ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

The delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.

FREQUENCY DOMAIN CHARACTERIZATION OF OPTIC FLOW AND VISION-BASED OCELLAR SENSING FOR ROTATIONAL MOTION

The structure of an animal’s eye is determined by the tasks it must perform. While vertebrates rely on their two eyes for all visual functions, insects have evolved a wide range of specialized visual organs to support behaviors such as prey capture, predator evasion, mate pursuit, flight stabilization, and navigation. Compound eyes and ocelli constitute the vision forming and sensing mechanisms of some flying insects. They provide signals useful for flight stabilization and navigation. In contrast to the well-studied compound eye, the ocelli, seen as the second visual system, sense fast luminance changes and allows for fast visual processing. Using a luminance-based sensor that mimics the insect ocelli and a camera-based motion detection system, a frequency-domain characterization of an ocellar sensor and optic flow (due to rotational motion) are analyzed. Inspired by the insect neurons that make use of signals from both vision sensing mechanisms, advantages, disadvantages and complementary properties of ocellar and optic flow estimates are discussed.

Texture-Detail Preservation Measurement in Camera Phones: An Updated Approach

Recent advances in mobile phone cameras have poised them to take over compact hand-held cameras as the consumer’s preferred camera option. Along with advances in the number of pixels, motion blur removal, face-tracking, and noise reduction algorithms have significant roles in the internal processing of the devices. An undesired effect of severe noise reduction is the loss of texture (i.e. low-contrast fine details) of the original scene. Current established methods for resolution measurement fail to accurately portray the texture loss incurred in a camera system. The development of an accurate objective method to identify the texture preservation or texture reproduction capability of a camera device is important in this regard. The ‘Dead Leaves’ target has been used extensively as a method to measure the modulation transfer function (MTF) of cameras that employ highly non-linear noise-reduction methods. This stochastic model consists of a series of overlapping circles with radii r distributed as r−3, and having uniformly distributed gray level, which gives an accurate model of occlusion in a natural setting and hence mimics a natural scene. This target can be used to model the texture transfer through a camera system when a natural scene is captured. In the first part of our study we identify various factors that affect the MTF measured using the ‘Dead Leaves’ chart. These include variations in illumination, distance, exposure time and ISO sensitivity among others. We discuss the main differences of this method with the existing resolution measurement techniques and identify the advantages. In the second part of this study, we propose an improvement to the current texture MTF measurement algorithm. High frequency residual noise in the processed image contains the same frequency content as fine texture detail, and is sometimes reported as such, thereby leading to inaccurate results. A wavelet thresholding based denoising technique is utilized for modeling the noise present in the final captured image. This updated noise model is then used for calculating an accurate texture MTF. We present comparative results for both algorithms under various image capture conditions.