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Title: Controlling the surface processes of X- and Z-type ligands to tailor the photophysics of II-VI semiconductor nanocrystals
Creator: Saniepay, Mersedeh
Date: 2018
Collection: Electronic Theses & Dissertations
Description: II−VI colloidal semiconductor nanocrystals (NCs), such as CdSe NCs, are often plagued by efficient nonradiative recombination processes that severely limit their use in energy-conversion schemes. While these processes are now well-known to occur at the surface, a full understanding of the exact nature of surface defects and of their role in deactivating the excited states of  NCs has yet to be established, which is partly due to challenges associated with the direct probing of the complex and...
Show moreII−VI colloidal semiconductor nanocrystals (NCs), such as CdSe NCs, are often plagued by efficient nonradiative recombination processes that severely limit their use in energy-conversion schemes. While these processes are now well-known to occur at the surface, a full understanding of the exact nature of surface defects and of their role in deactivating the excited states of  NCs has yet to be established, which is partly due to challenges associated with the direct probing of the complex and dynamic surface of colloidal NCs. In this dissertation, we report a detailed study of the surface of cadmium-rich zinc-blende CdSe NCs. The surfaces of these cadmium-richspecies are characterized by the presence of cadmium carboxylate complexes (CdX2) that act as Lewis acid (Z- type) ligands that passivate under-coordinated selenide surface species. The systematic displacement of CdX2 from the surface by N,N,N′,N′-tetramethylethylene-1,2-diamine (TMEDA) has been studied using a combination of 1H NMR and photoluminescence spectroscopies. We demonstrate the existence of two independent surface sites that differ strikingly in the binding affinity for CdX2 and that are under dynamic equilibrium with each other. A model involving coupled dual equilibria allows a full characterization of the thermodynamics of surface binding (free energy, as well as enthalpic and entropic terms), showing that entropic contributions are responsible for the difference between the two surface sites. Importantly, we demonstrate that cadmium vacancies only lead to important photoluminescence quenching when created on one of the two sites, allowing a complete picture of the surface composition to be drawn where each site is assigned to specific NC facet locale, with CdX2 binding affinity and nonradiative recombinationefficiencies that differ by up to two orders of magnitude.To understand the effect of steric hindrance and types of functional groups in different ligands on X-type ligand exchanges, using NMR, PL and UV-Vis absorption spectroscopy, we studied X-type exchanges on CdSe NCs capped with native carboxylates, with oleic acid, oleyl thiol, benzoic acid and benzenethiol ligands. We discussed the results and occurrence of undesired pathways including displacement of Z-type ligands, and suggested ligand exchange strategies that most likely lead to 100% X-type exchange.The structural complexity of surface of CdS NCs is also discussed in this dissertation. We demonstrate presence of two different sulfur surface defects on CdS NCs with ligand binding equilibrium constants that are two orders of magnitude apart and 20-60% smaller than those of selenium on similar size CdSe NCs. We also correlated the different surface defects to the PL quenching efficiency of CdS NCs.
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Title: DETECTION OF COHERENT ENERGY TRANSFER PATHWAYS IN PHOTOSYNTHESIS WITH TWO-DIMENSIONAL ELECTRONIC SPECTROSCOPY
Creator: Roscioli, Jerome Daniel
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Light harvesting proteins in photosynthetic organisms contain highly ordered arrays of chromophores responsible for the collection of energy from solar photons. The organization of the chromophores may lead to collective excitations (excitons) that are delocalized over many molecules in the array. The delocalized excitations allow for coherent, or wavelike, energy transfer between the chromophores, rather than a particle-like, incoherent, energy transfer process. It has been proposed that...
Show moreLight harvesting proteins in photosynthetic organisms contain highly ordered arrays of chromophores responsible for the collection of energy from solar photons. The organization of the chromophores may lead to collective excitations (excitons) that are delocalized over many molecules in the array. The delocalized excitations allow for coherent, or wavelike, energy transfer between the chromophores, rather than a particle-like, incoherent, energy transfer process. It has been proposed that these collective excitations may direct the flow of energy along the most efficient pathway to enhance the fitness of photosynthetic organisms. Photosynthetic organisms may also favor closely packed chromophore arrays because the structure is compact whilst optimizing large optical cross sections for absorption. Control of the coupling between chromophores may lead to a photoregulatory mechanism, which could control the energy transfer rate as a function of ambient light intensity fluctuations. Broad-band two- dimensional electronic spectroscopy (2DES) can be used to elucidate donor–acceptor pathways and mechanisms for both coherent and incoherent excitation energy transfer (EET) in photosynthetic light-harvesting proteins. In this dissertation, 2DES is applied to determine how quantum coherent energy transfer occurs between carotenoids and chlorophylls (Chls) in the peridinin-chlorophyll protein (PCP), a mid-visible peripheral light-harvesting protein in marine dinoflagellates that delivers excitation energy to photosystem II. PCP is unique in that it uses a carotenoid, peridinin, as the main light harvesting chromophore and that it can be reconstituted with different chlorophylls to change the energy landscape without causing structural changes. Through 2DES experiments on native PCP with Chl a, we show that although the collective excitations of chromophores are very short lived, they lead to an enhanced quantum yield compared to that for conventional, incoherent energy transfer mechanisms. Replacing the native Chl a acceptor chromophores with Chl b slows energy transfer from peridinin to Chl despite narrowing the donor–acceptor energy gap. The formyl substituent on the Chl b macrocycle hastens decoherence by sensing the surrounding electrostatic noise, leading to lower EET efficiencies. This work is significant because it improves our understanding of the role of coherent energy transfer in photosynthetic light harvesting. This information may prove useful when designing materials featuring strongly interacting electronic chromophores for the collection of solar energy for the generation of fuels or for use in photocatalysis.
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Title: I. amhb : (anti)aromaticity-modulated hydrogen bonding. ii. evaluation of implicit solvation models for predicting hydrogen bond free energies
Creator: Kakeshpour, Tayeb
Date: 2019
Collection: Electronic Theses & Dissertations
Description: My doctoral research under Professor James E. Jackson focused on hydrogen bonding (H-bonding) using physical organic chemistry tools. In the first chapter, I present how I used quantum chemical simulations, synthetic organic chemistry, NMR spectroscopy, and X-ray crystallography to provide robust theoretical and experimental evidence for an interplay between (anti)aromaticity and H-bond strength of heterocycles, a concept that we dubbed (Anti)aromaticity-Modulated Hydrogen Bonding (AMHB). In...
Show moreMy doctoral research under Professor James E. Jackson focused on hydrogen bonding (H-bonding) using physical organic chemistry tools. In the first chapter, I present how I used quantum chemical simulations, synthetic organic chemistry, NMR spectroscopy, and X-ray crystallography to provide robust theoretical and experimental evidence for an interplay between (anti)aromaticity and H-bond strength of heterocycles, a concept that we dubbed (Anti)aromaticity-Modulated Hydrogen Bonding (AMHB). In the second chapter, I used accurately measured hydrogen bond energies for a range of substrates and solvents to evaluate the performance of implicit solvation models in combination with density functional methods for predicting solution phase hydrogen bond energies. This benchmark study provides useful guidelines for a priori modeling of hydrogen bonding-based designs.Coordinates of the optimized geometries and crystal structures are provided as supplementary materials.
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Title: Structure and function study of HIV and influenza fusion proteins
Creator: Liang, Shuang
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Human immunodeficiency virus (HIV) and influenza virus are membrane-enveloped viruses causing acquired immunodeficiency syndrome (AIDS) and flu. The initial step of HIV and influenza virus infection is fusion between viral and host cell membrane catalyzed by the viral fusion protein gp41 and hemagglutinin (HA) respectively. However, the structure of gp41 and HA as well as the infection mechanism are still not fully understood. This work addresses (1) full length gp41 ectodomain and TM domain...
Show more"Human immunodeficiency virus (HIV) and influenza virus are membrane-enveloped viruses causing acquired immunodeficiency syndrome (AIDS) and flu. The initial step of HIV and influenza virus infection is fusion between viral and host cell membrane catalyzed by the viral fusion protein gp41 and hemagglutinin (HA) respectively. However, the structure of gp41 and HA as well as the infection mechanism are still not fully understood. This work addresses (1) full length gp41 ectodomain and TM domain structure and function and (2) IFP membrane location and IFP-membrane interaction. My studies of gp41 protein and IFP can provide better understanding of the membrane fusion mechanism and may aid development of anti-viral therapeutics and vaccine. The full length ectodomain and transmembrane domain of gp41 and shorter constructs were expressed, purified and solubilized at physiology conditions. The constructs adopt overall a helical structure in SDS and DPC detergents, and showed hyperthermostability with Tm > 90 °C. The oligomeric states of these proteins vary in different detergent buffer: predominant trimer for all constructs and some hexamer fraction for HM and HM_TM protein in SDS at pH 7.4; and mixtures of monomer, trimer, and higher-order oligomer protein in DPC at pH 4.0 and 7.4. Substantial protein-induced vesicle fusion was observed, including fusion of neutral vesicles at neutral pH, which are the conditions similar HIV/cell fusion. Vesicle fusion by a gp41 ectodomain construct has rarely been observed under these conditions, and is aided by inclusion of both the FP and TM, and by protein which is predominantly trimer rather than monomer. Current data was integrated with existing data, and a structural model was proposed. Secondary structure and conformation of IFP is a helix-turn-helix structure in membrane. However, there has been arguments about the IFP membrane location. 13C2H REDOR solid-state NMR is used to solve this problem. The IFP adopts major a helical, minor b strand secondary structure in PC/PG membrane. The a helical IFP's with respectively 13CO labeled Leu-2, Ala-7 and Gly-16 all show close contacts with the lipid acyl chain tail, suggesting IFP has strong interaction with the membrane. By screening the current IFP topology models, it either has a membrane-spanning confirmation, or it promotes lipid trail protrusion. IFP bounded lipid membrane structure was studied by paramagnetic relaxation enhancement (PRE) solid-state NMR to provide more information about the detailed IFP membrane location model. The T2 relaxation time and rate were measured for membrane with or without IFP and with or without Mn2+. Based on the results, it is concluded that IFP does not promote lipid protrusion at both gel phase and liquid phase, which is evidenced by that the R2 difference with and without Mn2+ is smaller for IFP free membrane than IFP bounded membrane, meaning IFP does not induce a smaller average distance between lipid acyl chain and aqueous layer. By integrating these results, a IFP membrane spanning model was proposed, in which IFP N-terminal helix adopts a 45° angle with respect to membrane normal."--Pages ii-iii.
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Title: Solid-state nuclear magnetic resonance studies of the structures, membrane locations, cholesterol contact, and membrane motions of membrane-associated HIV Fusion Peptide (HFP
Creator: Jia, Lihui (Scientist)
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Membrane fusion is the key step during HIV viral entry to cells, and the process is catalyzed by HIV membrane fusion protein gp41. HFP is the ~25-residue N-terminal domain of gp41 and is required for membrane fusion with significant decreases in fusion activity with point mutations. Both viral and host cell membrane contain ~30mol % cholesterol (CHOL), and HFP induced fusion is faster in membrane with CHOL. However, how HFP interacts with membrane lipids and CHOL is unknown. In this thesis,...
Show moreMembrane fusion is the key step during HIV viral entry to cells, and the process is catalyzed by HIV membrane fusion protein gp41. HFP is the ~25-residue N-terminal domain of gp41 and is required for membrane fusion with significant decreases in fusion activity with point mutations. Both viral and host cell membrane contain ~30mol % cholesterol (CHOL), and HFP induced fusion is faster in membrane with CHOL. However, how HFP interacts with membrane lipids and CHOL is unknown. In this thesis, we used the newly developed 13C-2H Rotational Echo Double Resonance (REDOR) solid-state NMR method to study the membrane location of HFP in chemically-native membrane environment. HFP is 13CO labeled at specific residue, and the membrane is deuterated at specific regions of the membrane using selective regions deuterated phosphatidylcholine (PC) and CHOL. We study HFP wild type, HFP_V2E and L9R mutants because these two mutants eliminate and decrease fusion respectively. HFP is predominantly β sheet structure in bilayer membrane for both HFP wild type and HFP_V2E mutant, HFP_L9R has a different structure and is likely helical. Both HFP and HFP_V2E mutant have major deeply-inserted membrane location contacting membrane center and minor shallowly-inserted membrane location contacting half way of one membrane leaflet. The HFP_V2E mutant has bigger fraction of molecules with shallower membrane location, which is consistent with the strong correlation between membrane location insertion depth and the peptide fusogenicity. HFP_L9R mutant has majorly deeply inserted into membrane.By comparing the HFP- PC and HFP- CHOL contact, there is preferential contact between HFP and CHOL vs PC at several residues including G5, G10 and G16. The free energy difference for contacting PC vs CHOL is ~ 0.57(5) kcal.mol-1 for T= 300K. HFP- CHOL contact geometry is successfully modeled by Swiss Dock and YASARA energy minimization with two strands antiparallel HFP (1→16/16→1 registry). There are two energetically favorable binding models between HFP and CHOL, from docking, energy minimization and consistency with REDOR results. The contact models reveal tilted and curved-up tail orientation of Chol_d7. Fusion may be catalyzed by matching the curvature of lipids contacting HFPs with the membrane curvature during the fusion intermediates like the stalk. Membrane motion perturbation by HFP is studied by static deuterium NMR from deuterium powder pattern spectrum, order parameter profile and T2 relaxation time. The DMPC-d54 spectrum becomes ~10% narrower in membrane without CHOL with 4% HFP and in membrane with 33% CHOL with 1% HFP. Accordingly, the order parameter of lipid acyl chain becomes ~ 1-10% disordered by HFP. However, the spectrum becomes 20% broader in membrane with 33% CHOL with 4% HFP, and the order parameter of lipid acyl chain becomes ~ 20- 30% ordered by HFP. With HFP at 37 °C, DMPC-d54 T2 decreases ~ 70 %, and the CHOL T2 decreases ~ 30%. T2 reduction is probably associated with increased membrane curvature induced by HFP. With greater membrane curvature, the C-D bond will experience more orientation diversity relative to the external magnetic field. Thus, the quadrupolar field will have greater change, leading to faster relaxation and shorter T2. Gp41_V2E mutant eliminates cell-cell fusion. Our CD spectroscopy studies show that the FPHM_V2E mutant is helical and the melting temperature is above 90 °C in 10mM Tris buffer + 0.2 % SDS at pH 7.4. Protein is trimer and induces no lipid mixing in PC: CHOL= 2:1 vesicles.
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Title: Solid state NMR studies of structure and dynamics of membrane associated influenza fusion peptide
Creator: Ghosh, Ujjayini
Date: 2016
Collection: Electronic Theses & Dissertations
Description: "This work seeks to delineate the role of influenza fusion peptide in the process of membrane fusion. Influenza fusion peptide is represented by the 203033 N-terminal residues of the HA2 subunit of the hemagglutinin (HA) protein. The influenza fusion peptide plays an important role in the membrane fusion between the host viral and the host cell endosomal membrane and has pH dependence. The influenza fusion peptide is the most conserved sequence in the in the influenza genome such that a...
Show more"This work seeks to delineate the role of influenza fusion peptide in the process of membrane fusion. Influenza fusion peptide is represented by the 203033 N-terminal residues of the HA2 subunit of the hemagglutinin (HA) protein. The influenza fusion peptide plays an important role in the membrane fusion between the host viral and the host cell endosomal membrane and has pH dependence. The influenza fusion peptide is the most conserved sequence in the in the influenza genome such that a modest mutation can arrest the fusion activity. It was shown that in detergents the structure of the 20 residue and the 23 residue influenza fusion peptide have different structures. However, influenza fusion peptide is a membrane peptide and induces fusion the lipid vesicles and not between the detergent micelles. In this work, solid state NMR was used to study the structure of the influenza fusion peptide in membranes and its correlation to the vesicle fusion. The influenza peptide was chemically synthesized chemically and was used as a model system to study the membrane fusion process. In PC:PG membranes, the influenza fusion peptide adopts closed and semiclosed structure. Both the closed and the semiclosed structure have a helix/turn/helix structure with an interhelical angle of 1030358° and 1403036° respectively. Unlike detergents, the structures of the 20 residue and the 23 residue are very similar in membranes with some minor differences. At low pH or the fusogenic pH, there is a higher fraction of the semiclosed fraction for both the influenza peptide constructs. For the longer peptide, higher fractions of the closed structures were determined. Vesicle fusion assays served as a surrogate for the virus/endosome fusion. Our data supported a iii strong positive correlation between the vesicle fusion and the hydrophobic surface area. Based on these data we proposed that the hydrophobic interaction between HAfp and the membrane is an important factor in HAfp-catalyzed fusion. Solid state NMR has been applied to study the structure and dynamics of lipid molecules in membrane with fusion peptide but the solid-state NMR data are typically the sum over all lipid molecules with only a small fraction of these molecules next to the fusion peptide. My second project primarily utilized 2 H NMR to study the dynamics of the influenza fusion peptide in membranes. This work describes the development and application of the cross polarization with solid or quadrupolar echo. The main idea of the work is to probe the motions of the lipids adjacent/close to the peptide. This method is applied to two different peptides, HIV-fusion peptide and influenza fusion peptide in presence of membranes. By comparing the conventional solid-echo experiment and the newly developed cross polarization with quadrupolar echo, I have seen differences in the lipid dynamics."--Pages ii-iii.
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Title: Investigation of folding, stability and function of alpha-helical membrane proteins under native condition
Creator: Guo, Ruiqiong
Date: 2018
Collection: Electronic Theses & Dissertations
Description: "Membrane proteins count for 2530%0303 of all proteins and carry out a variety of critical biological processes, such as nutrient transport, signal transduction, catalysis and generation of metabolic energy. Despite the importance, understandings of membrane protein folding lag far behind those of water-soluble proteins. The knowledge gap stems from inherent difficulties in controlling the reversible folding of membrane proteins in lipid bilayers, which is necessary for thermodynamic analysis...
Show more"Membrane proteins count for 2530%0303 of all proteins and carry out a variety of critical biological processes, such as nutrient transport, signal transduction, catalysis and generation of metabolic energy. Despite the importance, understandings of membrane protein folding lag far behind those of water-soluble proteins. The knowledge gap stems from inherent difficulties in controlling the reversible folding of membrane proteins in lipid bilayers, which is necessary for thermodynamic analysis of driving forces and mechanisms of folding. Steric trapping is a promising tool to reversibly control membrane protein folding. It utilizes the strong binding affinity between the biotin affinity tag and the bulky tag-binding protein streptavidin. In my Ph.D. research, I developed an array of novel methods by synthesizing a set of novel biotinylated protein probes, advancing the steric trapping method for a general application. Applying those methods to studying the folding of a helical-bundle membrane protein, rhomboid protease GlpG in detergent micelles, I mapped its folding energy landscape by revealing subglobal unfolding of the region encompassing the active sites, and quantifying a network of cooperative and localized interactions to maintain the stability. Combining computational methods, I elucidated the role of packing interactions in the stability and function of GlpG, showing that the advanced steric trap method can be used for studying driving forces in membrane protein folding. By using the novel biotinylated spin label, I was able to determine the inter-spin distance between the two biotinylated sites in the sterically trapped denatured state by double electron-electron resonance spectroscopy in the native lipid bilayer environments. These novel steric trapping methods can be applied to investigate a variety of problems in the folding and stability of membrane proteins directly under native lipid and solvent conditions."--Pages ii-iii.
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Title: The integration of computational methods and nonlinear multiphoton multimodal microscopy imaging for the analysis of unstained human and animal tissues
Creator: Murashova, Gabrielle Alyse
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Nonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption ...
Show moreNonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption (TPA) cross-section of the molecule being studied and is incredibly difficult to satisfy using typical incoherent light, say from a light bulb. Therefore, the stimulated emission of coherent photons by pulsed lasers are used for NMMM applications in biomedical imaging and diagnostics.In this dissertation, I hypothesized that due to the near-IR wavelength of the Ytterbium(Yb)-fiber laser (1070 nm), the four MP-two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF) and third harmonic generation (THG), generated by focusing this ultrafast laser, will provide contrast to unstained tissues sufficient for augmenting current histological staining methods used in disease diagnostics. Additionally, I hypothesized that these NMMM images (NMMMIs) can benefit from computational methods to accurately separate their overlapping endogenous MP signals, as well as train a neural network for image classification to detect neoplastic, inflammatory, and healthy regions in the human oral mucosa. Chapter II of this dissertation explores the use of NMMM to study the effects of storage on donated red blood cells (RBCs) using non-invasive 2PEF and THG without breaching the blood storage bag. Unlike the lack of RBC fluorescence previously reported, we show that with two-photon (2P) excitation from an 800 nm source, and three-photon (3P) excitation from a 1060 nm source, there was sufficient fluorescent signal from hemoglobin as well as other endogenous fluorophores. Chapter III employs NMMM to establish the endogenous MP signals present in healthy excised and unstained mouse and Cynomolgus monkey retinas using 2PEF, 3PEF, SHG, and THG. We show the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using NMMM with an ultrafast fiber laser centered at 1070 nm and a 303038 fs pulse. Two spectrally and temporally distinct regions were shown; one from the nerve fiber layer (NFL) to the inner receptor layer (IRL), and one from the retinal pigmented epithelium (RPE) and choroid. Chapter IV focuses on the use of minimal NMMM signals from a 1070 nm Yb-fiber laser to match and augment H&E-like contrast in human oral squamous cell carcinoma (OSCC) biopsies. In addition to performing depth-resolved (DR) imaging directly from the paraffin block and matching H&E-like contrast, we showed how the combination of characteristic inflammatory 2PEF signals undetectable in H&E stained tissues and SHG signals from stromal collagen can be used to analytical distinguish healthy, mild and severe inflammatory, and neoplastic regions and determine neoplastic margins in a three-dimensional (3D) manner. Chapter V focuses on the use of computational methods to solve an inverse problem of the overlapping endogenous fluorescent and harmonic signals within mouse retinas. The least-squares fitting algorithm was most effective at accurately assigning photons from the NMMMIs to their source. This work, unlike commercial software, permits using custom signal source reference spectra from endogenous molecules, not from fluorescent tags and stains. Finally, Chapter VI explores the use of the OSCC images to train a neural network image classifier to achieve the overall goal of classifying the NMMMIs into three categories-healthy, inflammatory, and neoplastic. This work determined that even with a small dataset (< 215 images), the features present in NMMMIs in combination with tiling, transfer learning can train an image classifier to classify healthy, inflammatory, and neoplastic OSCC regions with 70% accuracy.My research successfully shows the potential of using NMMM in tandem with computational methods to augment current diagnostic protocols used by the health care system with the potential to improve patient outcomes as well as decrease pathology departmental costs. These results should facilitate the continued study and development of NMMM so that in the future, NMMM can be used for clinical applications.
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Title: Utilizing fluidic platforms for the development of in vitro pharmacokinetic/pharmacodynamic models
Creator: Lockwood, Sarah Yvonne
Date: 2014
Collection: Electronic Theses & Dissertations
Description: The pharmaceutical industry is constantly developing new therapies and treatments, while the cost of the drug discovery process currently is estimated at two billion dollars, spent over a 12-15 year period. Adding to the cost associated with bringing a drug to market is the high attrition rate, with only 1 in every 10,000 compounds being approved by the Food and Drug Administration. Interest in reassessing existing research procedures for improved efficiency has recently been garnering...
Show moreThe pharmaceutical industry is constantly developing new therapies and treatments, while the cost of the drug discovery process currently is estimated at two billion dollars, spent over a 12-15 year period. Adding to the cost associated with bringing a drug to market is the high attrition rate, with only 1 in every 10,000 compounds being approved by the Food and Drug Administration. Interest in reassessing existing research procedures for improved efficiency has recently been garnering attention. Specifically, pharmacology studies, which utilize in vivo studies to obtain pharmacokinetic (PK) and pharmacodynamic (PD) information during the preclinical stage of the drug discovery process, have been a focal point. By complimenting the in vivo studies with in vitro models, an increase in efficiency is able to be realized by a reduction in consumed materials. In this dissertation, a diffusion-based dynamic in vitro (DDIV) PK model, fabricated on a microfluidic polydimethyl siloxane (PDMS) platform, was used to characterize the loading and elimination of a PK profile. However, challenges traditionally associated with the microfluidic devices, such as the fragility of the membrane due to device flexibility, reusability, and lack of automation make long-term PK studies incredibly difficult to perform, as well as reproduce. DDIV models fabricated on a rigid three-dimensional (3D) printed platform are rugged, reusable, and amenable to automation when integrated with a disposable cell culture insert. The 3D printed DDIV PK/PD device was characterized using fluorescein (332.31 g/mol) and validated using the antibiotic levofloxacin (361.37 g/mol). The loading profiles were achieved by flowing concentrated analyte through the device channels while adding buffer to the membrane insert to create a concentration gradient across the porous membrane, thereby allowing diffusion from the channel into the insert. Parameters related to the loading portion of a PK curve, such as loading time, flow rate, volume of the insert, and initial concentration in the channel were characterized. The profiles obtained during the characterization of the initial concentrations (7.5, 15, 30 μM) in the channel yielded a prediction model for both the concentration along the loading profile and the maximum concentration (Cmax) at a given loading time. Elimination of analyte from the membrane insert was proven to undergo first order rate kinetics. The elimination profile, and the resulting elimination rate constant are used to obtain the half-life. Ultimately, a prediction model for the half-life will be crucial to the characterization of the DDIV model, however preliminary gradient studies highlighted the importance of a correction factor pertaining to the amount of analyte absorbed by the device. Upon complete characterization, the reusable 3D printed DDIV PK/PD millifluidic device will allow researchers to mimic in vivo dosing regimens on an in vitro platform, resulting in a useful tool to be used in tandem with animal models.
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Title: A spectroelectrochemical investigation of the thermodynamic and structural properties of the 2-oxoglutarate-dependent oxygenase, taud
Creator: John, Christopher Wayne
Date: 2019
Collection: Electronic Theses & Dissertations
Description: 2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations making their method of action and thermodynamic properties of great interest to industrial synthesis. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. Unfortunately, the non-heme and less accessible active sites of these enzymes makes it a challenge to...
Show more2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations making their method of action and thermodynamic properties of great interest to industrial synthesis. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. Unfortunately, the non-heme and less accessible active sites of these enzymes makes it a challenge to study them. To counteract this issue, we develop a method that uses electrochemical mediators and combines normal pulse spectrovoltammetry (NPSV) with Fourier transform infrared (FTIR) for detection and subsequent global spectral regression analysis to resolve the structural and thermodynamic properties simultaneously. We develop comprehensive semiemipirical kinetic simulation models to investigate the thermodynamic and kinetic limitations of mediators/analyte interactions. These methods are first validated using methylene green and thionine acetate as mediators and myoglobin (Mb) as the analyte. Both the E½ and unbiased redox difference FTIR spectra of the Fe(II)/Fe(III) redox couple of Mb in reduction and oxidation NPSV modes were in good agreement with those reported earlier by independent techniques. The modeling effort yielded a flexible computational tool capable of quantitatively predicting the redox response in mediated electrochemical studies and defining its limitations. These methods are used to characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD), demonstrating that the FeIII/II transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement changes the apparent redox potential of the active site between -272 mV for reduction of the ferric state and 196 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex resulting in a maximal observed redox hysteresis in the wild type enzyme of 468 mV. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations. We use H99A, D101Q, H255Q, and Y73I variants of TauD to investigate the structural origin of the redox-linked reorganization and the relative contributions of the active site residues to the dynamic tuning of the redox potential of TauD. Extended time-dependent redox titrations show that, in all cases, reorganization occurs as a multi-step process, with individual phases exhibiting different sensitivities to ligand substitutions. The H99A variant shows the largest net redox change relative to the wild type protein, suggesting that redox-coupled protonation of H99 is required for TauD to support highly positive potentials. The effect of the D101Q substitution suggests that changes in the metal coordination of the carboxylate group may be secondary to changes involving H99 and are required for the ensuing reorganization steps. The H255Q substitution inhibits the conformational change, providing evidence for its involvement in the structural rearrangement. An investigation of the pD sensitivity of wild type TauD exposes a protonation event at the active site of TauD most likely attributable to H99 or H255. Ultimately, we propose H99 is protonated in the ferrous form of TauD and forms a hydrogen bond with the protein backbone. Oxidation of the enzyme results in the loss of this hydrogen bond allowing movement in the H99-T100-D101 chain so that D101 can form a bidentate ligand with the ferric iron center.
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Title: Vibronic coherence in light-harvesting proteins and semiconductor nanomaterials
Creator: Tilluck, Ryan
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Light harvesting in photosynthetic organisms employ complex arrays of highly ordered chromophores in proteins for the capture and transfer of energy from solar photons. The structural design of the light harvesting proteins leads to extensive delocalization across many molecules of the system. Recent studies demonstrate the involvement of quantum coherence in extraordinarily fast energy transfer pathways in photosynthetic light harvesting proteins. The extent of delocalization may also form...
Show moreLight harvesting in photosynthetic organisms employ complex arrays of highly ordered chromophores in proteins for the capture and transfer of energy from solar photons. The structural design of the light harvesting proteins leads to extensive delocalization across many molecules of the system. Recent studies demonstrate the involvement of quantum coherence in extraordinarily fast energy transfer pathways in photosynthetic light harvesting proteins. The extent of delocalization may also form vibronic excitons, vibronic excitons, arising from quantum coherent mixing of vibrations with the extensively delocalized electronic states. Vibronic excitons can greatly enhance the excitation energy transfer within light harvesting systems. In this dissertation, the detection and role of quantum coherence in the peridinin-chlorophyll protein (PCP), a mid-visible peripheral light-harvesting protein in marine dinoflagellates that delivers excitation energy to photosystem II, will be discussed. This dissertation will also discuss the role of vibronic coherences, involving organic surface ligands, in the hot carrier cooling process of CdSe semiconductor quantum dots (QDs). Optical broadband two-dimensional electronic spectroscopy (2DES) with ultrashort pulses has been employed on these systems to characterize nonradiative decay and energy transfer pathways in PCP and CdSe QDs. Understanding the advantage gained by the organism through the use of coherent energy transfer mechanisms may enable the development of more efficient light harvesting materials for use in photovoltaic cells or in photocatalysis.
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Title: Investigating blood flow and antibiotic dosing using traditional microfluidics and novel 3D printed devices
Creator: Meisel, Jayda Erkal
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Over the last 25 years, it has been established that the red blood cell (RBC) is a major determinant in blood flow, which it can modulate through release of adenosine triphosphate (ATP). Although RBCs store intracellular ATP in mM concentrations, measurements indicate that the cells release nM concentrations when stimulated by deformation, hypoxia (lowered oxygen tension), or incubation with pharmacological stimuli such as hydroxyurea (HU), which is the only approved drug for treatment of...
Show moreOver the last 25 years, it has been established that the red blood cell (RBC) is a major determinant in blood flow, which it can modulate through release of adenosine triphosphate (ATP). Although RBCs store intracellular ATP in mM concentrations, measurements indicate that the cells release nM concentrations when stimulated by deformation, hypoxia (lowered oxygen tension), or incubation with pharmacological stimuli such as hydroxyurea (HU), which is the only approved drug for treatment of sickle cell disease. Upon release, RBC-derived ATP can induce vessel dilation via activation of endothelial cell nitric oxide synthase (eNOS) to produce nitric oxide (NO). To probe the fate of increased ATP release from human RBCs incubated with the drug hydroxyurea, a traditional soft polymer platform was utilized to facilitate measurement of cell-to-cell communication between RBCs and a cultured endothelium. This device contained an array of micron-scale channels through which RBC samples were pumped. The sample flow was separated from a detection well by a porous polycarbonate membrane. Stimulated ATP released from the RBCs diffused across the membrane to the detection wells and was measured using the luciferin-luciferase chemiluminescence assay, integrated with a plate reader for detection. RBCs incubated with 100 uM of HU released on average 2.06 ± 0.37 times more ATP relative to the control sample. Through the use of various inhibitors, this increase in ATP release was subsequently demonstrated to depend on RBC deformability, RBC NOS activity, and the cystic fibrosis transmembrane conductance regulator protein (CFTR). The fate of the measured RBC-derived ATP was also investigated by probing ATP signalling to an adjacent cultured endothelium.ATP release from RBCs increases in response to hypoxia, or lowered oxygen tension; however, the dependence of RBC ATP release on oxygen tension has not been investigated. To enable measurement of RBC ATP release and oxygen tension in a flowing stream of RBCs, a 3D printed device was designed to accomodate commercial transwell inserts for ATP measurements, as well as threaded Clark-type electrodes for amperometric oxygen measurements. The device consisted of a channel 2 mm wide and 0.5 mm in height with two ports for analyte detection and one threaded port for an electrode. The Clark-type electrode was fabricated from gold and silver wires secured into a finger tight fitting. Oxygen standards and RBC samples were prepared using air and argon purged buffers. Using the 3D printed device, RBC ATP release and oxygen tension were measured simultaneously from prepared RBC samples. Relative to controls, RBC ATP release increased significantly in response to systematically lowered oxygen tension with a maximum increase of 2.38 ± 0.43 fold more ATP when exposed to 5.35 ± 0.12 ppm oxygen. ATP release saturated, i.e., was not significantly different, at lower oxygen tensions. This increase in ATP release was inhibited by incubating RBCs with the cell stiffening agent, diamide. The dependence of hypoxic RBC ATP release on the conformation of heme in hemoglobin (Hb) is demonstrated by converting measured oxygen tensions to Hb saturation.The 3D printed platforms presented herein were also utilized as in vitro tools to model pharmacokinetic dosing profiles, specifically with applications for studying antibiotic resistance. The World Health Organization, Centers for Disease Control, and the White House have issued reports that outline strategies to combat antibiotic resistance. The Spence lab has developed a 3D printed device to mimic in vivo drug dosing profiles on an in vitro platform for applications in drug discovery. This 3D printed diffusion-based dynamic dosing device mimics the dosing capabilities of the hollow fiber chamber reactor (HFCR). The in vitro 3D printed device contains 6 ports to house commercial polyester transwell membrane inserts (0.4 micron) and in house fabricated 0.2 micron pore size inserts (polyester), which can be loaded with a sample of Escherichia coli. Chemically competent, kanamycin resistant E. coli were dosed with the DNA gyrase and topoisomerase IV inhibitor levofloxacin, which reached a maximum concentration (Cmax) of 21.0 ± 5.7 uM (0.4 micron pore size) and 68.0 ± 7.1 uM (0.2 micron pore size) of levofloxacin in approximately 1 hour. After dosing, the viability of the bacteria samples was measured using standard plating methods.
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Title: Solid state nuclear magnetic resonance studies of structures and membrane locations of peptides
Creator: Xie, Li
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Solid state nuclear magnetic resonance (SSNMR) can be used to study the structures of molecules such as small peptides and large proteins. Different structures correspond to different chemical environments and thus yield different chemical shifts in NMR spectra. In addition, SSNMR can also be used to probe membrane locations of peptides and proteins and provide insights into their biological functions. This dissertation mainly focuses on the structural and membrane location studies of...
Show moreSolid state nuclear magnetic resonance (SSNMR) can be used to study the structures of molecules such as small peptides and large proteins. Different structures correspond to different chemical environments and thus yield different chemical shifts in NMR spectra. In addition, SSNMR can also be used to probe membrane locations of peptides and proteins and provide insights into their biological functions. This dissertation mainly focuses on the structural and membrane location studies of peptides by rotational-echo double-resonance (REDOR) SSNMR, which is a technique for measuring distances between two coupled hetero nuclei such as 13C and 15N.Influenza fusion peptide (IFP) is the N-terminal peptide of the HA2 subunit of the influenza hemagglutinin (HA) protein and this peptide plays an important role in the membrane fusion between the virus and the endosome of the host cell. There are 15 different HA subtypes. I studied the structure of membrane-associated H3 subtype IFP (H3_IFP) by 13C-15N REDOR SSNMR. SIMPSON simulations of the data indicate that H3_IFP adopts predominantly closed and semi-closed structures in membranes. Similar to IFP, human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal peptide of the viral gp41 fusion protein and this peptide plays a key role in the HIV-host cell membrane fusion. HFP adopts major antiparallel and minor parallel fÒ sheet structures in membranes. Earlier fluorescence spectroscopy and SSNMR studies support a strong positive correlation between the membrane insertion depth and fusogenicity of HFP. However, the 19F labeling in earlier membrane location studies of HFP may perturb the membrane bilayer integrity. Due to this concern, 13C-2H REDOR was developed to detect the residue-specific membrane location of HFP, where one residue of HFP is backbone 13CO labeled and the lipid is either perdeuterated or selectively deuterated in its acyl chains. Since 1H and 2H are chemically equivalent, there is no perturbation on membrane bilayer integrity as well as peptide-lipid interaction regardless of what fraction of deuterated lipid is used. The 13C-2H REDOR pulse sequence was optimized using the setup peptide I4. The membrane locations of two peptides, HFP and KALP, were studied by 13C-2H REDOR, where KALP is a designed transmembrane fÑ helical peptide. Fitting of the REDOR data revealed that both peptides have multiple locations within the membrane hydrocarbon core. The multiple locations are attributed to the snorkeling of lysine sidechains on both termini for KALP and to the distribution of antiparallel fÒ sheet registries for HFP. HFP has a ~0.7 fraction of deep insertion and a ~0.3 fraction of shallow insertion in the membrane. The predominant deep insertion of HFP may significantly perturb the membrane bilayer structure and lower the activation energy of membrane fusion, which is consistent with the observed positive correlation between the membrane insertion depth and fusion rate for different HFP constructs.
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Title: Comparison of vibrational energy transfer in micelles and vesicles
Creator: Pratama, Fredy Setiady
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Understanding intermolecular energy transfer is important from both fundamental and applied perspectives. It is a process that determines thermal conductivity, an important property for applications such as cooling and lubrication. Our interest in vibrational energy transfer lies in the organization of fluid systems at the molecular level can influence the efficiency of vibrational energy transfer. We use one type of amphiphile that can for different assemblies in aqueous solution to...
Show moreUnderstanding intermolecular energy transfer is important from both fundamental and applied perspectives. It is a process that determines thermal conductivity, an important property for applications such as cooling and lubrication. Our interest in vibrational energy transfer lies in the organization of fluid systems at the molecular level can influence the efficiency of vibrational energy transfer. We use one type of amphiphile that can for different assemblies in aqueous solution to determine how organization affects energy dissipation. Sodium decanoate was used as the amphiphile because it can form micelles or vesicles in aqueous solution, depending on the solution pH and the amphiphile concentration. Our results provide evidence that micelles and vesicles affect the dissipation of vibrational energy. Vibrational population relaxation data show the time constant for intermolecular energy transfer from perylene to the amphiphile aliphatic chain differs by a factor of two for micelles and vesicles, and is more efficient in micelles. Complementary measurements of transient heating in these same systems show that micelles experience higher temperature change than vesicles following the deposition of excess energy into the system by means of internal conversion from the S2 to S1 states of perylene. This finding indicates that the non-specific dissipation of energy from the amphiphile assembly to the aqueous bath is the same to within the experimental uncertainty for vesicles and micelles. This finding is in contrast to our findings for mode-specific vibrational energy transfer and is likely a consequence of the non-mode-specific nature internal conversion within the chromophore.
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Title: High resolution tertiary structure of the membrane-associated HIV fusion peptides by solid state nuclear magnetic resonance
Creator: Schmick, Scott
Date: 2012
Collection: Electronic Theses & Dissertations
Description: HIV gp41 protein catalyzes fusion between viral and host cell membranes, and its apolar N-terminal region or "fusion peptide" binds to host cell membranes and plays a key role in viral and host cell membrane fusion. Gp41 fusion can be dominantly inhibited by dilute amounts of V2E mutant gp41, but a structural basis for this inhibition has not been demonstrated. "HFP" is a construct containing the fusion peptide sequence that induces membrane vesicle fusion, and V2E mutant HFP (V2E-HFP) has...
Show moreHIV gp41 protein catalyzes fusion between viral and host cell membranes, and its apolar N-terminal region or "fusion peptide" binds to host cell membranes and plays a key role in viral and host cell membrane fusion. Gp41 fusion can be dominantly inhibited by dilute amounts of V2E mutant gp41, but a structural basis for this inhibition has not been demonstrated. "HFP" is a construct containing the fusion peptide sequence that induces membrane vesicle fusion, and V2E mutant HFP (V2E-HFP) has reduced membrane vesicle fusion rates. Earlier solid-state NMR (SSNMR) studies showed that when HFP or V2E-HFP are associated with membranes with ~30 mol% cholesterol (mHFP or mV2E-HFP), the apolar N-terminal regions of these constructs have predominant β strand secondary structure. In mHFP, a fraction of the strands form antiparallel β sheet structure with residue 161/116 or 171/117 registries of adjacent strands (i.e t = 16 and t = 17 registries). Other SSNMR and infrared studies have been interpreted to support a large fraction of approximately in-register parallel registry of adjacent strands. However, the samples had many isotopic labels and other structural models were also consistent with the data. The tertiary structure of mHFP was studied using SSNMR with the rotational-echo double resonance (REDOR) pulse sequence to measure a sample's average 13CO-15N dipolar couplings. Experimental data were collected for samples with sparser 13CO and 15N labeling and were compared to simulated NMR data. The in-register parallel β sheet fraction was ≤ 0.15, and a much greater fraction of antiparallel registries were identified. The accuracy of the quantitative measurements was enhanced by inclusion of "long range" natural abundance contributions in the data analysis, and the validity of this approach was supported by a negative control sample. Furthermore, mHFP samples were prepared with a single 13CO and a single 15N label for which the closest 13CO-15N interstrand proximity resulted from a distinct registry. These experimental data were compared to simulated data that incorporated fractional populations, ft, of 17 different registries. These ft, were globally fit using a χ2 metric which identified a broad distribution of antiparallel β sheet registries (11 < t < 21). Sequential hydrophobic residues in HFP result in intrastrand hydrophobic patches and interstrand overlap of these patches result in interstrand hydrophobic regions. These regions may insert into the vesicle membranes, and a hydrophobicity or insertion energy metric, , was developed to quantify each registry's insertion energy. In general, registries present in our NMR samples had a negative while registries that were not present generally had a positive . A similar set of experiments were run with mV2E-HFP, and mV2E-HFP had a narrower distribution of registries where the t = 20 registry was significantly more populated in mV2E-HFP than in mHFP. The hydrophobic residues of HFP are located within the first 12 N-terminal amino acids, and the t = 12 registry was more populated in mHFP than mV2E-HFP. The t = 12 registry localizes hydrophobic residues which may result in deeper membrane insertion and increased vesicle fusion rates compared to the t = 20 registry. The t = 20 registry delocalizes the interstrand proximity of N-terminal hydrophobic residues which may result in shallower membrane insertion and reduced membrane fusion rates. These results provide a new, experimentally-based structural model for transdominant inhibition where co-mixing of V2E mutant gp41 and wild type gp41 may energetically favor a non-native registry distribution shifted toward longer registries for the FP region of wild type gp41.
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Title: Synthetic modification of spin density in phenanthrenesemiquinones, Zn(II)-phenanthrenesemiquinone and Ni(Ii)-phenanthrenesemiquinone complexes
Creator: Morris, Larry D., III
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Contained within this body of work is a study that aims to quantify spin density delocalization in 3,6-R2-phenanthrenesemiquinone (3,6-R2-PSQ, where R = OMe and H) radicals and their metal complexes (where metals=ZnII, GaIII and NiII), of the form [M(tren)(PSQ)](BPh4)n, where M is a metal that is listed above, tren = tris(2-aminoethyl)amine, PSQ = phenanthrenesemiquinone and BPh4 = tetraphenylborate. Spin delocalization will be scrutinized by analyzing carbonyl carbon hyperfine coupling...
Show moreContained within this body of work is a study that aims to quantify spin density delocalization in 3,6-R2-phenanthrenesemiquinone (3,6-R2-PSQ, where R = OMe and H) radicals and their metal complexes (where metals=ZnII, GaIII and NiII), of the form [M(tren)(PSQ)](BPh4)n, where M is a metal that is listed above, tren = tris(2-aminoethyl)amine, PSQ = phenanthrenesemiquinone and BPh4 = tetraphenylborate. Spin delocalization will be scrutinized by analyzing carbonyl carbon hyperfine coupling constants by continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy, electron spin echo envelope modulation (ESEEM) spectroscopy, and hyperfine sublevel correlation (HYSCORE) spectroscopy. These examinations will reveal the influence molecular structure has on the hyperfine coupling at the carbonyl carbon nuclei. Spin delocalization has been quantified by isotopically labeling the carbonyl carbon nuclei with carbon-13 nuclei. 13C-labelled quinones were synthesized from a sequence of reactions, beginning with 13C-α-N,N’-dimethylformamide and 2,2’-dibromobiphenyl to form a diformyl-biphenyl. The carbonyl carbons were then coupled with hydrazine via a reductive cyclisation reaction, followed by oxidation with CrO3 to afford the quinone. CW-EPR of the 12C isotopologues revealed 1H fine structure in the organic radicals and the ZnII-PSQ complex. Inspection of the 13C-labeled compounds provided isotropic hyperfine coupling constants for all compounds studied except for NiII complexes; any fine structure from NiII CW-EPR spectra was unable to be resolved. The magnitude of all isotropic coupling constants fell within the range of 0 – 3 MHz. ESEEM and HYSCORE spectroscopies allowed for scrupulous investigation of the dipolar coupling. Carbonyl carbon dipolar couplings varied across the spectrum of compounds examined. This study shows promise for the use of ESEEM and especially HYSCORE spectroscopies for examining the spin properties of molecules in the field of molecular magnetic materials.
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Title: Protein and hydration shell dynamics of ZnII-substituted cytochrome c
Creator: Mueller, Jennifer Jo
Date: 2013
Collection: Electronic Theses & Dissertations
Description: This dissertation describes two studies that provide new information on the nature of the protein and solvent dynamics that is probed in ZnII-substituted cytochrome c (ZnCytc) by the intrinsic ZnII porphyrin, which serves as a probe of the surrounding protein and solvent. In the first study, the nature of the partially unfolded structures that are generated in ZnCytc upon optical excitation significantly above the vibronic origin of the Q (SShow moreThis dissertation describes two studies that provide new information on the nature of the protein and solvent dynamics that is probed in ZnII-substituted cytochrome c (ZnCytc) by the intrinsic ZnII porphyrin, which serves as a probe of the surrounding protein and solvent. In the first study, the nature of the partially unfolded structures that are generated in ZnCytc upon optical excitation significantly above the vibronic origin of the Q (S₀→S₁, &pi&rarr&pi* transition) band was investigated using continuous-wave fluorescence spectroscopy. The results show that step-like transitions of the fluorescence Stokes shift correspond to the activation threshold for changes in structure from the native state to a partially unfolded state associated with the &omega loop formed by residues 20-35, which is adjacent to the Cys14 and Cys17 thioether linkages from the porphyrin to the polypeptide backbone. The excitation energy for optical formation of the unfolded state is consistent with the previous determination by Englander and coworkers using hydrogen-exchange NMR spectroscopy in ferrocytochrome c in the presence of Gdm+.In the second study, the hydration shell of ZnCytc was probed using the indolecyanine dye Cy5 using femtosecond pump-continuum-probe spectroscopy. Cy5 was attached to a surface lysine residue by a flexible linker so that it senses the viscosity of the surrounding medium owing to its nonpolar solvation response. The main conclusion of this work is that the hydration shell is as much as 200 times as viscous as bulk water. A simple structural interpretation of this finding is that longer or more persistent chains of hydrogen-bonded water molecules are present than in the bulk.
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Title: Ultrafast spectroscopic studies of molecular interactions and vibrational energy relaxation dynamics in binary solvents and liposomes
Creator: Qiu, Chen (Of Michigan State University)
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Achieving a fundamental understanding of intermolecular interactions and energy transfer processes in fluid systems is key for studies on chemical reaction mechanisms, material properties and the biological dynamics. There is a well established body of work describing transient molecular organization and interactions between dissimilar molecules in neat liquids, the studies in multi-component liquid remain a challenge due to the transient and complex nature of solvent-solvent and solute...
Show moreAchieving a fundamental understanding of intermolecular interactions and energy transfer processes in fluid systems is key for studies on chemical reaction mechanisms, material properties and the biological dynamics. There is a well established body of work describing transient molecular organization and interactions between dissimilar molecules in neat liquids, the studies in multi-component liquid remain a challenge due to the transient and complex nature of solvent-solvent and solute-solvent interactions.To gain insight into the solution phase heterogeneity and molecular scale organization, picosecond laser technologies and time resolved spectroscopic approaches have been applied. We selected the ethanol/cyclohexane and n-butanol/cyclohexane binary solvent mixtures as model systems. The polycyclic aromatic hydrocarbon (PAH) perylene has been chosen as the probe molecule to examine local organization in binary solvent systems. The perylene ring breathing mode is nearly degenerate with the ethanol and n-butanol terminal methyl group rocking modes. Steady-state spectroscopic data show that there is a discontinuous dependence of the spectroscopic origin on the binary solvent systems examined. From both orientational and vibrational energy relaxation dynamics measurements of perylene as a function of solution composition, we observed molecular scale heterogeneity in both binary solvent systems. For the ethanol/cyclohexane system, both rotational diffusion and vibrational population relaxation time constants show a clear discontinuity between 5% and 7.5% (v/v) ethanol, suggesting a discontinuous change in the organization of the chromophore local environment. For n-butanol/cyclohexane system, the rotational diffusion results show that perylene reorients as an oblate rotor in neat n-butanol and cyclohexane, but as a prolate rotor in all binary mixtures, and the vibrational population relaxation data show that perylene experiences an n-butanol dominated environment when the n-butanol concentration is 5% (v/v) or above. Taken collectively, both studies demonstrate a non-uniform distribution of alcohol in the binary mixtures, providing experimental evidence on the existence of composition-dependent nano meter scale local organization in these systems.Another question we addressed in this dissertation using picosecond spectroscopy is the long-term stability of phospholipid vesicles in an aqueous environment, which is an important issue for studies that use phospholipid vesicles. Unilamellar vesicles containing 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), with and without cholesterol, formed by extrusion in aqueous buffer solution (pH 8) were shown to remain dimensionally stable for periods in excess of hundreds hours by dynamic light scattering (DLS) measurements. The rotational diffusion dynamics of perylene confined in the vesicle acyl chain region revealed structural evolution that was dependent on vesicle composition. We also found that the re-extrusion of the vesicles caused no change in the average diameter or size distribution, but did give rise to diminished organization in the lipid acyl chain region for these vesicles. These findings provide new insight from both macroscopic and microscopic perspectives on the structural stability of phospholipid vesicles.
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Title: Molecular dynamics in the hairpin ribozyme : calculational and experimental aspects
Creator: Ochieng, Patrick Omondi
Date: 2015
Collection: Electronic Theses & Dissertations
Description: ABSTRACTMOLECULAR DYNAMICS IN THE HAIRPIN RIBOZYME: CALCULATIONAL AND EXPERIMENTAL ASPECTSByPatrick Omondi OchiengThe increasing role of RNA therapy in targeting diseases has inspired several RNA studies and especially structural RNA. Of interest to many scientists is how such RNA can perform their work with limited functional groups available to RNA. The structural versatility of RNA seems to underscore the importance of dynamics in performing several functions. Ribozymes are a good example...
Show moreABSTRACTMOLECULAR DYNAMICS IN THE HAIRPIN RIBOZYME: CALCULATIONAL AND EXPERIMENTAL ASPECTSByPatrick Omondi OchiengThe increasing role of RNA therapy in targeting diseases has inspired several RNA studies and especially structural RNA. Of interest to many scientists is how such RNA can perform their work with limited functional groups available to RNA. The structural versatility of RNA seems to underscore the importance of dynamics in performing several functions. Ribozymes are a good example of structured RNA involved in RNA backbone cleavage with a range of strategies. Hairpin ribozyme invokes domain-domain docking to activate the cleavage process. The major loop rearrangements observed upon docking, as well as the kinetically unfavorable docking process, both argue for conformational selection by pre-organization of the catalytically-competent active site of the hairpin ribozyme. In this thesis, we sought to study the behavior of loop A in sampling the docked-like conformation as evidence for conformational selection. We addressed three major aims which involved (i) understanding the dynamics in loop A using molecular dynamics simulation as a tool for assessing conformational sampling (ii) determining the right loop A construct for NMR studies and resonance assignments for structure determination and (iii) elucidation of fast and slow dynamics in loop A using NMR relaxation techniques. In aim 1 (Chapter 2), molecular dynamics simulation was used to determine conformational heterogeneity in RNA based on alternate base-pair formation within a subset of residues in the loop region of domain A of the hairpin ribozyme. Three main conformers and several minor conformations were observed in our simulations as analyzed by the Markov State model analysis. RNA base residues and backbone dynamics played a major role in the conformational heterogeneity and ensemble in loop A. Of the conformations that were sampled, the most populated conformer, AA/CA, closely sampled conformational properties similar to the activated (docked) loop A conformation, suggesting the activating role induced by conformational heterogeneity. In aim 2 (Chapter 3), we determined the suitable loop A construct for NMR studies by NMR secondary structure analysis on various constructs of loop A. Using exchangeable and non-exchangeable NMR experiments we assigned certain specific proton, nitrogen and carbon resonances of loop A for structural determination and relaxation measurements.In aim 3 (Chapter 4), we assessed loop A dynamics using the 13C-NMR relaxation measurements. Fast internal motions in the order of ps - ns timescale were analyzed by Model-free approach using data from 13C R1, R1ρ, and heteronuclear NOE of loop A. Loop A was generally found to be a rigid molecule on this timescale with internal generalized order parameters, S2, of at least 0.9 in the helical regions. Several residues reported correlation times indicative of fast motions on the ps to ns timescale, while a few reported slow exchange in the μs-ms timescale.Our data underscores the importance of fast and slow dynamics in the formation of conformational states with structures similar to the activated form. These conformational variability and structural transitions seem to activate RNA thereby facilitating RNA-RNA interaction via the conformational selection mechanism.
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Title: Kinetic models for the prediction of weathering of complex mixtures on natural waters
Creator: McIlroy, John
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Models play a vital role in predicting environmental fates of pollutants, which is critical for effective remediation. However, many fate and transport models for complex mixtures, e.g. petroleum products, do not incorporate the individual compounds, which are responsible for toxicity and environmental persistence. In this research, a diesel/water microcosm mimicked an environmental fuel spill with simulated weathering by evaporation and irradiation. Temporal changes in composition were...
Show moreModels play a vital role in predicting environmental fates of pollutants, which is critical for effective remediation. However, many fate and transport models for complex mixtures, e.g. petroleum products, do not incorporate the individual compounds, which are responsible for toxicity and environmental persistence. In this research, a diesel/water microcosm mimicked an environmental fuel spill with simulated weathering by evaporation and irradiation. Temporal changes in composition were assessed by gas chromatography-mass spectrometry (GC-MS) and time of flight mass spectrometry (ToF-MS) with atmospheric pressure chemical ionization (APCI). During evaporation, first-order kinetic rate constants were calculated for selected compounds and employed to develop predictive models, based on GC retention indices. Models were initially developed for compounds from individual classes (normal alkane, branched alkane, alkyl benzene, and polycyclic hydrocarbon) and later expanded to include compounds from all classes (comprehensive model). Using the comprehensive model, the rate constants were predicted with an average error of 10%, whereas the class specific models resulted in less error (4 - 8%). A model was also developed that incorporated varying temperature (5 to 35 °C), allowing for the prediction of the rate constants over environmentally relevant temperatures (16 % error). Using the rate constant, the fraction remaining of individual compounds was determined. The fraction remaining of individual compounds was used to calculate the fraction remaining of the total fuel (± 6%), and was in good agreement with currently available evaporation models. The variable-temperature model successfully applied to predict the fraction remaining of other petroleum products, demonstrating applicability beyond diesel fuel. The variable-temperature model was also used to predict chromatographic profiles of a fuel after evaporation, estimated the length of time a fuel has been evaporated using the predicted chromatogram, and estimate the time to reach a specific percent evaporated for an individual compound or for the entire fuel. First-order kinetic rate constants were also determined for diesel fuel irradiated with simulated sunlight for 10 hours by GC-MS and APCI-ToF-MS. The decay of hydrocarbons and formation of oxygenated compounds began within the first hour of irradiation. Using GC-MS, a two-fold increase in the rate constant was observed during irradiation (0.004 - 1.211 h-1) than predicted from the variable-temperature evaporation model (0.000 - 0.379 h-1). Compounds unlikely to evaporate also decayed, indicating they were precursors to photooxidation. In the APCI-ToF-MS, rate constants were determined for decay of hydrocarbons (0.003 - 0.210 h-1) and formation of oxygenated compounds (0.002 - 1.173 h-1). The kinetic rate constants developed in this work provided valuable information about changes in individual compounds during the weathering of petroleum products. Predicting changes in individual compounds provides additional information not available in most current models impact assessment and guide remediation of petroleum releases.
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