due to damage on the cellular level. Consequently, the second part of the thesis focuses on the maintenance of the intracellular structure, in particular on the positioning of the nucleus. Namely, it was recently discovered that the shapes of the cells in the epithelium strongly correlate with the position and the shape of the nuclei, with the common assumption that it is the cytoskeleton that regulates the latter. However, mechanistic understanding of this process is not yet available. To get more insights, different components of the cytoskeleton were systematically manipulated to establish their role in the nuclear mechanics. Intermediate filaments were found to play only a little role in this process. Actin filaments push the nuclei towards the centre, while microtubules are responsible for pulling the nuclei out of the centre, in a myosin II-independent manner. The presented results deepen our understanding of the organisation of epithelial tissue and its maintenance. While in this thesis the focus lies on physical aspects of the response to mechanical stimuli, in the future, the coupling to biochemical signalling will be a relevant mechanism to be studied over all the time and length scales involved, in order to achieve full understanding of the maintenance of cell and tissue shape. The here elucidated biophysical response is an important step forward towards this goal.Epithelgewebe kleiden innere und äußere Hohlräume des Körpers aus, dabei agiert die Epithelschicht als Barriere zwischen dem Organismus und der Außenwelt und grenzt verschiede Organen und deren Bestandteilen voneinander ab. Epithelien haben die wichtige Funktion, äußere Veränderungen wahrzunehmen und darauf zu reagieren, indem sie zum Beispiel Signalkaskaden initiieren. Die Zellschichten des Epithels sind konstant Deformationen ausgesetzt, zum einen auf der Ebene des sich bewegenden und entwickelnden Organismus, zum anderen auf der Zellebene, bedingt durch interne Flüsse und Spannungen. Trotz des ständigen mechanischen Einflusses muss das Gewebe seine Form und Architektur aufrechterhalten und zwar auf allen Längenskalen – von der Struktur innerhalb der Zellen, über die Form der Zellen selbst und ihren Zusammenschluss mit anderen Zellen, bis hin zu der makroskopischen Form des Gewebes. Die veränderte Morphologie des Gewebes wird mit einer Vielzahl von pathologischen Befunden assoziiert. Dabei ist es unklar, ob die Krankheiten der Grund für die Veränderungen sind oder das Resultat der geänderten Morphologie sind. Der Grund für diese Ambiguität ist, dass das Zusammenspiel zwischen der Architektur, der Reaktion auf Deformation und der Funktion von Gewebe immer noch nicht hinreichend verstanden ist. Genau diese Beziehungen werden in dieser Doktorarbeit mit Hilfe von Epithel-Monolagen untersucht. Der erste Teil dieser Arbeit legt den Fokus auf die Frage, wie sich Dehnung entlang einer Richtung oder Scherdeformation auf die Aufrechterhaltung der internen Gewebestruktur auswirkt. Zu diesem Zweck werden MDCK II Monolagen auf elastischen Substraten gezüchtet. Diese Substrate lassen sich mit Hilfe von mechanischen Manipulatoren dehnen, die zusammen mit unseren Kooperationspartnern entwickelt wurden. In Übereinstimmung mit den Berichten in der Literatur zeigen unsere Experimente ein linear elastisches Verhalten auf kurzen Zeitskalen. Die Längenskala beläuft sich dabei über mehrere Zellen. Die lineare Antwort auf die Deformation wird auch durch die Tatsache ermöglicht, dass die topologische Umstrukturierung in nur sehr kleinem Maße von statten geht und die Proliferationsrate stark verringert ist. Wird die Dehnung des Gewebes allerdings für mehr als 30 Minuten beibehalten, wird ein aktiver Prozess in Gang gesetzt, der die Aufrechterhaltung der Gewebearchitektur zum Ziel hat. Zellen teilen sich häufiger und davon begleitet, kommt es vermehrt zu topologischen Änderungen in der lokalen Nachbarschaft der Zellen. Abhängig von der Deformations-Amplitude braucht das Gewebe 48 bis 72 Stunden, um die ursprüngliche Architektur wiederherzustellen. Dabei findet ein Großteil der Restrukturierung in den ersten sechs Stunden statt. Auf diesen langen Zeitskalen, führt die Dehnung mit kleinen Amplituden zu einem umfassenden Wachstum der Gewebe, während eine schnelle Änderung der Fläche um mehr als 30% eine Verzögerung in der Gewebeentwicklung zur Folge hat. Die Gründe für diese Verzögerung sind vermutlich Beschädigungen auf Zellebene oder einzelner Proteine in der Zelle. Die Instandhaltung intrazellulärer Strukturen, insbesondere der Positionierung des Zellkerns, stellt den Schwerpunkt des zweiten Teils dieser Doktorarbeit dar. Es wurde kürzlich entdeckt, dass die Form der Zellen innerhalb des Epithels stark mit der Position und der Form der Zellkerne korreliert. Dabei wird im Allgemeinen angenommen, dass das Zytoskelett eine entscheidende Rolle spielt. Allerdings ist das mechanistische Verständnis dieses Prozesses noch unklar. Um einen Beitrag zu dieser Fragestellung zu leisten, wurden verschiedene Komponenten des Zytoskeletts systematisch manipuliert, um so ihren Beitrag zur Mechanik des Zellkerns zu ergründen. Intermediärfilamente spielen eine untergeordnete Rolle in diesem Prozess. Dahingegen schieben Aktinfilamente den Zellkern in Richtung Mitte der Zellen, während Mikrotubuli dafür verantwortlich sind, den Zellkern aus der Zellmitte in die Zellperipherie zu ziehen. Der Beitrag von Myosin II ist dabei zu vernachlässigen. Die hier präsentierten Ergebnisse vertiefen unser Verständnis für die Organisation und Aufrechterhaltung der Struktur von Epithelien. Während physikalische Reaktionen auf mechanische Stimuli in dieser Arbeit den Schwerpunkt bilden, müssen zukünftig auch die Verbindung mit biochemischen Signalwegen untersucht werden. Dabei ist es für das umfassende Verständnis der Aufrechterhaltung von Zell- und Gewebeformen wichtig, dass die Singnaltransduktion auf allen Zeit- und Längenskalen untersucht wird. Die hier erläuterte biophysikalische Antwort der Epithelgewebe auf mechanische Reize ist bereits ein wichtiger Schritt für das Erreichen dieses Ziels

Mechanical Regulation of Epithelial Tissue Homeostasis

Despite recent efforts to understand homeostasis in epithelial tissues, there are many unknowns surrounding this steady state. It is considered to be regulated by mechanoresponse, but unlike for single cells, this remains heavily debated for tissues. Here, we show that changes in matrix stiffness induce a non-equilibrium transition from tubular to squamous Madin-Darby Canine Kidney II tissues. Nonetheless, despite different cell morphologies and densities, all homeostatic tissues display equivalent topologies, which, hence, must be actively targeted and regulated. On the contrary, the mechanoresponse induces dramatic changes in the large-scale organization of the colonies. On stiff gels, this yields an unreported cooperative state of motile cells displaying higher densities than in the arrested homeostatic state. This suggests a more complex relation between cell density and motility than previously anticipated. Our results unequivocally relate the mechanosensitive properties of individual cells to the evolving macroscopic structures, an effect that could be important for understanding the emergent pathologies of living tissues

Does Coffee Intake Reduce Postoperative Ileus After Laparoscopic Elective Colorectal Surgery?: A Prospective, Randomized Controlled Study: The Coffee Study

BACKGROUND Postoperative ileus after colorectal surgery is a frequent problem that significantly prolongs hospital stay and increases perioperative costs. OBJECTIVE The aim was to evaluate the effect of standardized coffee intake on postoperative bowel movement after elective laparoscopic colorectal resection. DESIGN This is a prospective randomized controlled trial that was conducted between September 2014 and December 2016. SETTINGS This study was performed in a public cantonal hospital in Switzerland with accreditation for colon and rectum cancer surgery. PATIENTS Patients who underwent elective colorectal surgery were included. INTERVENTIONS Patients were randomly assigned either to the intervention group receiving coffee or the control group receiving tea. A total of 150 mL of the respective beverage was drunk 3 times per day every postoperative day until discharge. MAIN OUTCOME MEASURES The primary end point was time to first bowel movement. Secondary end points included the use of laxative, insertion of a nasogastric tube, length of hospital stay, and postoperative complications. RESULTS A total of 115 patients were randomly assigned: 56 were allocated to the coffee group and 59 to the tea group. After coffee intake, the first bowel movement occurred after a median of 65.2 hours versus 74.1 hours in the control group (intention-to-treat analysis; p = 0.008). The HR for earlier first bowel movement after coffee intake was 1.67 (p = 0.009). In the per-protocol analysis, hospital stay was shorter in the coffee group (6 d in the coffee group vs 7 d in the tea group; p = 0.043). LIMITATIONS The rate of protocol violation, mostly coffee consumption in the tea arm, was relatively high, even if patients were clearly instructed not to consume coffee if they were in the tea arm. CONCLUSIONS Coffee intake after elective laparoscopic colorectal resection leads to faster recovery of bowel function. Therefore, coffee intake represents a simple and effective strategy to prevent postoperative ileus. See Video Abstract at http://links.lww.com/DCR/A955

Formation of liquid crystalline phases in aqueous suspensions of platelet-like tripalmitin nanoparticles

Suspensions of platelet-like shaped tripalmitin nanocrystals stabilized by the pure lecithin DLPC and the lecithin blend S100, respectively, have been studied by small-angle x-ray scattering (SAXS) and optical observation of their birefringence at different tripalmitin (PPP) concentrations φ PPP . It could be demonstrated that the platelets of these potential drug delivery systems start to form a liquid crystalline phase already at pharmaceutically relevant concentrations φ PPP of less than 10 wt. %. The details of this liquid crystalline phase are described here for the first time. As in a previous study [A. Illing et al. , Pharm. Res.21, 592 (2004)] some platelets are found to self-assemble into lamellar stacks above a critical tripalmitin concentration φstPPP of 4 wt. %. In this study another critical concentration φlcPPP≈7 wt. % for DLPC and φlcPPP≈9 wt. % for S100 stabilized dispersions, respectively, has been observed. φlcPPP describes the transition from a phase of randomly oriented stacked lamellae and remaining non-assembled individual platelets to a phase in which the stacks and non-assembled platelets exhibit an overall preferred orientation. A careful analysis of the experimental data indicates that for concentrations above φlcPPP the stacked lamellae start to coalesce to rather small liquid crystalline domains of nematically ordered stacks. These liquid crystalline domains can be individually very differently oriented but possess an overall preferred orientation over macroscopic length scales which becomes successively more expressed when further increasing φ PPP . The lower critical concentration for the formation of liquid crystalline domains of the DLPC-stabilized suspension compared to φlcPPP of the S100-stabilized suspension can be explained by a larger aspect ratio of the corresponding tripalmitin platelets. A geometrical model based on the excluded volumes of individual platelets and stacked lamellae has been developed and successfully applied to reproduce the critical volume fractions for both, the onset of stack formation and the appearance of the liquid crystalline phase

Liquid Crystalline Phase Formation in Suspensions of Solid Trimyristin Nanoparticles

The presence of liquid crystalline phases in suspensions of solid lipid nanoparticles can increase the risk of their gelling upon administration through fine needles. Here we study the formation of liquid crystalline phases in aqueous suspensions of platelet-like shaped solid lipid nanoparticles. A native lecithin-stabilized trimyristin (20 wt %) suspension was investigated at different dilution levels by small-angle X-ray scattering (SAXS) and visual inspection of their birefringence between two crossed polarizers. For trimyristin concentrations φ_MMM < 6 wt %, the dispersed platelets are well separated from each other whereas they start to self-assemble into stacked lamellae for 6 wt % ≤ φ_MMM < 12 wt %. For φ_MMM ≥ 12 wt %, the SAXS patterns become increasingly anisotropic, which is a signature of an evolving formation of a preferred orientation of the platelets on a microscopic scale. Simultaneously, the suspensions become birefringent, which proves the existence of an anisotropic liquid crystalline phase formed in the still low viscous liquid suspensions. Spatially resolved SAXS scans and polarization microscopy indicate rather small domains in the (sub)­micrometer size range in the nematic liquid crystalline phase and the presence of birefringent droplets (tactoids). The observed critical concentrations for the formation of stacks and the liquid crystalline phase are significantly higher as for equivalent suspensions prepared from triglycerides with longer chains. This can be explained with the lower aspect ratio of trimyristin platelets. Special emphasis is put on the isotropic–liquid crystalline phase transition as a function of the ionic strength of the dispersion medium and φ_MMM. Higher salt concentrations allow shifting of the phase transition to higher trimyristin concentrations. This can be attributed to a partial screening of the repulsive forces between the platelets, which allows higher packing densities within the platelet stacks and of remaining isolated platelets

Fewer nutrient deficiencies after laparoscopic sleeve gastrectomy (LSG) than after laparoscopic Roux-Y-gastric bypass (LRYGB)-a prospective study

Deficiencies in micronutrients after bariatric operations are frequent, despite routine supplementation. Main outcome measures were pre- and postoperative frequency of nutrient deficiencies and success rate of their treatment