Matching Items (3)
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- All Subjects: Cyanines
- All Subjects: Functional genomics
- Creators: Vaiana, Sara
Description
CpG methylation is an essential requirement for the normal development of mammals, but aberrant changes in the methylation can lead to tumor progression and cancer. An in-depth understanding of this phenomenon can provide insights into the mechanism of gene repression. We present a study comparing methylated DNA and normal DNA wrt its persistence length and contour length. Although, previous experiments and studies show no difference between the physical properties of the two, the data collected and interpreted here gives a different picture to the methylation phenomena and its effect on gene silencing. The study was extended to the artificially reconstituted chromatin and its interactions with the methyl CpG binding proteins were also probed.
ContributorsKaur, Parminder (Author) / Lindsay, Stuart (Thesis advisor) / Ros, Robert (Committee member) / Tao, Nongjian (Committee member) / Vaiana, Sara (Committee member) / Beckenstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2012
Description
Fluorescence spectroscopy is a powerful tool for biophysical studies due to its high sensitivity and broad availability. It is possible to detect fluorescence from single molecules allowing researchers to see the behavior of subpopulations whose presence is obscured by “bulk” collection methods. The fluorescent probes used in these experiments are affected by the solution and macromolecular environments they are in. A misunderstanding of a probe’s photophysics can lead researchers to assign observed behavior to biomolecules, when in fact the probe is responsible. On the other hand, a probe’s photophysical behavior is a signature of the environment surrounding it; it can be exploited to learn about the biomolecule(s) under study. A thorough examination of a probe’s photophysics is critical to data interpretation in both cases and is the focus of this work. This dissertation investigates the photophysical behavior of symmetric and asymmetric cyanines in a variety of solution and biomolecular environments. Using fluorescent techniques—such as time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS)—it was found that cyanines are influenced by the local environment. In the first project, the symmetric cyanines are found to be susceptible to paramagnetic species, such as manganese(II), that enhance the intersystem crossing (ISC) rate increasing triplet blinking and accelerating photobleaching. Another project found the increase in fluorescence of Cy3 in the protein induced fluorescence enhancement (PIFE) technique is due to reduced photoisomerization caused by the proximity of protein to Cy3. The third project focused on asymmetric cyanines; their photophysical behavior has not been previously characterized. Dy630 as a free dye behaves like Cy3; it has a short lifetime and can deactivate via photoisomerization. Preliminary experiments on Dy dyes conjugated to DNA show these dyes do not photoisomerize, and do not show PIFE potential. Further research will explore other conjugation strategies, with the goal of optimizing conditions in which Dy630 can be used as the red-absorbing analogue of Cy3 for PIFE applications. In summary, this dissertation focused on photophysical investigations, the understanding of which forms the backbone of rigorous fluorescent studies and is vital to the development of the fluorescence field.
ContributorsCiuba, Monika A (Author) / Levitus, Marcia (Thesis advisor) / Liu, Yan (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2017
Description
Proteins are essential for most biological processes that constitute life. The function of a protein is encoded within its 3D folded structure, which is determined by its sequence of amino acids. A variation of a single nucleotide in the DNA during transcription (nSNV) can alter the amino acid sequence (i.e., a mutation in the protein sequence), which can adversely impact protein function and sometimes cause disease. These mutations are the most prevalent form of variations in humans, and each individual genome harbors tens of thousands of nSNVs that can be benign (neutral) or lead to disease. The primary way to assess the impact of nSNVs on function is through evolutionary approaches based on positional amino acid conservation. These approaches are largely inadequate in the regime where positions evolve at a fast rate. We developed a method called dynamic flexibility index (DFI) that measures site-specific conformational dynamics of a protein, which is paramount in exploring mechanisms of the impact of nSNVs on function. In this thesis, we demonstrate that DFI can distinguish the disease-associated and neutral nSNVs, particularly for fast evolving positions where evolutionary approaches lack predictive power. We also describe an additional dynamics-based metric, dynamic coupling index (DCI), which measures the dynamic allosteric residue coupling of distal sites on the protein with the functionally critical (i.e., active) sites. Through DCI, we analyzed 200 disease mutations of a specific enzyme called GCase, and a proteome-wide analysis of 75 human enzymes containing 323 neutral and 362 disease mutations. In both cases we observed that sites with high dynamic allosteric residue coupling with the functional sites (i.e., DARC spots) have an increased susceptibility to harboring disease nSNVs. Overall, our comprehensive proteome-wide analysis suggests that incorporating these novel position-specific conformational dynamics based metrics into genomics can complement current approaches to increase the accuracy of diagnosing disease nSNVs. Furthermore, they provide mechanistic insights about disease development. Lastly, we introduce a new, purely sequence-based model that can estimate the dynamics profile of a protein by only utilizing coevolution information, eliminating the requirement of the 3D structure for determining dynamics.
ContributorsButler, Brandon Mac (Author) / Ozkan, S. Banu (Thesis advisor) / Vaiana, Sara (Committee member) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2016