Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 140-172).

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 173 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 79-89). Also available on the World Wide Web.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 160 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Roger Bradley. Includes bibliographical references (leaves 122-140).

abstract: This dissertation features a compilation of studies concerning the biophysics of multicellular systems. I explore eukaryotic systems across length scales of the cell cytoskeleton to macroscopic scales of tissues. I begin with a general overview of the natural phenomena of life and a philosophy of investigating developmental systems in biology. The topics covered throughout this dissertation require a background in eukaryotic cell physiology, viscoelasticity, and processes of embryonic tissue morphogenesis. Following a brief background on these topics, I present an overview of the Subcellular Element Model (ScEM). This is a modeling framework which allows one to compute the dynamics of large numbers of three-dimensional deformable cells in multi-cellular systems. A primary focus of the work presented here is implementing cellular function within the framework of this model to produce biologically meaningful phenotypes. In this way, it is hoped that this modeling may inform biological understanding of the underlying mechanisms which manifest into a given cell or tissue scale phenomenon. Thus, all theoretical investigations presented here are motivated by and compared to experimental observations. With the ScEM modeling framework I first explore the passive properties of viscoelastic networks. Then as a direct extension of this work, I consider the active properties of cells, which result in biological behavior and the emergence of non-trivial biological phenotypes in cells and tissues. I then explore the possible role of chemotaxis as a mechanism of orchestrating large scale tissue morphogenesis in the early embryonic stages of amniotes. Finally I discuss the cross-sectional topology of proliferating epithelial tissues. I show how the Subcellular Element Model (ScEM) is a phenomenological model of finite elements whose interactions can be calibrated to describe the viscoelastic properties of biological materials. I further show that implementing mechanisms of cytoskeletal remodeling yields cellular and tissue phenotypes that are more and more biologically realistic. Particularly I show that structural remodeling of the cell cytoskeleton is crucial for large scale cell deformations. I provide supporting evidence that a chemotactic dipole mechanism is able to orchestrate the type of large scale collective cell movement observed in the chick epiblast during gastrulation and primitive streak formation. Finally, I show that cell neighbor histograms provide a potentially unique signature measurement of tissue topology; such measurements may find use in identifying cellular level phenotypes from a single snapshot micrograph. / Dissertation/Thesis / Ph.D. Physics 2011

INTRODUÇÃO: O carcinoma espinocelular de origem bucal (CEC) apresenta uma alta taxa de mortalidade devido à invasividade das células tumorais. A migração celular, principal evento da invasão e metástase, pode ser regulada tanto por fatores intrínsecos, como adesão e contratilidade celular, quanto extrínsecos, como composição, densidade e remodelagem da matriz extracelular (MEC). OBJETIVO: Avaliar o papel de elementos intrínsecos e extrínsecos sobre o processo invasivo do carcinoma espinocelular de origem bucal. MÉTODOS: Foi realizada imuno-histoquímica para as proteínas: Miosina II (isoformas A, B e C), metaloproteinases de matriz (1, 2, 9 e 14); imunofluorescência as proteínas: e-caderina, n-caderina, FAK, paxilina, vinculina e fibronectina em amostras de CEC oral. Foi realizado ensaio de migração nas seguintes condições: 1 – matriz 2D com o substrato de fibronectina, ou laminina ou matrigel; 2 – matriz 3D com colágeno na presença ou não de fibronectina ou laminina; 3 – matriz 3D com diferentes concentrações de colágeno (0,6; 1,2 e 1,8 mg/ml) + fibronectina na presença ou não de um inibidor de MMP. Foi realizado análise de adesão celular utilizando-se o microscópio TIRF e o microscópio confocal, tanto em matrizes 2D quanto 3D. Foram realizados esferoides celulares para avaliar a contratilidade celular, através do plaqueamento das células em gel de agarose e a utilização de drogas que inibem ou que induzem a contratilidade, bem como a partir de células transfectadas com versões fosfomiméticas para a cadeia leve de miosina. Foi realizado ainda western blotting para proteínas: e-caderina, FAK, vinculina, paxilina, N-caderina, integrinas e as isoformas de miosina II, bem como foi avaliado os níveis de ativação das proteínas da família RhoGTPase, as quais estão envolvidas no controle da migração celular. RESULTADOS: A expressão das MMPs analisadas e das isoformas de miosinas foi maior nas zonas de invasão tumoral, sendo que o CEC oral também apresenta uma maior expressão de proteínas associadas à adesão com a MEC. A migração celular foi afetada pela densidade e a composição da MEC, bem como pela atividade das MMPs. Adicionalmente, a modulação das proteínas de adesão célula-matriz altera a velocidade de migração, a direcionalidade dessa migração e também a forma de migração, mudando de uma migração coletiva para uma migração individual. O aumento na contratilidade células resulta numa dispersão celular enquanto que a diminuição da contratilidade resulta numa melhor adesão célula – célula. CONCLUSÕES: O comportamento das células tumorais pode ser modulado através de fatores extrínsecos como, por exemplo, a alteração no microambiente tumoral, seja ela por mudança no substrato ou na densidade da matriz, e também dos fatores intrínsecos como a alteração nos níveis de miosina. / INTRODUCTION: Oral squamous cell carcinoma (OSCC) presents high mortality index due to the invasive phenotype of tumor cells. Cell migration is the main event in cell invasion and metastasis and it can be regulated by intrinsic factor, such as adhesion and cell contractility, and extrinsic factors, such as density and extracellular matrix (EMC) remodeling. OBJECTIVE: Analyze the role of intrinsic and extrinsic factor during the invasive process of oral squamous cell carcinoma. METHODS: We performed immunostaining in OSCC samples for the following proteins: myosin II (isoforms A, B and C), matrix metalloproteinase (1, 2, 9 and 14) e-cadherin, n-cadherin, FAK, paxillin, vinculin and fibronectin. We also performed migration assays with OSCC cell line in the following conditions 1 – 2D matrix with fibronectin or laminin or matrigel; 2 – 3D matrix with collagen in the presence or not of fibronectin or laminin; 3 – 3D matrix with different collagen concentration (0,6; 1,2 e 1,8 mg/ml) with fibronectin in the presence or not of the MMP inhibitor. In order to analyze cell adhesion, it was performed Total Internal Reflectance Fluorescence and Confocal microscopy, in 2D and 3D matrix. To analyze cell contractility, cells were plated in agarose gel in order to produce spheroids, which were treated with drugs that inhibit or induce cell contractility or cells were previously transfected with Myosin Light Chain phosphomimetics mutants. It was also performed western blotting to: e-cadherin, n-cadherin, FAK, paxillin, vinculin and myosin II isoforms, as well as it was analyze the levels in RhoGTPase family, which are involved in cell migration control. RESULTS: The expression to MMPs and myosin II isoforms were higher at invasion zone of the tumor, and the OSCC presented higher expression of proteins associated to adhesion to ECM. Cell migration was affected by the EMC composition and density and by MMP activity. Also, the modulation of cell-matrix adhesion proteins altered migration speed, cell directionality as well as influenced the switch between collective and single cell migration. The increase in cell contractility resulted in cell dispersion while the decrease in cell contractility resulted in a better cell-cell adhesion. CONCLUSIONS: The behavior of cell tumor can be modulate by extrinsic factors, for example, the change in tumor microenvironment, by the change in the EMC substrate or density and by intrinsic factors such as the alteration in myosin levels.

La migration cellulaire collective (MCC) est un processus fondamental qui intervient au cours du développement, de la réparation tissulaire, de l'invasion tumorale et de la formation de métastases. Les cellules qui migrent collectivement possèdent deux types d'interaction avec leur environnement : i) l'un avec leur substrat et ii) l'autre avec les cellules voisines en migration. Deux grandes familles de protéines permettent ces interactions ainsi que la génération de forces mécaniques: i) la famille des intégrines (les récepteurs de la matrice extracellulaire) et ii) la famille des cadhérines (formant les jonctions intercellulaires). Les cadhérines classiques sont impliquées dans la formation des jonctions intercellulaires et sont les principaux acteurs de la MCC au cours du développement normal et tumoral. La transmission de force entre les cellules en migration est nécessaire à leur cohésion et à la communication des cellules entre elles. Des études récentes montrent que les cadhérines sont nécessaires à la transmission des forces au substrat. Néanmoins, les processus par lesquels les cadhérines agissent sur ses forces dans le contexte d'une MCC restent inexplorés. Nous avons identifié l'expression de la P-cadhérine comme étant associée à l'agressivité du rhabdomyosarcome de type alvéolaire (ARMS), sous type ayant le plus mauvais pronostic car très invasif. Nos données, ainsi que de récentes études qui démontrent que la P-cadhérine est impliquée dans l'agressivité des tumeurs du sein, nous ont conduits à étudier le rôle de cette cadhérine dans la migration cellulaire, processus majeur dans le développement tumoral. Nous nous sommes intéressés à l'impact de l'expression de la P-cadhérine sur la migration des myoblastes murins normaux C2C12. Pour ce faire nous utilisons un test de migration in vitro en 2D proche du test de blessure qui consiste à retirer une barrière physique induisant la migration des cellules vers l'espace libre ainsi créé. Nous avons pu monter que l'expression de la P-cadhérine dans les myoblastes C2C12 augmente les paramètres caractéristiques d'une MCC in vitro : la vitesse, la polarité, la persistance et la directionalité de la migration des cellules du front et au sein du feuillet. De plus, à l'aide de techniques microscopiques de mesure des forces nous avons montré une augmentation des forces intercellulaires allant du front vers le feuillet cellulaire au cours de la migration des cellules exprimant la P-cadhérine. Cela suggère une augmentation de la cohésion cellulaire. L'ensemble de ces résultats démontrent clairement que l'expression de la P-cadhérine induit une MCC. Nous avons aussi mesuré et cartographié les forces de traction au substrat connues pour être le moteur de la migration cellulaire. Nos données indiquent que l'expression de la P-cadhérine augmente l'anisotropie de ces forces de traction ainsi que leur intensité, et ce, uniquement au front de migration. L'expression de la P-cadhérine remodèle et stimule la dynamique des plaques focales d'adhérence à cet endroit.Afin de mieux comprendre comment la P-cadhérine modifie la dynamique des adhésions focales et augmente les forces de traction, nous avons étudié l'activité spatiotemporelle des petites protéines G de la famille Rho. Nous montrons que l'expression de la P-cadhérine active Rac1 et Cdc42 au front de migration, entrainant ainsi le remodelage et l'organisation des plaques focales d'adhérence à cet endroit. L'inhibition de Rac1 et Cdc42 bloque la MCC induite par la P-cadhérine. Pour conclure, en combinant la mesure des paramètres de migration cellulaire avec la mesure des forces mécaniques intercellulaires et au substrat, nous avons démontré que la P-cadhérine induit un comportement collectif des cellules et ce dépendamment de l'activité de Rac1 et de Cdc42. De plus nous mettons en avant l'existence de propriétés mécano-transductrices de cette cadhérine au cours de la MCC. / Collective cell migration (CCM), the coordinated movement of multiple cells that are connected by cell-cell adhesion, is a fundamental process in development, tissue repair and tumor invasion and metastasis. Cells part of a moving collective have two different types of interactions, i) one with the substratum, and ii) one with surrounding moving cells. Two protein families allow these interactions and also the generation of mechanical forces: i) typically integrins on the underlying extracellular matrix (ECM) and ii) cadherins at intercellular adhesion sites. Classical cadherins are involved in cell-cell adhesion and are major drivers of collective cell migration in embryonic development and tumorigenesis.Mechanical coupling between migratory cells may result in the production of force-dependent signals by which the cells influence each other. Moreover, whereas recent data showed that cadherin-dependent cell-cell adhesions are important for the force transmission to the ECM, how intercellular adhesion impacts on cell-ECM forces in the context of collective cell migration is totally unexplored. We identified P-cadherin expression to be associated with alveolar rhabdomyosarcoma (ARMS) aggressiveness, tumors with a bad prognosis due to the propensity for early and wide dissemination. Our data and recent findings showing that P-cadherin is associated with breast tumor invasiveness and aggressiveness, led us to investigate the role of P-cadherin in cell migration. We analyzed cell migration of normal mouse C2C12 myoblasts that express P-cadherin using a “wound-healing like assay” in which migration is analyzed after removal of a physical barrier. We observed that P-cadherin expression in C2C12 myoblasts increased the speed, polarity, persistence and directionality of migration toward the free space of both cells at the border and cells into the sheet. Using monolayer stress microscopy we showed that P-cadherin increases inter-cellular stresses and force transmission across the cell sheet. According to those observations we concluded that P-cadherin induces CCM.Traction forces exerted by the cells on the substrate are important for cell migration. Using traction force microscopy, we demonstrated that P-cadherin expression increases the traction forces anisotropy specifically at the multicellular leading row. To better understand how these mechanical signals induce CCM, we studied both the organization of the focal adhesions and the spatio-temporal activity of Rho GTPase. We showed that P-cadherin expression activates Rac1 and Cdc42 which induces extensive focal adhesions remodeling at the leading edge of cells at the leading row. Rac1 and Cdc42 inhibition impaired P-cadherin-induced CCM, focal adhesion remodeling and forces generation. In conclusion, combining a detailed measurement of the parameters of cell migration with physical measure of the intercellular stresses and traction forces, we have shown that P-cadherin promotes collective behavior of cells during migration through Rac1 and Cd42 activity. Also, those results provide evidence for mechano-transmission properties of P-cadherin during collective cell migration.

Collective cell migration is ubiquitous in biology, from development to cancer; it is influenced by heterogeneous cell types, signals and matrix properties, and requires large scale regulation in space and time. Understanding how cells achieve organized collective motility is crucial to addressing cellular and tissue function and disease progression. While current two-dimensional model systems recapitulate the dynamic properties of collective cell migration, quantitative three-dimensional equivalent model systems have proved elusive. The overarching hypothesis of this work is that cell collectives are heterogeneous in nature; and that the influence of biochemical, physical, and mechanical factors combined leads to diverse physical behaviors. The central goal of this work is to establish standard tools for the understanding of 3D collective cell motility by treating individual cell-collectives as independent entities. An experimental model studies cell collectives by tracking individual cells within cell cohorts embedded in three dimensional collagen scaffolding. A computational model of 3-dimensional multi-scale self-propelled particles recreates experimental data and accounts for intercellular adhesion dynamics. A custom algorithm identifies cellular cohorts from experimental and simulated data so these may be treated as independent entities. A second custom algorithm quantifies the temporal and spatial heterogeneity of motion in cell cohorts during ‘motility events’ observed in experiments and simulations. The results show that cell-cohorts in 3D are dynamic with spatial and temporal heterogeneity; cohesive motility events can emerge without an external driving agent. Simulated cohorts are able to recreate experimental motility event signatures. Together these model systems and analytical techniques are some of the first to address collective motility of adhesive cellular cohorts in 3-dimensions.

Wound healing in the corneal epithelium is an essential process to maintain corneal clarity and organism health. The earliest events of cellular injury response include the release of nucleotides and the activation of P2 purinergic receptors. While the purinergic receptor P2X7 has been shown to promote cell migration, its role in corneal epithelial wound healing is still poorly understood. The goal of this work is to better understand the role of P2X7 in the injury response. We analyzed P2X7 expression after epithelial injury in rat corneal organ cultures and found that the receptor localizes to the leading edge of the corneal epithelium. However, overall mRNA and protein expression of P2X7 decreased after injury. Inhibition of P2X7 activation significantly delayed wound closure and prevented the leading edge-localization after injury. We found that P2X7 inhibition altered the wound-induced calcium wave in epithelial cells and altered the number and distribution of focal adhesions in the migrating cells. Live cell imaging of epithelial cells showed that P2X7 inhibition led to altered actin rearrangement, with thick actin bundles in the treated cells. In order to determine the importance of P2X7 in epithelial differentiation and stratified cell migration, we developed a stratified culture model. The cells in the stratified model expressed proliferative and differentiation markers similar to organ cultured corneas, as well as similar P2X7 expression and localization after injury. Together, these results show the importance of P2X7 in the overall purinergic response to injury, and provide tools to study P2X7 in stratified corneal cell migration. To determine if P2X7 may contribute to pathologic delayed wound healing in diseases such as type 2 diabetes, we analyzed P2X7 expression in diabetic human corneas and diabetic model rodent corneas. We showed that P2X7 expression is significantly elevated in unwounded diabetic corneas, and that wound healing is delayed in the rodent model. These data show that elevated P2X7 expression may contribute to the delayed healing in disease and may be a possible therapeutic target.