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- Creators: Vaiana, Sara
- Creators: Rege, Kaushal
Description
Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.
ContributorsRamos, James (Author) / Rege, Kaushal (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Garcia, Antonio (Committee member) / Arizona State University (Publisher)
Created2014
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
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