On the bead modeling methodology, or BMM, a macromolecule is modeled as a rigid, non-overlapping bead array with arbitrary radii. The BMM approach was pioneered by Kirkwood and coworkers (Kirkwood, J.G., Macromolecules, E.P. Auer (Ed.), Gordon and Breach, New York, 1967; Kirkwood, J.G., Riseman, J., J. Chem. Phys., 1948, 16, 565) and applied to such transport properties as diffusion, sedimentation, and viscosity. With the availability of computers, a number of investigators extended the work to account for the detailed shape of biomolecules in the 1970s. A principle objective of my research has been to apply the BMM approach to more complex transport phenomena such as transport in a gel, electrophoresis (free solution and in a gel), and also transport in more complex media (such as the viscosity of alkanes and benzene). Variables considered by the BMM include the number of beads (N), the radii of the beads, net charge and charge distribution, conformations, salt type, and salt concentration. The BMM has been extended to: (1) account for the existence of a gel; (2) characterize the charge and secondary structure of macromolecules; (3) account more accurately for hydrodynamic interaction (remove the orientationnal preaveraging approximation of hydrodynamic interaction); (4) study the effect of ion relaxation for particles in arbitrary size, shape, and charge; (5) consider the salt dependence of electrokinetic properties; (6) account for the formation of possible complex between guest ions and BGE ions. We also did diffusion constant measurement by NMR for amino acids and short peptides in 10%D2O-90% H2O at room temperature and applied to our modeling study by BMM.

Implanted tissue engineered substitutes constitute dynamic systems, with remodeling mediated by both the implanted cells and the host. Thus, there exists a significant need for methods to monitor the function and morphology of tissue engineered constructs. Noninvasive monitoring using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and imaging can prove to be the solution to this problem. Spectroscopy allows for assessment of cellular function through the monitoring of inherent metabolic markers, such as total-choline, while high resolution imaging enables the evaluation of construct morphology and interfacial remodeling. We applied these 1H NMR methods to monitor betaTC3 mouse insulinoma cells within hydrogel-based materials as a model pancreatic tissue substitute. In vitro research established a strong correlation between total-choline, measured by 1H NMR spectroscopy, and viable betaTC3 cell number, measured by MTT. Extending these methods to in vivo monitoring, however, was met with additional challenges. First, the implanted cells needed to be contained within a planar construct above a threshold density to allow for adequate quantification of the total-choline peak. Secondly, cell-free buffer zones between the implanted cells and the host tissue needed to be incorporated to prevent host tissue signal contamination. Finally, quantitative techniques needed to be developed to accurately account for contaminating signal from diffusing molecules. To overcome these challenges, a disk-shaped agarose construct, initially containing a minimum of 4 million betaTC3 cells and coated with an outer layer of pure agarose, was fabricated. Mathematical simulations aided the implant design by characterizing diffusive transport of nutrients and metabolites into and out of the construct. In vivo 1H NMR studies of these constructs implanted in mice established a strong correlation between total-choline, measured noninvasively using 1H NMR spectroscopy, and viable cell number, measured invasively using MTT. This study establishes total-choline as a reliable marker for noninvasively quantifying dynamic changes in viable betaTC3 cell number in vivo. 1H NMR imaging was used to monitor the implants structural integrity over time, while also assessing the hosts fibrotic response. We expect these studies to establish quantitative criteria for the capabilities and limitations of NMR methodologies for monitoring encapsulated insulinomas, as well as other tissue implants.

Osteoarthritis (OA) is a disease characterized by the progressive degradation of articular cartilage. Current strategies for the disease are mainly towards relieving symptoms. This study was aimed to investigate the therapeutic potentials of Bone Morphogenetic Protein-9 (BMP-9), Raloxifene (Ral) and Pluronic F-68 (PLF-68) with a three-dimensional in vitro OA model. Articular chondrocytes isolated from rats were cultured in growth media and embedded in agarose to obtain agarose-chondrocyte discs. Dynamic hydrostatic mechanical stress was applied to discs. The discs were incubated with Aza-C for 48 hours for OA development. After its removal, chondrocytes were treated with different doses of BMP-9, Ral and PLF-68 for 10 days. The efficacies of treatments were evaluated by measuring cell number, glycosaminoglycan and collagen amount, and mechanical properties of the v discs. Measurements of these properties were performed with MTT, quantitative colorimetric assays, histochemical staining and mechanical tests, respectively. According to comparative results with healthy groups and controls (osteoarthritic chondrocytes without any treatment), it was found that BMP-9 had negative effect on osteoarthritic chondrocytes. On the other hand, Ral showed positive results related with matrix synthesis and mechanical properties especially at 5 &amp / #956 / M dose suggesting that it holds promise for the treatment of OA. The therapeutic effect of Ral on OA was documented for the first time in literature. The potential of PLF-68 for treatment of OA was also supported by this study considering its positive effects on cell number, collagen synthesis and mechanical properties. Yet, further investigations are also suggested for conclusive results on this agent.

The development of a chip-based sensor array composed of individually addressable polystyrene-polyethylene glycol and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. The cavities possess pyramidal pit shapes with trans-wafer openings that allow for both fluid flow through the microreactors/analysis chambers as well optical access to the chemically sensitive microspheres. Identification and quantification of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data is extracted from the array efficiently using a charge-coupled device (CCD) allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, sugars and antibody reagents. The application of artificial neural network analyses to the microbead array is demonstrated in the context of pH measurements. To assess the utility of the analysis and gain an understanding of the molecular level design of the sensor, parameters such as the choice of the indicator dyes, array size, data pre-processing techniques, as well as different network types and architectures were evaluated. Additionally, the development of miniaturized chromatographic systems localized within individual polymer microspheres and their incorporation into an array is reported. The integrated chromatographic and detection concept is based on the creation of distinct functional layers within the microspheres. Such beads have been incorporated into the array platform and used for speciation and concentration determination of aqueous metal cation solutions. / text

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Agarose gel was used as a template to prepare zirconium titanium mixed oxide pellets with bimodal porosity. The materials were fully characterized to assess the effect ofZr:Ti ratio on the physical properties. It was found that the metal oxide ratio had an impact on surface acidity, the number of surface hydroxyl groups, the surface area the crystallinity and the mesopore diameter. The oxides were tested for the adsorption of vanadium ions to determine which Zr mole fraction exhibited the highest loading capacity and the fastest kinetics. A comparative study demonstrated that a hierarchical pore structure had enhanced mass transport properties over a monomodal pore structure of similar Zr:Ti composition. / Three porous zirconium titanium oxides (25 mol% Zr) were synthesized using sol-gel chemistry. One of the materials was templated from agarose gel, the second was produced without the use of a template and the third was templated from stearic acid. All three materials varied in pore architecture. Surface modification was performed post-synthetically using propionic acid (a monomer), glutaric acid (a dimer) and three molecular weights of poly(acrylic acid). Higher loading within the inorganic support was obtained for the polymers than for the smaller molecules. It was found that the pore architecture had a strong bearing on the quantity of polymer incorporated into the metal oxide framework and some effect on the rate of polymer adsorption. Thus there is great value in using templates to control pore structure. The materials were subjected to irradiation with 60Co γ-rays to determine the stability of the inorganic support and the organic functionality. / Hybrid materials were prepared by coating five distinct macroporous commercial membranes with zirconium titanium oxide using sol-gel chemistry. Calcination of these templated materials produced oxide membranes which had a suite of macropore and mesopore architectures, pore volumes and surface areas. These differences in physical properties were used to conduct a fundamental study on the relationship between the mesopore size and volume and the capacity for polymer incorporation. It was found that the polymer loading capacity was highly dependent on the pore size and pore volume. As surface area increased, loading capacity decreased, indicating that much of the increased internal surface was inaccessible to the macromolecules. Thus, mesopore diameter and pore volume must be considered when designing a mesoporous solid support. / Hierarchically porous zirconium titanium oxide and carbon zirconium titanium oxide beads with adjustable meso- and macroporosity were prepared in a one-pot, engineering-friendly process. Poly(acrylonitrile) and block copolymer Pluronic F127 were used as structure directing agents. These millimeter sized spheres were fabricated through drop-wise addition of the template-metal alkoxide solution into either water or liquid nitrogen. Carbon zirconium titanium oxide beads were produced by carbonizing the beads at 550 °C in an inert atmosphere. The (carbon) zirconium titanium oxide beads were assessed for surface accessibility and adsorption rate by monitoring the adsorption of uranyl from solution. / Porous metal oxide monoliths, specifically silica, titania, zirconia and mixed oxides containing aluminum and yttrium, were prepared in a one-pot synthesis. Macroporosity was induced using the phase separation of furfuryl alcohol. These materials have a suite of mesopore and macropore structures, the domains of which can be controlled by adjusting the synthesis conditions. These conditions were studied in detail to optimize the pore interconnectivity, the monolith stability, the pore volume and the surface area.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

O uso de DNA plasmidial (pDNA) visando a aplicações terapêuticas tem aumentado nos últimos anos. A cromatografia aparece como a técnica de purificação mais comum para obtenção de amostras de pDNA com o elevado grau de pureza exigido. Porém, as resinas cromatográficas disponíveis apresentam ainda uma série de desafios, nomeadamente no desenvolvimento de ligantes específicos e matrizes capazes de acomodar este tipo de molécula. Relativamente à apuração de novos ligantes, alguns estudos têm mostrado o potencial do aminoácido arginina para estabelecer interações específicas e preferenciais com o pDNA. Por outro lado, resinas monolíticas surgem como suportes interessantes devido às suas excelentes propriedades de transferência de massa e altas capacidades de adsorção. Neste estudo, diferentes ligantes baseados em arginina (arginina, di-arginina e tri-arginina) foram imobilizados em resinas de agarose previamente ativadas. Um primeiro estudo de adsorção em batelada foi realizado a fim de avaliar e compreender os mecanismos envolvidos no processo de adsorção dos ácidos nucleicos pDNA e RNA em resina com o aminoácido arginina. Na sequência, apresentamos uma proposta inovadora para o uso de ligantes de arginina em resinas de agarose, em um único passo de purificação em modo negativo a seguir ao passo de concentração por isopropanol. A capacidade da resina para o pDNA foi substancialmente maior do que a obtida para o mesmo tipo de resina no modo positivo, com notória vantagem de capacidade no uso de di-arginina face a arginina com rendimentos próximos de 100% do plasmídeo carregado. Os ligantes di-arginina e tri-arginina foram também imobilizados em resinas monolíticas. Em comparação com o aminoácido arginina, a imobilização dos homopeptídeos nas resinas monolíticas levou ao aumento da capacidade de adsorção (cerca de 2,5 vezes superior) e especicificidade de interações, mostrando-se como uma estratégia promissora para processos de purificação de pDNA. / The use of plasmid DNA (pDNA) for therapeutic applications has increased in recent years. Chromatography appears as the most common purification technique to obtain samples of pDNA with the high degree of purity required. However, the available chromatographic resins still present a series of challenges, namely in the development of specific ligands and matrices capable of accommodating this type of molecule. Regarding the determination of new ligands, several studies have shown the potential of the arginine amino acid to establish specific and preferential interactions with the pDNA. On the other hand, monolithic resins appear as interesting approaches due to their excellent mass transfer properties and high adsorption capacities. In this study, different arginine based ligands (arginine and di-arginine) were firstly immobilized on activated agarose resins. The first part of the work describes the adsorption equilibrium of plasmid DNA adsorption process, as well as the interaction with its main impurity (RNA) on arginine supports in a batch format, in order to compare and gather crucial information about adsorption mechanisms involved in this type of affinity system. Then, a new use for chromatographic bead matrixes based on arginine ligands was proposed, working as an adsorption matrix pDNA purification in negative mode after isopropanol concentration of the sample. The arginine based supports capacity for pDNA under negative mode for pDNA was substantially higher than that obtained with the same type of resin in the conventional positive mode, with a notable advantage of using di-arginine with recovery yields near 100%. The homopeptides (di-arginine and tri-arginine) were also immobilized on functionalized monolithic resins (BIA Separations, Slovenia). Effectively, the immobilization of the arginine homopeptides made the monolithic resins more functional compared to the (mono)arginine based resin, exhibiting greater binding capacities (around 2,5 times higher) and interaction intensities, proving to be a promising strategy for purification processes of pDNA.

This diploma thesis is focused on the study of the behaviour of carbon fibers in the hydrogel, especially the viscoelastic modules od these gels ort he coefficient of color transmission through the gel. The main goals were to optimize the samples and measure their viscoelastic modules on a rheometer, in several different modes. This was followed by simulation of physiological conditions for selected samples and their measurement on the rheometer. There was also an experiment of continuous diffusion in cuvettes, in which the penetration of the dye through the gel with the addition of fibers and for comparison also without them. Last but not least diffusion pairs were prepared.

The content of this diploma thesis was testing the applicability of available structural analysis techniques on a appropriate model hydrogel system. The main aim was to evaluate the advantages and disadvantages of selected structural analysis techniques and the influence of the composition change of the hydrogel system on its internal structure. Semi-interpenetrated hydrogels based on agarose of different concentration were chosen as model system, this type of material was chosen for simple and repeatable preparation and also for its application potential. Electron microscopy (SEM, cryoSEM), mercury porosimetry and turbidimetry were chosen as structural analysis techniques. Experimental results could be used to improve knowledge about the influence of hydrogel composition on its structure and the comparison of chosen techniques will be used for the choice of appropriate structural analysis in the future, which will be applicable to other hydrogel systems as well.