Browsing by Author "Kolomeisky, Anatoly"
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Item Contrast Agent Development for Molecular Imaging of Cancer(2021-12-03) Pollard, Alyssa Camille; Pagel, Mark D; Kolomeisky, AnatolyThis dissertation focuses on the design, synthesis, and application of contrast agents for molecular imaging. Specifically, these agents have been designed to probe specific tumor biomarkers such as pH or cancer metabolism to provide information related to tumor biology. Two sets of positron emission tomography/magnetic resonance imaging (PET/MRI) co-agents have been designed to measure tumor extracellular pH. One set of agents is metal-based using gadolinium or a radiometal, and the other introduces a fluorine-18 or fluorine-19 tag. While both the radiometal and fluorine-based co-agents were able to measure pH in solution, the fluorine-based co-agents reached a lower standard error of 0.06 pH units. In addition, the fluorine-based co-agents demonstrated high stability in solution and in vivo, while the radiometal-based agents underwent dechelation of the radiometal upon injection. Finally, the fluorine-based agents were able to measure tumor pH in a 4T1 breast cancer cell line. To apply the PET/MRI co-agents, extensive work had to first be performed to characterize the small animal PET/MRI system and determine how the two modalities affected the other’s image quality. After correcting for PET quantification, radioactivity could be accurately measured by the PET detector with errors of less than 5% for fluorine-18 and less than 20% for gallium-68. Despite the larger errors for gallium-68, they did not largely influence pH measurements using 68Ga-labeled PET co-agents. The power of PET/MRI was also utilized to identify lung tumor metastases and measure [18F](2S,4R)-4-fluoroglutamine ([18F]FGln) uptake in a clear cell renal cell carcinoma lung metastasis model. Due to the high anatomical contrast of PET/MRI, lung tumors were clearly identified versus normal lung tissue, and 1.5-fold higher [18F]FGln uptake was observed in lung tumors versus the healthy lungs in a set of control mice. In addition, we found that [18F]FGln uptake was decreased after treatment with the glutaminase inhibitor CB-839. Finally, a novel fullerene-based nanomaterial with potential biomedical applications was radiolabeled with copper-64 to answer questions about its in vivo biodistribution and pharmacokinetic profile using PET imaging. Interestingly, this highly water-soluble fullerene conjugated with serinolamide groups displayed rapid renal clearance in mice.Item Embargo Covalent and Non-Covalent Functionalization of ssDNA-Wrapped Single-Wall Carbon Nanotubes: Computational and Experimental Studies(2023-04-13) Alizadehmojarad, Ali A.; Weisman, Bruce; Kolomeisky, AnatolySingle-wall nanotubes (SWCNTs) are nanomaterials with a wide range of optical and electronic properties that depend on their physical structure, which is indexed by a pair of integers (n,m). The discovery and interpretation of nanotube fluorescence has taken SWCNT research to a new level by enabling novel studies of structure-specific reactions and processes. These require the suspension of individualized SWCNTs in liquids through non-covalent functionalization by dispersants such as polymers, conventional surfactants, and single-stranded DNA (ssDNA). Understanding the specific interactions between nanotubes and coatings is important for advancing both basic and applied research. Both computational and experimental investigations have been conducted to understand the mechanism of separation and sorting of specific (n,m) species coated by DNA oligonucleotides. In addition to non-covalent SWCNT functionalization, covalent functionalization has also added a new research dimension and the ability to modify important properties of pristine nanotubes. In this thesis, novel computational and experimental methods were developed to further understand non-covalent and covalent functionalization of SWCNTs dispersed in DNA oligonucleotides and in a conventional surfactant. Computational studies were performed to understand how a DNA oligo distinguishes two enantiomers of an (n,m) species. Replica exchange molecular dynamics (REMD) simulations revealed that this recognition is directly correlated with the nanotube surface area exposed to the environment when wrapped by a DNA oligo. In the case of covalent functionalization of guanine nucleobases to SWCNTs, steered MD (SMD) simulations showed that the nucleotides in the middle of a DNA strand are found in closer proximity to nanotube surface than those at the strand end. These observations are aligned with experimental findings suggesting that a greater degree of guanine functionalization is achieved with 31-nucleotide DNA oligos containing only one guanine in the middle than DNA oligos with one guanine near the end of a DNA strand. A novel experimental method was developed to measure extinction coefficient of SWCNTs in the UV region. These results have enabled the simple determination of total SWCNT concentrations in aqueous dispersions. The DNA/SWCNT mass ratio was then quantitatively evaluated to provide an important parameter for understanding conformations of DNA oligos wrapped around SWCNTs. Through this method, the dependence of DNA/SWCNT mass ratio on DNA oligo base sequence was experimentally determined. Those conformations around different (n,m) species were then further explored using standard MD simulations. In this way, the strength of interactions between nanotubes and DNA oligos was investigated, revealing that thymine-rich DNA oligos interact with nanotube surfaces more strongly than cytosine-rich oligos. The guanine functionalization of SWCNTs was conducted using excitation of metalloporphyrin photosensitizers with violet light. Guanine functionalization of nanotubes was monitored by red-shifts in their absorption and fluorescence spectra. The great advantage of using metalloporphyrin dyes instead of rose bengal as a photosensitizer is that they are more photostable and cause less interference with SWCNT absorption and fluorescence spectra. Consequently, kinetic studies of guanine functionalization of SWCNTs were performed to find the correlation between kinetic parameters and either dye concentration or guanine contents of the DNA oligos. Quantum chemistry methods were used to predict find the most stable product of covalent attachment of guanine to SWCNT side walls and to correlate the formation enthalpy of each adduct with nanotube diameter. The semi-empirical quantum chemistry results show that 4,5-guanine peroxide (GPO) attached to either ortho L30 or ortho L-30 positions on the nanotubes’ surface give the most energetically stable products of guanine functionalization. Excited state calculations then predict that 4,5-GPO attached at the ortho L30 positions results in the most red-shifted (6,5) absorption peak compared to other adducts. In a separate experimental study, the first structure-selective guanine functionalization of SWCNTs was achieved using near-infrared or visible photochemistry. Specific (n,m) SWCNT species were excited by monochromatic excitation at their characteristic E11 or E22 transitions. Energy transfer to dissolved O2 then generated singlet oxygen, which led to formation of guanine peroxide (GPO) and its bonding to SWCNT side walls. The guanine functionalization was selective for the excited nanotubes. Finally, MD simulations were used to investigate the noncovalent adsorption of the surfactant sodium dodecyl sulfate (SDS) on SWCNT surfaces. This study uncovered four different SDS morphologies, characterized their structures, and found the combinations of SDS concentrations and nanotube diameters leading to the different morphologies.Item Data-driven Construction of Coarse-grained Protein Models(2023-09-26) Yang, Wangfei; Clementi, Cecilia; Kolomeisky, AnatolyWith the rapid development of computational power over the past few decades, computational simulation has grown increasingly important in protein-related research due to its high resolution and predictability. However, simulating large proteins over extended time scales using all-atomic resolution models and explicit solvents remains infeasible due to the substantial computational burden. Consequently, researchers have turned to developing coarse-grained (CG) models, which reduce degrees of freedom to accelerate simulations. To develop a CG model, two tasks must be completed. One involves determining which degrees of freedom will be retained in the CG model, known as the CG mapping. The other task is accurately capturing equivalent interactions at the CG resolution using an appropriate force field. In this dissertation, two data-driven methods are developed for the aforementioned tasks. The first method, Variational Approach for Markov Processes (VAMP)-based coarse-graining, provides a criterion to evaluate different CG mappings under a specific resolution directly from finer-grained simulation data. This evaluation is accomplished without constructing a complete CG model and running CG simulations. Additionally, based on this method, a Markov Chain Monte Carlo sampling method is developed to attain the optimal CG mapping without enumerating all possibilities. The second method optimizes the CG force field according to the native structure based on the energy landscape theory. Specifically, this method is used to extend an existing coarse-grained model, the Associative memory, Water-mediated, Structure, and Energy Model (AWSEM), to include protein-metal ion interactions. The use of these methods enables the construction of accurate coarse-grained protein models, making it possible to simulate larger protein systems over longer time scales.Item Measuring forces at the leading edge: a force assay for cell motility(Royal Society of Chemistry, 2013) Farrell, Brenda; Qian, Feng; Kolomeisky, Anatoly; Anvari, Bahman; Brownell, William E.Cancer cells become mobile by remodelling their cytoskeleton to form migratory structures. This transformation is dominated by actin assembly and disassembly (polymerisation and depolymerisation) in the cytoplasm. Synthesis of filamentous actin produces a force at the leading edge that pushes the plasma membrane forward. We describe an assay to measure the restoring force of the membrane in response to forces generated within the cytoplasm adjacent to the membrane. A laser trap is used to form a long membrane nanotube from a living cell and to measure the axial membrane force at the end of the tube. When the tube, resembling a filopodium, is formed and in a relaxed state the axial membrane force exhibits a positive stationary value. This value reflects the influence of the cytoskeleton that acts to pull the tube back to the cell. A dynamic sawtooth force that rides upon the stationary value is also observed. This force is sensitive to a toxin that affects actin assembly and disassembly, but not affected by agents that influence microtubules and myosin light chain kinase. We deduce from the magnitude and characteristics of dynamic force measurements that it originates from depolymerisation and polymerisation of F-actin. The on- and off-rates, the number of working filaments, and the force per filament (2.5 pN) are determined. We suggest the force-dependent transitions are thermodynamically uncoupled as both the on- and off-rates decrease exponentially with a compressive load. We propose kinetic schemes that require attachment of actin filaments to the membrane during depolymerisation. This demonstrates that actin kinetics can be monitored in a living cell by measuring force at the membrane, and used to probe the mobility of cells including cancer cells.Item Embargo Molecular coarse-graining for classical and quantum systems(2024-04-19) Zaporozhets, Iryna; Kolomeisky, Anatoly; Clementi, CeciliaUnderstanding the intricate molecular mechanisms underlying biological processes is crucial for tackling multiple biomedical challenges. Molecular dynamics serves as a "computational microscope", offering insights into biomolecular processes with unparalleled spatial and temporal resolution. Yet capturing these processes on biologically relevant scales poses significant computational challenges, especially when additional phenomena, such as nuclear quantum effects (NQEs), must be considered. However, many processes of interest can be described by a smaller set of collective variables instead of the intractably large number of degrees of freedom arising in atomistic simulation. The idea behind coarse-graining is to integrate out the irrelevant degrees of freedom and model the target system at a lower resolution while preserving the target properties. This thesis contributes to the development and application of coarse-grained models to increase the computational efficiency of biomolecular simulation and extend the range of molecular processes that can be investigated computationally. First, we applied a structure-based coarse-grained model combined with all-atom simulations to elucidate the helix formation mechanism following the chromophore isomerization in cyanobacteriochrome Slr1393-g3. Our findings indicate a destabilization of the helical state in the 15-Z configuration compared to the 15-E configuration, which has implications for future experimental investigations. This project also highlights the need for improved coarse-grained models. Second, the ODEM optimization framework was used to parameterize protein structure-based models using experimental data. The results suggest that incorporating many-body terms to describe nonbonded interactions is crucial to accurately reproduce the protein thermodynamics. This result underscores the importance of using neural networks' potential in approximating coarse-grained force-fields for future research. Next, a combination of coarse-graining, path integral quantum mechanics, and machine learning was used to develop potentials that incorporate NQEs into all-atom simulation at the cost of classical molecular dynamics. We developed separate models to approximate quantum dynamics and quantum statistics, which demonstrated good performance when applied to test systems. These approaches have the potential to obtain an accurate incorporation of NQEs in biomolecular simulation. Finally, we discuss how the developed approaches contribute to the bigger goal of effective and accurate methods for computational elucidation of biomolecular processes.Item Nanopores: Modeling the Separation Mechanism and Application of Carbon Nanotubes in Sensing(2018-11-12) Agah, Shaghayegh; Kolomeisky, Anatoly; Pasquali, MatteoTransport and separation through biological membranes exhibit fast, efficient and selective behaviors. These unique features have brought a significant interest recently in using artificial and biological nanopore membranes for separation and sensing pur- poses. Molecular transport through nanopores is a complex process that involves various interactions between the molecules and the channel. These interactions lead to a highly efficient and selective separation. The goal of this thesis is to under- stand this complicated phenomenon and develop a model that captures this behav- ior. Based on this modeling, the mechanism can be mimicked in different applications varying from molecular separation in nanopore sensing and drug delivery technologies to chemical and pharmaceutical separations. We construct a theoretical framework which models the general mechanism of selectivity in the translocation dynamics of molecular mixtures through a nanopore. By developing a discrete-state stochastic model, the effects of the molecule-channel interactions on molecular selectivity and flux are demonstrated. Our framework shows that the amount and the position of the interaction inside the channel has a significant effect on the separation efficiency. In addition, an efficient algorithm is developed to implement our theoretical framework for long channels. The proposed algorithm optimizes the magnitude and the position of the interaction in channels with two to ten binding sites. Due to the exponential nature of the problem, it is computationally infeasible to apply our algorithm for channels with more than ten binding sites. To address this issue, we use machine-learning techniques and extend our results for longer channels and predict the best position for molecular-pore interaction. Our results demonstrate that the best interaction location depends on the length of the nanopore and the mass transport rates inside and outside of the channel. Depending on these factors, the best interaction location may vary between the entrance and the middle of the pore. Sensing is another important application of nanopores. Nanopores have been shown to have the ability of single-molecule sensing of various biological molecules in a rapid fashion and at a low cost. We discuss the recent progress in the nanopore- sequencing field with a focus on the nature of nanopores as well as sensing mechanisms during the translocation. The current challenges such as the fast translocation speed and low sensitivity of the sensing techniques, and alternative methods based on using Single Walled Carbon Nanotubes (SWCNTs) are also explored. Carbon nanotubes (CNTs) demonstrate enhanced water and ion flow, have wide variety of lengths and diameters, and manifest excellent electrical and optical properties. These features make CNTs one of the best candidates for artificial biochannels and nanopore devices. Our molecular dynamic simulation study shows that it is beneficial to use SWCNT for nanopore sequencing application. This is due to the possibility of slowing down the DNA transport through the tube by two orders of magnitude. Additionally, we propose to use the photoluminescence (PL) properties of SWCNTs as a sensing method. Therefore, the PL spectrum characteristics of SWCNTs at the presence of different defect densities are studied to investigate whether the carbon nanotube photoluminescence quantum yield can be enhanced at the single molecular level for future sensing technologies.