The genetic disorder sickle cell anemia causes hemolytic anemia and sickle pain crisis, episodes of microvascular occlusion resulting in painful ischemic tissue damage. Pain crisis is thought to occur when sickle erythrocytes adhere in the post-capillary venule, partially occluding the vessel. The resulting slowed blood flow causes more extensive cell adherence and entrapment of rigid, deoxygenated erythrocytes until the vessel is entirely occluded. It was hypothesized that the inflammatory mediators histamine and tumor necrosis factor-, factors known to cause endothelial expression of adhesive ligands, might significantly increase sickle erythrocyte adhesion, and thus be capable of initiating sickle pain crisis. It was also hypothesized that the perfusion shear stress environment of the endothelium, known to be oscillatory and reduced in sickle cell patients, was a significant mediating factor of sickle cell adhesion. An in-vitro flow chamber using cultured endothelial cells and erythrocytes from blood samples of sickle cell anemic patients was used to quantify sickle erythrocyte adherence to stimulated and unstimulated endothelial cells under shear stresses from 1.0 to 0.1 dyne/cm2. Results showed that both endothelial stimulation and reduction of the perfusion shear stress increased sickle erythrocyte adherence. In combination, the use of inflammatory stimulation with reduced shear stress resulted in further increased adhesion, but only when above the range of 0.1 V 0.2 or 0.4 dyne/cm2, depending on the inflammatory mediator. Adhesion below this level of shear is not significantly increased by endothelial stimulation. The mechanism by which histamine mediates adhesion was investigated, and found to involve the endothelial H2 and H4 receptors and expression of the P-selectin ligand. These data suggest that irregular flow, typical of sickle microvasculature, may act in conjunction with the pro-inflammatory state of sickle vasculature and the histaminergic nature of some pain treatments to initiate or propagate sickle vaso-occlusion. Findings concerning histamine, tumor necrosis factor-alpha, and shear stress effects on adherence are discussed in relation to their possible applicability to patient health, future studies are outlined to confirm the relation of in vitro data to in vivo patient condition, and proposals are made for applying these methodologies to other potential mediators of sickle erythrocyte adhesion.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

O carcinoma espinocelular (CEC) de cabeça e pescoço é uma neoplasia maligna que implica em baixas taxas de sobrevida e prognóstico desfavorável. A perda de adesão do epitélio e a migração celular são processos biológicos envolvidos na carcinogênese. O objetivo desse estudo foi descrever o padrão de expressão da molécula cinase de adesão focal (FAK) e suas formas fosforiladas, FAK-Tyr576 e FAK-Tyr925, em amostras de CEC no centro do tumor (CT) e zona de invasão (ZI) e no tecido epitelial não neoplásico adjacente a estes tumores (EA). De acordo com a graduação histopatológica, 48,1% do total das amostras de CEC são de grau moderado. Segundo o sistema pTNM, 62,9% eram T1 e T2, 64,8% não apresentaram metástase regional e não houve metástase à distância. O tempo máximo de acompanhamento foi de 5,9 anos e obteve-se a proservação final de 46 dos 54 pacientes, onde foi observado que 58,1% apresentaram boa evolução e 41,9% exibiram evolução ruim. A média em anos para que os pacientes obtivessem o status de evolução ruim foi de 5,01. Quanto ao estadiamento clínico, houve maior imunomarcação no estádio IVa quando comparado ao estádio II no EA da FAK Tyr-576 e ocorreu maior expressão no estádio IVb quando comparado ao estádio I no CT da FAK Tyr-576. Os pacientes que apresentaram evolução ruim após o período máximo de acompanhamento tiveram alta expressão no EA e na ZI da FAK Tyr-576 e no CT da FAK Tyr-925. Na ZI, o aumento de expressão da FAK está associado ao aumento da expressão da FAK Tyr-576. À medida que há aumento da expressão no EA há aumento da expressão na ZI na FAK Tyr-576. E quando há aumento da expressão no EA há aumento da marcação na ZI na FAK Tyr-925. Apesar do papel de todas as FAKs fosforiladas não estar claro, mas sabendo-se que são essas FAKs que estão ativas no câncer, a partir dos dados do presente trabalho pode-se sugerir que a avaliação da marcação FAK Tyr -576 no EA, no CT e na ZI venha a fazer parte da rotina no diagnóstico histopatológico e das margens da peça operatória, como norteadores da conduta terapêutica e do protocolo de acompanhamento. / Squamous cell carcinoma (SCC) of the head and neck is a malignant neoplasm that implies low rates of survival and poor prognosis. The loss of adhesion and cell migration are biological processes involved in carcinogenesis. The aim of this study was to describe the pattern of expression of focal adhesion kinase molecule (FAK) and its phosphorylated forms, FAK-Tyr576, FAK-Tyr925, in SCC samples in the center of the tumor (CT) and invasion zone (ZI) and non-neoplastic epithelial tissue adjacent to these tumors (EA). According to histopathological grading, 48,1% of SCC samples are moderate. According to the system pTNM, 62,9% were T1 and T2, 64.8%, had no regional metastasis and no distant metastasis. The maximum time of follow up was 5,9 years and was obtained as the final proservation 46 of 54 patients, where it was observed that 58,1% of patients had good prognosis and 41,9% of patients had poor prognosis. The average years for patients to obtain the poor prognosis of status was 5.01. As for clinical staging, there was greater immunostaining in the stadium IVa compared to stage II in EA Tyr-576. And FAK expression was higher in stage IVb when compared to CT in stage I of FAK Tyr-576. Patients with poor prognosis after the maximum follow-up period had high expression in EA and ZI of FAK Tyr-576 and CT of FAK Tyr-925. In ZI, increased expression of FAK is associated with increased FAK Tyr-576 expression. As there is increased expression in EA's increased expression in ZI in FAK Tyr-576. And when there is increased expression in EA's increased marking the ZI in FAK Tyr-925. Despite the role of all phosphorylated FAKs not clear, but given that these are FAKs that are active in cancer, from the present data it can be suggested that the evaluation of expression of FAK Tyr 576 in EA, CT and ZI will be part of the routine histopathological diagnosis and surgical specimen margins, as guiding the therapeutic management and monitoring protocol.

O carcinoma espinocelular (CEC) de cabeça e pescoço é uma neoplasia maligna que implica em baixas taxas de sobrevida e prognóstico desfavorável. A perda de adesão do epitélio e a migração celular são processos biológicos envolvidos na carcinogênese. O objetivo desse estudo foi descrever o padrão de expressão da molécula cinase de adesão focal (FAK) e suas formas fosforiladas, FAK-Tyr576 e FAK-Tyr925, em amostras de CEC no centro do tumor (CT) e zona de invasão (ZI) e no tecido epitelial não neoplásico adjacente a estes tumores (EA). De acordo com a graduação histopatológica, 48,1% do total das amostras de CEC são de grau moderado. Segundo o sistema pTNM, 62,9% eram T1 e T2, 64,8% não apresentaram metástase regional e não houve metástase à distância. O tempo máximo de acompanhamento foi de 5,9 anos e obteve-se a proservação final de 46 dos 54 pacientes, onde foi observado que 58,1% apresentaram boa evolução e 41,9% exibiram evolução ruim. A média em anos para que os pacientes obtivessem o status de evolução ruim foi de 5,01. Quanto ao estadiamento clínico, houve maior imunomarcação no estádio IVa quando comparado ao estádio II no EA da FAK Tyr-576 e ocorreu maior expressão no estádio IVb quando comparado ao estádio I no CT da FAK Tyr-576. Os pacientes que apresentaram evolução ruim após o período máximo de acompanhamento tiveram alta expressão no EA e na ZI da FAK Tyr-576 e no CT da FAK Tyr-925. Na ZI, o aumento de expressão da FAK está associado ao aumento da expressão da FAK Tyr-576. À medida que há aumento da expressão no EA há aumento da expressão na ZI na FAK Tyr-576. E quando há aumento da expressão no EA há aumento da marcação na ZI na FAK Tyr-925. Apesar do papel de todas as FAKs fosforiladas não estar claro, mas sabendo-se que são essas FAKs que estão ativas no câncer, a partir dos dados do presente trabalho pode-se sugerir que a avaliação da marcação FAK Tyr -576 no EA, no CT e na ZI venha a fazer parte da rotina no diagnóstico histopatológico e das margens da peça operatória, como norteadores da conduta terapêutica e do protocolo de acompanhamento. / Squamous cell carcinoma (SCC) of the head and neck is a malignant neoplasm that implies low rates of survival and poor prognosis. The loss of adhesion and cell migration are biological processes involved in carcinogenesis. The aim of this study was to describe the pattern of expression of focal adhesion kinase molecule (FAK) and its phosphorylated forms, FAK-Tyr576, FAK-Tyr925, in SCC samples in the center of the tumor (CT) and invasion zone (ZI) and non-neoplastic epithelial tissue adjacent to these tumors (EA). According to histopathological grading, 48,1% of SCC samples are moderate. According to the system pTNM, 62,9% were T1 and T2, 64.8%, had no regional metastasis and no distant metastasis. The maximum time of follow up was 5,9 years and was obtained as the final proservation 46 of 54 patients, where it was observed that 58,1% of patients had good prognosis and 41,9% of patients had poor prognosis. The average years for patients to obtain the poor prognosis of status was 5.01. As for clinical staging, there was greater immunostaining in the stadium IVa compared to stage II in EA Tyr-576. And FAK expression was higher in stage IVb when compared to CT in stage I of FAK Tyr-576. Patients with poor prognosis after the maximum follow-up period had high expression in EA and ZI of FAK Tyr-576 and CT of FAK Tyr-925. In ZI, increased expression of FAK is associated with increased FAK Tyr-576 expression. As there is increased expression in EA's increased expression in ZI in FAK Tyr-576. And when there is increased expression in EA's increased marking the ZI in FAK Tyr-925. Despite the role of all phosphorylated FAKs not clear, but given that these are FAKs that are active in cancer, from the present data it can be suggested that the evaluation of expression of FAK Tyr 576 in EA, CT and ZI will be part of the routine histopathological diagnosis and surgical specimen margins, as guiding the therapeutic management and monitoring protocol.

O carcinoma espinocelular (CEC) de cabeça e pescoço é uma neoplasia maligna que implica em baixas taxas de sobrevida e prognóstico desfavorável. A perda de adesão do epitélio e a migração celular são processos biológicos envolvidos na carcinogênese. O objetivo desse estudo foi descrever o padrão de expressão da molécula cinase de adesão focal (FAK) e suas formas fosforiladas, FAK-Tyr576 e FAK-Tyr925, em amostras de CEC no centro do tumor (CT) e zona de invasão (ZI) e no tecido epitelial não neoplásico adjacente a estes tumores (EA). De acordo com a graduação histopatológica, 48,1% do total das amostras de CEC são de grau moderado. Segundo o sistema pTNM, 62,9% eram T1 e T2, 64,8% não apresentaram metástase regional e não houve metástase à distância. O tempo máximo de acompanhamento foi de 5,9 anos e obteve-se a proservação final de 46 dos 54 pacientes, onde foi observado que 58,1% apresentaram boa evolução e 41,9% exibiram evolução ruim. A média em anos para que os pacientes obtivessem o status de evolução ruim foi de 5,01. Quanto ao estadiamento clínico, houve maior imunomarcação no estádio IVa quando comparado ao estádio II no EA da FAK Tyr-576 e ocorreu maior expressão no estádio IVb quando comparado ao estádio I no CT da FAK Tyr-576. Os pacientes que apresentaram evolução ruim após o período máximo de acompanhamento tiveram alta expressão no EA e na ZI da FAK Tyr-576 e no CT da FAK Tyr-925. Na ZI, o aumento de expressão da FAK está associado ao aumento da expressão da FAK Tyr-576. À medida que há aumento da expressão no EA há aumento da expressão na ZI na FAK Tyr-576. E quando há aumento da expressão no EA há aumento da marcação na ZI na FAK Tyr-925. Apesar do papel de todas as FAKs fosforiladas não estar claro, mas sabendo-se que são essas FAKs que estão ativas no câncer, a partir dos dados do presente trabalho pode-se sugerir que a avaliação da marcação FAK Tyr -576 no EA, no CT e na ZI venha a fazer parte da rotina no diagnóstico histopatológico e das margens da peça operatória, como norteadores da conduta terapêutica e do protocolo de acompanhamento. / Squamous cell carcinoma (SCC) of the head and neck is a malignant neoplasm that implies low rates of survival and poor prognosis. The loss of adhesion and cell migration are biological processes involved in carcinogenesis. The aim of this study was to describe the pattern of expression of focal adhesion kinase molecule (FAK) and its phosphorylated forms, FAK-Tyr576, FAK-Tyr925, in SCC samples in the center of the tumor (CT) and invasion zone (ZI) and non-neoplastic epithelial tissue adjacent to these tumors (EA). According to histopathological grading, 48,1% of SCC samples are moderate. According to the system pTNM, 62,9% were T1 and T2, 64.8%, had no regional metastasis and no distant metastasis. The maximum time of follow up was 5,9 years and was obtained as the final proservation 46 of 54 patients, where it was observed that 58,1% of patients had good prognosis and 41,9% of patients had poor prognosis. The average years for patients to obtain the poor prognosis of status was 5.01. As for clinical staging, there was greater immunostaining in the stadium IVa compared to stage II in EA Tyr-576. And FAK expression was higher in stage IVb when compared to CT in stage I of FAK Tyr-576. Patients with poor prognosis after the maximum follow-up period had high expression in EA and ZI of FAK Tyr-576 and CT of FAK Tyr-925. In ZI, increased expression of FAK is associated with increased FAK Tyr-576 expression. As there is increased expression in EA's increased expression in ZI in FAK Tyr-576. And when there is increased expression in EA's increased marking the ZI in FAK Tyr-925. Despite the role of all phosphorylated FAKs not clear, but given that these are FAKs that are active in cancer, from the present data it can be suggested that the evaluation of expression of FAK Tyr 576 in EA, CT and ZI will be part of the routine histopathological diagnosis and surgical specimen margins, as guiding the therapeutic management and monitoring protocol.

Cell adhesion to the extracellular matrix through cell-surface integrin receptors is essential to development, wound healing, and tissue remodeling and therefore represents a central theme in the design of bioactive surfaces that successfully interface with the body. This is especially significant in the areas of integrative implant coatings since adhesion triggers signals that regulate cell cycle progression and differentiation in multiple cellular systems. The interactions of osteoblasts with their surrounding extracellular matrix are essential for skeletal development and homeostasis and the maintenance of the mature osteoblastic phenotype. Our objective was to engineer integrin-specific bioactive surfaces that support osteoblastic differentiation and promote osseointegration by mimicking these interactions. We target two specific integrins essential to osteoblast differentiation the type I collagen receptor alpha2beta1 and the fibronectin receptor alpha5beta1. The central hypothesis of this project was that the controlled presentation of type I collagen and fibronectin binding domains onto well-defined substrates would result in integrin-specific bioadhesive surfaces that support osteoblastic differentiation, matrix mineralization, and osseointegration. We have demonstrated that these biomimetic peptides enhance bone formation and mechanical osseointegration on titanium implants in a rat tibia cortical bone model. We have also shown that the presentation of multiple integrin-binding ligands synergize to enhance intracellular signaling and proliferation. Finally, we demonstrate the advantage of the short biomimetic peptides over the native ECM proteins. This research is significant because it addresses current orthopaedic implant limitations by specifically targeting cellular responses that are critical to osteoblastic differentiation and bone formation. This biomolecular approach provides a versatile and robust strategy for developing bioactive surfaces that enhance bone repair and osseointegration of orthopaedic implants.

Cell adhesion to the extracellular matrix (ECM) provides tissue structure and integrity as well as triggers signals that regulate complex biological processes such as cell cycle progression and tissue-specific cell differentiation. Hence, cell adhesion is critical to numerous physiological and pathological processes, including embryonic development, cancer metastasis, and wound healing, as well as biotechnological applications, such as host responses to implanted devices and integration of tissue-engineered constructs. During the adhesion process, integrin surface receptors bind ECM proteins, cluster, and associate with the actin cytoskeleton. Subsequent strengthening of the integrin/actin cytoskeleton interaction occurs via complexes of proteins known as focal adhesions. Due to the close association between biochemical and biophysical processes within adhesion complexes, mechanical analyses can provide important new insights into structure/function relationships involved in regulating the adhesion process. The objective of this project was to investigate the role of the protein tyrosine kinase FAK in cell adhesion strengthening. Our central hypothesis was that FAK regulates adhesion strengthening by modulating interactions between integrins and FA structural components. Using a novel combination of genetically engineered cells to control the interactions of FAK, a spinning disk adhesion assay with micropatterned substrates to obtain reproducible and sensitive measurements of adhesion strength, and quantitative biochemical assays for analyzing changes in adhesive complexes, we demonstrate that FAK modulates adhesion strengthening via two distinct mechanisms: (1) FAK expression results in elevated integrin activation leading to regulation of strengthening rate and (2) FAK regulates steady-state adhesion strength via vinculin recruitment to focal adhesions. We also show that the autophosphorylation and catalytic sites of FAK are critical to this regulation of adhesion strengthening. This work is significant because it both identifies functional mechanisms of FAK and provides the first evidence that focal adhesion signaling regulates the adhesion strengthening process. Furthermore, this research demonstrates that the dependency of migration on adhesion strength is highly complex and establishes a need for adhesion strengthening metrics in analyzing the functional mechanisms of molecules within adhesion complexes.

Cell adhesion is essential to cell motility and tissue integrity and is regulated by the Integrin family of transmembrane receptors. Integrin binds to ligand extracellularly and provide anchor to the intracellular cytoskeleton via adhesion scaffolding proteins. In order to link cell to the surrounding matrix Integrin needs to be activated. Intracellular activation signals induce perturbations in Integrin cytoplasmic domain that are translated into a conformational change in extracellular region for high affinity ligand binding. Integrin engagement by matrix, in turn, triggers the assembly of adhesion complexes. Such early adhesions promote cytoskeletal organization with subsequent contractile activity that exerts forces against initial Integrin-matrix adhesions. In response to force, Integrin strengthens the interaction with matrix through its clustering and successive recruitment of additional adhesion components. These bidirectional regulatory loops mediated by such interactions are largely dependent on the unique function of Integrin adhesion components. / We demonstrate a novel role for the PDZ/LIM domain protein Zasp as a core component of Integrin adhesions. Specifically, Zasp colocalizes with Integrins at focal adhesion in cultured cells and myotendinous junctions in Drosophila embryos. In both cases elimination of Zasp modifies Integrin function causing consequently defects in cell spreading and muscle attachment. Zasp supports Integrin adhesion to the extracellular matrix that is required to withstand tensile forces exerted during cell spreading and muscle contraction. Furthermore, we found that the distribution of Zasp in muscle Z-lines is essential to orchestrate the cross-linking of alpha-Actinin and Actin filaments. Disruption of Zasp leads to loss of muscle cytoarchitecture, pointing to a larger role for Zasp in sarcomere assembly. Finally, we demonstrate that Zasp, in addition to alpha-Actinin, physically interacts with the Integrin- and Actin-bound cytoskeletal protein Talin. / Collectively, our results point to a dual role for Zasp as a structural scaffold. First it regulates Integrin adhesion to the extracellular matrix by interacting with the head domain of Talin at the myotendinous junctions. Second, Zasp controls sarcomere assembly by tethering the presarcomeric alpha-Actinin component to the tail domain of Talin. Zasp finding as a crucial adhesion component provides further insights on the mechanism underlying Integrin-mediated adhesion.

Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al.2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection.<p>In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOáIIbâ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin â3 subunit. We delineated IBS2 to a single amphipathic á-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation(L2094I2095/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the á helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K2085, K2089) exposed on the solvent face of á helix 50, which are directly involved in the talin IBS2-integrin interaction.<p>In the second part of this study, we investigated the functional role of talin IBS2 in spreading defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA mutant talin was unable to fully rescue the spread phenotype of these cells. These results provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the actin cytoskeleton. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Orientadores: Fernando Ferreira Costa, Nicola Amanda Conran Zorzetto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T23:11:17Z (GMT). No. of bitstreams: 1 Sakamoto_TatianaMary_D.pdf: 9561485 bytes, checksum: f03777ea2386a5e59f30b09eefcc7fb1 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Fisiopatologia Médica / Doutora em Ciências