DNA molecules stretching and deformation in hydrodynamic flow field has been investigated for many years with various experimental and theoretical methods. This research was performed experimentally in hybridized microchannels made of poly(dimethylsiloxane)(PDMS) and glass. We will directly measure the hydrodynamic stretching DNA molecules in elongation flow by CLSM (Confocal Laser Scanning Microscope). The deformation of YOYO-labeled lambda phage DNA molecules in flows was visualized with CLSM. The relation between DNA contour length and relaxation time in different models of stretching flows was also be presented. Furthermore, the distribution of stretching DNA molecules in microchannels was derived to analyze and compared with the DNA migration velocity and stress relaxation modulus.

In this study, sharp/gradual converging-diverging microchannels with contraction/ expansion ratio of 4:1/1:4 was designed to generate elongational flow with uniform velocity in the centerline. The £f-DNA stained with YOYO-1 was observed in the flow. MPIV was built to measure the velocity distribution and local strain rate was estimated by MPIV measurements. The deformation and conformation of individual DNA molecules in the flow was visualized with confocal laser scanning microscopy (CLSM). The goal of the present work was to develop a method for stretching DNA molecules, in order to perform analysis of coil-stretch transition of DNA. By measuring dynamic properties and relaxation time of DNA molecules stretched by pressure driven at various flow rate and viscosity, we have shown how one could investigate the influence of hydrodynamic interactions in the case of stretching of DNA molecules.

Imagens multiespectrais são utilizadas em diferentes aplicações, que vão desde sensoriamento remoto a processos médicos. No caso de imagens multiespectrais oriundas de microscopia confocal de varredura à laser (Confocal Laser Scanning Microscopy-CLSM), a extração da informação se inicia pela conversão das assinaturas espectrais, em uma imagem RGB. Esta imagem é a referência para a seleção da região de interesse, da qual se obtém a assinatura espectral média, originada do arquivo multiespectral (LSM). Mesmo utilizando um padrão muito bem estabelecido de conversão, alguns pontos devem ser considerados: i) o processo de conversão reduz a informação, a uma ordem de 10-145%; ii) a cor é uma experiência sensorial, subjetiva e pessoal, interferindo na seleção da região de interesse e; iii) a assinatura é obtida pela média espectral, da região de interesse, selecionada manualmente.Assim, esta tese de doutorado propõem um método de mapeamento e visualização das informações de imagens multiespectrais, combinando um algoritmo de agrupamento não supervisionado(kmeans) e um algoritmo que define uma paleta de cores coerentes com a informação espectral das regiões mapeadas. Aplicou-se o método em três casos de estudos de tecidos vegetais: i) no pré-tratamento de paredes celulares da cana-de-açúcar; ii) na plasticidade foliar do Jacaranda caroba e; iii) no uso de assinaturas espectrais na classificação de plantas do Cerrado. Os resultados demonstraram que o método é bastante robusto, permitindo de forma inovadora a: visualização, análise e comparação de imagens multiespectrais qualitativa e quantitativamente, e que seu uso é viável em qualquer área de pesquisa que utilize imagens multiespectrais. / Multispectral images are used in different applications, ranging from remote sensing images to medical images. In the case of multispectral images derived from confocal laser scanning microscopy (CLSM), the extraction of information begins with the conversion of spectral signatures in an RGB image. This is the reference for selecting the region of interest, from which it gets the average spectral signature, originated from multispectral file (LSM). Even using a very well established pattern of conversion, some points should be considered: i) the conversion process reduces the information on the order of 10-145%; ii) the color is a sensory experience, subjective and personal, interfering in the selection of the interest region and; the signature is obtained by the spectral average, from interest region which is selected manually. Thus, this doctoral thesis proposes a method of mapping and visualization of multispectral imaging information, combining an unsupervised clustering algorithm (kmeans) and an algorithm that defines a consistent color palette with the spectral information of mapped regions. The proposed method was applied in three cases plant tissue studies: i) in the pre-treating the cell walls of sugarcane; ii) in the leaf plasticity of Jacaranda caroba; iii) in the use of spectral signatures in the Cerrado plant classification. The results showed that the proposed method is quite robust. It presents innovation to the visualization and analysis of multispectral images and makes possible a qualitative and quantitative comparison of a group of multispectral images. Besides that, its use is feasible in any area of research, which are using multispectral images.

Imagens multiespectrais são utilizadas em diferentes aplicações, que vão desde sensoriamento remoto a processos médicos. No caso de imagens multiespectrais oriundas de microscopia confocal de varredura à laser (Confocal Laser Scanning Microscopy-CLSM), a extração da informação se inicia pela conversão das assinaturas espectrais, em uma imagem RGB. Esta imagem é a referência para a seleção da região de interesse, da qual se obtém a assinatura espectral média, originada do arquivo multiespectral (LSM). Mesmo utilizando um padrão muito bem estabelecido de conversão, alguns pontos devem ser considerados: i) o processo de conversão reduz a informação, a uma ordem de 10-145%; ii) a cor é uma experiência sensorial, subjetiva e pessoal, interferindo na seleção da região de interesse e; iii) a assinatura é obtida pela média espectral, da região de interesse, selecionada manualmente.Assim, esta tese de doutorado propõem um método de mapeamento e visualização das informações de imagens multiespectrais, combinando um algoritmo de agrupamento não supervisionado(kmeans) e um algoritmo que define uma paleta de cores coerentes com a informação espectral das regiões mapeadas. Aplicou-se o método em três casos de estudos de tecidos vegetais: i) no pré-tratamento de paredes celulares da cana-de-açúcar; ii) na plasticidade foliar do Jacaranda caroba e; iii) no uso de assinaturas espectrais na classificação de plantas do Cerrado. Os resultados demonstraram que o método é bastante robusto, permitindo de forma inovadora a: visualização, análise e comparação de imagens multiespectrais qualitativa e quantitativamente, e que seu uso é viável em qualquer área de pesquisa que utilize imagens multiespectrais. / Multispectral images are used in different applications, ranging from remote sensing images to medical images. In the case of multispectral images derived from confocal laser scanning microscopy (CLSM), the extraction of information begins with the conversion of spectral signatures in an RGB image. This is the reference for selecting the region of interest, from which it gets the average spectral signature, originated from multispectral file (LSM). Even using a very well established pattern of conversion, some points should be considered: i) the conversion process reduces the information on the order of 10-145%; ii) the color is a sensory experience, subjective and personal, interfering in the selection of the interest region and; the signature is obtained by the spectral average, from interest region which is selected manually. Thus, this doctoral thesis proposes a method of mapping and visualization of multispectral imaging information, combining an unsupervised clustering algorithm (kmeans) and an algorithm that defines a consistent color palette with the spectral information of mapped regions. The proposed method was applied in three cases plant tissue studies: i) in the pre-treating the cell walls of sugarcane; ii) in the leaf plasticity of Jacaranda caroba; iii) in the use of spectral signatures in the Cerrado plant classification. The results showed that the proposed method is quite robust. It presents innovation to the visualization and analysis of multispectral images and makes possible a qualitative and quantitative comparison of a group of multispectral images. Besides that, its use is feasible in any area of research, which are using multispectral images.

A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

In this study, we design different inscribed/circumscribed circular torus-type microchannels to investigate the stretching behavior of DNA molecules. Strain rate and relaxation time play an important role in DNA stretching. In order to perform an analysis of the coil-stretch transition of DNA, we develop a method of stretching DNA molecules by using £gPIV and CLSM measurements. £gPIV is designed to measure the velocity distribution, after which the local strain rate can be estimated. The hydrodynamic stretching of DNA molecules in the elongation flow is observed using a confocal laser scanning microscope (CLSM). The relaxation time of the DNA molecules is then estimated according to the CLSM images analysis. At present, our experiments using the electro-osmotic flow (EOF) driven at various electric fields and viscosities to stretch DNA molecules show how one can investigate the influence of hydrodynamic interactions in the case of stretching of DNA molecules.

In this study, an electrokinetics-induced elongation flow was created inside a gradual converging-diverging microchannel with different temperature (25, 35, 45, 55¢XC). The conformation of DNA molecules, local strain rate, and the relaxation time play important roles in determining the extent of DNA stretching. By using £gPIV/£gLIF measurements, the velocity/temperature distributions in microchannels can be secured. The local strain rate was estimated by £gPIV measurements. We observe the hydrodynamic stretching DNA molecules in elongation flow by confocal laser scanning microscope (CLSM). Through CLSM images analysis, relaxation time of DNA molecules was estimated. Finally, dynamic properties and stretching ratio of DNA molecules stretched by EOF driven at various electric field and temperature ware measured. The thermal effect and the electric field on the conformation were also studied and discussed.

Non-destructive investigation methods where applied to study evaporation dynamics and other dynamic processes of volatile and non-volatile fluids from different porous membranes. While evaporation dynamics from porous alumina didn't show any irregularities, it was shown that evaporation of ethanol from porous glasses can be divided into two different phases - linear endothermal evaporation and adiabatic burst evaporation.