This study was conducted to determine if electroporation could deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. The long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. The ability of electroporation to introduce biologically active molecules, e.g., plasmid DNA and siRNA, into these monolayers, to either express a protein of interest or modify cellular function, was also studied. Results showed that intracellular uptake of calcein, a small tracer molecule, and bovine serum albumin, a globular protein, occurred uniformly throughout the monolayers and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. Barrier function recovery depended on the electroporation conditions applied, with some monolayers recovering normal physiologic function within a day. Electroporation also increased the permeability of the monolayers to calcein and BSA, possibly through a combination of increased paracellular and transmonolayer transport. When compared to cationic lipid transfection (lipofection), transfection of intestinal epithelial monolayers with reporter plasmids by electroporation was more efficient in situations where high concentrations of DNA, and as a result, higher levels of expression were needed. Although uptake of DNA was high after electroporation and increased with increasing amounts of DNA, overall expreseion efficiency was still low (~3%). Electroporation-mediated transfection of intestinal epithelial monolayers with a plasmid that expressed inflammation inhibitor protein, IκВα was not always successful, probably because of low levels of protein expression. Introduction of the much smaller siRNA molecules into the monolayers by electroporation, on the other hand, was very successful at inhibiting the production of the nuclear envelope proteins lamin A and lamin C. The results of these experiments demonstrated that electroporation can introduce a wide variety of molecules intracellularly into model intestinal epithelia. These results should be useful to identify optimal electroporation conditions for transporting drugs, proteins, and genes into intestinal and, possibly, other epithelia for local drug and gene therapy, as well as for development of improved models of intestinal epithelium.

In earlier research in our lab, the manipulation of microtubules on gold patterned silicon wafers was achieved by E-beam lithography, Poly (ethylene glycol) self assembled monolayers (PEG-SAMs) and electrophoresis. To develop a technique for delicate single microtubule manipulation, further studies need to be done on PEG-SAMs and electrophoresis. As a foundation of this goal, we examined the electric field in an aqueous solution between two planar electrodes and the compatibility of the antifouling property of PEG-SAMs with the electric field. For this purpose, the distribution of microbeads was analyzed using a Boltzmann distribution. The amount of adsorbed microtubules on a PEG-SAM was examined to test the compatibility of the antifouling property of a PEG-SAM with concomitant exposure to electric field. It is shown that the product of the electric field and the effective charge of the microbead does not have a linear relation with the applied electric potential but an exponentially increasing function with respect to the potential. The antifouling property of the PEG-SAM was not retained after an exposure to the electric field.

The thesis is divided into two parts. One is about the preparation of discotic liquid crystals Acid-6. The other is about the growth of Acid-6 thin film by thermal evaporation on silicon oxide surfaces and modified silicon oxide surfaces such as self-assembled monolayer(SAM) in different temperature. The surface morphology and molecular orientation of the thin film were studied by Atomic Force Microscopy(AFM) , X-ray Diffraction (XRD) , and then they were applicated to organic thin film transistor and measured properties by Semiconductor Parameter Analyzer. In the second part of our research, our expectative characteristics was not observed in different temperature and substructure. In the other side, we guessed that because discotic liquid crystals Acid-6 is negative semiconductor materials, so it is susceptible to hydrosphere, thus we can¡¦t observe the electric characteristic of OTFT in the atmosphere. In addition, due to discotic liquid crystals Acid-6 have biggish moleculer weight, thus its viscosity was so big that cause the diameter of Acid-6 crystals to be too small. Therefore, it influenced the carrier mobility. Finally, from the aspect of procedure about fabrication of the devices we can discuss whether this parameter of this device can apply to OTFT.

Controlling the orientation of liquid crystal molecules in LC displays is extremely important for optimizing device performance. The method most commonly used in industry today involves rubbing the surface of the polymer-coated glass substrates used in the displays with a velvet cloth to create microscopic grooves. Berreman theory states that the liquid crystal molecules then align along the direction of the grooves. Alternatively, some literature shows that the friction caused by rubbing aligns the polymer chains in the surface layer which then attract and align the liquid crystal molecules along the direction of the chains. Even now, it is still unclear exactly how the process of rubbing the surface causes the liquid crystal molecules to align in an orderly manner. This thesis describes a systematic study of the physical and chemical influence of the substrate on the alignment and orientation of liquid crystal molecules. We used Fourier Transform Infrared spectroscopy (FTIR) to identify surface chemistry, contact angle measurements to determine the surface energy, and atomic force microscopy (AFM) to observe the alignment of liquid crystal on the surfaces. In the course of this study, we have gained insight into how the physical and chemical properties of the surface affect the molecular arrangement in the solid-liquid interface. Our results can be applied not only to LCD technology, but more generally to biochips and biosensor devices.

Atomic force microscopy (AFM) is an important technology that allows researchers to probe local surface properties at nanometer length scales. In addition to surface topography, the AFM can probe many types of tip-surface interactions (including adhesion and friction) to gain a better understanding of the chemical properties of surfaces. This thesis contains two experiments which utilize AFM to in addition to several other techniques to study (1) Self Assembled Monolayer (SAM) formation and corrosion and (2) intermolecular and surface/molecular effects on gramicidin film formation and molecular orientation. In the first experiment, N-octadecyltrichlorosilane (OTS) molecules were self-assembled onto silicon samples. We observed that OTS required a very short time (about 15 seconds) to complete the formation of the monolayer on surface. However, this SAM film was highly susceptible to corrosion by the strong oxidant (KMnO4), resulting in a chemical change to the film from hydrophobic functional groups (CH3) to hydrophilic functional groups (OH). In subsequent experiments, we observed that if the SAMs were formed using longer exposure times (about 24 hours), they were highly resistant to corrosion. Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Photoelectron Spectroscopy (XPS) also showed that the 24 hour growth SAM films were densely packed. These results indicate that SAM films based on organosilane molecules can protect the surface from corrosion, and further that more densely packed SAMs exhibit better anti-corrosion performance than less dense films. In the second experiment, the antibacterial peptide Gramicidin was used to study how intermolecular and surface energy properties can influence the aggregation and film formation of molecules on several surfaces. Gramicidin has a unique physical and chemical structure with hydrophobic side chain and hydrophilic ends. Here, we have used three different substrates (Silicon, Mica, and Graphite) to study intermolecular interactions, aggregation, and orientation of Gramicidin peptide. Langmuir-Blodgett methods were also used to study aggregation and molecular orientation at the solid-liquid interface.

Self assembled monolayer films (SAMs) deposited on silicon surfaces have gained considerable interest due to their ability to modify surface properties for advanced applications in sensors, MEMS, and NEMS devices. These molecular films are typically deposited on silicon surfaces from solution using a variety of solvents, which can influence the molecular packing and quality of the films. To better understand these effects, we have performed a systematic solvent effect study of the growth of n-Octadecyltrichlorosilane (OTS) on silicon substrates using chloroform, dichloromethane, toluene, benzene and hexadecane. The films were characterized using contact angle measurements, Fourier Transform Infrared Spectroscopy (FTIR), and Atomic Force Microscopy (AFM) to evaluate the SAM growth rate and film quality. Lateral Force Microscopy (LFM) and transmission FTIR were used to characterize the molecular packing. Finally, we used AFM to make adhesion measurements on the films and correlated these results with friction data. These techniques provide a means to characterize the local nanoscale packing of the films. The Hertzian contact model was used to model and describe the adhesion and friction result. Our results show that using hexadecane as the solvent produced OTS films with the highest density molecular packing. By comparing to Langmuir-Blodgett SAM film deposition methods, we show that it is the intermolecular interaction between the solvent molecules and OTS that determines this density. Thus, the structure and chemical properties of the solvent molecule strongly influences the molecular packing, quality, and performance of the SAM film.

This dissertation investigates the photophysics of bis(diarylamino)biphenyl (TPD) and silver nanoparticles (AgNP). A main goal of this work was to develop an understanding of the relaxation pathways involved in the deactivation of photoexcited TPD chromophores in close proximity to silver nanoparticles. The TPD chromophores were attached to the silver nanoparticle core via an alkylthiol group. The TPD-AgNP systems were synthesized and characterized using Transmission Electron Microscopy (TEM), UV-visible absorption, infrared spectroscopy, and Nuclear Magnetic Resonance (NMR) spectroscopy, Inductively Coupled Plasma - Emission Spectroscopy (ICP-ES) and Thermogravimetric Analysis (TGA). Time-resolved photophysical processes in these systems were studied using femtosecond transient absorption spectroscopy. Initial studies of the interaction of the TPD and AgNP addressed the linker length dependence of the dye excited state decay kinetics, wherein alkyl linker chains of 3, 4, 8 and 12 carbon atoms were used. These results showed that an ultrafast deactivation of the excited state of the TPD chromophore, which is three orders of magnitude faster than that of the free chromophore in solution, occurred in all of the systems. However, an unexpected new transient species was observed for the systems with three and four carbon linker chains. Further studies showed this species to be spectroscopically very similar to the TPD radical cation, suggesting a charge separation pathway in the excited state relaxation. Possible pathways for formation of the cation-like state were examined through comparisons to the photophysics of alkyl substituted TPD in solution and in solid films, investigation of the pulse energy and TPD surface coverage dependence of the yield of the cation-like TPD species, transient absorption anisotropy decay dynamics, and kinetic modeling studies. Taken together, these investigations provide support for exciton-exciton annihilation being responsible for the formation of cation-like species. The packing of the TPD chromophores is concluded to be of critical importance in the generation of the cation like species but it is also possible that proximity to the silver nanoparticle plays a role in facilitating charge separation as well.

<p>This project involves the fabrication and testing of microporous, polymer membranes designed to remove minute amounts of toxic air pollutants such as formaldehyde from air streams. The hypothesis to be tested is that active, the silver contained within the porous polymer membranes results in high formaldehyde retention.</p><p>Monolayers consist of different sizes of sPS particles are assembled first on the silicon wafers by spin coating method and convective assembly method, respectively. Then each kind of monolayer with one dimension of sPS particles is deposited with a nanometer scaled silver thin film with a bench top metal evaporator. The porous membranes are produced by assembly of close-packed colloidal crystals of silver capped polystyrene template particles and subsequent infiltration with polyurethane prepolymer. The prepolymer is cured by UV exposure. The sPS particles are removed from the particle polymer membrane by treatment with organic solvents resulting in the formation of inverse opal structures. Silver does not dissolve in the organic solvents and cannot leave the pores due to the small size of connecting holes in an inverse opal. All the monolayers, cylindrical colloidal crystals and microcapillaries after infiltration of polyurethane had been characterized by optical microscope, and the porous membranes had been characterized by SEM.</p><p>The application of porous membranes with silver caps is to absorb formaldehyde in the air, while in fact the silver caps are oxidized and become Ag2O, which will initiate a gas-phase/solid reaction with formaldehyde. In the future, TiO2 will be applied together with Ag2O, since TiO2 is another good absorbent for formaldehyde</p>

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.