Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / No presente trabalho foi realizado um estudo integrado de fisiologia, bioquÃmica e proteÃmica comparativa em feijÃo de corda, uma cultura de grande valor econÃmico, com o objetivo de entender respostas de aclimataÃÃo das plantas à salinidade. Para tanto, dois experimentos foram conduzidos em casa de vegetaÃÃo, sob condiÃÃes hidropÃnicas, utilizando dois cultivares de feijÃo de corda (Vigna unguiculata) com tolerÃncia diferencial ao estresse salino: PitiÃba (tolerante) e TVu 2331 (sensÃvel). No primeiro experimento foram avaliadas as mudanÃas fisiolÃgicas (crescimento, trocas gasosas, teor relativo de Ãgua, teor de clorofila, fluorescÃncia da clorofila) e bioquÃmicas (acÃmulo de Ãons e solutos orgÃnicos em folhas e raÃzes e padrÃo de expressÃo protÃico foliar) induzidas por concentraÃÃes crescentes de salinidade (NaCl a 50, 75 e 100 mM). Os resultados demonstraram a existÃncia de respostas contrastantes entre os cultivares estudados, especialmente com relaÃÃo à inibiÃÃo do crescimento da parte aÃrea e ao acÃmulo de solutos compatÃveis, os quais foram maiores no TVu. Contudo, a salinidade nÃo alterou os parÃmetros de fluorescÃncia da clorofila em ambos os cultivares. ConcentraÃÃes crescentes de NaCl alteraram de forma diferencial o padrÃo de expressÃo de proteÃnas nas folhas, com maiores alteraÃÃes na dose 100 mM de NaCl. Apesar disso, no segundo experimento, a concentraÃÃo moderada de sal com NaCl a 75 mM foi escolhida como referÃncia para o estudo das mudanÃas no proteoma durante o estresse salino e apÃs um perÃodo pÃs-estresse (recuperaÃÃo). A salinidade modificou a expressÃo de 22 âspotsâ protÃicos no PitiÃba, dos quais 10 (6 que aumentaram e 4 que diminuÃram) tiveram suas identidades determinadas por espectrometria de massas acoplada a cromatografia lÃquida (LC-ESI-MS/MS). No TVu foram observadas mudanÃas em 27 âspotsâ, sendo determinada a identidade de 9 (5 que aumentaram e 4 que diminuÃram). AlÃm destes, 5 âspotsâ que mantiveram suas taxas de expressÃes em condiÃÃes de salinidade no PitiÃba tambÃm foram identificados. A maioria das proteÃnas identificadas està relacionada com o processo de fotossÃntese, realizando funÃÃes enzimÃticas ou participando da constituiÃÃo molecular dos fotossistemas. ProteÃnas com papel protetor contra o estresse, como chaperonas e enzimas do estresse oxidativo (SOD) foram identificadas, assim como a calreticulina, que provavelmente està envolvida em processos de transduÃÃo de sinal. Em adiÃÃo, foi possÃvel observar que durante a recuperaÃÃo das plantas os mecanismos de homeostase Ãs novas condiÃÃes restabeleceram os nÃveis de algumas proteÃnas, o que sugere a participaÃÃo delas no processo de aclimataÃÃo ao estresse salino. No geral, os resultados fornecem informaÃÃes adicionais que podem levar a uma melhor compreensÃo das bases moleculares da tolerÃncia ou sensibilidade de plantas de feijÃo de corda à salinidade. / In the present work, an integrated physiological, biochemical and proteomic analysis on cowpea (an economically important species) was carried out in order to understand the responses of plants to salinity. Two experiments were conducted in a greenhouse under hydroponic conditions, using two cultivars of cowpea (Vigna unguiculata) with differential tolerance to salt stress: Pitiuba (tolerant) and TVu 2331 (sensitive). In the first experiment, we evaluated the physiological (growth, gas exchange, relative water content, chlorophyll content, chlorophyll fluorescence) and biochemical (ions and organic solutes accumulations) changes induced by increasing levels of salinity (50, 75 and 100 mM NaCl), as well as the influences of stress two-dimensional (2D) protein patterns in leaves tissues. The results showed the existence of contrasting responses between the cultivars studied, especially with respect to inhibition of shoot growth and the accumulation of compatible solutes, which were higher in TVu. However, salinity did not alter the fluorescence parameters in both genotypes. The global analysis of 2D protein patterns showed that increasing levels of NaCl differentially alter the expression pattern of proteins in leaves, with larger changes in dose 100 mM NaCl. Nevertheless, in the second experiment, the moderate dose of salt with 75 mM NaCl was chosen as the reference dose for the study of changes in the proteome during salt stress and recovery. Salinity altered the expression of 22 protein spots in Pitiuba. Of these spots, the identities of 10 (6 up-regulated and 4 down-regulated) were determined by liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS). In TVU changes were observed in 27 spots being the identity determined for 9 (5 up-regulated and 4 down-regulated). Besides these, 5 spots that kept their rates expressions in salinity conditions in PitiÃba were also identified. The majority of the identified proteins is related to the process of photosynthesis, performing enzymatic functions or participating in the molecular constitution of photosystems. Proteins with protective role against stress such as chaperones and enzymes of oxidative stress (SOD) were identified, as well as calreticulin, which are probably involved in signal transduction network. In addition, during recovery treatments homeostasis mechanisms of plants to new conditions restored the levels of certain proteins, suggesting their participation in the process of acclimation to salt stress. Overall, results provide some additional information that can lead to a better understanding of the molecular basis of salt tolerance or sensitivity in cowpea plants.

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Nanofabrication techniques were used to manufacture nanometer scale protein templates. The fabrication approach employs electron beam lithography (EBL) patterning on poly(ethylene glycol) (PEG) thiol (CH3O(CH2CH2O)17NHCO(CH2)2SH) self-assembled monolayers (SAM) on Au. The PEG SAM prevented protein surface adhesion and binding sites for protein were created in the SAM by EBL. Subsequent to EBL, the patterns in the PEG SAM were backfilled with 40-nm NeutrAvidin-coated fluorescent spheres (FluoSpheres). The spontaneous and directed immobilization of the spheres from a solution to the patterns resulted in high resolution protein patterns. The FluoSpheres could be arranged in any arbitrary pattern with ultimately only one or a few FluoSpheres at each binding site. Growth dynamics and SAM morphology of PEG on Au were studied by atomic force microscopy (AFM). PEG SAMs on three types of Au with different microstructure were examined: thermally evaporated granular Au and two types of Au films produced by hydrogen flame annealing of granular Au, Au(111) and "terraced" Au (crystal orientation unknown). The different Au surfaces' substructure affected the morphology and mechanical properties of the PEG SAM. On Au(111), AFM imaging revealed monolayer formation through three distinct steps: island nucleation, island growth, and coalescence. The fine-structure of the SAM revealed dendritic island formation - an observation which can be explained by attractive intermolecular interactions and diffusion-limited aggregation. Island growth was not observed on the "terraced" Au. AFM studies of EBL patterned PEG SAMs on Au(111) revealed two different patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature depth is directly dose dependent. At higher doses electron beam induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. In addition to PEG SAMs, fibronectin monolayers on SiO2 surfaces were patterned by EBL. The areas exposed with EBL lose their functionality and do not bind anti-fibronectin. With this approach we constructed fibronectin templates and used them for cell studies demonstrating pattern dependent cell geometries and cell adhesion. / QC 20101008

<p>Nanofabrication techniques were used to manufacture nanometer scale protein templates. The fabrication approach employs electron beam lithography (EBL) patterning on poly(ethylene glycol) (PEG) thiol (CH3O(CH2CH2O)17NHCO(CH2)2SH) self-assembled monolayers (SAM) on Au. The PEG SAM prevented protein surface adhesion and binding sites for protein were created in the SAM by EBL. Subsequent to EBL, the patterns in the PEG SAM were backfilled with 40-nm NeutrAvidin-coated fluorescent spheres (FluoSpheres). The spontaneous and directed immobilization of the spheres from a solution to the patterns resulted in high resolution protein patterns. The FluoSpheres could be arranged in any arbitrary pattern with ultimately only one or a few FluoSpheres at each binding site.</p><p>Growth dynamics and SAM morphology of PEG on Au were studied by atomic force microscopy (AFM). PEG SAMs on three types of Au with different microstructure were examined: thermally evaporated granular Au and two types of Au films produced by hydrogen flame annealing of granular Au, Au(111) and "terraced" Au (crystal orientation unknown). The different Au surfaces' substructure affected the morphology and mechanical properties of the PEG SAM. On Au(111), AFM imaging revealed monolayer formation through three distinct steps: island nucleation, island growth, and coalescence. The fine-structure of the SAM revealed dendritic island formation - an observation which can be explained by attractive intermolecular interactions and diffusion-limited aggregation. Island growth was not observed on the "terraced" Au.</p><p>AFM studies of EBL patterned PEG SAMs on Au(111) revealed two different patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature depth is directly dose dependent. At higher doses electron beam induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern.</p><p>In addition to PEG SAMs, fibronectin monolayers on SiO2 surfaces were patterned by EBL. The areas exposed with EBL lose their functionality and do not bind anti-fibronectin. With this approach we constructed fibronectin templates and used them for cell studies demonstrating pattern dependent cell geometries and cell adhesion.</p>

Cell adhesion to extracellular matrices (ECM) is critical to many cellular processes including differentiation, proliferation, migration, and apoptosis. Alterations in adhesive mechanisms are central to the behavior of cells in pathological conditions including cancer, atherosclerosis, and defects in wound healing. Although significant progress has been made in identifying molecules involved in adhesion, the mechanisms that dictate the generation of strong adhesive forces remain poorly understood. Specifically, the role of nanoscale geometry of the adhesive interface in integrin recruitment and adhesion forces remains elusive due to limitations in the techniques available for engineering cell adhesion environments. The objective of this project was to analyze the role of nanoscale geometry in cell adhesion strengthening to ECM. Our central hypothesis was that adhesive interactions are regulated by integrin clusters whose recruitment is determined by the nanoscale geometry of the adhesive interface and whose heterogeneity in size, spacing, and orientation modulates adhesion strength. The objective of this project was accomplished by 1) developing an experimental technique capable of producing nanoscale patterns of proteins on surfaces for cell adhesion arrays, 2) assessing the regulation of integrin recruitment by geometry of the adhesive interface, and 3) determining the functional implications of adhesive interface geometry by systematically analyzing the adhesion strengthening response to nanoscale patterns of proteins. A printing technique was developed that patterns proteins into features as small as 90nm with high contrast and high reproducibility. Cell adhesion arrays were produced by directly immobilizing proteins into patterns on mixed-SAMs surfaces with a protein-resistant background. Colocalization analysis of integrin recruitment to FN patterns demonstrated a concentrating effect of bound integrins at pattern sizes with areas equivalent to small nascent focal adhesions. At adhesion areas below 333 × 333 nm2, the frequency of integrin recruitment events decreased significantly indicating a threshold size for integrin clustering. Functionally, pattern sizes below the threshold were unable to participate in generation of adhesion strength. In contrast, patterns between the threshold and micron sizes showed a relationship between adhesion strength and area of individual adhesion points, independent of the total available adhesion area. These studies introduce a robust platform for producing nanoscale patterns of proteins in biologically relevant geometries. Results obtained using this approach yielded new insights on the role of nanoscale organization of the adhesive interface in modulating adhesion strength and integrin recruitment.