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The process of reversible growth arrest, otherwise known as cellular quiescence or the G₀ phase denoted by withdrawal from the cell cycle, is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to/dependent on the limiting factor or circumstance inducing growth arrest. Here I characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition and oxygen limitation (hypoxia). Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients is the limiting factor inducing growth arrest. Contact inhibition and hypoxia induced FABP4 expression, whereas cycling control CEF and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. The GAS gene p20k lipocalin has been shown to exhibit similar expression patterns to FABP4, with its regulation determined by the presence of the transcription factor C/EBP-β. CEF overexpressing C/EBP-β also showed strong FABP4 induction. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that C/EBP-β bound directly to the FABP4 promoter in both normoxic and hypoxic cells, although only the latter condition induced FABP4 protein expression. In summary, these results suggest that FABP4 is induced during growth arrest specifically when oxygen is the limiting factor, as induction was not seen during growth arrest mediated by starvation-induced endoplasmic reticulum (ER) stress, where nutrients was the limiting factor. The induction of these hypoxia-responsive genes suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes. Additionally, FABP4 was shown to be associated with growth arrest and the promotion of cell survival and proliferation, as depicted by proliferation assays. Lastly, C/EBP-β not only strongly induced FABP4 expression, but directly bound to the FABP4 promoter. This suggests that C/EBP-β is a regulator of FABP4, although there may be other interacting factors acting as activators or repressors as this FABP4-C/EBP-β interaction was observed in conditions permissive and non-permissive to FABP4 expression. / Thesis / Master of Science (MSc) / The process of reversible growth arrest is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to the limiting factor or circumstance inducing growth arrest. Here we characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition (CI) and hypoxia. Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients availability is the limiting factor. CI and hypoxia induced FABP4 expression, whereas control and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. When overexpressing C/EBP-β, CEF showed strong FABP4 induction. Additionally, a direct interaction with the FABP4 promoter was observed in both normoxic and hypoxic cells, although only the latter condition induced expression. In summary, the induction of this hypoxia-responsive gene suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes and that this induction may be regulated by C/EBP-β.

Contact inhibition of locomotion (CIL), a migratory mechanism, first introduced by Abercrombie and Heaysman in 1953 is now a fundamental driving force in developmental, repair and disease biology. Much of what we know of CIL stems from studies done on 2D substrates which are unable to provide the essential biophysical cue of fibrous extracellular matrix curvature. Here we inquired if the same rules are applicable for cells attached to and migrating persistently on suspended and aligned ECM-mimicking nanofibers. Using two elongated cell shapes (spindle attached to one fiber, and parallel attached to two fibers), we quantitate CIL rules for spindle-spindle, parallel-parallel and spindle-parallel collisions. Two approaching spindles do not repolarize upon contact but rather continue to migrate past one another. Contrastingly, approaching parallel cells establish distinct CIL, with only one cell repolarizing upon contact followed by migration of both cells as a cohesive unit in the repolarization direction. Interestingly, for the case of spindle and parallel cell collision, we find the parallel cell to shift the morphology to that of spindle and continue persistent movement without repolarization. To account for effect of cell speed, we also quantitate CIL collisions between daughter and non-dividing cells. While spindle-spindle collisions result in cells still walking by, for parallel-parallel collisions, we capture rare events of a daughter cell pushing the non-dividing cell. With increasing population numbers, we observe formation of cell streams that collapse into spheroids. Single cells are able to invade along fibers from the spheroids and are then subject to same CIL conditions, thus providing a platform with cyclic CIL. The presented coupling of experimental and analytical framework provides new insights in contextually relevant CIL and predictive capabilities in cell migration decision steps. / Master of Science / Contact inhibition of locomotion (CIL) is a migratory process that can lead to a change in migration direction through protrusion inhibition of single cells. First described in 1953, the traditional model of CIL shows that on a 2D substrate, two migrating cells experience a decrease in protrusive behavior upon contacting each other, followed by repolarization, and migration away from one another. However, a cell's extracellular matrix (ECM) is fibrous in nature, and how cells maintain standard CIL rules in fibrous environments remains unclear. Here, using suspended, aligned nanofibers created using a non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) method, we investigate CIL decision steps of two fibroblast cells approaching each other in two shapes: spindle cells attached to single fibers, and parallel cells attached to two fibers. Most spindle cells approaching each other do not switch direction upon contact, but rather continue to migrate past each other, termed a walk past. Contrastingly, approaching parallel cells display unique CIL whereby only one cell repolarizes and reverses its migration direction. Subsequently, both cells remain in contact while migrating in the repolarization direction. Interestingly, we report that both spindle and parallel CIL are also affected by speed post cell division. Altogether, for the first time, we introduce a platform to understand cell shape driven CIL geometrical rules in ECM mimicking environments.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. A hallmark of epithelial cells is strong cell-cell junctions. These junctions include E-Cadherin, a type of adherens junction that is critical for both barrier function and contact inhibition. Prior experiments by other groups have shown that adherens junctions are subject to mechanical tension. Externally applied forces (e.g. stretch) results in changes in E-Cadherin forces that coordinate proliferation. My current work tests the hypothesis that E-Cadherin forces mediate the spatial regulation of cell proliferation even in the absence of externally applied forces.

COPD is characterized by tissue inflammation and impaired remodeling that suggests fibroblast maintenance of structural homeostasis is dysregulated. Thus, we performed in vitro wound healing experiments on normal and diseased human lung fibroblasts and developed an ABM of fibroblasts closing a scratched monolayer using NetLogo to evaluate differences due to COPD or cigarette smoke condensate exposure. This ABM consists of a rule-set governing the healing response, accounting for cell migration, proliferation, death, activation and senescence rates; along with the effects of heterogeneous activation, phenotypic changes, serum deprivation and exposure to cigarette smoke condensate or bFGF. Simulations were performed to calibrate parameter-sets for each cell type using in vitro data of scratch-induced migration, viability, senescence-associated beta-galactosidase and alpha-smooth muscle actin expression. Parameter sensitivities around each calibrated parameter-set were analyzed. This model represents the prototype of a tool designed to explore fibroblast functions in the pathogenesis of COPD and evaluate potential therapies.

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成2年11月22日 竹中徳哉氏の博士論文として提出された

High-throughput cell culture is an emerging technology that shows promise as a tool for research in tissue engineering, drug discovery, and medical diagnostics. An important, but overlooked, challenge is the integration of experimental methods with information processing suitable for handling large databases of cell-cell and cell-substrate interactions. In this work the traditional global descriptions of cell behaviors and surface characteristics was shown insufficient for investigating short-distance cell-to-cell and cell-to-surface interactions. This problem was addressed by introducing individual-cell based local metrics that emphasize cell local environment. An individual-cell based local data analysis method was established. Contact inhibition of cell proliferation was used as a benchmark for the effectiveness of the local metrics and the method. Where global, summary metrics were unsuccessful, the local metrics successfully and quantitatively distinguished the contact inhibition effects of MC3T3-E1 cells on PLGA, PCL, and TCPS surfaces. In order to test the new metrics and analysis method, a model of cell contact inhibition was proposed. Monte Carlo simulation was performed for validating the individual-cell based local data analysis method as well as the cell model itself. The simulation results well matched with the experimental observations. The parameters used in the cell model provided new descriptions of both cell behaviors and surface characteristics. Based on the viewpoint of individual cells, the local metrics and local data analysis method were extended to the investigation of cell-surface interactions, and a new high-throughput screening and knowledge discovery method on combinatorial libraries, local cell-feature analysis, was developed. PLGA/PCL combinatorial libraries were used as a prototype and a shaper and holder phenomenon involving MC3T3-E1 cells interacting with PCL islands was discovered. In summary, the viewpoint of individual cells casts new light on the study of cell-cell and cell-surface interactions and represents a novel methodology for developing new data analysis and knowledge discovery methods. The results of contact inhibition study and the shaper and holder model provide new knowledge, while the local data analysis method as well as the cell model of contact inhibition suggested novel approaches to study cell-cell and cell-surface interactions.

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T19:11:01Z (GMT). No. of bitstreams: 1 Prado_Mairado_D.pdf: 14853217 bytes, checksum: f86be389a870042c299930884a318e11 (MD5) Previous issue date: 2012 / Resumo: Introdução: O objetivo do presente trabalho foi avaliar a interação entre as diferentes substâncias químicas auxiliares, utilizadas em endodontia, e seus efeitos nas etapas do tratamento endodôntico. Métodos: Soluções de hipoclorito de sódio (NaOCl) a 0,16%, 1%, 2,5% e 5,25%, clorexidina 2% solução e gel, EDTA 17%, ácido cítrico 10% e ácido fosfórico 37% foram utilizadas isoladamente ou associadas entre si na proporção 1:1. As mesmas foram analisadas quimicamente por espectrometria de massa; e microbiologicamente pelo método de difusão em ágar, contra diferentes patógenos. Adicionalmente, foram avaliados onze protocolos de irrigação em relação à formação de smear layer química por microscopia eletrônica de varredura. Por fim, foram avaliados 9 protocolos de irrigação associados a dois diferentes sistemas de obturação, guta-percha/ AH Plus e Resilon/ Real Seal SE, em relação à microinfiltração coronária e à resistência de união da dentina aos materiais obturadores. Resultados: O NaOCl, em todas as concentrações, associado à clorexidina em ambas as formulações, levou à formação de precipitado, assim como a associação entre a clorexidina e o EDTA e entre clorexidina e solução salina. Todas as associações avaliadas apresentaram algum grau de inibição contra os patógenos testados. Irrigação intermediária, com 10 mL de água destilada, entre as soluções de NaOCl e clorexidina, não foram capazes de inibir a formação de smear layer química, assim como irrigações com EDTA e ácido cítrico. Dos diferentes protocolos de irrigação avaliados, aqueles que apresentaram uma irrigação final com solução de clorexidina 2% tiveram níveis reduzidos de microinfiltração coronária. Em relação à resistência de união à dentina, no sistema obturador guta-percha/AH Plus, os grupos em que se utilizou as associações NaOCl/ácido fosfórico e clorexidina/EDTA apresentaram maiores valores de resistência de união, ao passo que no sistema Resilon/Real Seal SE, os maiores valores foram encontrados nos grupos clorexidina/ácido fosfórico. A utilização da clorexidina como irrigante final não afetou negativamente os sistemas obturadores avaliados. Conclusões: A interação entre as substâncias químicas auxiliares pode levar à formação de precipitados. As diferentes substâncias químicas auxiliares, quando associadas, possuem atividade antimicrobiana. Irrigações intermediárias entre as diferentes substâncias químicas auxiliares são necessárias para reduzir ou até mesmo impedir a formação de precipitados, visualizados na superfície dentinária como uma smear layer química. Durante o preparo químicomecânico, as diferentes substâncias químicas auxiliares geram modificações na superfície dentinária que influenciam na microinfiltração coronária e na resistência de união dos sistemas obturadores guta-percha/AH Plus e Resilon/Real Seal SE / Abstract: Introduction: The aim of the present study was to evaluate the interaction among different chemical auxiliary substances used in endodontics and their effects on different steps of endodontic treatment. Methods: 0.16%, 1%, 2.5% and 5.25% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine solution and gel, 17% EDTA, 10% citric acid, 37% phosphoric acid, distilled water, and saline solution were used both associated with each other (1:1 ratio) and not associated. The solutions were chemically examined with mass spectrometry. They were microbiologically examined using the Agar Diffusion Method, against different pathogens. In addition, eleven protocols were analyzed in regards to chemical smear layer with scanning electron microscopy. Finally, nine irrigation protocols (associated with two filling systems, i.e. gutta-percha/AH Plus and Resilon / Real Seal SE) were examinated regarding coronal microleakage and were examined as well regarding their bond strength with dentin. Results: NaOCl, at all concentrations, associated with both chlorhexidine formulations, led to precipitate formation, similar to the association between chlorhexidine/EDTA, and the chlorhexidine/saline solution association. All associations had some degree of inhibition against the evaluated pathogens. Intermediate flush, with 10 mL of distilled water, between NaOCl and chlorhexidine did not inhibit chemical smear layer formation, similar to intermediate flushes with EDTA and citric acid. Among the different irrigation protocols that were evaluated, when final flush with 2% chlorhexidine was present, the lowest levels of coronal microleakage were found. Regarding the bond strength to dentin, in the gutta-percha/AH Plus system, the groups with NaOCl/phosphoric acid and chlorhexidine/EDTA associations showed higher bond strength values. In the Resilon/Real Seal SE system, the highest values were found in the chlorhexidine/phosphoric acid groups. The use of chlorhexidine as a final flush did not negatively affect the filling systems evaluated. Conclusions: The interaction among the auxiliary chemical substances may lead to precipitate formation. The association between the different substances has antimicrobial activity. Intermediate flushes are necessary to reduce or even avoid the formation of chemical smear layer on the dentin surface. During the chemo-mechanical preparation, the various auxiliary chemical substances used do modify the dentine surface. These modifications have influence in the coronal microleakage and in the bond strength of the filling systems gutta-percha/AH Plus and Resilon / Real Seal SE / Doutorado / Endodontia / Doutora em Clínica Odontológica

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