19 research outputs found
Spatial quantification and classification of skin response following perturbation using organotypic skin cultures
No full textAbstract
Motivation: For a mechanistic understanding of skin and its response to an induced perturbation, systems biology is gaining increasing attention. Unfortunately, quantitative and spatial expression data for skin, like for most other tissues, are almost not available.
Results: Integrating organotypic skin cultures, whole-slide scanning and subsequent image processing provides bioinformatics with a novel source of spatial expression data. We here used this approach to quantitatively describe the effect of treating organotypic skin cultures with sodium dodecyl sulphate in a non-corrosive concentration. We first measured the differentiation-related spatial expression gradient of Heat-Shock-Protein 27 in a time series of up to 24 h. Secondly, a multi-dimensional tissue classifier for predicting skin irritation was developed based on abstract features of these profiles. We obtained a high specificity of 0.94 and a sensitivity of 0.92 compared with manual classification. Our results demonstrate that the integration of tissue cultures, whole-slide scanning and image processing is well suited for both the standardized data acquisition for systems biological tissue models and a highly robust classification of tissue responses.
Contact: [email protected]
Supplementary information: Supplementary data are available at Bioinformatics online.</jats:p
A molecular mechanotransduction pathway regulates collective migration of epithelial cells
No full textCollective movement of epithelial cells drives essential multicellular organization during various fundamental physiological processes encompassing embryonic morphogenesis, cancer and wound healing. Yet the molecular mechanism that ensures the coordinated movement of many cells remains elusive. Here we show that a tumour suppressor protein, merlin, coordinates collective migration of tens of cells, by acting as a mechanochemical transducer. In a stationary epithelial monolayer and also in three-dimensional human skin, merlin localizes to cortical cell-cell junctions. During migration initiation, a fraction of cortical merlin relocalizes to the cytoplasm. This relocalization is triggered by the intercellular pulling force of the leading cell and depends on the actomyosin-based cell contractility. Then in migrating cells, taking its cue from the intercellular pulling forces, which show long-distance ordering, merlin coordinates polarized Rac1 activation and lamellipodium formation on the multicellular length scale. Together, these results provide a distinct molecular mechanism linking intercellular forces to collective cell movements in migrating epithelia
Wound healing revised: A novel reepithelialization mechanism revealed by in vitro and in silico models
No full textWound healing is a complex process in which a tissue's individual cells have to be orchestrated in an efficient and robust way. We integrated multiplex protein analysis, immunohistochemical analysis, and whole-slide imaging into a novel medium-throughput platform for quantitatively capturing proliferation, differentiation, and migration in large numbers of organotypic skin cultures comprising epidermis and dermis. Using fluorescent time-lag staining, we were able to infer source and final destination of keratinocytes in the healing epidermis. This resulted in a novel extending shield reepithelialization mechanism, which we confirmed by computational multicellular modeling and perturbation of tongue extension. This work provides a consistent experimental and theoretical model for epidermal wound closure in 3D, negating the previously proposed concepts of epidermal tongue extension and highlighting the so far underestimated role of the surrounding tissue. Based on our findings, epidermal wound closure is a process in which cell behavior is orchestrated by a higher level of tissue control that 2D monolayer assays are not able to capture
Abstract 4923: Understanding cancer invasion by the systems biological analysis of collective cell migration in 3D tissue models
No full textCharacterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin
No full textMost molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging
Analyse des Reepithelialisierungsmechanismus auf zellulärer Ebene mittels organotypischer in vitro Wundheilungsmodelle aus systembiologischer Perspektive
No full textSeit mehr als 40 Jahren werden in der Literatur kontroverse Mechanismen diskutiert, welche die Reepithelialisierung und die damit einhergehende Bildung einer epidermalen Migrationszunge postulieren. Die diskutierten Mechanismen lassen hierbei die kollektive Zellmigration als möglichen Einflussfaktor während der Reepithelialisierung vollkommen außer Acht. Die, im Rahmen dieser Arbeit, etablierten organotypischen Wundheilungsmodelle ermöglichten die Analyse der Reepithelialisierung aus einer systembiologischen Perspektive und führten zu einem neuen
Reepithelialisierungmechanismus, der Proliferation sowie kollektive Zellmigration als essentielle Faktoren in sich vereint. Die Proliferationsanalyse zeigte hierbei, dass sich die proliferative Aktivität als konzentrische Welle von den unverwundeten Bereichen in Richtung
des Wundareals bewegt, wobei die Majorität der neu produzierten Zellen nicht von der Wunde selbst, sondern auf Basis dieser Proliferationswelle generiert wird. Darüber hinaus konnte durch Polaritätsanalyse gezeigt werden, dass diese neu generierten Zellen mittels kollektiver Migration in das Wundareal einwanderten um die Migrationszunge zu verlängern. Hierbei migrierten basale Keratinozyten des umliegenden, unverwundeten Gewebes sowie in der Migrationszunge selbst unter den differenzierten, suprabasalen Keratinozyten hindurch. Eine neu entwickelte doppelte Fluoreszenzfärbestrategie ermöglichte es, die räumliche Verteilung migrierender Keratinozyten innerhalb der sich bildenden Migrationszunge zu
verfolgen. Hierbei zeigte sich, dass im Laufe der Reepithelialisierung alle fluoreszenzgefärbten Zellen im suprabasalen Kompartiment der Migrationszunge akkumulierten um einen sich kontinuierlich verlängernden Schutzschild zu bilden, welcher durch die kollektive Migration basaler Keratinozyten vorangetrieben wird. Zusammenfassend basiert dieser Reepithelialisierungsmechanismus zur Verlängerung der EET auf drei Prozessen: (i) Der kollektiven Migration basaler Keratinozyten, (ii) dem Lifting Mechanismus, welcher für die Ausbildung eines mehrschichtigen Epithels sorgt, sowie (iii) der aktiven Migration der Keratinozyten an der Migrationsfront der EET. Der im Rahmen dieser Arbeit postulierte Reepithelialisierungsmechanismus stellt somit die in der Literatur diskutierten Mechanismen in Frage und führt zu einem fortschrittlichen Erklärungsmodell der Reepithelialisierung epidermaler Wunden.
Darüber hinaus wurden in dieser Arbeit mittels Multiplex Technologie Wundheilungassoziierte Signalwege untersucht. Zu diesem Zweck wurde ein zweites, neuartiges Wundheilungsmodell auf Basis epidermaler, dermaler Kokulturen entwickelt. Dieses Modell erlaubte die Interaktion zwischen Keratinozyten und Fibroblasten während der Kultivierung, ermöglichte jedoch durch einfache Separation beider Zelltypen eine voneinander unabhängige proteomische Analyse. Auf Basis dieser Untersuchungen konnte nachgewiesen werden, dass die mitogen aktivierten Proteinkinasen p38 und Erk1/2 sowie
der Transkriptionsfaktor STAT3 eine entscheidende Rolle während der Reepithelialisierung spielen. Zeigten Erk1/2 und STAT3 eine durch Fibroblasten induzierbare Aktivität, so konnte die Phosphorylierung von p38 auf den Wundstimulus zurückgeführt werden. Überdies wiesen p38 und STAT3 Migrations-assoziierte Effekte auf, während die Phosphorylierung von Erk1/2 zu einer möglichen Steigerung proliferativer Aktivität der Keratinozyten führte. Die Analyse des Kulturüberstandes ermöglichte es weiterhin Zytokine zu identifizieren, welche potentiell für die Aktivierung der jeweiligen Signalwege verantwortlich sein könnten.
Zusammenfassend wurden im Rahmen dieser Dissertation organotypische in vitro Wundheilungsmodelle etabliert, welche die Analyse der Reepithelialisierung, als auch Wundheilung-assoziierter Signalwege ermöglichten. Auf Basis dieser Ergebnisse konnte ein neuartiger Reepithelialisierungsmechanismus postuliert werden, der Proliferation, Differenzierung und kollektive Zellmigration in sich vereint und entscheidend zu dem Verständnis der Reepithelialisierung während der Wundheilung beträgt. Zusätzlich konnten durch die Anwendung der Multiplex-Technologie mit p38, Erk1/2, sowie STAT3 essentielle Signalwege identifiziert werden, deren zukünftige Pertubation wichtige Werkzeuge zur Modulation der Wundheilung darstellen
Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.
No full textCutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration
Untersuchungen zu den Praefationes von Seneca pater
No full textThe paper is divided in two major parts. As a commentary on the work of Seneca the Elder has not existed, in the first part commentary work on the text of the Praefationes had to be done. Based on this preliminary work, it was possible in a second large part to consider important issues together and looking to provide literature in historical context. Why Seneca chooses this unique structure of his work, the significance of his own sons as recipients of this work, which political attitude of the author is evident in this difficult time.Die Arbeit ist ein zwei große Teile gegliedert, die sich natürlich aus der Themenstellung ergeben haben. Da ein wissenschaftlicher Kommentar zu dem Werk von Seneca Pater bisher nicht vorlag, musste in einem ersten Teil der Text der Praefationes sprachlich und inhaltlich erfasst und kommentiert werden. Aufbauend auf diesen Vorarbeiten war es möglich in einem zweiten großen Teil, wichtige Themen zusammenschauend zu betrachten und in literaturhistorischen Zusammenhang zu stellen. Dabei wurden unter anderem die Fragen behandelt, warum Seneca diesen einzigartigen Aufbau seines Werkes wählt, welche Bedeutung die eigenen Söhne als Adressaten dieses Werkes haben, welche politische Einstellung des Autors in dieser zum Teil innenpolitisch schwierigen Zeit erkennbar wird. Da Senecas Werk das einzige ist, das uns heute noch detailreiche Auskunft über die Deklamation in der frühen Kaiserzeit gibt, trägt jede Beschäftigung mit seinem Werk zu einer Aufarbeitung der Entwicklung der römischen Literatur in der nachklassischen Phase bei
Beyond Territoriality: The Case of Transnational Human Rights Litigation
No full textCases for civil damages that have been brought before Western courts by victims of torture and persecution against states officials or corporations, challenge the principles of state sovereignty and jurisdictional competence. While national courts can in cases of serious crimes hear cases that grow out of acts committed in another country, the same is not true for cases for civil compensation. A persisting and rising number of private law cases that attempts to empower disenfranchised victims of crime and abuse, points to the necessity of reconsidering the prevailing procedural and substantial obstacles that govern the so-far unsuccessful civil law suits. The law of transnational civil litigation [TCL] emerged with the US American decision in Filartiga in 1980 and perhaps culminated in the US Supreme Court's Decision in Sosa v. Alvarez-Machain in 2004. TCL has become a laboratory for our inquiry into the relationship between laws that were developed within and for the nation-state on the one hand and an increasingly globalized political and legal human rights discourse, on the other. As such, TCL is a case in point for the dramatically changing nature of norm-creation, law, and law enforcement in an era of globalization.law; fundamental/human rights; sovereignty; globalization
