O Trabalho de pesquisa consiste em desenvolver um sistema de avaliação de qualidade de imagens radiológicas que não apresente os problemas dos métodos atualmente disponíveis e principalmente ofereça para o radiologista urna relação entre as características do seu sistema e a nitidez da imagem. Os fatores que mais influenciam a qualidade da imagem são a característica de campo e o tamanho do ponto focal. Estes assuntos foram, portanto, principalmente estudados na revisão bibliográfica, atualmente utilizados imagem junto com os principais métodos para avaliação da qualidade da imagem. O novo método, desenvolvido utilizando simulação computacional, apresenta a imagem do ponto focal e do objeto radiografado, de qualquer forma e qualquer tamanho, para qualquer posição do campo de raios-X. A imagem do objeto é dada com os contornos em tons de cinza que representam bem a nitidez da imagem real. / The aim of this work is the development of a system to evaluate radiologic images quality, without the constraints of the current available methods. Furthermore, the new proposed technique should present to the radiologist a relationship between the radiologic system characteristics and its image sharpness. The main parameters which affect the radiologic image quality are the field characteristic and the focal spot size. Therefore, both issues were studied and presented in the text, beside the main methods currently used to evaluate the image quality. The new method, which uses computational simulation, shows the images of focal spot and of the radiographed object, taking into account objects of any shape and size, and for any X-ray field location. The image is shown with grey scale boundaries which are a good representation of the actual image.

Made available in DSpace on 2014-06-12T15:07:44Z (GMT). No. of bitstreams: 2 arquivo1550_1.pdf: 769292 bytes, checksum: 706bd0d94cb68ca3e146e48b7d3f3e6a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / Esta dissertação analisa uma organização à luz de dois modelos teóricos, o de estrutura organizacional de Mintzberg e o de análise multidimensional-reflexivo de Alves, baseada em elementos da cultura organizacional. Foram pesquisadas a situação existente e a situação desejada da organização, a partir da percepção dos stakeholders internos e externos da ADVB Pernambuco - Associação dos Dirigentes de Vendas e Marketing do Brasil. As análises das perspectivas interna e externa da identidade da organização subdividiu-se na identificação da percepção que os, stakeholders internos e externos, tinham sobre o seu estado atual e sobre e a projeção do seu futuro. Foram observadas as diferenças (os gaps) entre essas percepções. A contribuição pretendida para a ADVB-PE, é de uma compreensão mais ampla sobre a organização em análise e o seu direcionamento no contexto mercadológico em que encontra inserida, aumentando as suas chances de sobrevivência e desenvolvimento. Na coleta de dados foi utilizada uma abordagem qualitativa tendo como técnica de pesquisa o grupo focal e a estratégia de estudo de caso. Na condução do grupo focal foram adotados elementos do método sugerido por Moingeon e Ramanantsoa no intuito de atingir mais profundidade nas respostas. Os resultados remetem à conclusão de que os dois modelos permitiram analisar a organização conforme os objetivos propostos, embora o modelo de Alves tenha se mostrado mais satisfatório para o exercício desta análise

Abstract Reindeer antler was studied for its possible use as a bone implant material. A molecular biological study showed that antler contains a growth factor promoting bone formation. Ectopic bone formation assay showed that antler is not an equally effective inducer as allogenic material. Ectopic bone formation assay was optimised for biocompatibility studies of orthopaedic NiTi implants. Ti-6Al-4V and stainless steel were used as reference materials. The assay showed differences in bone mineral densities, with superior qualities in NiTi. The rate of endochondral ossification varied between the implants, NiTi ossicles had larger cartilage and bone areas than ossicles of the two other materials. The cytocompatibility of NiTi was studied with three different methods. Cell viability, cell adhesion and TGF-β1 concentration were assessed in ROS-17/2.8 cell cultures. Cells grown on NiTi had better viability than cells grown on pure nickel or stainless steel. Cell attachment on the materials was studied with paxillin staining of focal contacts. The number of focal contacts was clearly higher in cells grown on NiTi than in cells grown on pure titanium, pure nickel or stainless steel. TGF-β1 concentration was measured with ELISA. The results showed that there was only some minor variation between NiTi, pure titanium and stainless steel. Nickel showed a lower TGF-β1 concentration. Taken together, these results suggest that NiTi is well tolerated by ROS-17/2.8 cells. The cytocompatibility of stainless steel is not so good as that of NiTi. The same tests were used to study the effects of the surface roughness of the implant on cytocompatibility. Three different surface roughness grades were compared in cell cultures on NiTi and titanium alloy discs. Titanium alloy was subjected to two different heat treatments, to compare the effects of the treatments on cytocompatibility. The studies showed that NiTi had a lesser impact on cell viability and attachment than titanium alloy. Further, rough NiTi was found to be a better tolerated surface than the others. In this study, heat treatment of titanium alloy at +850° C did not interfere with cell viability or attachment, as did the +1050° C treatment of the alloy. On the contrary, TGF-β1 concentrations decreased on the +850° C treated alloy and were approximately same on the +1050° C treated alloy and on NiTi.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.

Proteins represent major constituent of the extracellular matrix which plays an important role in the formation, maintenance and remodeling of tissues, this project focuses on adsorption of two specific serum proteins fibronectin (FN) and vitronectin (VTN) responsible for mediating cell matrix interaction through integrin binding, tripeptide Arg-Gly-Asp (RGD) sequence found in these protein features are recognized by αβV3 integrin which ultimately helps in clot formation.

Cells have the ability to sense the rigidity of the extracellular matrix which directly affects the control of cellular functions in development, wound healing and malignant transformation. Polydimethylsiloxane elastomers are useful model biomaterials for mechanotransduction studies because they possess several advantages including ease of fabrication, tunable elasticity and modifiable surface chemistry. In this work, we are investigating the influence of matrix stiffness on adhesion strength and the mechanosensory structures that regulate these processes. In addition, the effect of surface modifications to this elastic substrate system on other physical properties such as local stiffness and topography will be analyzed. Based on previous research, we hypothesized that cell adhesion dependent processes will be regulated by matrix stiffness, but that surface chemistry influences on protein adsorption could provide overriding regulatory signals. The results of this research will provide insight into the interconnected processes of mechanosensing and cell adhesion strengthening, and reveal criteria for designing instructive biomaterials with specific mechanical and chemical properties.

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.

Made available in DSpace on 2014-06-12T23:03:31Z (GMT). No. of bitstreams: 2 arquivo9005_1.pdf: 752915 bytes, checksum: 8e613912c3a5b16cf267e01b803842c8 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / O objetivo deste estudo foi compreender os índios Xukuru, a partir da noção de continuidade do self que, segundo Chandler e Colaboradores, diz respeito ao processo de se reconhecer a mesma pessoa ou de resolver o paradoxo de continuar o mesmo, apesar das mudanças inevitáveis ocorridas no decorrer do tempo. Chandler e Lalonde (1998, 2008, no prelo ) apontam em suas pesquisas que os aborígines do Canadá foram perdendo, ao longo da história de colonização do seu povo, o senso de pertencimento e envolvimento em relação ao local onde vivem. Nesse sentido, a percepção de uma identidade desconexa do contexto cultural pode favorecer dificuldades para resolver o dilema da continuidade na mudança. Sendo assim, os indivíduos necessitam de uma justificativa para persistirem através do tempo, mesmo diante de tantas transformações. Ao refletir dessa forma, pode-se compreender que a continuidade passa a ser um aspecto constitutivo do self e da cultura, necessitando assim de conexão entre passado, presente e futuro. Quando uma perturbação tal dificulta esta conexão, o suicídio, transtornos psiquiátricos e até envolvimento com drogas, aparecem como uma atitude que refletem uma falta de perspectiva e compromisso com si mesmo e com a cultura a qual pertence. De forma semelhante ao que se observou em relação aos sujeitos da pesquisa de Chandler, o povo Xukuru passou por intensas mudanças, ao longo de sua história, resultando em conflitos e mortes. Em consequência, eles foram expulsos de seu território, só retomando há pouco mais de duas décadas, podendo estes acontecimentos terem favorecido processos de dificuldade em resolver o dilema de continuar o mesmo frente às mudanças inevitáveis da vida. Sendo assim, a análise dessa população, a partir de uma perspectiva dialógica, enfrenta o desafio de abarcar a dinâmica e poder construtivo do diálogo. Os resultados emergiram da discussão de um grupo focal (GF), composto de onze índios Xukuru de Pesqueira, Pernambuco. Esta discussão, construída coletivamente, versou sobre o dilema continuidade versus mudança desses indivíduos. O diálogo foi analisado de acordo com as relações estabelecidas entre o posicionamento das falas dos participantes, destacando-se o dilema proposto, o framing e a circulação de ideias. Observamos que o dilema proposto a continuidade e mudança desses indivíduos assumiu a forma de uma discussão aberta. O framing caracterizou-se como um diálogo de reflexões de suas vidas e tradições. Com relação à circulação de ideias, identificamos cinco tópicos contendo alusão a diversos conteúdos semânticos relativos à continuidade desses indivíduos frente às mudanças resultantes do processo de colonização sofrido . Investigamos a trajetória tomada por estes tópicos e suas modificações no diálogo sobre o dilema referido. A análise aponta para o uso de citações literais de frases relembrados por eles, ao falar sobre o cacique falecido da aldeia. Foram identificados padrões globais de temas que exploram um conhecimento social compartilhado que sugerem representações sociais, razoavelmente estáveis, dos fatos traumáticos ocorridos. Evidenciou-se um padrão temático recorrente: a perseguição sofrida e as lutas travadas para retomada do território e de seus direitos. Todavia, sugere-se que os indivíduos diferem nas possibilidades de resolverem o dilema de continuarem os mesmos apesar das marcantes mudanças sofridas. Encontraram-se padrões de resistência à mudança ao lado de uma maior flexibilidade em aceitá-las e, assim, desenvolverem perspectivas para o futuro

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle cells (VSMCs). However, it is often assumed that FAs in non-migratory, differentiated vascular smooth muscle cells (dVSMCs) embedded in the wall of healthy blood vessels are static structures. Recent work from our lab has demonstrated agonist-induced actin polymerization and Src-dependent focal adhesion phosphorylation in dVSMCs, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the alpha-agonist phenylephrine. VASP and Zyxin displayed the largest redistributions while beta-integrin and FAK showed undetectable redistribution. Vinculin, metavinculin, Src, CAS, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and EEA1. Endosomal disruption with the small molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B and colchicine, indicating that the redistribution is actin and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus, these studies are consistent with the concept that FAs in dVSMCs, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.

The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.