Browsing by Subject "Spectroscopy"

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Developing Stimulated Raman Spectroscopic Techniques For Imaging Below the Optical Diffraction Limit
(2020-05) Graefe, Christian
Stimulated Raman spectroscopy (SRS) is a technique that amplifies the normally weak Raman scattering process using an additional laser beam, resulting in increased signal amplitudes. For this reason, it has been developed as a biological imaging platform with the potential to be used as an alternative to fluorescence microscopy due to its chemical specificity. This eliminates the need for fluorescent tags, which can photobleach or disrupt the structure or dynamics of the system of interest. However, due to the optical diffraction limit SRS cannot compete with the spatial resolution that super-resolution fluorescence techniques are capable of. An SRS-based technique capable of breaking the diffraction limit would therefore allow for nanoscale research to occur on systems for which super-resolution fluorescence is not an option. To that end, we developed a method to improve spatial resolution in SRS using a toroidal beam to deplete SRS signal. As a result, signal is only generated in a reduced area at center of the beam. Initial experiments demonstrated up to 97% depletion of the signal and explored the properties of the depletion process. Additionally, we improved spatial resolution by approximately a factor of two using the toroidal beam to deplete signal while scanning the laser beams over the edge of a diamond plate. While the proof-of-concept experiments were successful, they were performed with a laser with high peak power and a relatively low repetition rate of 1 kHz. These high powers were not compatible with soft matter samples, causing significant photodamage. We therefore adapted super-resolution SRS on laser with a 2.04 MHz repetition rate to average faster and increase the peak power flexibility. Experiments on the 2.04 MHz laser corroborated many proof-of-concept results, including resolution improvement by about a factor of two. However, depletion iv was not achieved with the same efficiency and further improvements in resolution were not forthcoming. This is likely due to the inconsistent phase of the laser’s fundamental pulse profile, highlighting the importance of consistent and reproducible pulses when driving sensitive nonlinear optical processes. Additionally, we demonstrate the use of a new Raman tag using carboranes. By scanning a thin film of a carborane-terminated poly(N-isopropylacrylamide) (pNIPAAm), we show that their high density of B-H bonds and their unique vibrational frequency in the cell silent region make carboranes useful Raman imaging tags that expand multiplexing options. Carboranes’ role as reversible addition-fragmentation chain transfer (RAFT) polymerization agents make them especially good endogenous probes for polymers produced in this manner. Finally, we discuss planned experiments to further improve signal-to-noise ratio (SNR) and explore the mechanism of signal depletion. We also discuss applications of Raman imaging in lipid dynamics, using both diffraction-limited and sub-diffraction techniques. We propose possible methods to compare results from Raman and fluorescence microscopy to determine the impact of fluorescent tags on dynamics. In the research described herein, we develop and explore new Raman imaging methods and highlight the potential power of super-resolution SRS as a versatile chemical imaging tool.
Driven by Light: An Ultrafast Look into the Bright Future of Photosensitizers
(2024-04) Schaffner, Jacob
This thesis investigates various strong light-absorbing molecules that have potential applications in furthering our progress into replacing fossil fuels with clean energy resources and remediating harmful chemicals in the environment. The research presented in this thesis employs a range of spectroscopic techniques, complemented with computational predictions, to characterize the light absorption and excited state dynamics of newly developed chromophores that have shown promise in these various applications. Chapter 3 investigates a BODIPY-fullerene dyad designed to be used in organic photovoltaics as a triplet sensitizer to form longer-lived excitons. This triplet sensitization occurs via a ping-pong energy transfer mechanism between the BODIPY and fullerene, resulting in a long-lived BODIPY triplet (>1 µs). Chapters 4 and 5 investigate the MB-DIPY chromophore that could potentially displace fullerene as a strong and more versatile electron acceptor in organic photovoltaics. In Chapter 4, the redox potentials and photophysics of four MB-DIPY analogs are explored. The MB-DIPYs had comparable reduction potentials to fullerene and demonstrated efficient intersystem crossing to form long-lived triplet states (>10 µs). In Chapter 5, the MB-DIPY is functionalized with ferrocene, a strong electron donor, and demonstrated sub-ps charge-transfer from the ferrocene to the MB-DIPY followedby charge recombination in 12 ps. Chapters 6 and 7 investigate the Rh-Ga and Co-Ga heterobimetallic photocatalysts that can access challenging bonds via a photoredox mechanism. The excited state nature of these photocatalysts is first explored in Chapter 6. The results were consistent with the naked anionic catalyst being the active participant in the photocatalytic cycle. Chapter 7 investigates the reactivity of the photocatalysts with a chloroadamantane substrate. The results suggested that the substrate binds to the anionic rhodium photocatalyst and that the photocatalytic reactivity is not diffusion-limited. In contrast, the anionic cobalt catalyst was converted into the chlorinated precatalyst upon the addition of the substrate, demonstrating that the chemical reactivity of the rhodium and cobalt photocatalysts differ with this substrate.
Elucidating the Structural Dynamics of Muscle Myosin Using Novel Methods in Electron Paramagnetic Resonance
(2016-11) Binder, Benjamin
Muscle contraction is fundamentally driven by an interaction between two proteins: actin and myosin. Myosin is a molecular motor, and assumes the active role in this relationship, coupling energy from hydrolysis of ATP with conformational changes to generate force on actin. In the context of a muscle fiber, this force causes filaments of myosin and actin to slide past one another in an ordered lattice, drawing the ends of individual contractile units (called sarcomeres) together. Concerted shortening of sarcomeres along the length of a fiber results in large-scale shortening of the entire fiber. Although muscle myosin has been the focus of intense study for many years, crucial details regarding its mechanism remain unknown. In particular, few structures of actin and myosin together have been reported—this is largely due to the inherent difficulties of handling large, filamentous protein complexes in traditional methods for structure determination. Myosin's interactions with actin are absolutely essential for macroscopic function, and this lack of structural information has created a knowledge gap: there is an abundance of functional and kinetic data for myosin in both normal and pathological states, but often no direct insight into the underlying structural causes for the observed behavior. In the present work, I seek to address this knowledge gap by providing high-resolution insight into the structural states of actin-bound myosin. My work is based on the hypothesis that allosteric coupling in myosin's catalytic domain (the domain responsible for actin binding, ATP hydrolysis, and initiation of force-generating conformational change) is accomplished via subtle internal rearrangements of individual structural elements. Furthermore, I hypothesize that these changes can be detected and quantified by innovative applications of site-directed spectroscopy. In Chapter 4, I establish a method using electron paramagnetic resonance (EPR) of a bifunctional spin label to probe nucleotide-dependent changes in the actomyosin complex. In Chapter 5, this method is expanded to include two complementary EPR techniques, ultimately providing sufficient constraints for direct modeling of nucleotide-dependent changes. Following these results, Chapter 6 addresses the ongoing development and further application of these methods within myosin and other protein systems.
Excited State Dynamics of Model Photovoltaic Materials
(2021-01) Swedin, Rachel
Investigation of new materials for potential use in organic photovoltaics and dye-sensitized solar cells found unique systems that maintained a relatively long-lived (ns and longer) charge separated state or energy transferred state. The molecules studied in this thesis show promise for use in organic photovoltaics or dye-sensitized solar cells. Further studies in the solid state of these molecules are required to determine their efficiency andtheir ability to function in a photovoltaic module. Chapter 1 gives an overview of the status of energy usage in the world and how photovoltaics fit into it. This chapter also explains the key scientific concepts used for interpretation of experiments. Chapter 2 goes through an in-depth description of the experimental techniques and processes used in this thesis. Chapter 3 examines a thiophene- and furan-based dye when in an equimolar mixture with varying sizes of ZnO nanoparticles. A long-lived charge-separated state is found when both dyes are coordinated to the ZnO nanoparticles, showing a spectral signature of electron transfer from the thiophene and furan-based dyes to the ZnO. The charge-separated state exists beyond the time delay for the experiment (3.5 ns), indicating promise for a dye-sensitized solar cell containing these molecules. Investigation into BODIPY molecules for use as an absorber in organic photovoltaics begins with Chapter 4. In Chapter 4, the electron transfer properties from the catechol group to multiple BODIPY derivatives are identified. It is found that rapid electron transfer from the catechol, linked at the boron hub of the BODIPY, to the BODIPY deactivated the excited state from further interaction with surrounding systems, a detail missed in other publications with the same catechol attached to the boron-hub of BODIPY. This conclusion was carried into Chapter 5 where use of the catechol to bridge the fullerene to the BODIPY leads to no interaction with fullerene. This is because the catechol rapidly transferred an electron to the BODIPY and deactivated further electron transfer. Ferrocene added to the BODIPY-fullerene molecule out-competed the catechol for electron transfer to the BODIPY derivative, making a ~200 ps lived electron transfer state. The catechol linker is not the only bridge studied between a BODIPY derivative and fullerene. A pyridone ring connected at the alpha and position of the BODIPY is also used to bridge to fullerene. In this study the fullerene functioned as a triplet sensitizer for the BODIPY, leading to a microsecond lived BODIPY triplet. Lastly, a zinc phthalocyanine is studied when coordinated to a BODIPY derivative and fullerene through a pyridine ring. Spectral and redox evidence shows electron transfer from the phthalocyanine occurred, soon followed by recombination. Energy transfer from the BODIPY to the phthalocyanine was also present, followed by electron transfer back to the BODIPY before decaying to the ground state.
Exciton Dynamics in Alternative Solar Cell Materials: Polymers, Nanocrystals, and Small Molecules
(2014-07) Pundsack, Thomas
To keep fossil fuel usage in 2040 even with 2010 usage, 50% of global energy will need to come from alternative sources such as solar cells. While the photovoltaic market is currently dominated by crystalline silicon, there are many low-cost solar cell materials such as conjugated polymers, semiconductor nanocrystals, and organic small molecules which could compete with fossil fuels. To create cost-competitive devices, understanding the excited state dynamics of these materials is necessary.The first section of this thesis looks at aggregation in poly(3-hexylthiophene) (P3HT) which is commonly used in organic photovoltaics. The amount of aggregation in P3HT thin films was controlled by using a mixture of regioregular and regiorandom P3HT. Even with few aggregates present, excited states were found to transfer from amorphous to aggregate domains in <50 fs which could indicate efficient long-range energy transfer.To further study P3HT aggregation, a triblock consisting of two P3HT chains with a coil polymer between them was investigated. By changing solvents, aggregation was induced in a stable and reversible manner allowing for spectroscopic studies of P3HT aggregates in solution. The polarity of the solvent was adjusted, and no change in excited state dynamics was observed implying the excited state has little charge-transfer character.Next, the conduction band density of states for copper zinc tin sulfide nanocrystals (CZTS NCs) was measured using pump-probe spectroscopy and found to be in agreement with theoretical results. The density of states shifted and dilated for smaller NCs indicative of quantum confinement. The excited state lifetime was found to be short (<20 ps) and independent of NC size which could limit the efficiency of CZTS photovoltaic devices.Finally, triplet-triplet annihilation (TTA) was studied in platinum octaethylporphyrin (PtOEP) thin films. By analyzing pump-probe spectra, the product of TTA in PtOEP thin films was assigned to a long-lived metal-centered state. To elucidate the mechanism of TTA, the annihilation dynamics were modeled using second order kinetics as well as Förster and Dexter energy transfer. Dexter energy transfer provided the best fits and the most reasonable fitting parameters.
Femtosecond stimulated Raman spectroscopy – guided library mining leads to efficient singlet fission in rubrene derivatives
(2021-09-28) Bera, Kajari; Douglas, Christopher J; Frontiera, Renee R; rrf@umn.edu; Frontiera, Renee R; Frontiera lab
From structure and dynamics to novel therapeutic development for muscular dystrophy.
(2012-07) Lin, Ava Yun
Dystrophin is defective in Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are debilitating X-linked diseases that currently have no cure. Dystrophin links the actin cytoskeleton at its N-terminus and a glycoprotein complex (DGC) embedded in the sarcolemma at its C-terminus, apparently providing mechanical stability to the muscle during contraction. Due to the large size (427 kD) and filamentous nature of dystrophin, studies of its function and attempts to develop effective therapeutics have developed slowly, despite intensive efforts. Utrophin (395 kD) is a homolog of dystrophin that has shown therapeutic promise in mdx mice, which lack dystrophin. Utrophin is endogenously expressed in the cytoskeleton of fetal and developing muscle but is replaced by dystrophin as the muscle matures (8-10). Both dystrophin and utrophin belong to the spectrin superfamily of actin-binding proteins, which carry out diverse functions in the cytoskeleton of most cells. Of the many proteins included in this superfamily, dystrophin and utrophin are among the least studied in terms of structural dynamics, limiting the understanding of their function at the sarcolemma. In order to target the root of dystrophin malfunction in muscular dystrophy, we need to better understand the native functions of dystrophin and utrophin. Lack of structural information about dystrophin and its interactions adds to the complexity of tying clinical presentations to the diverse disease-causing mutations, and hinders therapeutic advancement in gene or drug therapy. There are numerous mouse-model studies, but there are varied results across several parameters tested, and no construct or drug has been found that restores normal muscle force in the mdx mouse. Exon-skipping morpholinos are expensive to produce with variable delivery and efficacy to muscle groups and require a customized oligo design for each mutation, making it difficult to test them individually in mouse models. In order to (a) understand disease mechanisms and (b) design better therapies rationally, we need more fundamental information about the structures and interactions of specific regions of dystrophin and utrophin. That is the goal of this project.
Heterogeneous protein distribution during rapid and equilibrium freezing
(2013-04) Twomey, Alan Michael
Interactions between proteins and ice were studied in situ using FTIR and confocal Raman microspectroscopy under equilibrium and non-equilibrium conditions over a range of temperatures. During quasi-equilibrium freezing of aqueous solutions of dimethyl sulfoxide (DMSO) and bovine serum albumin, preferential exclusion of albumin and/or DMSO was observed. It was hypothesized that the albumin may be adsorbed onto the ice interface or entrapped in the ice phase. To investigate protein-ice interactions during freezing under non-equilibrium conditions, confocal Raman microspectroscopy was used to map the distribution of albumin and the cryoprotective agent trehalose. Microheterogeneity was found in the composition of the freeze-concentrated liquid phase that indicated that albumin was preferentially distributed near or at the boundary of the ice phase. The observed microheterogeneity did not occur under all freezing protocols, which suggests that the technique developed here could be used to develop freezing protocols that would reduce harmful protein-ice interactions.
Integrated Fluorescence Spectroscopy for FRET Analysis of Novel Ionic Strength Sensors in the Presence of a Hofmeister Series of Salts
(2019-07) Miller, Robert
Living eukaryotic cells are complex, crowded, and dynamic organisms that continually respond to environmental and intracellular stimuli. In addition, these cells have heterogeneous ionic strength with compartmentalized variation of both intracellular concentrations and types of ions. The underlying mechanisms associated with ionic strength variations that trigger different biological functions and response to environmental cues remain largely unknown. Therefore, there is a need to develop a quantitative method for mapping the compartmentalized ionic strength and their temporal fluctuations within living cells. In this work, we investigate a class of novel ionic- strength sensors that consists of tethered mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) via a linker of varied amino acids. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing fluorescence resonance energy transfer (FRET) based on both the linker amino acids and the environmental ionic strength. The energy transfer efficiency and the donor-acceptor distance of these sensors can be quantified noninvasively using integrated fluorescence methods in response to intracellular ionic strength in living eukaryotic cells. We employed time-resolved fluorescence methods to monitor the excited-state dynamics of the donor in the presence and absence of the acceptor as a function of the environmental ionic strength using potassium chloride (KCl, 0–500 mM). Towards mapping out the response to of these sensors towards biologically relevant salts, we carried out time- resolved fluorescence for FRET analysis of these sensors as a function of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). We also used these results towards technique development for FRET analysis based on time-resolved fluorescence polarization anisotropy. Our results show that the energy transfer efficiency of these sensors is sensitive to both the linker amino acid sequence and the environmental ionic strength. These studies in a controlled environment complement previous steady-state spectroscopy analysis of these sensors in a cuvette with the advantage of the compatibility of our approach with fluorescence lifetime imaging microscopy on living cells.
Iron and Carbon Speciation in Non-Buoyant Hydrothermal Plumes along the East Pacific Rise: A Chemistry Love Story
(2018-09) Hoffman, Colleen
In the ocean, iron (Fe) is an important micronutrient for phytoplankton growth. Phytoplankton play a vital role in the global carbon (C) cycle, accounting for 50% of the total photosynthesis on the planet (Field et al. 1998; Moore et al. 2013; Fitzsimmons et al. 2014). When they die, phytoplankton sink and can become buried in the sediments of the deep ocean, removing C from the atmosphere and surface water. While Fe is an abundant element overall in the Earth’s crust (Edwards et al. 2004), it is extremely diluted in the surface ocean. Iron-poor surface waters limit phytoplankton growth (Vraspir and Butler 2009) and their ability to remove C from the atmosphere and surface ocean. Over the past few decades, research has focused on constraining the global Fe cycle and its impacts on the global C cycle (Tagliabue et al. 2010). Hydrothermal vents have become a highly debated potential source of Fe to the surface ocean. Initially, the hypothesis stated that hydrothermal Fe would all be oxidized and deposited locally upon being expelled from the vent. Therefore, hydrothermal vent would have a negligible effect on global biogeochemical cycles (Elderfield and Schultz 1996). However, as a variety of new sampling techniques were developed to preserve reduction-oxidation (redox) states and increase the ability to collect trace-metal clean samples (Johnson et al. 1997; Johnson et al. 2007; Breier et al. 2009), it was discovered that hydrothermal Fe could be protected from oxidation and removal to the sediments and be a potential source of Fe to the deep ocean and surface waters in some locations (Toner et al. 2009a; Toner et al. 2012a). With the amount of Fe released through hydrothermal venting to the ocean per year being similar in magnitude to that delivered by global riverine run-off (Elderfield and Schultz 1996), hydrothermal vents could be an unrecognized nutrient source to the surface ocean and play a role in global C cycling. Two main mechanisms, nanoparticles with slow settling rates, and complexation reactions with organic functional moieties, have been hypothesized to transport solid and aqueous phase Fe over long distances (Bennett et al. 2008; Toner et al. 2009a; Yücel et al. 2011). During the time interval of this dissertation, studies have investigated a large hydrothermal Fe source emanating from the East Pacific Rise (EPR; Resing et al. 2015; Fitzsimmons et al. 2017; Lee et al. 2018). This has informed current working geochemical models about the complexity of reaction pathways and transport mechanisms active in hydrothermal plumes with implications for basin-scale transport and bioavailability of hydrothermal Fe (Tagliabue and Resing 2016; Tagliabue et al. 2017). This dissertation will focus on investigating the chemical speciation and transport mechanisms of Fe in non-buoyant plume particles along the East Pacific Rise. Therefore, further informing the global impact of hydrothermal venting within ocean basins.
On The Allosteric Mechanisms Of Paradoxical Activation By Raf Inhibitors
(2024-04) Rasmussen, Damien
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
Radiative characterization of heterogeneous media for solar thermochemical applications
(2013-02) Ganesan, Krithiga
Transport radiative properties of ceria ceramics as a function of wavelength and morphology are presented. Experimental facilities for measuring transmitted and re- flected radiant energy were developed. A Monte Carlo ray-tracing technique was devel- oped to infer transport scattering coefficient and absorption coefficient from measured transmittance and reflectance. An experimental setup to measure normal-emittance of materials employed in solar thermochemical applications is also designed. This experi- mental setup can achieve temperatures on the order of 1500 K and facilitate maintaining an inert, oxidizing or reducing environment to control composition of materials. Transmittance and reflectance of two types of morphologies were examined. Sin- tered ceria discs with pores and grains of size a few microns and a random grain and pore structure, and a three-dimensionally ordered macro-porous (3DOM) ceria with nano-structured features were considered. The 3DOM samples were also ther- mochemically cycled for 60 cycles consisting of high-temperature reduction and low- temperature oxidation. Two porosities were considered for the sintered ceria discs with porosity, p = 0.08 (“dense” samples), and p = 0.72 (“porous” samples) and porosi- ties of the packed bed before and after themochemical cycling were 0.9 and 0.83 re-spectively. Morphological characterization was performed by Scanning Electron Mi- croscopy (SEM), and porosity was verified by mass and volume measurements of the samples. Sintered ceria discs had extremely high optical thickness, with low and uncertain values of directional-hemispherical transmittance, and bi-normal transmittance on the order of 0.001 %. The transport radiative properties did not show a strong wavelength dependence after a wavelength of 0.5 μm. The transport scattering coefficient was on the order of 30mm−1 for the porous ceramics and 15mm−1 for the dense ceramics after a wavelength of 0.5 μm. The absorption coefficient was on the order of 0.005 mm−1 for the dense ceramics and 0.03 mm−1 for the porous ceramics in the spectral range of weak absorption. Experimental uncertainty resulted in only approximate determination of transport radiative properties, particularly for the dense ceramics. For the 3DOM packed bed, measured transmittance was of higher accuracy lead- ing to lower uncertainty in measurements and identification procedure. The transport scattering coefficient showed a stronger spectral dependence than that of the sintered ceramics especially after a wavelength of 0.5 μm. Thermochemical cycling resulted in changes in the 3DOM morphology due to sintering of walls of the 3DOM struc- ture. The morphology of 3DOM ceria after thermochemical cycling resembled sintered structures and this change also impacted the predicted radiative properties. The trans- port scattering coefficient showed strong dependence on morphology, with a drastic increase by a factor of four for 3DOM packed bed after thermochemical cycling. The absorption coefficient was only dependent on porosity, and did not show any change after thermochemical cycling, because the porosity only decreased weakly as a result of thermochemical cycling. Predicted radiative properties indicate that an approximation applicable in the lim- its of large optical thickness, such as the Rosseland approximation can be employed to model heat and mass transfer rates in the ceria-based redox thermochemical cycles. Approximate models for optically thick media can greatly reduce computational cost of determination of radiative heat flux in such combined heat transfer problems. Mor- phological features that promote effective absorption of solar radiation in the visible spectrum and confinement of infrared radiation in the reactor are suggested.
Raman spectroscopy data and phonon calculations for ScV6Sn6 in P6/mmm and R-3m structures, 2023
(2023-06-27) Ritz, Ethan T; Birol, Turan; Gu, Yanhong; Musfeldt, Janice L; eritz@umn.edu; Ritz, Ethan T; Birol Research Group, University of Minnesota; Musfeldt Group, University of Tennessee Knoxville
We use density functional theory (DFT) to calculate the phonon frequencies and the distortions associated with them in the compound ScV6Sn6 in the P6/mmm and R-3m space groups, then compute the overlap between the Raman-active phonons in each structure. This data includes scripts to generate the DFT submission files, the results of those simulations, as well as MATLAB scripts to plot the results. We also include experimental Raman spectroscopy data at temperatures from 5.5 K to 300 K.
Rational design of loss-of-function phospholamban mutants to tune SERCA function.
(2012-04) Ha, Kim N.
Unphosphorylated phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), the enzyme that regulates cardiac muscle relaxation in humans. In its phosphorylated state, PLN (pS16-PLN, pT17-PLN, and pS16pT17-PLN) does not inhibit SERCA. Dysfunctions in SERCA:PLN interactions and in the PLN phosphorylation mechanism have been implicated in cardiac disease and targeting PLN is becoming a viable avenue for treating heart disease. Specifically, innovative genetic treatments using recombinant adeno-associated virus (rAAV) with S16E-PLN, a pseudo-phosphorylated form of PLN, have shown a remarkable efficacy in reducing the progression of cardiac failure in both small and large animals. The following thesis summarizes efforts to rationally design PLN mutants to tune SERCA function. Using a combination of NMR spectroscopy and biochemical assays, we have built a structure-dynamics-function correlation that shows PLN can be tuned to augment SERCA function by acting on the conformational coupling between the cytoplasmic and transmembrane domain and by pseudo-phosphorylation. Additionally, to better understand the role of mutation in PLN:SERCA interactions, we also investigated a mutant of PLN (R9C) known to be linked to hereditary dilated cardiomyopathy, showing that the mutation disrupts the pentamer-monomer equilibrium, and that these effects are exacerbated under oxidizing conditions. Insights to these issues will provide better paradigms with which to design therapeutic mutants of PLN for treatment of heart failure.
A Self-Consistent Theoretical Framework for Estimating Outflow Rates, Lyman Alpha Escape, and Lyman Continuum Escape
(2023-04) Carr, Cody
Over the past two decades, both theory and observations have made tremendous progress revealing the inner workings of galaxy formation. In the current paradigm, theory predicts pristine inflows of gas, left over from the Big Bang and captured in the gravitational potential wells of dark matter halos, to be constantly feeding star formation and super massive black holes. This in turn leads to various forms of feedback (e.g., supernovae, stellar winds, radiation pressure, relativistic jets, cosmic rays, etc.) which then drive massive outflows of processed gas back out of the galaxy. In this way, feedback acts to regulate star formation. While we have drawn back the curtain to reveal the big picture behind galaxy formation, many open questions remain. We don’t yet know which sources of feedback are the primary drivers of outflows, the efficiency at which they operate, or how they influence their surroundings. Making precise measurements of the properties of flows will be essential to answering these questions. Traditionally, the properties of flows are measured from absorption and emission lines imprinted on the spectra of background sources, which encodes information about the density and velocity of the intervening gas. Extracting flow properties from absorption lines is easier said than done, however. The difficulty reflects the complex physics governing the radiation transfer underlying the lines and early attempts at modeling the lines have been limited. In this thesis, we present novel semi-analytical line transfer (SALT) models designed to predict the spectra of galactic flows to reveal their properties. The models are based on the transport of radiation through an extended moving medium and represent a major improvement to prior models. We demonstrate the model’s effectiveness by showcasing various comparison tests between SALT predictions and those of idealized numerical radiation transfer codes as well as numerical simulations of galaxy formation. In doing so, we develop a self-consistent theoretical framework linking simulations to observations. After demonstrating the effectiveness of the model, we show results from various applications including constraints on outflow rates and predictions of the ionizing escape fraction from star forming galaxies.
Structural dynamics of the myosin force-generating region.
(2009-12) Agafonov, Roman
Myosin is a molecular motor that generates force on actin using energy from ATP hydrolysis. Myosin plays a key role in muscle contraction and is responsible for a variety of motility processes at the cellular level. It works cyclically, changing its conformation during the power stroke and the recovery stroke. X-ray crystallography has provided information about the structural organization of myosin in different biochemical states (as defined by bound nucleotide), inspiring several structural models that could explain the molecular mechanism of myosin's function. Spectroscopy, in combination with site-directed labeling and transient experiments, can test and refine these models and provide information about myosin's dynamic properties. The goal of this project was to determine the structural dynamics of the myosin force-generating domain and study coupling mechanisms between this domain and the myosin active site. We have chosen Dictyostelium discoideum (Dicty) as our experimental system since it provides multiple advantages in comparison with the muscle myosin. In particular, it is possible to manipulate the Dicty DNA sequence, engineering labeling sites at desired locations and introducing functional mutations at the points of interest. As a first part of the project, we have tested Dicty myosin in comparison with myosin purified from rabbit skeletal muscle, and have shown that structural changes in the force-generating domain of Dicty and rabbit myosin are identical. We then focused on specific elements within the force-generating domain, relay helix and relay loop, as these elements appeared to be crucial for interdomain coupling and force generation. Using time-resolved EPR and FRET, we have developed a spectroscopic approach to determine the conformation of the relay helix. We have also developed a novel technique that we called transient time-resolved FRET [(TR)2FRET], which allowed us to monitor structural changes within the relay helix in real time. We then studied the relationship between the state of the myosin active site (which is determined by the bound ligand) and the structure of the relay helix. To obtain insights about regulatory mechanisms, we have investigated the effect of a mutation that is known to abolish myosin motor function, despite leaving enzymatic activity intact. These experiments revealed important coupling mechanisms between the relay loop and relay helix, providing a structural explanation for the previously observed functional effects and a model for power stroke activation in myosin.
Studies toward the total synthesis of rac-Leuconolam, modified Julia Olefination approach to access functionalized steroidal side chains, proton-NMR Studies of Mosher-like esters, and exploring a non-enzymatic Diels Alder reaction to account for the methyl sarcophytoate core
(2012-09) Izgu, Enver Cagri
This Ph.D. thesis is composed of five chapters, two of which are closely related and are presented at the begining. In Chapter-I, an extensive study toward the total synthesis of a plant natural product, namely leuconolam, will be discussed. In the course of this project, two different routes have been explored, where novel synthetic methods have been developed. In particular, some of the key bond-forming events such as Ireland-Claisen rearrangement, arene-alkene coupling (via either Stille reaction or C-H bond functionlization) and allylative ring closure are highlighted.A side project that has emerged during my investigations in Chapter-I will be covered in Chapter-II. This work focuses on the synthesis of two new organometallic reagents and their utility in organopalladium mediated cross-couling reactions with various alkenyl and aryl halides. Chapter-III encompasses the studies in the area of steroid chemistry, more specifically, in chemical construction of important steroid side chains. In order for a convergent strategy, a modified Julia olefination method has been performed on a common sulfone donor with a series of uselful aldehyde acceptors. Biologically relevant derivatives of alkyl and alkoxy branched side chains have been successfully synthesized. In Chapter-IV, synthetic and spectroscopic studies in Mosher ester analysis technique will be discussed. This NMR based tool is critical in determining the absolute configuration of a stereogenic carbon center and is commonly used by organic chemists. Finally, in Chapter-V, our efforts in generating a reactive pyrylium dienophile to facilitate a Diels-Alder reaction will be outlined.
Surface-Enhanced Raman Spectroscopy as a Probe to Understand Plasmon-Mediated Photochemistry
(2019-09) Brooks, James
The development of plasmonic nanostructures as light-activated photocatalysts has proven to be a promising research avenue due to their ability to access and drive unfavorable chemical reactions. Theses chemical reactions are fueled by the presence of surface plasmons, which are the collective oscillation of the free electron density on the material’s surface. Once a surface plasmon is photoexcited, their initial energy rapidly decays into multiple different pathways, such as enhanced electromagnetic fields, an abundance of hot carriers, and dramatically elevated local thermal environments. To better understand the various chemistries that are enabled by plasmonic materials and the associated mechanisms driving these processes, we have employed surface-enhanced Raman spectroscopy to interrogate a plethora of plasmon-molecule coupled systems. Our initial studies investigated the relationship between the plasmonic local fields and a well-established plasmon-driven photochemical reaction. We found that there were no observable correlations between the two in our studies and identified a competing degradation pathway for the studied analytes. In addition to exploring well-studied plasmon-induced chemical photoreactions, we have highlighted two new reactions that were accessed on the gold film-over-nanosphere substrates. First, we were able to induce and subsequently monitor a selective intramolecular methyl migration on N-methylpyridinium using surface-enhanced Raman spectroscopy. This work emphasizes the growing potential of initiating highly-selective chemistries with plasmonic materials for synthetic or redox purposes. The second previously unreported plasmon-driven reaction involves the double cleavage of the C-N bond on a pair of viologen derivatives. While these viologens have traditionally been employed as robust redox species, the unique and highly-powerful plasmonic local fields allowed the viologens to access an entirely new reaction pathway to transform into 4,4’-bipyridine. Lastly, we discuss our experimental approaches towards transiently studying the mechanism behind plasmon-mediated hot electron transfer. Using ultrafast surface-enhanced Raman spectroscopy, we interrogated the transient dynamics that occurred between surface plasmons and a bevy of electron accepting chemical adsorbates. Ultimately, the primary goal of this work is to provide a quantitative description of the transient interactions, which will assist in increasing the reported efficiencies and yields of plasmon-mediated chemical reaction and inspire the rational design of plasmonically-powered devices.
Synthesis and Characterization of a Dicobalt Catalyst for the Silylation of Dinitrogen
(2016-06) Siedschlag, Randall
Several dicobalt compounds were synthesized and characterized. Through these studies, dinitrogen binding was found at to happen in three oxidation states of the dicobalt core. This finding lead to the exploration of dinitrogen fixation, specifically the reduction of dinitrogen to tris(trimethylsiyl)amine. The complex was found to generate a turnover number (TON) of 195 for the catalytic silylation of dinitrogen, putting as the top performing catalyst for this process. Mechanistic insight was gained through computational modeling. The modeling studies lead to a road map for the isolation of potential intermediates along the catalytic pathway. All of these studies will be discussed within.
Time Resolved Vibrational Spectroscopies as a Tool for Exploring Dynamics of Confined Systems
(2022-01) Pyles, Cynthia
This thesis examines a variety of vibrational probe-containing molecules such as triphenyl hydrides, CO2, and metal carbonyls with the goal of better understanding the dynamics for each system. Particular emphasis is placed on understanding how the behavior of a restricted probe, such as one dissolved in a rigid polymer or confined to a nanopore, may differ from the same probe placed in bulk solvent or a more rubbery polymer. The first study described herein scrutinized the vibrational heavy atom effect and its impact on ultrafast vibrational dynamics. A series of three triphenyl hydride compounds was investigated in a range of solvents by Fourier transform infrared (FTIR), infrared (IR) pump-probe, and two-dimensional infrared (2D-IR) spectroscopies. The mass of the central atom in the three compounds was varied systematically down the group 14 elements of silicon, germanium, and tin while keeping the rest of the molecule unaltered. Interestingly, frequency-frequency correlation functions obtained from 2D-IR spectra indicated that an increasingly large central atom produces small, but measurable changes in the dynamics of the solvation shell surrounding each compound. Next, CO2 (g) was examined via 2D-IR spectroscopy as a precursory study to understanding its behavior inside polymers. Processes which lead to dephasing of the vibrational echo such as collisions were largely circumvented by using CO2 diluted in N2 under ambient pressure and temperature. Off diagonal features in the 2D-IR spectra were observed which correspond to population and coherence exchange between rovibrational transitions. Then, CO2 (g) was dissolved inside polymers such as poly(methyl methacrylate), poly (methyl acrylate), and poly(dimethylsiloxane). These polymers with differing properties were chosen to study the impact of the glass transition on the dynamics of the dissolved CO2 probe. Interactions between the polymeric backbone and probe also impacted the dynamics. The parameters obtained from 2D-IR studies directly correlated with the diffusivity of CO2 through the polymer matrices. Next, I inspected CO2 (g) adsorbed to microporous systems such as MIL-53(Al) and ZIF-8. Preliminary FTIR studies suggest that these samples could possess a wealth of dynamic information despite narrow FTIR peaks, much like CO2 dissolved in polymers. Experimental limitations regarding these novel systems are briefly discussed. Lastly, I compared the dynamics of three ruthenium-bound carbonyl complexes: Ru3CO12 in bulk THF, [HRu3(CO)11]- entrapped in an aluminum sol-gel, and [NEt4][HRu3(CO)11] in bulk THF. Ru3CO12 is catalytically inactive but becomes active upon incorporation into an alumina sol-gel matrix. Pump probe and 2D-IR studies indicated that the changed dynamics are primarily due to an altered solvent shell which most likely exhibits long-range ordering. Though it is uncertain whether the increased catalytic activity of [HRu3(CO)11]- is due to the presence of the hydride or this newly ordered solvent shell, the results nonetheless showcase 2D-IR’s efficacy in sensing dynamics of confined environments.

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