<p> This thesis focuses on the use of time-resolved fluorescence anisotropy (TRF A) for the analysis of peptide-silica and protein-silica interactions. Previous studies from our group have shown that strong ionic binding of the cationic probe rhodamine 6G (R6G) to the anionic surface of silica particles in water provides a convenient labeling procedure to study both particle growth kinetics and surface modification by time-resolved fluorescence anisotropy (TRF A). The decays for R6G dispersed in diluted Ludox silica sols usually fit to a sum of picosecond and nanosecond decay components, along with a significant residual anisotropy component. The first objective of my work was to assess the nature of the R6G:silica interaction to determine the origin of the nanosecond decay component, and ultimately validate the model used to fit the TRFA data and gain further insight into the physical meaning of the anisotropy decay parameters. Our results show the origin of the nanosecond decay component ( ¢2) is due to the presence of a subpopulation of small nanoparticles in the Ludox sol. </p> <p> With the correct physical model in place, we have been able use TRFA ofR6G in aqueous Ludox to monitor peptide adsorption onto the silica particles in situ. Steady-state anisotropy and TRF A of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE) and N-acetylated Glu-Trp-Glu (Ac-EWE). R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the addition of cationic peptides blocking the adsorption of the dye to the silica surface. The study shows that the competitive binding method can be used to assess the binding of various biologically relevant compounds onto silica surfaces, and demonstrates the potential of TRF A for probing peptide: silica and protein: silica interactions. </p> <p> We have also extended the application of TRF A to monitor protein adsorption onto plain and modified silica particles using a recently reported cationic long-lifetime quinolinium dye, CG437, which strongly binds to anionic silica particles through electrostatic interactions. In this case, alterations in the rotational correlation time of Ludox particles resulting from increases in the diameter of the rotating body upon binding of protein to the silica surface were monitored. The study shows that TRFA analysis of long-lived cationic probes such as CG437 can provide an effective method to investigate interactions between proteins and modified silica surfaces, extending the utility of the TRF A method. </p> / Thesis / Master of Science (MSc)

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

No presente trabalho, foram desenvolvidas nanocápsulas de núcleo lipídico (LNCs) para posterior revestimento com manana através da utilização de duas metodologias diferentes, sendo ambas por interações eletrostáticas. Na primeira metodologia, soluções de manana em diferentes concentrações foram gotejadas sobre a formulação LNC-Quit+ e na segunda metodologia a LNC-Quit+ foi injetada nas soluções de manana. Através de análises de diâmetro, mobilidade eletroforética, potencial zeta e viscosidade, foi possível notar que a primeira metodologia não apresenta reprodutibilidade, demonstrando valores aleatórios, ao passo que, na segunda metodologia os valores são reprodutíveis para a concentração de 0,5 μg/mL final de manana. Também foi possível observar que as nanopartículas revestidas com manana não apresentam estabilidade após 24 horas, sugerindo-se que a manana se desprende da superfície da partícula. Nas duas metodologias foram utilizadas concentrações diferentes de material de partida, e com isso, foi criada a metodologia 2B para que as anteriores pudessem ser comparadas. Com estes resultados tem-se um caminho promissor para estudos futuros e o aprimoramento deste tipo de revestimento. / In the present work, lipid core nanocapsules (LNCs) was developed for subsequent coating with mannan by using two different methodologies and both by electrostatic interactions. In the first methodology, mannan solutions at different concentrations were drop wise on LNC-Quit+ formulation and the second methodology, LNC-Quit+ was injected in mannan solutions. Through analysis of diameter, electrophoretic mobility, zeta potential and viscosity, it was possible to notice that the first methodology was not reproductible, showing random values, whereas, in the secont methodology the values was reproductible for the 0,5 μg/mL concentration. It was also possible to observe that the particle coated with mannan has no stability after 24 hours, suggesting that mannan detaches from the particle surface. In this methodologies, two different concentrations of raw materials are used, and with that, it was created the methodology 2B so that the results could be compared. With these results, it was a promising path way for future studies and enhancement of this type of coating.

No presente trabalho, foram desenvolvidas nanocápsulas de núcleo lipídico (LNCs) para posterior revestimento com manana através da utilização de duas metodologias diferentes, sendo ambas por interações eletrostáticas. Na primeira metodologia, soluções de manana em diferentes concentrações foram gotejadas sobre a formulação LNC-Quit+ e na segunda metodologia a LNC-Quit+ foi injetada nas soluções de manana. Através de análises de diâmetro, mobilidade eletroforética, potencial zeta e viscosidade, foi possível notar que a primeira metodologia não apresenta reprodutibilidade, demonstrando valores aleatórios, ao passo que, na segunda metodologia os valores são reprodutíveis para a concentração de 0,5 μg/mL final de manana. Também foi possível observar que as nanopartículas revestidas com manana não apresentam estabilidade após 24 horas, sugerindo-se que a manana se desprende da superfície da partícula. Nas duas metodologias foram utilizadas concentrações diferentes de material de partida, e com isso, foi criada a metodologia 2B para que as anteriores pudessem ser comparadas. Com estes resultados tem-se um caminho promissor para estudos futuros e o aprimoramento deste tipo de revestimento. / In the present work, lipid core nanocapsules (LNCs) was developed for subsequent coating with mannan by using two different methodologies and both by electrostatic interactions. In the first methodology, mannan solutions at different concentrations were drop wise on LNC-Quit+ formulation and the second methodology, LNC-Quit+ was injected in mannan solutions. Through analysis of diameter, electrophoretic mobility, zeta potential and viscosity, it was possible to notice that the first methodology was not reproductible, showing random values, whereas, in the secont methodology the values was reproductible for the 0,5 μg/mL concentration. It was also possible to observe that the particle coated with mannan has no stability after 24 hours, suggesting that mannan detaches from the particle surface. In this methodologies, two different concentrations of raw materials are used, and with that, it was created the methodology 2B so that the results could be compared. With these results, it was a promising path way for future studies and enhancement of this type of coating.

No presente trabalho, foram desenvolvidas nanocápsulas de núcleo lipídico (LNCs) para posterior revestimento com manana através da utilização de duas metodologias diferentes, sendo ambas por interações eletrostáticas. Na primeira metodologia, soluções de manana em diferentes concentrações foram gotejadas sobre a formulação LNC-Quit+ e na segunda metodologia a LNC-Quit+ foi injetada nas soluções de manana. Através de análises de diâmetro, mobilidade eletroforética, potencial zeta e viscosidade, foi possível notar que a primeira metodologia não apresenta reprodutibilidade, demonstrando valores aleatórios, ao passo que, na segunda metodologia os valores são reprodutíveis para a concentração de 0,5 μg/mL final de manana. Também foi possível observar que as nanopartículas revestidas com manana não apresentam estabilidade após 24 horas, sugerindo-se que a manana se desprende da superfície da partícula. Nas duas metodologias foram utilizadas concentrações diferentes de material de partida, e com isso, foi criada a metodologia 2B para que as anteriores pudessem ser comparadas. Com estes resultados tem-se um caminho promissor para estudos futuros e o aprimoramento deste tipo de revestimento. / In the present work, lipid core nanocapsules (LNCs) was developed for subsequent coating with mannan by using two different methodologies and both by electrostatic interactions. In the first methodology, mannan solutions at different concentrations were drop wise on LNC-Quit+ formulation and the second methodology, LNC-Quit+ was injected in mannan solutions. Through analysis of diameter, electrophoretic mobility, zeta potential and viscosity, it was possible to notice that the first methodology was not reproductible, showing random values, whereas, in the secont methodology the values was reproductible for the 0,5 μg/mL concentration. It was also possible to observe that the particle coated with mannan has no stability after 24 hours, suggesting that mannan detaches from the particle surface. In this methodologies, two different concentrations of raw materials are used, and with that, it was created the methodology 2B so that the results could be compared. With these results, it was a promising path way for future studies and enhancement of this type of coating.

No description available.

Current research efforts have focused on the synthesis of novel, segmented, cross-linked networks and thermoplastics for emerging technologies. Tailoring macromolecular structures for improved mechanical performance can be accomplished through a variety of synthetic strategies using step-growth polymerization. The synthesis and characterization of novel Michael addition networks, ionene families, and ion-containing polyurethanes are described, with the underlying theme of fundamentally investigating the structure-property relationships of novel, segmented macromolecular architectures. In addition, it was discovered that both covalent and electrostatic crosslinking play an important role in the mechanical properties of all types of polymers described herein. Novel cross-linked networks were synthesized using quantitative base-catalyzed Michael chemistry with acetoacetate and acrylate functionalities. These novel synthetic strategies offer unique thermo-mechanical performance due to the formation of a multiphase morphology. In order to fundamentally elucidate the factors that influence the kinetics of the Michael addition reaction a detailed analyses of model compounds were conducted in the presence of an in-situ IR spectrometer to optimize reaction conditions using statistical design of experiments. Networks were then prepared based on these optimized conditions. The mechanical performance was evaluated as a function of molecular weight between crosslink points. Furthermore, the incorporation of hydrogen bonding within the monomer structure enhanced mechanical performance. The changes in morphological, thermal, and mechanical properties evaluated using dynamic mechanical analysis (DMA) and tensile behavior are described. In addition, the use of preformed urethane segments provides a safer method for incorporating hydrogen bonding functional groups into macromolecules. In order to compare the thermomechanical and morphological properties of ion-containing polyurethanes to non-charged polyurethanes, poly(tetramethylene oxide)-based polyurethanes containing either a novel phosphonium diol or 1,4-butanediol chain extenders were prepared using a prepolymer method. The novel phosphonium polyurethane was more crystalline, and it was presumed that hydrogen bonding in the non-charged polyurethane restricted polymer mobility, and reduced PTMO crystallinity, and hydrogen bonding interactions were significantly reduced due to the presence of phosphonium cations. These results correlated well with mechanical property analysis. The phase separation and ionic aggregation were demonstrated via wide-angle X-ray scattering, small-angle X-ray scattering, scanning transmission electron microscopy, and energy-dispersive X-ray spectroscopy during STEM imaging, as described herein. In addition, a novel polyurethane containing imidazolium cations in the hard segment was synthesized and behaved very similarly to the phosphonium cation-containing polyurethane. Ammonium ionenes, which contain quaternary nitrogen in the macromolecular repeating unit, have many potential uses in biomedical applications. They offer interesting coulombic properties, and the charge density is easily controlled through synthetic design. This property makes ionenes ideal polyelectrolyte models to investigate the influence of ionic aggregation on many physical properties. Ammonium ionenes were prepared via the Menshutkin reaction from 1,12-dibromododecane and 1,12-bis(N,N-dimethylamino)dodecane. The absolute molecular weights were determined for the first time using an on-line multi-angle laser light scattering (MALLS) in aqueous size exclusion chromatography (SEC). Tensile testing and DMA were used to establish structure-property relationships between molecular weight and mechanical properties for a series of 12,12-ammonium ionenes. Furthermore, degradation studies in the presence of base support the possibility for water-soluble coatings with excellent mechanical durability that are amenable to triggered depolymerization. A novel synthetic strategy was utilized to prepare chain extended 12,12-ammonium ionenes containing cinnamate functional groups. In the presence of UV light, the polymers chain extended, and the resulting ionenes possessed enhanced thermomechanical properties and increased molecular weight. In addition, the novel synthesis of imidazolium ionenes was demonstrated, and the charge density was tuned for appropriate applications using either low molecular weight segments or oligomeric precursors. The change in charge density had a profound role in imidazolium ionene thermal and mechanical behavior. / Ph. D.

Wide literature precedence exists for polymers containing electrostatic interactions and polymers containing hydrogen bonding motifs, however the combination of electrostatic and hydrogen bonding interactions is not widely investigated in current literature. Polyelectrolytes containing hydrogen bonding groups are expected to exhibit properties of both classes of supramolecular interactions. A series of adenine- and thyminecontaining PDMAEMA and tert-butyl acrylate copolymers were synthesized to investigate the effect of incorporating hydrogen bonding groups into a polyelectrolyte. Incorporation of the styrenic nucleobases significantly affected the solubility of these copolymers on aqueous solutions and showed salt-triggerability with higher contents of these groups. Polyelectrolytes are capable of binding and condensing DNA through electrostatic interactions with the negatively charged phosphate groups of the DNA backbone; however a high degree of cytotoxicity is also often observed for these gene delivery systems. The high level of cytotoxicity is attributed to high degree of cationic character for the polyplexes formed with these systems according to the proton-sponge hypothesis. One method of reducing the overall cationic character for these systems is incorporation of non-electrostatic binding mechanisms such as hydrogen bonding. A series of nucleobase-containing PDMAEMA copolymers were utilized in order to investigate the effect of incorporation of these groups on the cell viability, binding efficiency, and transfection efficiency of PDMAEMA. / Master of Science

Master of Science / Department of Grain Science and Industry / X. Susan Sun / Soybean protein modified with sodium bisulfite behaves like latex adhesives, with adhesive strength comparable to formaldehyde-based adhesives. β-conglycinin and glycinin are two major protein components of the adhesive system. The objective of this research was to investigate the effect of sodium bisulfite on the physicochemical, morphological, and adhesion properties of glycinin and β-conglycinin in order to better understand the function of glycinin and β-conglycinin in the formation of the soy latex adhesive. Sodium bisulfite broke the disulfide bonds that linked acidic and basic polypeptides of glycinin, and the reducing effect was enhanced with increasing sodium bisulfite concentration. Although cleavage of disulfide bonds was expected to destabilize proteins, the thermal stability of glycinin increased as the sodium bisulfite concentration increased. Sodium bisulfite modified glycinin had higher surface hydrophobicity, which facilitated hydrophobic interations between molecules and aggregation of glycinin. The balance between hydrophobic interactions and electrostatic forces makes glycinin form unique chain-like structures. Adhesive performance of glycinin dropped significantly at lower sodium bisulfite concentration and then increased as sodium bisulfite concentration increased up to 24 g/L. Excess sodium bisulfite was detrimental to adhesive strength and water resistance. High-molecular-weight aggregates were observed in unmodified β-conglycinin, but these aggregates were dissociated by sodium bisulfite treatment. Similar to glycinin, the thermal stability of β-conglycinin was improved by the modification. However, the denaturation enthalpy of β-conglycinin decreased significantly at high level of sodium bisulfite (36 g/L). The turbidity at pH 4.8 also dropped extensively at the concentration of 36 g/L. The contact angle of β-conglycinin reached its minimum at 6 g/L sodium bisulfite on cherry wood and 24 g/L on glass. Morphology study proved that sodium bisulfite modification made the β-conglycinin solution more dispersed. At pH 9.5, water resistance of β-conglycinin was improved to a small extent by 6 g/L sodium bisulfite. At pH 4.8, adhesive performance was enhanced by 3 g/L and 6 g/L sodium bisulfite. High level of sodium bisulfite at 36 g/L reduced the adhesive performance of β-conglycinin drastically.