Yes / Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 106 cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis. / Replicel Life Sciences Inc, Vancouver, Canada.

Every year there are nearly two million new cases of invasive breast cancer worldwide and over 500,000 deaths, the majority from metastatic sites. While cellular changes during tumorigenesis and progression have been studied, our understanding of extracellular matrix remodeling, at the fiber level, by individual and collective cellular cohorts remains limited. Furthermore, recent studies suggest that there is a correlation between the organization of collagen I fibers perpendicular to the tumor and patient survival. However, the underlying mechanism of this alignment remains unknown. The central hypothesis proposed in this dissertation is that breast cancer tumors reorganize collagen I fibers perpendicular to the tumor surface via integrin β1 and matrix metalloproteinases (MMPs). To investigate this hypothesis, we developed a novel in vitro assay that replicates collagen I fiber alignment previously reported in vivo and a new quantitative collagen I fiber orientation algorithm. Our studies using multicellular aggregates, derived from the triple negative breast cancer cell line MDA-MB-231, embedded into collagen I matrices and confocal reflectance microscopy provide novel insights into how the local microenvironment is affected and into local orientation of the collagen I fibers near the spheroid-collagen I interface. These results agree well with our computational studies. Furthermore, the viability of the algorithm is demonstrated using both in silico and in vitro derived images, and shows that this algorithm is more accurate than similar algorithms previously published. Using the developed in vitro assay and computational algorithm it is also demonstrated that knocking down integrin β1 reduces the amount of collagen I aligned perpendicularly to the tumor surface, while inhibiting MMP activity using the broad spectrum MMP inhibitor GM6001 increases the amount of collagen I aligned perpendicularly to the tumor surface at early time points. The work presented here has implications in three-dimensional multicellular assays, accurate fiber orientation analysis, and understanding the role of integrins in matrix reorganization and cancer cell metastasis. / 2019-08-09T00:00:00Z

Being the longest-lived rodent, naked mole-rats (NMR; Heterocephalus glaber) are an exceptional model for biogerontological research. However, unlike other rodents, not much is known about their wound healing process. To investigate that, full-thickness wounds were created in the back skin of naked mole rats. Our initial data confirmed that wound closure in NMR skin was achieved primarily by reepithelialization and granulation tissue formation, with only ~26% wound contraction, making them an excellent model to study human cutaneous wound healing. Similar to mice and human skin, changes in wound epithelial tongue included progressive enlargement of wound epithelium, increased proliferation and changes in the expression pattern of epidermal markers including K14, K17, integrin α6 and E-cadherin. Further analysis revealed characteristics of reduced scarring in NMR wounds including low collagen I to III ratio, increased HA expression (HMW) and increased fibronectin expression. Transcriptional profiling of TGFβ isoforms and different pro/anti-inflammatory cytokines revealed a balance in the expression and repression of different cytokines, potentially contributing into reduced scarring. Comparison of RNA-seq data from NMR and human fullthickness wounds revealed a delay in the activation of important biological processes and pathways in NMR skin in response to injury. Further analysis based on cultured human and NMR cells revealed differential regulation of TGFβ signalling pathway between both species. 3-D collagen gel contraction assay revealed that NMR fibroblast showed noticeable contraction but independently of TGFβ treatment, while human fibroblast showed marked increased in gel contraction in the presence of TGFβ. In conclusion, NMR can serve as a very useful model to study human cutaneous wound healing. The reduced scarring in NMR could be a result of multiple factors including HMW-HA, balanced cytokine expression and differential regulation of different TGFβ cytokines as observed in the in vitro studies.

The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.

Tout au long de la vie d’un organisme, l’architecture du système nerveux est mise à l’épreuve par des processus de maturation, de croissance, de stress mécaniques et de vieillissement. Bien que certaines molécules de maintenance de l’organisation des ganglions et fascicules neuronaux aient été identifiés chez le nématode C. elegans, les mécanismes assurant la protection à long terme de l’architecture du système nerveux restent mal compris. Chez les mutants de maintenance neuronale sax-7/L1CAM, certaines structures neuronales se développent initialement normalement, mais se désorganisent avec le temps. Un criblage génétique effectué au laboratoire a indiqué l’implication du gène mig-6/Papiline dans la maintenance neuronale: la perte de fonction de mig-6 supprime la désorganisation neuronale progressive des mutants sax-7. De plus, l’organisation neuronale des mutants mig-6 est mieux préservée dans un contexte de stress mécanique que chez le type sauvage. Un équilibre entre l'adhésion cellulaire et la flexibilité du milieu semble donc clé. Par ailleurs, les cellules gliales sont en relation étroite avec les neurones, mais leur implication dans la maintenance neuronale reste inexplorée. Ainsi, lors de ces travaux, la question principale est d’étudier la contribution de la matrice extracellulaire et de cellules gliales dans un contexte de maintenance de l’architecture du système nerveux chez C. elegans. Les résultats révèlent que MIG-6/Papiline régule l’état de la matrice extracellulaire en modifiant l’organisation du collagène IV, un composant abondant et conservé des membranes basales. Cette modification du collagène IV semble compenser les défauts d’adhésion cellulaire présents chez les mutants de maintenance sax-7/L1CAM et contrer un déplacement des ganglions neuronaux lors d’un stress mécanique accru. L’exploration de cellules gliales en contexte de maintenance neuronale a mis en évidence certains défauts des mutants de maintenance sax-7/L1CAM. Comprendre les principes généraux du maintien de l'architecture et de la connectivité neuronale pourrait aider à identifier des facteurs clés influençant l'apparition et la progression de neuropathologies. / Throughout life, the architecture of the nervous system is challenged by processes of maturation, growth, mechanical stress and aging. Although neuronal maintenance mechanisms of ganglia and fascicles organization involving conserved factors have been identified in the nematode C. elegans, little is known about processes that aim for the long-term protection of the nervous system architecture. In sax-7/L1CAM neuronal maintenance mutants, some neuronal ganglia and fascicles initially develop normally, but become disorganized over time. A genetic screen performed in the laboratory indicated the involvement of mig-6/Papilin in neuronal maintenance: loss of mig-6 function suppresses progressive neuronal disorganization in sax-7 mutants. Moreover, the neuronal organization of mig-6 mutants is better preserved under mechanical stress than in the wild-type strain. A balance between the adhesion of neurons to their environment and the flexibility of the surrounding extracellular matrix thus seems of importance. Furthermore, glial cells are closely related to neurons, but their involvement in the maintenance of the organization of neuronal structures remains unexplored. The main question of this work is to study the contribution of the extracellular matrix and of two types of glial cells in the context of maintenance of the nervous system architecture in C. elegans. Our results reveal that MIG-6/Papilin regulates the state of the extracellular matrix by altering the organization of collagen IV, an abundant and conserved component of basement membranes, thus compensating for cell adhesion defects in sax-7/L1CAM maintenance mutants and counteracting a neural ganglia displacement upon increased mechanical stress. Our exploration of glial cells in the context of neuronal maintenance also revealed defects in sax-7/L1CAM maintenance mutants. Understanding the general principles of maintenance of neuronal architecture and connectivity could help identify key factors influencing the onset and progression of neuropathologies.

This thesis ventures with the extracellular matrix protein (ECM) fibronectin (FN) as an interface protein in the interaction between cells and materials to design microenvironment for future use in tissue engineering. It is studied the FN adsorption and conformations, cell behaviour to different FN conformation, cell adhesion, reorganisation and remodelling of FN at the material interface, the role of growth factors (GF) and their interactions with components of the extracellular matrix (ECM), the immunology cell response, and the stem cell fate influenced by the extrinsic signals coming from the engineered microenvironments using ECM's proteins. To investigate the FN response, in terms of adsorbed amount and conformation to different chemical properties of the material, model surfaces were used. Self assembled monolayers (SAM) with different percentages of two different chemical groups were used: CH3 and OH. FN adsorption, initial cell adhesion and signalling (focal adhesions, integrin expression and phosphorylation of FAK) is related with the reorganisation and secretion of FN and matrix degradation. It is shown that matrix degradation at the cell material interface depends on surface chemistry in metalloproteinase-dependent way. A direct relationship between FN activity at the cell-material interface and metalloproteinase 9 (MMP9) expression was found, being the product of a sequence of events that include integrin expression, focal adhesion formation, matrix reorganisation and focal adhesion kinase (FAK) phosphorylation. Two different materials with subtle variations in their chemical composition were employed as a drastically different FN conformation: from a globular conformation on PMA (poly (methyl acrylate)) to the formation of a well-interconnected FN network (similar to the FN physiological fibrillar network) triggered by PEA (poly (ethyl acrylate)). The formation of focal adhesions (vinculin), FAK expression and phosphorylation, specific integrin binding, protein and gene expression for ¿5 and ¿v was studied, seeking to correlate cell adhesion with matrix degradation. It is demonstrated that the material-driven FN fibrillogenesis on PEA triggers proteolytic activity: MMP activity is higher as a compensatory mechanism to the inability of cells to reorganise this FN network. Looking into the role of protein-material interactions and stem cell fate, and with the knowledge on PEA, we engineer different synergistic microenvironments to direct cell and stem cell fate. FN has a growth factor (GF) binding domain on its molecule (FNIII12-14) and has been demonstrated to produce a synergistic response when occurs at the same time the recognition of the cell binding domain (FNIII9-10). It is demonstrated that this domain is available on the FN coated PEA, and exploiting these interactions between PEA, FN and GF, it is developed a microenvironment to control cell behaviour and tissue repair. It is studied the BMP2 binding and presentation, the effect of BMP2 presentation on MSC proliferation and differentiation. These systems allow not only enhanced activity of GF compared to soluble administration, but also reduce GF doses, improving safety and cost effectiveness. Finally, the immunological reaction of the microenvironment developed is studied using dendritic cells, beside the conformational structure of ECM protein importance in DC integrin-based activation it is studied, helping to establish the field of adhesion-based modulation of DC as a general mechanism that has previously not been defined. The microenvironment didn't induce any maturation in DC, while different FN conformation shows differences in DC morphology and citokine level production (IL-10 and IL-12). / En esta tesis se estudia la interacción de una proteina de la matriz extracelular, fibronectina (FN) como interfase en la interacción entre células y materiales, para diseñar microambientes con el propósito de ser usados en el futuro en ingeniería tisular. Se estudia la adsorción y conformación de FN y la relación con el diferente comportamiento celular: la adhesión celular, la reorganización y remodelado de la FN en la interfase célula-material, el papel que juegan los factores de crecimiento y sus interacciones con los componentes de la matriz extracelular, la respuesta immunológica y el destino celular de células madre influenciadas por las señales extrínsecas provenientes de microambientes elaborados a partir de proteínas de la matriz extracelular. Con el objetivo de investigar la respuesta a la FN en términos de conformación y cantidad absorbida a diferentes propiedades químicas del material, se usaron materiales modelo: monocapas autoensambladas (self-assembled monolayers, SAM). Las químicas estudiadas fueron CH3 and OH. La adsorption de FN, adhesion y señalización (adhesiones focales, expresión de interinas y fosforilación de quinasas de adhesiones focales (FAK)) se estudiaron en relación a la reorganización y secreción de FN y degradación de la matriz extracelular. Se demuestra que la degradación de la matriz extracelular en la interfase célula-material depende de la química de la superficie, a través de las metaloproteinasas. Se ha descubierto una relación directa entre la actividad de la FN que se encuentra en el material y la expresión de metaloproteinasa 9 (MMP9), a través de la expresión de integrinas, formación de adhesiones focales, reorganización de la matriz extracelular y fosforilación de FAK En el siguiente capítulo se emplean materiales poliméricos con una sutil diferencia en la composición química, provocando una diferencia drástica en la conformación de la FN: se pasa de una conformación globular en PMA (polimetil acrilato) a una conformación en forma de red interconectada en PEA (polietil acrilato). Con el propósito de relacionar la adhesión celular con la degradación de la matriz extracelular, se estudia la formación de adhesiones focales (vinculina), la expresión y fosforilación de FAK, la unión específica de integrinas y la expresión de las integrinas ¿5 and ¿v. Se demuestra que la formación de una red de FN sobre PEA induce la actividad proteolítica: la actividad de las MMPs es mayor, actuando como mecanismo compensatorio a la incapacidad de reorganización de la red de FN. Haciendo uso de la conformación de la FN sobre PEA, se estudiaron las interacciones entre la proteína-material y el destino celular de células madres. La FN posee un dominio de unión de factores de crecimiento (FNIII12-14) y se ha demostrado que se produce una respuesta sinérgica cuando el reconocimiento ocurre junto con el dominio de unión celular (FNIII9-10). En esta tesis se demuestra que el dominio de unión de factores de crecimiento está disponible en la conformación que adquiere sobre PEA y se diseñan microambientes para controlar el comportamiento celular y regeneración de tejido. Se estudia la unión y presentación de BMP2 y su efecto en la diferenciación de células madre mesenquimales. Los microambientes desarrollados, ademas de mejorar la actividad de los factores de crecimiento comparado con la administración soluble, también reduce la cantidad de factores de crecimiento que se tendría que administrar, mejorando la seguridad y efectividad. Finalmente se estudió la reacción inmunológica a los microambientes desarrollados usando células dendríticas, estudiando además la influencia de la estructura de la conformación de las proteínas en la activación de las células dendríticas a través de las integrinas. Los microambientes no indujeron ninguna maduración de células dendríticas, mientras que la conformación de la FN muestra control / En aquesta tesi s'estudia la interacció entre una proteïna de la matriu extracel.lular, fibronectina (FN) com interfase en la interaccio entre cèl·lules i materials, per a dissenyar microambients amb el propòsit d'utilitzar-se al futur en enginyeria tissular. S'estudia l'adsorció i conformació de la FN i la relació amb el diferent comportament cel·lular: l'adhesió cel·lular, la reorganització i remodelat de la FN a la interfase cèl·lula-material, el paper que juguen els factors de creixement i les seus interaccions amb els components de la matriu extracel·lular, la resposta immunològica i el destí cel·lular de cèl·lules mare influenciades pels senyals extrínseques provinents de microambients elaborats a partir de proteïnes de la matriu extracel·lular. Amb l'objectiu d'investigar la respostar a la FN en termes de conformació i quantitat absorbida a diferents propietats químiques del material, s'utilitzaren materials model: monocapes autoacoblades (self-assembled monolayers, SAM). Les químiques estudiades van ser CH3 and OH. L'absorció de FN, adhesió i senyalització (adhesions focals, expressió d'integrines i fosforilació de quinases d'adhesions focals (FAK)) es van estudiar en relació a al reorganització i secreció de la FN i degradació de la matriu extracel·lular. Es demostra que la degradació de la matriu extracelular en la interfase cèl·lula-material depèn de la química de la superficie, a través de les metal·loproteïnases. S'ha descobert una relació directa entra l'activitat de la FN que es troba en el material i l'expressió de metaloproteinasa 9, a través de l'expressió d'integrines, formació d'adhesions focals, reorganització de la matriu extracel·lular i fosforilació de FAK. Al següent capítol es fan servir materials polimèrics amb una subtil diferència en la composició química, provocant una diferència dràstica en la conformació de la FN: es passa d'una conformació globular en PMA (polimetil acrilat) a una conformació en forma de xarxa interconnectada en PEA (polietil acrilat). Amb el propòsit de relacionar l'adhesió cel·lular amb la degradació de la matriu extracel·lular, s'estudia la formació d'adhesions focals (vinculina), l'expressió i fosforilació de FAK, la unió específica d'integrines i l'expressió de les integrines ¿5 and ¿v. Es demostra que la formació d'una xarxa de FN sobre PEA indueix l'activitat proteolítica: l'activitat de les MMPs és més gran, actuant com a mecanisme compensatori a la incapacitat de reorganització de la xarxa de FN. Fent ús de la conformació de la FN sobre PEA, es van estudiar les interaccions entre la proteïna-material i el destí cel·lular de cèl·lules mares. La FN posseeix un domini d'unió de factors de creixement (FNIII12-14) i s'ha demostrat que es produeix una resposta sinèrgica quan el reconeixement ocurreix juntament amb el domini d'unió cel·lular (FNIII9- 10). En aquesta tesi es demostra que el domini d'unió de factors de creixement està disponible a la conformació que adquireix sobre PEA i es dissenyen microambients per controlar el comportament cel·lular i regeneració de teixit. S'estudia la unió i presentació de BMP2 i el seu efecte en la diferenciació de cèl·lules mare mesenquimals. Els microambientes desenvolupats, a més de millorar l'activitat dels factors de creixement comparat amb l'administració soluble, també redueix la quantitat de factors de creixement que s'hauria d'administrar, millorant la seguretat i efectivitat. Finalment es va estudiar la reacció immunològica als microambients desenvolupats usant cèl·lules dendrítiques, estudiant a més la influència de l'estructura de la conformació de les proteïnes en l'activació de les cèl·lules dendrítiques a través de les integrines. Els microambients no van induir cap maduració de cèl·lules dendrítiques, mentre que la conformació de la FN mostra controlar la morfologia de les cèl·lules dendrítiques i / Llopis Hernández, V. (2017). Material-driven fibronectin fibrillogenesis to engineer cell function [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90412

The neuropilin (Nrp) family consists of multifunctional cell surface receptors with critical roles in a number of different cell and tissue types. A core aspect of Nrp function is ligand-dependent cellular adhesion and migration, where it controls the multistep process of cellular motility through integration of ligand binding, receptor coupling and signaling via the coordinated action of its extracellular and intracellular domains. While Nrp regulates cellular adhesion and motility in the cardiovascular and nervous systems under physiological conditions, the emerging pathological role of Nrp in tumor cell migration and metastasis has been identified and provides motivation for continued efforts toward developing Nrp inhibitors. At the molecular level, the role of Nrp in adhesion and migration is intimately connected to the control of adhesive interactions and cytoskeletal reorganization. The adhesive “interactome” for Nrp draws much attention because of its lack of enzymatic activity and inability to transduce signals on its own. It is an active area of research and is still expanding dramatically. Nrp has been well defined as a co-receptor for vascular endothelial growth factor receptor (VEGFR)/vascular endothelial growth factor (VEGF) signaling through enhancing receptor-ligand interaction in angiogenesis. Here, we contribute to this concept through characterization in more biochemical detail about Nrp-1/VEGF physical interactions. VEGF has been shown to compete with Sema3 for binding to Nrp-1 b1 ligand binding pocket. This competition fine-tunes VEGF-induced angiogenesis. Our data provides a molecular mechanism for high affinity Sema3F binding to Nrp-1 in the b1 domain. As to the VEGFR-independent function, Nrp/integrin association has been demonstrated. The functional integration has been shown for Nrp/integrin in angiogenic sprouting. Both proteins are highly expressed in endothelial tip cells to mediate endothelial cell migration during angiogenesis and knockdown of either one in mice leads to embryonic lethality due to similar defects in vascular development. To identify the structure and function correlation, we characterized in more detail about Nrp-1/integrin physical interactions with biochemical and cell-based assays. Through an integrated approach of biochemical, molecular and cellular methods, we defined the direct physical interactions between Nrp-1 and integrins. We have also extended this work to demonstrate the functional importance and contribution of the interactions in integrin-mediated cell adhesion on extracellular matrix (ECM) in angiogenesis and platelet function during wound healing and provide a molecular basis for the integration of Nrps/integrins in cell migration, adhesion to ECM, breast cancer initiation and breast cancer stem cell fate determination.

There is a basic rule to mammalian neocortical expansion: as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.

Der Fokus der vorliegenden Arbeit lag auf dem Teilvorgang der Migration epithelialer Zellen auf annähernd physiologischem Matrix-Untergrund. Im Zellkulturmodell wurde die migratorische Aktivität der aus dem humanen Mammakarzinom isolierten, schwach invasiven epithelialen Zelllinie MCF-7 untersucht. Es stellte sich die grundsätzliche Frage, ob eine Wnt-bedingte Invasionssteigerung, welche in vorangegangenen Untersuchungen gezeigt worden war, durch die gesteigerte migratorische Aktivität der Tumorzellen verursacht ist. Es sollten zwei an der Tumorprogression (Wnt5b) bzw. an deren Inhibition (Dkk-2) beteiligte Proteine auf den Invasions-Teilvorgang der Migration hin näher beleuchtet werden. Dazu wurden Untersuchungen in einem modifizierten Tumor-Migrationsassay auf extrazellulärer Matrix (ECM) durchgeführt. In Analogie zu den Vorgängen bei der Wundheilung wurde ein in Anlehnung an die bereits seit langem verwendete Methode des Scratchassays sowie an den etablierten Migrationsassay ein Konfrontationsassay auf ECM konzipiert. Der Einfluss von Wnt5b und Dkk-2 auf die Migration der schwach invasiven, epithelialen Zelllinie MCF-7 wurde schließlich im Migrationsassay sowie im neu etablierten Konfrontationsassay auf ECM näher untersucht. Es wurden dazu Messungen zur Migrationsaktivität der eingesetzten Zellen unter Stimulation mit Wnt5b und Dkk-2 erhoben. Jedoch zeigte sich in beiden Assay-Modellen auf ECM kein signifikanter Einfluss der beiden untersuchten Proteine auf das Migrationsverhalten der MCF-7-Zellen. Diese Ergebnisse demonstrieren, dass die Beobachtungen zum Teilaspekt der Migration nicht automatisch Rückschlüsse auf die Invasion zulassen. Letztere ist ein komplex zusammengesetzter Gesamtvorgang, dessen einzelne Bestandteile es noch weiter zu erforschen gilt.