Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [82]-96).

Orientadores: José Dirceu Ribeiro, Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T08:13:24Z (GMT). No. of bitstreams: 1 Grotta_MilenaBaptistella_D.pdf: 2330139 bytes, checksum: 5a69a2a222d3f7028574efcf65a49676 (MD5) Previous issue date: 2011 / Resumo: Introdução: A prevalência da obesidade e da asma tem aumentado muito durante as últimas décadas. Investigações atuais sugerem que a obesidade está associada à asma em numerosos estudos. Os mecanismos pelos quais a obesidade interfere nos sintomas da asma ainda são controversos. Objetivo: avaliar o efeito da obesidade na atividade eosinofílica através da quimiotaxia e adesão em crianças e adolescentes asmáticos e não asmáticos em associação a dosagem das adipocinas séricas. Método: Incluídos 32 asmáticos obesos (AO) e não obesos (ANO), 5 não asmáticos obesos (NAO) e 5 não asmáticos não obesos (NANO). Colhido sangue periférico para isolamento de eosinófilos através do gradiente de Percolll seguido de separação imunomagnética. Para quimiotaxia utilizada câmara de microquimiotaxia com 48 poços em triplicata com MEM (espontânea), eotaxina, PAF (fator de ativação plaquetária) e RANTES (regulated on activation, normal T cell expressed and secreted). Leitura através de filtro com contagem em microscópio óptico. A adesão foi realizada com placas de fibronectina em triplicata com MEM e eotaxina. Leitura através da absorbância das amostras desconhecidas com as da curva padrão de células. Feito dosagem de leptina e adiponectina por ELISA. Resultado: A quimiotaxia espontânea foi maior entre os AO e ANO (p=0,02) e entre os AO e NANO (p=0,03). Com eotaxina houve aumento para o grupo AO em relação aos ANO e aos NANO, e do grupo dos NAO em relação aos ANO e NANO (p<0,0001). Para o PAF, o grupo AO mostrou-se maior do que NANO (p=0,02). O RANTES apresentou significância entre os NAO em relação aos ANO e NANO (p=0,01). Na adesão espontânea dos eosinófilos a fibronectina, não foi encontrado relevância entre os grupos. Com eotaxina, houve maior adesão para os AO em relação aos NANO (p=0,04). Maior concentração de leptina no grupo dos AO e NAO (p=0,0001). Não foi encontrado diferença entre os grupos para adiponectina total. Conclusão: Este é o primeiro estudo a demonstrar uma maior atividade eosinofílica (quimiotaxia e adesão) em crianças e adolescentes obesos asmáticos atópicos em relação aos não obesos e aos voluntários saudáveis com associação ao aumento de leptina sérica / Abstract: Background: The prevalence of obesity and asthma has increased over the past several decades. Recent investigations suggest relationship between asthma and obesity in many studies, but the mechanisms are unclear. The aim of this study was evaluate the obesity effect in eosinophil activity by chemotaxis and adhesion in asthmatic and non-asthmatic children and adolescents in association with serum adipokines measurement Method: 32 asthmatic obese (AO) and asthmatic non obese (ANO), 5 non asthmatic obese (NAO) and 5 non asthmatic non obese (NANO) were included. Peripheral blood was collected and eosinophils were purified using a Percoll gradient followed by immunomagnetic cell separator. Chemotaxis was performed with microchemotaxis chamber in triplicate with MEM (spontaneous), eotaxin, PAF (platelet-activating factor) and RANTES (regulated on activation, normal T cell expressed and secreted). The measurement was done by optical microscope count. The adhesion was performed by fibronectin plates in triplicate with MEM and eotaxin. Eosinophilic adhesion was calculated by comparison between absorbance of unknown samples with the standard curve. Leptin and adiponectin were quantified by ELISA. Results: spontaneous chemotaxis was higher between AO and ANO (p=0.02) and between AO and NANO (p=0.03).With eotaxin, the increased was between AO and ANO and between AO and NANO, and between ANO and NANO (p<0.0001).With PAF, AO was higher than NANO (p=0.02). RANTES was increased among NAO and ANO and NANO (p=0.01). In spontaneous adhesion, there was no difference between groups. With eotaxin, the adhesion was higher between AO and NANO (p=0.04). Leptin was higher in AO and NAO than the others (p=0.0001). There was no difference among groups for total adiponectin. Conclusion: This is the first study that showed higher eosinophilic activity (chemotaxis and adhesion) in atopic obese asthmatic children and adolescents in relationship to asthmatic non obese and healthy volunteers / Doutorado / Saude da Criança e do Adolescente / Doutor em Saude da Criança e do Adolescente

Weanalyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDAMB- 231, MDA-MB-436) that exhibit a shift in E-, N- and P-cadherin levels characteristic of an epithelial−mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. Wedevelop a unique set of methods to measure cell–cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations.Wefind that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours.Wefind that surface densities of adhesive molecules do not correlate with measured cell–cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, theDAHdoes not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial−mesenchymal transition.

Thesis--University of Oklahoma. / Includes bibliographical references (leaves 51-55).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on November 5, 2008). Research advisor: Jie Zheng, Ph.D. Document formatted into pages (xiii, 140 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 105-116).

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.

ABSTRACT INTERACTION BETWEEN MICRO AND NANO PATTERNED POLYMERIC SURFACES AND DIFFERENT CELL TYPES &Ouml / z&ccedil / elik, Hayriye Ph.D., Department of Biology Supervisor: Prof. Dr. Vasif Hasirci Co-Supervisor: Dr. Celestino Padeste August 2012, 139 pages Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are &ldquo / softer&rdquo / . In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was &gt / 1.5 &mu / m. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

The colonization of immersed surfaces by a myriad of marine organisms is a complex, multi-stage, species-specific process giving rise to economic and environmental costs. This unwanted accumulation of organisms in the marine environment, called biofouling, has been attacked from different fronts, going from the ‘problem-elimination-as-problem-solving’ strategy (essentially through the use of biocides) to more elaborated and environmentally-friendly options based on the principle of ‘non-stick’ or ‘easy foul-release’ surfaces, which do not jeopardize marine life viability. Several marine organisms rely on proteinaceous adhesives to secure a holdfast to surfaces. Proteolytic enzymes have been demonstrated to be effective agents against settlement and settlement consolidation onto surfaces of marine bacteria, algae, and invertebrates, their proposed mode-of-action being the enzymatic degradation of the proteinaceous components of the adhesives. So far, however, the evidence remains inconclusive since most of the published investigations refer to commercial preparations where the enzyme is mixed with other components, like additives, which obviously act as additional experimental variables. This work aims at providing clear, conclusive evidence about the potential of serine proteases to target the adhesives produced by a group of model marine biofoulers. The strategy towards the goal consisted in the preparation and characterization of maleic anhydride copolymer nanocoatings modified by a surface-bound enzyme, Subtilisin A, the active constituent of the commercial preparations reported as effective against biofouling. The enzyme-containing maleic anhydride copolymer films were characterized (enzyme surface concentration, activity, stability, roughness and wettability) and thereafter tested in biological assays with three major biofoulers: spores of the green alga Ulva linza, cells of the pennate diatom Navicula perminuta, and cyprid larvae of the barnacle Balanus amphitrite. The purpose of the biological assays was to elucidate the efficacy of the immobilized catalyst to discourage settlement and/or to facilitate removal of these organisms from the bioactive layers. Results confirmed the initial hypotheses related to the enzymatic degradation of the biological adhesives: the immobilized protease was effective at reducing the adhesion strength of Ulva spores and Navicula diatoms in a manner that correlated with the enzyme activity and surface concentration, and deterred settlement of Balanus amphitrite barnacle cyprids even at the lowest surface activity tested. By facilitating the removal of biofilm-forming diatoms and of spores of the troublesome alga Ulva linza, as well as by interfering with the consolidation of adhesion of the calcareous Balanus amphitrite macrofouler, the enzyme-containing coatings here disclosed are considered to constitute an appealing and promising alternative to control marine biofouling without jeopardizing marine life.

Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes’ surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.