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No description available.

<p> Downregulation of E-cadherin is a frequent event in epithelial cancers and it correlates with weakened cell-cell adhesion and the induction of an epithelial-to-mesenchymal transition (EMT). It is postulated that E-cadherin downregulation liberates the catenin p120 and allows p120's translocation to the nucleus where it interacts with and functionally regulates the novel BTB/POZ transcription factor, Kaiso. Kaiso mediates transcriptional repression of various tumourigenesis-associated genes via methylated CpG dinucleotides or a sequence-specific Kaiso binding site (KBS). The Kaiso/p120 interaction has been detected in E-cadherin expressing cells of various origins, but is seldom detected in N-cadherin expressing cells or cells that have undergone EMT. We hypothesize that p120 and Kaiso play a role in EMT by modulating the expression of EMT-associated genes. We demonstrated that TGF-β-induced EMT occurs in a dose- and time-dependent manner in NMuMG cells but not in FHL-124 cells. In both cells lines, the Kaiso/p120 interaction occurred irrelevant of EMT induction by TGF-β. In NMuMG cells, the expression of p120 increased with EMT induction, while the expression of Kaiso remained unchanged. Finally, misexpression of Kaiso and p120 in mammary epithelial cells affected TGF-β-mediated EMT induction by delaying the upregulation of the positive mesenchymal markers, N-cadherin and α-SMA.</p> / Thesis / Master of Science (MSc)

The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, Epilife®, compared to Dulbecco's modification of Eagle's medium (DMEM) with fetal bovine serum (FBS), and to characterize cultured canine corneal cells. Superficial keratectomies were performed on three dogs. Samples were trypsinized to separate cell layers. Post-trypsinization, immunohistochemistry confirmed that epithelial cells had been released from the stroma. Both cell populations (presumed epithelial cells and stromal tissues) were cultured in DMEM with FBS or Epilife®. First passage cells were fixed for immunocytochemistry and prepared for PCR. Immunocytochemical staining for pancytokeratin, vimentin, and E-cadherin was evaluated, and immunofluorescence for zonula occludens-1 was attempted. Amplification of cytokeratin 5 (CK5) mRNA was assessed by PCR. Primary presumed epithelial cells grew faster when cultured in DMEM with FBS compared to Epilife®. Stromal tissue segments in Epilife® medium failed to adhere to culture plates, indicating that this medium may inhibit attachment and growth of non-epithelial tissues. Staining of corneal tissue segments confirmed that epithelial layers were pancytokeratin and E-cadherin positive, while stromal cells were vimentin positive. Immunocytochemistry of cultured cells revealed that epithelial cells stained positively for pancytokeratin, vimentin, and E-cadherin, while stromal cells remained only vimentin positive. Greater amplification of CK5 mRNA occurred from epithelial cells grown in Epilife® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, Epilife® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS. / Master of Science

Les épithéliums jouent le rôle fondamental de barrière physique et chimique chez les Métazoaires. Les jonctions adhérentes, par le biais de la protéine transmembranaire E-cadhérine (E-cad), assurent une grande partie de l’adhésion intercellulaire. Malgré cette robustesse, les épithéliums peuvent subir des remodelages considérables pendant l’embryogenèse ou la cicatrisation. Lors de la gastrulation de l’embryon de Drosophile, l’épithélium ventro-latéral (la bandelette germinale) subit une élongation le long de l’axe antéropostérieur induite par l’intercalation cellulaire. Le remodelage polarisé des jonctions cellulaires est à la base de ce phénomène: les jonctions parallèles à l’axe dorsoventral (DV) rétrécissent et forment de manière irréversible de nouvelles jonctions parallèles à l’axe antéropostérieur (AP). Ce remodelage dépend de l’enrichissement du moteur moléculaire Myosine II (MyoII) dans les jonctions DV, qui induit une anisotropie de tension. Les protéines des jonctions adhérentes (E-cad, &#946;-catenin) sont, elles aussi, polarisées : elles sont enrichies dans les jonctions AP. Néanmoins, nous ne savions pas si cette polarité de l’adhésion avait un rôle dans le remodelage des jonctions, et nous ne connaissions pas les mécanismes contrôlant cette localisation asymétrique. L’un des mécanismes les mieux connus de la modulation de l’adhésion cellulaire est l’endocytose des protéines d’adhésion. A ce titre, je me suis intéressé au rôle de l’endocytose Clathrine dépendante (ECD) pendant l’intercalation cellulaire. J’ai ainsi pu montrer que l’ECD de E-cad est régulée à la hausse dans la bandelette germinale au niveau jonctionnelle, plus particulièrement au niveau des jonctions DV (qui rétrécissent). L’ECD d’E-cad est nécessaire à l’intercalation et à la distribution polarisée d’E-cad. Elle est régulée par l’organisation de l’actine: la formine Diaphanous ainsi que le moteur moléculaire Myosine II accélèrent le recrutement de la machinerie d’endocytose (AP2 et Clathrine) et régulent la polarité de l’ECD dans l’embryon. Elles sont contrôlées par RhoGEF2, qui est enrichie dans les jonctions DV, et induisent l’endocytose par un mécanisme de clustering latéral d’E-cad. Dans la seconde partie de ma thèse, je me suis intéressé au couplage entre E-cad et la dynamique de MyoII. En effet, l’intercalation dépend aussi de flux contractiles de MyoII qui ont lieu préférentiellement en direction des jonctions DV. J’ai ainsi pu montrer que la direction des flux est induite par les anisotropies de forces d’ancrage de MyoII. Les faibles niveaux d’E-cad et le fort taux d’endocytose dans les jonctions DV augmentent la probabilité de générer une anisotropie d’ancrage et induisent davantage de flux de MyoII vers les jonctions DV. Ce projet met en lumière le rôle fondamental du couplage entre E-cad et MyoII dans la régulation de la morphogenèse. / Epithelia build up strong mechanical and chemichal barriers in Metazoans. Adherens junctions, through the adhesion provided by the transmembrane protein E-cadherin (E-cad), are essential for the mechanical integrity of the tissue. Yet, epithelia can be dramatically remodeled during embryogenesis or wound healing. During gastrulation of Drosophila embryo, the ventrolateral epithelium (the germ band) undergoes a massive elongation along the anteroposterior (AP) axis, driven by cell-cell intercalation. This is based on the polarized remodeling of intercellular junctions whereby junctions parallel to the dorsoventral axis (DV) shrink and form new junctions along AP axis. This remodeling is mediated by the planar polarized enrichment of Myosin II (MyoII) in DV junctions, which generates high tension. Adhesion proteins are also planar polarized, E-cad is enriched in AP junctions, but we did not know if this polarity contributed to cell-cell intercalation and the mechanism driving this polarity. As such, I have studied the role of Clathrin mediated endocytosis (CME) during germ band extension. I have shown that E-cad CME is specifically upregulated at the junction plane in the germ band, and planar polarized (enriched in DV shrinking junctions). It is required for cell-cell intercalation and the planar polarized distribution of E-cad. E-cad CME is regulated by the concerted action of the Formin Diaphanous and Myosin-II, which accelerates CME through the lateral clustering of E-cad. They are controlled by RhoGEF2, which is also enriched in DV junctions. In the second part of my PhD, I have studied the coupling between E-cad and MyoII dynamics. Indeed, planar polarized contractile flows of MyoII are required for DV junction shrinkage, but we did not know the mechanism driving the polarity of these flows. I have shown that the transient anisotropy of anchoring forces between two facing junctions triggers flow. As such, the low steady state amount of E-cad and the high rate of CME in DV junctions trigger more anisotropy and polarize the flow. These results outline the strong crossregulation between E-cad and MyoII and their concerted action in morphogenesis.

Introdução: A endometriose é uma doença ginecológica comum caracterizada pela presença de estroma e/ou glândula endometrial fora da cavidade uterina, e que não possui sua etiopatogenia bem estabelecida. A transição epitélio-mesênquima (TEM) é um processo que consiste em uma série de mudanças no fenótipo de células epiteliais que fazem com que estas células assumam características de células mesenquimais. Assim como observado na TEM, as células endometriais no contexto da endometriose apresentam capacidade migratória, invasibilidade e elevada resistência à apoptose. As moléculas de adesão têm adquirido crescente relevância na TEM, pois relacionam-se à perda de adesão célula-célula com o aumento da invasão e metástase. O objetivo deste estudo foi investigar a expressão de marcadores relacionados com a TEM na endometriose superficial, ovariana e profunda. Pacientes e Métodos: Foram selecionadas 103 mulheres que preenchiam os critérios de inclusão estabelecidos, constituindo 2 grupos de estudo independentes entre si: 18 mulheres com endometriose peritoneal, ovariana e profunda concomitantes; 85 mulheres com endometriose ovariana e/ou profunda, dividido em 44 mulheres com endometriose ovariana e 41 com endometriose intestinal. Através de reações de imunoistoquímica, a expressão proteica dos marcadores e-caderina, n-caderina, betacatenina, receptor de estrogênio e receptor de progesterona foram avaliados nos tecidos de interesse em cada grupo de estudo. Além dos locais de doença, as mulheres foram avaliadas quanto à relação com a fase do ciclo e à classificação histológica da doença. Resultados: As lesões de endometriose de ovário mostraram uma menor expressão de n-caderina em comparação às lesões de intestino e peritônio (p=0,032). O receptor de estrogênio e receptor de progesterona se mostraram significativamente menos expressos no componente epitelial da doença de ovário do que no epitélio da endometriose de peritônio e intestino (p=0,002; p=0,48). A expressão da n-caderina apresentou uma correlação direta com a expressão do receptor de estrogênio no estroma da endometriose de intestino (p=0,036). Conclusão: Estes resultados sugerem que a transição epitélio-mesênquima esteja envolvida na etiopatogenia da endometriose, demonstrando que a doença de ovário se comporta de maneira diferente da doença superficial e da doença infiltrativa profunda, sendo a n-caderina um importante fator envolvido neste processo possivelmente influenciada pela ação do estrogênio / Background: Endometriosis is a common gynecological disease defined as the presence of ectopic endometrial glands and stroma outside the uterine cavity, and its pathogenesis is not well established. The epithelial to mesenchymal transition (EMT) is a process consisting of a series of changes in the phenotype of epithelial cells that make these cells assume the characteristics of mesenchymal cells. As observed in the EMT, endometrial cells in the context of endometriosis have the capacity of migration, invasiveness and high resistance to apoptosis. . The adhesion molecules have become progressively relevant in EMT, in view of the cell-to-cell adhesion loss, with increased invasion and metastasis. The goal of this study was to investigate the expression of markers related to EMT in superficial, ovarian and deep endometriosis. Patients and Methods: 103 women were selected who met the inclusion criteria, constituting two independent study groups: 18 women with peritoneal, ovarian and deep concomitant endometriosis, 85 women with ovarian and / or deep endometriosis, divided in 44 women with ovarian endometriosis and 41 with intestinal endometriosis. Through immunohistochemical reactions, the protein expression of e-cadherin, ncadherin, beta-catenin, estrogen receptor and progesterone receptor markers were evaluated in tissues of interest in each study group. In addition to the sites of the disease, menstrual phase and histological classification (well-differentiated, undifferentiated, mixed pattern and stromal) of the disease were recorded. Results: The ovarian endometrisis showed less n-cadherin marker than lesions of the peritoneum and bowel (p=0,032). Ovarian endometriosis also showed markedly decreased expression of estrogen and progesterone receptors in epithelial cells, compared with peritoneal and deep endometriosis (p=0,002; p=0,48). The expression of N-cadherin showed a direct correlation with estrogen receptor expression in the stroma of bowel endometriosis (p = 0.036). Conclusion: These results suggest that epithelial to mesenchymal transition involved in the pathogenesis of endometriosis, demonstrating that the ovary disease behaves differently disease than peritoneal and deep disease, so that the n-cadherin is an important factor involved in this process, possibly influenced by the action of estrogen

Introdução: A endometriose é uma doença ginecológica comum caracterizada pela presença de estroma e/ou glândula endometrial fora da cavidade uterina, e que não possui sua etiopatogenia bem estabelecida. A transição epitélio-mesênquima (TEM) é um processo que consiste em uma série de mudanças no fenótipo de células epiteliais que fazem com que estas células assumam características de células mesenquimais. Assim como observado na TEM, as células endometriais no contexto da endometriose apresentam capacidade migratória, invasibilidade e elevada resistência à apoptose. As moléculas de adesão têm adquirido crescente relevância na TEM, pois relacionam-se à perda de adesão célula-célula com o aumento da invasão e metástase. O objetivo deste estudo foi investigar a expressão de marcadores relacionados com a TEM na endometriose superficial, ovariana e profunda. Pacientes e Métodos: Foram selecionadas 103 mulheres que preenchiam os critérios de inclusão estabelecidos, constituindo 2 grupos de estudo independentes entre si: 18 mulheres com endometriose peritoneal, ovariana e profunda concomitantes; 85 mulheres com endometriose ovariana e/ou profunda, dividido em 44 mulheres com endometriose ovariana e 41 com endometriose intestinal. Através de reações de imunoistoquímica, a expressão proteica dos marcadores e-caderina, n-caderina, betacatenina, receptor de estrogênio e receptor de progesterona foram avaliados nos tecidos de interesse em cada grupo de estudo. Além dos locais de doença, as mulheres foram avaliadas quanto à relação com a fase do ciclo e à classificação histológica da doença. Resultados: As lesões de endometriose de ovário mostraram uma menor expressão de n-caderina em comparação às lesões de intestino e peritônio (p=0,032). O receptor de estrogênio e receptor de progesterona se mostraram significativamente menos expressos no componente epitelial da doença de ovário do que no epitélio da endometriose de peritônio e intestino (p=0,002; p=0,48). A expressão da n-caderina apresentou uma correlação direta com a expressão do receptor de estrogênio no estroma da endometriose de intestino (p=0,036). Conclusão: Estes resultados sugerem que a transição epitélio-mesênquima esteja envolvida na etiopatogenia da endometriose, demonstrando que a doença de ovário se comporta de maneira diferente da doença superficial e da doença infiltrativa profunda, sendo a n-caderina um importante fator envolvido neste processo possivelmente influenciada pela ação do estrogênio / Background: Endometriosis is a common gynecological disease defined as the presence of ectopic endometrial glands and stroma outside the uterine cavity, and its pathogenesis is not well established. The epithelial to mesenchymal transition (EMT) is a process consisting of a series of changes in the phenotype of epithelial cells that make these cells assume the characteristics of mesenchymal cells. As observed in the EMT, endometrial cells in the context of endometriosis have the capacity of migration, invasiveness and high resistance to apoptosis. . The adhesion molecules have become progressively relevant in EMT, in view of the cell-to-cell adhesion loss, with increased invasion and metastasis. The goal of this study was to investigate the expression of markers related to EMT in superficial, ovarian and deep endometriosis. Patients and Methods: 103 women were selected who met the inclusion criteria, constituting two independent study groups: 18 women with peritoneal, ovarian and deep concomitant endometriosis, 85 women with ovarian and / or deep endometriosis, divided in 44 women with ovarian endometriosis and 41 with intestinal endometriosis. Through immunohistochemical reactions, the protein expression of e-cadherin, ncadherin, beta-catenin, estrogen receptor and progesterone receptor markers were evaluated in tissues of interest in each study group. In addition to the sites of the disease, menstrual phase and histological classification (well-differentiated, undifferentiated, mixed pattern and stromal) of the disease were recorded. Results: The ovarian endometrisis showed less n-cadherin marker than lesions of the peritoneum and bowel (p=0,032). Ovarian endometriosis also showed markedly decreased expression of estrogen and progesterone receptors in epithelial cells, compared with peritoneal and deep endometriosis (p=0,002; p=0,48). The expression of N-cadherin showed a direct correlation with estrogen receptor expression in the stroma of bowel endometriosis (p = 0.036). Conclusion: These results suggest that epithelial to mesenchymal transition involved in the pathogenesis of endometriosis, demonstrating that the ovary disease behaves differently disease than peritoneal and deep disease, so that the n-cadherin is an important factor involved in this process, possibly influenced by the action of estrogen

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.

No description available.

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.