Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length.

No description available.

No description available.

<p>Nucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.</p><p>To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons. </p><p>We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter, wider structure compared to the B-form helix, its force spectra exhibit a shorter initial length before the overstretching force plateau, compared to B-form DNA. Contrarily, the Z-form double helix has a narrower, more extended helical structure than B-form DNA, and we see this fact manifest in the force spectra of Z-DNA, which has a longer initial length before the overstretching force plateau. Also, interestingly, we find that neither A, nor Z-DNA force spectra display the second melting force plateau. Indicating this plateau is not necessarily cause by melting of strands apart, but rather a feature of B-DNA. </p><p>To better understand the forces that stabilized these different structures, specifically base stacking, we also mechanically characterize different single-stranded helical polynucleotides using AFM based force spectroscopy. We expand on previous studies by confirming that single helical polynucleotides undergo a force transition at a force of ~20 pN as they are uncoiled, and also demonstrating, that when stretched beyond this force transition, the molecules behave differently depending on base sequence and backbone sugar. Specifically, the force spectra of poly-adenylic acid possess a linear force region, which persists to ~300 pN, after the force plateau. We also observe that poly-deoxyadenylic acid is comparatively stiffer than other polynucleotides after undergoing two force transitions. By supplementing our force spectroscopic data with MD simulations and NMR spectroscopy, we find that base stacking in adenine is quite strong, persisting above 100 pN. We find that initial helical structure, which is defined by base stacking and backbone sugar, guides the stretching pathway of the polynucleotides. This finding can possibly be extrapolated to the elasticity of double-stranded DNA.</p> / Dissertation

Atomic force microscopy (AFM) is a particularly useful tool, for obtaining structural information about drug-nucleic acid interactions. The mode of drug binding intercalation versus groove binding can be determined from images acquired on individual DNA molecules as the length of a DNA molecule increases in direct proportion to the number of intercalators bound to it. The efforts of this research were directed toward elucidating the mode of binding of a series of drugs based on polymers of naphthalenetetracarboxyl diimide (NDI) interacting with a linearized DNA plasmid. During the course of the investigation of these drugs, DNA intercalation was confirmed as the mode of binding and the binding affinity estimated. Unexpectedly, concentration-dependent formation of secondary DNA structures including condensates was observed. DNA toroids, spheres, and rods were imaged and measured. Conformations that are believed to be intermediate condensate forms were also identified at lower poly-NDI concentrations. Models for the DNA condensation process have been proposed. Ultimately, this research furthers the understanding of DNA condensation which can be applied to gene delivery systems and anti-viral agents. It may also help direct the development of better drugs based on the insight of poly-intercalators interactions with DNA.

The image resolution of atomic force microscopy (AFM) is still less superior to that of the electron microscopy (EM). However AFM operated in liquids complemented by Tapping-mode (TM) detection proves to be more suitable for imaging biomolecules in physiological-like environments. Nevertheless, manipulation of AFM in solution turned out to be non-trivial, several technical difficulties were encountered. In the thesis, I report using divalent cation-containing buffer as a feasible method to immobilize DNA molecules effectively for imaging in liquid media. AFM operating conditions, such as cantilever oscillating drive frequency, setpoint amplitude, feedback control parameters and scan rates were studied to obtain the optimized function. Various AFM images of Ssp I-linearized pUC19 DNA/EcoR I restriction enzyme complexes were captured, revealing the molecular details of their complex machineries. For example, the intermediate stage of the enzyme cleavage action was displayed by images showing that DNA was bent by an acute angle at the active site in the presence of one single EcoR I molecule. Some evidence for a jumping, sliding or intersegmental transfer mechanism is achieved. To trace the enzyme-DNA interaction dynamic in real time, preliminary results were obtained, but further improvements are required.

High-resolution lattice periodicity images of a variety of well-defined surfaces, including graphite, mica, and Au(111), validated the good stability of our atomic force microscope (AFM) system. Combining self-assembled monolayer (SAM) and AFM technology, we demonstrated the capabilities of pattern fabrication as well as modification of surface functionality. AFM-based nanolithography operating conditions, such as scan rate, deflection setpoint, and number of scan were studied to obtain the optimized quality of the fabricated patterns. Thiolated-DNA probe molecules could be patterned at a nanometer scale on a gold substrate. However, we found that the surface coverage began to drop notably with the probe length (number of DNA bases). Therefore, the displaced DNA molecules during nanoshaving were reversibly adsorbed, and patterning became unreliable. We were unsuccessful in detecting the subsequent hybridization reactions at these nanopatterns from AFM measurements. To realize the DNA hybridization, further studies on the incubation temperature, probe length and even DNA sequences are required to demonstrate that this AFM-based gene diagnostic method is truly operational.

The fabrication characteristic of focused ion beam (FIB) for fused quartz was investigated. With the progress of nanotechnology, new technologies and devices are invented constantly. In nanofabrication, FIB has several advantages such as high material removal rate, high resolving power and direct fabrication in some selected areas without etching mask. Therefore, it had been studied in detail to fabricate nano-structures by FIB. In this study, we found out the effect of nano-machining by adjusting the parameters of FIB system such as: beam current, overlap, and dwell time. The fabricated features together with their surface morphology and profile were investigated by scanning electron microscope (SEM) and atomic force microscope (AFM). Results show that when beam current was smaller, overlap was 50% and dwell time was 10£gs could get best performance by FIB.

This study successfully demonstrates the electrical control of the surface wettability of liquid crystal and polymer composite film. The application of external voltages significantly affects the surface wettability of the film. This study uses atomic force microscopy to quantitatively characterize the fundamental mechanism responsible for the structurally driven changes in surface properties at various applied voltages. The surface wettability transitions of the film are electrically driven, as shown by reorganized liquid crystal molecules. Measurements of the voltage-dependent surface wettability of the composite film suggest novel approaches to supporting control applications of future electro-optical nanotechnology devices, including liquid lenses, windshields and displays.

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.