Thesis (M.S. in genetics and cell biology)--Washington State University, May 2008. / Includes bibliographical references.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001? / Copy of author's previously published work inserted. Bibliography: leaves 139-160.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A presenÃa de metÃstase permanece como a principal causa de morte pelo cÃncer. Diante da ausÃncia de terapia farmacolÃgica para o tratamento de tumores secundÃrios, a pesquisa de novas drogas com potencial antimetastÃtico à de suma importÃncia para o desenvolvimento de novos fÃrmacos anticÃncer. Neste quadro, decidimos avaliar o potencial antimetastÃtico da biflorina, uma o-naftoquinona isolada das raÃzes da Capraria biflora. Em ensaio de proliferaÃÃo celular por Alamar blue, observamos que esta quinona possui atividade citotÃxica contra melanoma humano (MDAMB-435) a partir da concentraÃÃo 5 ÂM em 24h de exposiÃÃo. PorÃm, nessa mesma dose, nÃo houve efeito citotÃxico em 12h de exposiÃÃo. Ensaios de azul de tripan e cristal violeta mostraram que nas concentraÃÃes de 1,0; 2,5 e 5,0 ÂM durante 12h de exposiÃÃo a biflorina nÃo possui efeito citotÃxico. Utilizando as concentraÃÃes de 1,0; 2,5 e 5,0 ÂM (12h exposiÃÃo) foram realizados ensaio de migraÃÃo e invasÃo celular. Nestes ensaios observamos que a biflorina diminui a motilidade e a invasividade da cÃlula MDAMB-435. Em anÃlise morfolÃgica das cÃlulas, utilizando coloraÃÃo de May-Grunwald-Giemsa e coloraÃÃo de actina por faloidina, observamos que a biflorina altera a organizaÃÃo do citoesqueleto de actina, com a presenÃa de cÃlulas menores, retraÃdas e cÃlulas maiores com expansÃes filamentosas semelhantes à filopÃdios. Em ensaio de Western blot observou-se a diminuiÃÃo na expressÃo da molÃcula de adesÃo N-caderina e inibiÃÃo da via de sinalizaÃÃo PI3K/Akt. Estes resultados conferem à biflorina um potencial antimetastÃtico bastante promissor. / Metastasis remains the leading cause of death from cancer. Due to the absence of pharmacological therapy for the treatment of secondary tumors, the search for new drugs with antimetastatic potential is important to the development of new anticancer drugs. In this context we decided to evaluate the antimetastatic potential of biflorin, an o-naphthoquinone isolated from roots of Capraria biflora. In cell proliferation assay using Alamar blue, we found that this quinone has cytotoxic activity against human melanoma cells line (MDAMB-435) at 5 ÂM concentration during 24 hours of exposure. However, with this same dose, there was no cytotoxic effect within 12 hours of exposure. Trypan blue and crystal violet assay showed that biflorina has no cytotoxic effect at 1.0, 2.5 and 5.0 ÂM during 12 hours of exposure. Migration assay and cell invasion assay were performed using concentrations of 1.0, 2.5 and 5.0 ÂM (12h exposure). In these trials we found that biflorin decreases cell motility and invasiveness. In morphological analysis of cells stained using May-Grunwald-Giemsa and actin stain by phalloidin, we observed that biflorin alters the organization of the actin cytoskeleton, with the presence of smaller, retracted and larger cells. In Western blot assay we observed a decrease in the expression of the adhesion molecule N-cadherin and inhibition of PI3K/AKT signaling pathway. These results give biflorin as an agent with promising antimetastatic potential.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. Their extracellular structure is composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. Furthermore, selectins play a role beyond fixing cells to a specific location by regulating important signaling pathways in the migrating cell during physiological and pathological processes. These interactions start mainly with the binding of the lectin domain of selectins and ligand on cells. Therefore, structural/functional studies to date have mainly focused on the direct interactions of the lectin domain of E-selectin with its ligands while other domains and conformational dynamics received less attention. For this purpose, we produced a number of different recombinant E-selectin proteins with and without artificial oligomerization and with changes in the SCR units in addition to proteins where strategic residues will be mutated to change the conformation of the selectin to an extended conformer. Moreover, double cysteine mutant candidates were produced for maleimide labeling for the real-time SM-FRET (single molecule fluorescence resonance energy transfer) studies to assess conformational dynamics of E-selectin. Using a comprehensive set of static- and flow-based assays, we concluded that SCR domains play a role by enhancing the interaction of recombinant E-selectin proteins with E-selectin ligand, while dimerization and extension of the lectin domain improve the binding. However, our double cysteine mutants purification and labeling requires further optimization to be utilized to study the conformational dynamics of E-selectin binding to its ligands using SM-FRET and force microscopy. Furthermore, our experiments extend to highlight the importance of phosphatases in regulating signaling pathways that are affected by E-selectin binding to migrating cells. Collectively, these studies are beneficial to understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells.

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. An excellent model tissue is the corneal epithelium, which is an avascular stratified squamous tissue that responds to growth factors and nucleotides when the epithelial barrier is damaged. One signal that has a ubiquitous response in epithelial wound healing is the cellular release of the nucleotide ATP, which may occur because of mechanics forces and/or change in cell shape. Within milliseconds to seconds after injury, extracellular ATP binds to purinoreceptors and triggers a transient Ca2+ wave, which is used by cells to transduce mechanical signals into chemical signals and alter signaling pathways. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. In this study we developed a novel method to identify and characterize the degree of cell-cell communication that occurs through sustained Ca2+ mobilizations after injury, which are concentrated along the epithelial wound edge and reduced in cells distal to the injury. Using MATLAB analyses, we generated profiles of the sustained Ca2+ mobilizations, and demonstrated that the Ca2+ response was replicated in ex vivo organ culture models. The sustained Ca2+ mobilizations were present also after stimulation with either BzATP or UTP, which are agonists of P2X7 and P2Y2 respectively. The probability that cells would communicate was greater in response to BzATP compared to UTP. The specificity of these ligands was demonstrated using competitive inhibitors of P2Y2 and P2X7 receptors, AR-C 118925XX and A438079, respectively. An inhibitor of pannexin-1, 10Panx, attenuated both wound closure and BzATP agonist-initiated response. These sustained mobilizations are correlated with changes in cellular morphology and motility, which were prominent in cells at the leading edge of the wound during cell migration. Together, our results demonstrate that the sustained Ca2+ mobilizations mediated by purinoreceptors and pannexins are a vital component in regulating the long-term response to injury, as studied in organ culture.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18478号 / 農博第2078号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4862(農学部図書室) / 31356 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 小川 順, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM