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Browsing by Author "Crans, Debbie C., committee member"

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    A study of magnetostructural parameters related to spin crossover and single molecule magnetism
    (Colorado State University. Libraries, 2013) Fiedler, Stephanie R., author; Shores, Matthew P., advisor; Kennan, Alan J., committee member; Anderson, Oren P., committee member; Crans, Debbie C., committee member; Patton, Carl E., committee member
    Herein are described several methods to probe transition metal complexes that were designed by systematic structural modifications to allow for comparison of the resultant magnetic properties. In Chapter 1, a brief introduction is presented to introduce the broader goal of our research: controlling spin on the synthetic level. The introduction provides background regarding spin crossover and single molecule magnetism as well as some previous research to put our projects in context relative to endeavors by other researchers. In Chapter 2, heteroleptic complexes of the form [Fe(H2bip)2(pizR)]Br2 and [Fe(H2bip)2(pizR)](BPh4)2 are described, which have the opportunity to chelate an anion via hydrogen bonding to the H2bip ligand. The third ligand, pizR, is varied between two ligands that we predict will have similar ligand field strengths: pizH and pizMe. Because pizH has an additional hydrogen-bonding site, while pizMe does not, we selected these ligands in order to understand the effect of hydrogen bonding on the anion-binding/spin-state switching event independent from ligand field strength. From these studies, the pizH anion hydrogen bond is observed in crystallographic studies, but does not affect the anion-binding or spin-state switching properties in solution. In Chapter 3, we further investigate the geometry of the pizR ligand in Fe(II) complexes. What began as attempts to study hydrogen bonding in solution revealed unexpected structural distortions of the ligand that are correlated to the spin state of the complexes. The R-substituted nitrogen atom on the imidazoline moiety of the pizR ligand switches between a planar geometry, which is observed for high-spin species, and a pyramidalized geometry, which is observed for low-spin species. We reason that this occurs as a result of the weak-field, non-pizR ligands that influence the ligand field in the high-spin species. Chapters 4 and 5 delve deeper into understanding the relationship between structural parameters and magnetic properties in complexes with non-covalent interactions. In Chapter 4, a series of complexes with metallophilic Pt-Pt interactions show antiferromagnetic magnetic coupling of non-bonded transition metals through a Pt-Pt bond. By comparing complexes with Pt-Pt interactions to those without Pt-Pt interactions, we are able to determine that the Pt-Pt bond is a unique superexchange pathway for the transition metal coupling. Off-set complexes, exhibiting two Pt S interactions instead of one Pt-Pt interaction, do not show evidence of magnetic coupling between transition metals. Furthermore, by comparing magnetic properties of complexes where the apical ligand varies, we determine that the presence or absence of intermolecular interactions is largely independent from the strength of coupling through the Pt-Pt bond. In Chapter 5, an asymmetric trinuclear manganese complex with unique magnetic exchange properties and two high-spin square planar complexes of iron and cobalt, are investigated. The trinuclear manganese complex consists of a central octahedral Mn(II) ion that is coupled antiferromagnetically to another octahedral Mn(II) ion and ferromagnetically to a terminal tetrahedral Mn(II) ion. The different coupling is rationalized as a result of the change in geometry, which affects the orbital overlap that is predicted for each pair of ions. The high-spin square-planar Fe(II) and Co(II) complexes illustrate an unusual pairing of spin-state with square-planar geometry. Moreover, the Fe(II) complex exhibits signs of easy-axis molecular anisotropy and slow-relaxation of magnetization, albeit in the presence of a magnetic field. Lastly, in Chapter 6, we investigate a trinuclear Fe(III) complex bridged by a triethynylmesitylene ligand. The magnetic properties of the complex are compared to a previous Fe(III) complex bridged by a triethynylbenzene ligand. Steric interactions between the aromatic core of the ethynylmesitylene ligand and the auxiliary dimethylphosphinoethane ligands on Fe(III) are predicted to engender a ligand conformation to promote strong orbital overlap. Magnetic susceptibility data for the two complexes both exhibit ferromagnetic coupling between metal centers as expected. Further studies are necessary to confirm the observed behavior, but the new triethynylmesitylene complex appears to have slightly stronger coupling than the previous triethynylbenzene complex.
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    Biophysical studies of motions and interactions of membrane proteins
    (Colorado State University. Libraries, 2011) Winter, Peter William, author; Barisas, B. George, advisor; Roess, Deborah A., advisor; Crans, Debbie C., committee member; Bamburg, James R., committee member
    We have utilized a variety of biophysical techniques to quantitatively examine the motions and interactions of transmembrane proteins on living cells at the single-molecule level. These include both widefield and confocal optical microscopic methods such as single particle tracking, Förster resonance energy transfer and ratiometric imaging of phase-sensitive probes of lipid order, together with spectroscopic fluctuation methods such as fluorescence correlation spectroscopy and photon counting histogram analysis. Our studies indicate that; 1. Luteinizing hormone receptors on CHO cells and KGN human granulosa cells exhibit restricted lateral diffusion and increased self-association after exposure to human chorianic gonadotropin; 2. In addition to insulin receptor and IGF1 receptor homodimers, rat basophilic leukemia 2H3 cells express significant levels of insulin receptor-IGF1 receptor heterodimers; 3. Clustering of insulin receptors after exposure to insulin on rat basophilic leukemia 2H3 cells is affected by disruption of actin-filaments but not by extraction of membrane cholesterol; 4. Chromium Picolinate and Bis(maltolato)oxovandium(IV) both restrict the lateral diffusion of insulin receptors on rat basophilic leukemia cells and; 5. Individual FcE receptors on rat basophilic leukemia cells exhibit orientation fluctuations on millisecond timescales.
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    Development of biomass-derived furanic monomers for biorenewable polyesters and polyurethanes
    (Colorado State University. Libraries, 2019) Wilson, Jedediah Forrest, author; Chen, Eugene Y.-X., advisor; Reynolds, Melissa M., committee member; Ackerson, Chris J., committee member; Radford, Donald W., committee member; Crans, Debbie C., committee member
    Development of Biomass-Derived Furanic Monomers for Biorenewable Polyesters and Polyurethanes This dissertation describes the development of difuranic diol monomers through the N-heterocyclic carbene (NHC) catalyzed cross-coupling of the biomass-derived platform chemicals, 5-hydroxymethylfurfural (HMF) and furfural (FF), and their subsequent utilization in the synthesis of renewable polyesters and polyurethanes with tunable thermal and mechanical properties through the use of soft and rigid co-monomers. The resulting polymers can undergo reversible cross-linking with bis-maleimide cross-linkers through the thermally reversible Diels-Alder reaction involving both the internal and pendent furan rings. The ability to construct a thermally reversible cross-linked network, coupled with formation of a significant amount (up to 34%) of stable carbonaceous materials when heating the polymers to 700 °C, demonstrates some promising features of this class of new difuranic polymers. To address the need to enhance the molecular weight of the current furan-based polymers produced by the step-growth polycondensation process, alternative monomer structures have been designed to adopt the chain-growth mechanism. The first such alternative monomer belongs to a class of furan-derived lactones as candidates for ring-opening polymerization (ROP), which have been shown to produce high molecular weight polyesters because they follow the chain-growth mechanism. Two synthetic routes have been explored to produce such lactone monomers, and their polymerization behavior has been subsequently examined. The second such alternative is centered on a multifunctional furan acrylate monomer, methacrylate furan aldehyde (MFA). The studies tested a hypothesis that auto-tandem or cascading reaction involving the aldehyde functionality in MFA would undergo a benzoin condensation, then the consequent diacrylate would have the appropriate functionality for NHC catalyzed tail-to-tail coupling resulting in a proton transfer polymerization (HTP). It was found that the benzoin condensation was successful but an oxidation occurred at the α-hydroxy of the furoin diacrylate resulting in a highly electrophilic diketone furil diacrylate. Exploration of the coupling mechanism suggests that the enolate acts as a base catalyzing the oxidation. Through careful analysis of the adducts formed when the NHC was reacted with the furil diacrylate showed that the NHC had strong affinity for the diketone moiety thus blocking the HTP pathway. Overall, this work added significantly to our understanding of furans as monomers, NHC catalysis in furan monomer synthesis as well as polymerizations, and enhanced our ability to control thermal and mechanical properties of furan containing polymers.
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    Effect of aging on gene expression by granulosa cells from mares
    (Colorado State University. Libraries, 2019) Ashwish, Nadya M., author; Roess, Deborah A., advisor; Barisas, George, committee member; Crans, Debbie C., committee member; Miller, Charles W., committee member
    Changes in gene expression in granulosa cells from mares may result from aging or development of metabolic disease as well as from other causes. We used qRT-PCR to quantify expression of equine insulin receptor (IR), insulin-like growth factor receptor (IGF-1R), glucose transporter type 4 (GLUT4) and adenosine monophosphate-activated protein kinase (AMPK) subunit genes in granulosa cells from young (4-9 years), middle-aged (10-16 years) and older (>16 years) mares. Granulosa cells were isolated following follicular aspiration and incubated in the presence or absence of 10% fetal bovine serum (FBS) and 30mM glucose for 24 hrs. Cells were then treated for 1 hr with insulin (100nM), IGF-1 (10nM), epidermal growth factor (EGF; 1μM), progesterone (P4; 100 nM) or human chorionic gonadotropin (hCG; 100 nM). ∆Ct, where Δct is equal to the CT value for the gene of interest minus the CT value for a house keeping gene, values for IGF-1R or IR expression did not differ significantly for any age group. However, there were statistically significant differences in the ratio of ∆Ct for IR relative to ∆Ct for IGF-1R for individual young, middle-aged and old mares. Young animals expressed increased IGF-1R relative to IR after pre-incubation of cells in either – fetal bovine serum FBS/30mM glucose or + FBS/30mM glucose media followed by treatment with insulin, IGF-1, or EGF. Conversely, middle-aged animals expressed increased IR relative to IGF-1R following preincubation in both medium and following treatment with insulin or IGF-1. Finally, older animals expressed approximately equal amounts of IR relative to IGF-1R ( FBS/-30mM glucose) or increased IR relative to IGF-1R. When cells were pre-incubated in +FBS/+30mM glucose medium and treated with insulin or IGF-1, the ratio of IR expression relative to IGF1R expression changed and there was increased IGF-1R relative to IR. Changes in the relative numbers of IR and IGF-1R monomers expressed from IR and IGF-1R genes may affect the particular receptor dimers assembled from these monomers. In evaluating fold change 2-∆∆Ct in gene expression, we observed increases in IGF-1R expression (+FBS/+30mM glucose; p<0.03), GLUT4 expression (+FBS/+30mM glucose; p<0.05) and AMPKα2 expression (-FBS/-30mM glucose; p<0.03) in older mares relative to young animals. Together, these results indicate that young animals differ from older animals in several ways. Young animals maintain stable ratios of ∆CT for IGF-1R to ∆CT for IR and these ratios are not affected by treatment with insulin, IGF-1 or EGF. Thus, it is likely that young animals maintain higher levels of IGF-1R homodimers and hybrid receptors formed from IGF-1R and insulin receptor monomers and that receptor numbers, including insulin receptors, remain stable, particularly when circulating glucose and insulin levels are high. Older animals, on the other hand, are more labile with respect to ∆CT for IGF-1R relative to ∆CT for IR. Exposure of granulosa cells pre-incubated in +FBS/+30mM glucose medium to high levels of glucose, insulin, IGF-1 or EGF demonstrated increased IGF-1R expression. This may decrease numbers of insulin-responsive insulin homodimers, increasing hybrid receptors and IGF-1R homodimers and thus decreasing insulin responsiveness and insulin effects on metabolism. Moreover, when there are significant effects on expression of IR, IGF1R or GLUT4 in older animals, there are accompanying increases in expression of some AMPK subunit genes in older animals indicating additional effects of aging on overall metabolism.
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    Effect of aging on metabolic status and hormone responsiveness in the ovary
    (Colorado State University. Libraries, 2019) Elrmly, Mustafa Ali, author; Barisas, B. George, advisor; Roess, Deborah A., advisor; Crans, Debbie C., committee member; Carnevale, Elaine, committee member
    There were significant effects of mares' ages on gene expression in granolosa cells. Most of the effects we noted were on the expression of AMPK subunits, italics indicating genes rather than gene protein products, although there were significant effects of animal age on GLUT4 and IGF-1R expression as well. In general, increased expression of selected AMPK subunits occurred in older animals and, with one exception, in cells pre-treated with medium that did not contain FBS or additional glucose. Although the underlying cause of increased expression of the AMPK subunits was not determined in this study, various cellular stresses such as nutritional deprevation can lead to AMPK activation. This may be sufficient in aging animals to drive an increase in AMPK subunit expression. In addition to AMPK effects, there was a decrease in IGF-1R expression in older animals although this occurred only in cells pre-treated with medium containing FBS and additional glucose. There was also decreased expression of GLUT4 following hCG treatment and, again, this occurred only when cells were pre-treated with FBS and additional glucose. We then evaluated membrane lipid order to determine whether animal age affects the membrane structure of granulosa cells. Membrane lipid structure including formation of raft microdomains may affect signaling by receptors involved in reproductive and metabolic functions. Insulin receptors, IGF-1 receptors and EGF receptors require highly ordered membrane microdomains for signal transduction. In general, plasma membranes from older animals had more ordered membranes, perhaps due to an increase in in vivo cholesterol synthesis with age. Finally, we examined hormone responsiveness of granulosa cells from young and old mares fed a normal hay-based diet or antioxidant-enhanced commericial diet to evaluate hormone responsiveness with aging. Mares fed an enhanced antioxidant-rich diet showed increased insulin responsiveness, which suggests that at least some effects of aging, namely reduced insulin responses can be treated with an improved diet.
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    Effects of luteinizing hormone receptor expression levels on receptor aggregation and function
    (Colorado State University. Libraries, 2019) Althumairy, Duaa, author; Barisas, B. George, advisor; Roess, Deborah A., advisor; Crans, Debbie C., committee member; Miller, Charles W., committee member
    Luteinizing hormone receptors (LHR) are G protein-coupled receptors (GPCR) found primarily in female and male reproductive organs where they play a critical role in ovulation and sperm maturation, respectively, as well as maintenance of sex hormone production in both sexes. The role of oligomerization in LHR function is of considerable interest and not well understood. The oligomerization state of LHR has been suggested to have a significant role in signaling, desensitization and internalization of this receptor after activation by either luteinizing hormone (LH) or human chorionic gonadotropin (hCG) [2-8]. Overexpression of membrane proteins such as LHR may result in molecular crowding and may lead to increased protein oligomerization [10]. We hypothesize that LHR are present in the plasma membrane as constitutive small clusters or, alternatively, exist as dimers or mixture of monomers and dimers in the absence of hormone and then undergo varying degrees of aggregation after binding hCG. These differences in LHR organization depend on expression levels of LHR which may, in turn, affects LHR activity. In this project, we examined the effect of LHR expression levels on receptor oligomerization using polarized homo-transfer fluorescence resonance energy transfer (homo-FRET) methods to evaluate receptor interactions in cell lines stably expressing averages of 10,000 receptors per cell, 32,000 receptors per cell, 123,000 receptors per cell or 560,000 receptors per cell. In addition, we measured levels of cyclic adenosine monophosphate (cAMP), a second messenger involved in signal transduction which is produced in response to activation of LHR. This study demonstrated that the oligomerization state of LHR depends on the expression level of LHR, i.e. the number of receptors per cell or the concentration of LHR per unit area. Although LHR appear as in dimers or oligomers in the plasma membrane when receptor expression levels are low, it is clear that, with increased expression levels, LHR are found in larger structures exhibiting lower values for initial anisotropy. The effect of hCG binding on LHR was dependent on the expression level of receptors in the absence of bound hormone. The greatest effect of hCG occurred in cells expressing low numbers of LHR per cell where receptors were able to undergo further aggregation in response to hormone binding. The effect of hCG on highly expressed LHR was negligible; LHR, when highly expressed, were already extensively aggregated and did not undergo measurable changes in their aggregation state. Deglycosylated human chorionic gonadotropin (DG-hCG) had modest effects on cells expressing comparatively few LHR per cell since these receptors were already present in small clusters. Depletion of plasma membrane cholesterol using MβCD caused a decrease in intracellular cAMP level accompanied by decrease in cluster size of LHR as expression level of LHR increased. Together these results are important to our understanding of the roles that the expression levels of LHR, the oligomerization state of LHR and the plasma membrane play in LHR function. The organization of lipids in the bulk membrane and in membrane microdomains affects the ability of LHR to signal as does protein density, particularly when receptor crowding has occurred. These studies also suggest, more generally, that protein organization in the plasma membrane may function as an important pharmacological target that merits further exploration.
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    Energy storage and conversion materials: Part 1, Synthesis and characterization of ruthenium tris-bipyridine based fullerene charge transfer salts as a new class of tunable thermoelectric materials; Part 2, Synthesis and characterization of polymer thin films for use as a lithium ion battery separator
    (Colorado State University. Libraries, 2013) Bates, Daniel James, author; Elliott, C. Michael, advisor; Prieto, Amy L., advisor; Finke, Richard G., committee member; Van Orden, Alan, committee member; Crans, Debbie C., committee member; Dandy, David S., committee member
    To view the abstract, please see the full text of the document.
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    Evaluating luteinizing hormone receptor signaling using the cyclic AMP reporter ICUE3
    (Colorado State University. Libraries, 2018) Hadhoud, Giuma E., author; Roess, Deborah A., advisor; Barisas, George, advisor; Crans, Debbie C., committee member
    The luteinizing hormone (LH) receptor is a member of the G protein-coupled receptor family and the subfamily of glycoprotein hormone receptors. The LH receptor plays a vital role in normal development and in the function of the gonads. The LH receptor is expressed in interstitial cells, thecal cells, and granulosa cells and in cells making up the corpus luteum. Signal transduction by LH receptors is dependent on hormone activation of LH receptors and subsequent activation of G proteins. Evaluating the cyclic adenosine monophosphate (cAMP) level in response to binding of ligand to LH receptors depends on activation of adenylyl cyclase by G proteins and production of cAMP. In this project, fluorescence resonance energy transfer (FRET) methods were used to evaluate cAMP levels in individual cells in response to binding of ligand to LH receptors. These studies used ICUE3, an EPAC-based reporter molecule for cAMP in cells. ICUE3 is an engineered molecule that contains a FRET donor and acceptor pair as well as a membrane-targeting sequence. FRET occurs when the donor fluorophore in an excited electronic state transfers its excitation energy to a nearby acceptor chromophore. In the absence of bound cAMP, the fluorescence donor in ICUE3 transfers energy to the fluorescence acceptor. When ICUE3 binds cAMP, the donor-acceptor distance increases and the FRET signal is reduced. cAMP levels were evaluated in individual cells expressing ICUE3 and LH receptor under different conditions including exposure of cells to human chorionic gonadotropin (hCG). In some experiments, CHO cells co-expressing ICUE3 and constitutively-active LH receptors, LH receptors yoked to a single-chain modified form of hCG (yLHR), were used to determine whether the presence of a constitutively-active receptor increased basal cAMP levels in CHO cells. Our results show that ICUE3 is a reliable reporter molecule that measures basal cAMP levels in untreated cells and is responsive to changes in intracellular cAMP levels in response to forskolin treatment or, in the presence of a functioning LH receptor, to hCG.
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    Investigating luteinizing hormone receptor signal transduction using the cyclic AMP reporter ICUE1
    (Colorado State University. Libraries, 2010) Huang, Xin, author; Roess, Deborah A., advisor; Barisas, B. George, committee member; Crans, Debbie C., committee member
    Mechanisms involved in initiation of signaling by LH receptors have been under active investigation because they play important roles in human fertility and development of gonadal tumors. Assay of cAMP levels in response to hormone treatment is usually used to demonstrate LH receptor activation and has historically relied on biochemical methods. ICUEl is an Epac-based cAMP reporter which undergoes conformational changes after binding cAMP. Unlike traditional biochemical assays, ICUEl combined with FRET techniques is capable of real-time monitoring of cAMP levels in individual cells. In this project, Epac reporters have been used to evaluate LH receptor activity in cells expressing constitutively-active LH receptors or cells where transactivation of LH receptors is reported to occur. For the investigation of constitutively active LH receptors, DNA of ICUEl and yoked LH receptor were co-transfected into CHO cells and expressed on the cell membrane. For the study of LH receptor trans-activation, CHO cells were developed using two plasmids encoding LH receptors with defects either in ligand binding or coupling of LH receptor to Gs. Our results show ICUEl offers an useful tool for evaluating cAMP levels in real-time using single cell imaging methods. Further application of this technique to studies evaluating cAMP level in cells where ligand binding to receptor can be visualized using, for example, quantum dots or nano-gold particles linked to individual molecules of hCG, will be of interest as will studies of cAMP levels during LH receptor desensitization and resensitization in response to brief pulses of hCG.
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    Membrane fluidity of RBL-2h3 cells treated with insulin and BMOV using time-correlated single photon counting fluorescence anisotropy
    (Colorado State University. Libraries, 2013) Roan, Chelsea Rene' Lenne, author; Roess, Deborah A., advisor; Barisas, B. George, advisor; Crans, Debbie C., committee member
    Transition metal compounds have been shown to be insulin-enhancing but the mechanism of action has not been fully elucidated. With obesity, diabetes and other metabolic derangements increasing in developed countries, understanding the effects these compounds will better target drug therapy. Previous investigations have focused on vanadium and have studied the effects on protein-protein interactions in the insulin signaling pathway. In this paper, we propose that the mechanism of action may also include interactions with the plasma membrane. Lipids as bioactive molecules are on the horizon as the next great area of exploration in biochemistry and molecular biology. Within the insulin signaling pathway, the insulin receptor functions optimally in areas of specialized lipid packing that are characterized as small detergent insoluble regions enriched in sphingomyelin and cholesterol and termed lipid rafts. These lipid rafts are a subset of microdomains within the plasma membrane. Obesity and excess lipids have been shown to increase inflammation via increases in free fatty acids, cytokines, TNF-α, and reactive oxygen species resulting in the peroxidation of membrane lipids. We propose that one cause of insulin resistance, a failure of insulin receptors to respond to insulin, is due to disruption of the membrane lipids resulting in an increase in membrane fluidity. This disruption results in displacement of insulin receptors out of specialized lipid rafts. We propose that treatment with vanadium will result in an increase in membrane rigidity favoring lipid raft formation and restoration of insulin receptors to a platform favoring optimal signaling. Time-correlated single photon counting fluorescence anisotropy was used to measure the membrane fluidity of RBL-2H3 cells treated with insulin and the vanadium compound bis(maltolato)oxovanadium(IV) (BMOV).
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    Oh cryoprotectant, wherefore art thou cryoprotectant? Investigation of the permeation of common cryoprotectants into live rice callus cells by coherent Raman microscopy
    (Colorado State University. Libraries, 2023) Samuels, Fionna M. D., author; Levinger, Nancy E., advisor; Van Orden, Alan, committee member; Crans, Debbie C., committee member; Wilson, Jesse W., committee member
    Conserving a diverse selection of plant species is vital as climate change begins shifting the planet's ecosystems in earnest. Many plants can be preserved through maintaining their seeds in cool, dark chambers, like that of the Svalbard Seed Vault, but others do not reliably reproduce through seeds. This can include wild plants that lack seeds entirely, ferns for example, or agricultural plants where clonal propagation is the only way to conserve desirable traits, grapes, hops and bananas for example. Rather than collecting and saving seeds from these plants, researchers collect tissue samples, cool and preserve them in liquid nitrogen then warm and regrow the plants years later. To maximize post-freezing viability, tissue samples are exposed to mixtures of cryoprotectants. Only a few cryoprotectant formulations have been used almost exclusively since their development in the early 1990s, including Plant Vitrification Solution 2 (PVS2) and Plant Vitrification Solution 3 (PVS3). Unfortunately, these formulations are not universally protective—some plant species respond extremely well to exposure while others are killed. When PVS2 or PVS3 fail to preserve a species, researchers must either empirically develop a new formulation or method of cryopreservation or settle for not conserving the species. A lack of mechanistic understanding of how these formulations work to protect tissue from extreme cold prevents straightforward development of new formulations. Since the development of PVS2 and PVS3, advances in instrumental techniques have opened the door to improved physical characterizations of the components of these mixtures. Vibrational microscopies, like Raman or infrared microscopy, allow the direct visualization of cryoprotectants interacting with living cells. By exciting vibrations unique to the bonds in a molecule, these techniques can effectively image nearly unadulterated molecules. Through deuteration, cryoprotectants can be imaged without disturbing other molecules in the cell. The work presented in this dissertation demonstrates how deuterated dimethyl sulfoxide (d6-DMSO), deuterated ethylene glycol (d6-ethylene glycol) and deuterated glycerol (d8-glcyerol) can be directly imaged inside living rice callus cells. Readers are first introduced to the cell system, rice callus cells, and the analytical technique, coherent anti-Stokes Raman scattering (CARS) microscopy. Then, they will learn about initial static experiments searching for the location of d6-DMSO within the callus cells. The next two chapters explore the real-time permeation of d6-DMSO, d6-ethylene glycol and d8-glcyerol individually and then solvated in PVS2. The final chapter describes future experiments I think should come from this work that will increase fundamental understanding of cryoprotectant-cell interactions and streamline the process of developing new cryoprotectant formulations.
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    Organocatalytic, Michael-Stetter reaction and rhodium(I)-catalyzed hydroheteroarylation of acrylates with benzoxazoles: reaction development and investigations into origins of enantioselectivity
    (Colorado State University. Libraries, 2015) Filloux, Claire M., author; Rovis, Tomislav, advisor; McNaughton, Brian R., committee member; Prieto, Amy L., committee member; Crans, Debbie C., committee member; Hentges, Shane T., committee member
    The chapters that follow describe two independent investigations. Both relay the development of experimental methods for the catalytic, asymmetric addition of carbon-hydrogen bonds to alkenes. In the first chapter, nucleophilic amine and N-heterocyclic carbene cocatalysts cooperate in the organocatalytic, cascade synthesis of benzofuranone products in good yields and high enantioselectivities. Importantly, the cascade protocol is found to outperform a two-pot procedure in which reaction intermediates are isolated and purified before the second step. Mechanistic studies reveal that additives and geometry of an olefin intermediate crucially influence reaction enantioselectivity. In the second method, a bulky Rh(I)-bisphosphine complex catalyzes the asymmetric, intermolecular addition of benzoxazoles to methacrylate derivatives in fair to excellent yields and good to excellent enantioselectivities. Detailed deuterium labeling and epimerization studies provide considerable insight into the reaction mechanism: C-H activation is reversible; migratory insertion is likely enantiodetermining; and the bulky- bisphosphine ligand likely boosts reactivity and selectivity by discouraging deleterious ligation of benzoxazole starting materials to on- or off-cycle rhodium complexes and by impeding coordination-induced product epimerization.
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    Part I: The total synthesis of (±)-securinine and (±)-allosecurinine and synthetic strategies for a second generation synthesis of the securinega alkaloids and Part II: The use of (+)-K252a in the semi-synthesis of indolocarbazole natural products and novel analogs thereof
    (Colorado State University. Libraries, 2014) Levine, Samantha Roslyn, author; Ferreira, Eric M., advisor; Rovis, Tomislav, committee member; Crans, Debbie C., committee member; Strauss, Steven H., committee member; Lenaerts, Anne J., committee member
    In part I, the total syntheses of (±)-securinine ((±)-1.001) and (±)-allosecurinine ((±)-1.003) are described. The syntheses feature the use of a Rh-initiated O—H insertion/Claisen rearrangement/1,2-allyl migration, which would allow for an enantioselective synthesis when an enantioenriched allylic alcohol (ie: (+)-1.137) is used. Three more steps generates the common intermediate imine 1.144, which upon reduction gives a pair of diastereomers. Protection of the free amine and a second reduction gives 1.146a and 1.146b, which were advanced to (±)-1.003 and (±)-1.001 respectively. Additional investigations into improving the endgame and devising a more streamlined synthesis were conducted. This focused on reducing the number of oxidation state changes at C13. Part II of this dissertation details efforts to employ (+)-K252a (2.016) as a starting material for the synthesis of potential Hox-A14 inhibitors based on the indolocarbazole scaffold. It also covers the development of a novel method of selectively protecting the amide of 1.016 with a DMB group. This protected analog was employed as the starting material for a potential synthesis of the recently isolated indolocarbazoles Streptocarbazole A (2.019) and B (2.020). The proposed route to these compounds is via C—N bond migration on ketone 2.138 or dimethylketal 2.158. Substrates 2.138 and 2.158 have been synthesized. Preliminary investigations into conditions for the desired rearrangement have been conducted.
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    Rotation of cell surface and dissolved biomolecules examined by fluorescence imaging, time-tagged single-photon counting, and fluorescence depletion anisotropy
    (Colorado State University. Libraries, 2022) Pace, Jason M., author; Barisas, B. George, advisor; Crans, Debbie C., committee member; Roess, Deborah A., committee member; Van Orden, Alan K., committee member
    In this dissertation, I discuss our studies examining protein rotation both in solution and on single cells. Chapter I gives background on physics of rotational diffusion, the application of these measurements to cellular systems, and a general overview of the field, including a survey of techniques that have been used to measure rotation of membrane proteins. In the next two chapters, I discuss our research on the effect of various cell treatments known to perturb the dynamics of membrane proteins on the rotation of the high-affinity Type I IgE receptor (FcεRI) expressed on RBL-2H3 cells. I investigated effects on receptor rotation resulting from treatment with IgE antibody as well as from four treatments with IgE and an additional agent including DNP-BSA, paraformaldehyde, MβCD, and cytochalasin D. These agents have varied effects that I expect to cause a significant perturbation of the rotational dynamics of the receptor. These effects range from receptor crosslinking by DNP-BSA and paraformaldehyde which would be expected to hinder receptor rotation to effects on membrane cholesterol content and the underlying cytoskeleton in the cases of MβCD and cytochalasin D, the effects of which are more uncertain and thus of particular interest. I have investigated these phenomena using a single-particle fluorescence imaging approach and, alternatively, a time-tagged single photon counting approach. These topics are the subject of Chapters II and III respectively. These two approaches, while both designed with the intent to investigate the rotational dynamics of membrane proteins using fluorescence microscopy, share little in common with regards to their methods of data collection and analysis. The concepts behind them are completely different and they use an entirely different set of analysis programs. Chapter IV consists of a published manuscript entitled "Continuous fluorescence depletion anisotropy measurement of protein rotation" which describes our work using a newly-developed pump-probe technique to examine protein rotation in solution and extends this to single-cell measurements. In the continuous variant of fluorescence depletion anisotropy used here, the intensity and polarization of a laser beam are modulated continuously by a programmed acousto-optic modulator and Pockels cell respectively to produce the desired excitation waveform. We have used this method to examine rotation of eosin conjugates of carbonic anhydrase, BSA, and immunoglobulin G in 90% glycerol at varying temperatures. We have also explored the potential application of this method to single-cell measurements and recorded preliminary results on eosin-IgE-bound FcεRI. Generally, we found good agreement with time-resolved phosphorescence anisotropy measurements of rotation of solution-phase molecules and of cell surface FcεRI. Chapter V discusses future avenues worth exploring which would improve upon the methods presented in Chapters II and III. These include faster cameras to access shorter timescales, gold nanorods to improve the signal-to-noise ratio, and a method to obtain a true anisotropy in a microscope.
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    Signaling complexes formed by luteinizing hormone receptor trans-activation
    (Colorado State University. Libraries, 2019) Shanta, Hanan Ali A., author; Barisas, B. George, advisor; Roess, Deborah A., advisor; Crans, Debbie C., committee member; Miller, Charles W., committee member
    Signal transduction by luteinizing hormone (LH) receptors depends on hormone activation of these receptors, a process important for mammalian reproduction. The LH receptor, a member of the G protein-coupled receptor (GPCR) family, undergoes hormone-induced LH receptor dimerization and/or oligomerization and translocation into small membrane compartments where receptors are confined and exhibit slow lateral diffusion. However, the organization of the signaling complex confined within these structures is not clear. In this project, we used single particle tracking methods to evaluate the lateral motions of wild type receptor FLAG-LHR-YFP and mutant receptors defective in hormone binding (LHR-hCG,+cAMP) or defective in signal transduction (LHR+hCG,-cAMP) after exposure to human chorionic gonadotropin (hCG). These studies showed that, when wild type LH receptors and mutant receptors are coexpressed and treated with 100 nM hCG, there are decreases in receptor lateral diffusion, the number of receptor-occupied membrane microdomains and the size of receptor-containing membrane microdomains. These results suggest that wild type LH receptors are capable of both cis-activation of nearby wild type LH receptors and transactivation of LHR-hCG,+cAMP , a receptor that is not able to bind hCG. We then investigated interactions between wild type LH receptors and mutant receptors using homo-transfer fluorescence resonance energy transfer (FRET) methods. We showed that LH receptors associate with one another and that the extent of self-association increases in response to increasing hCG concentrations. Using homo-transfer FRET methods, we showed that mutant LH receptors are trans-activated by wild type receptors and undergo aggregation in response to 100 nM hCG despite being unable to bind hCG directly. Finally, we evaluated cAMP levels in cis-activated and trans-activated LH receptors using ICUE3, an EPAC-based reporter molecule for cAMP. We determined that increases in intracellular cAMP occur in cells expressing wild type receptors and exposed to increasing concentrations of hCG. Similarly, cells co-expressing mutant receptors exhibit increased cAMP when there is a 1:10 transfection ratio of LHR+hCG,-cAMP to LHR-hCG,+cAMP indicating that trans-activation is occurring. Disruption of membrane microdomains by pre-treatment of cells with 10 nM methyl-β-cyclodextrin for an hour has a negative effect on cAMP levels which indicates the importance of cholesterol-containing microdomains in signal transduction by LH receptors. Together these results demonstrate that trans-activated LH receptors can undergo receptor aggregation in response to hormone binding and can signal effectively despite the absence of a signal-transduction sequence in the mutant receptor.
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    Total synthesis of hapalindoles J and U, formal synthesis of haplaindole O, synthesis of the proposed biosynthetic precursor to hapalindole K and work towards the ambiguine family of alkaloids
    (Colorado State University. Libraries, 2011) Rafferty, Ryan J., author; Williams, Robert M., advisor; Shi, Yian, committee member; Crans, Debbie C., committee member; Prieto, Amy L., committee member; Thamm, Douglas H., committee member
    Herein I discussed the total synthesis of hapalindoles J and U, the formal synthesis of hapalindole O, the proposed biosynthetic precursor to hapalindole K and efforts towards other hapalindole and ambiguine families of alkaloids. The hapalindoles and ambiguines both possess a highly functionalized 6:6:6:5, which I accessed over six synthetic steps via a developed silyl strategy with an overall 54% yield. Hapalindole J was synthesized in an overall 11% yield over eleven synthetic steps and hapalindole U in an overall 25% yield over thirteen synthetic steps from commercially available materials utilizing the silyl strategy developed. A formal synthesis of hapalindole O, intercepting Natsume's total synthesis, was accomplished as well via the developed silyl strategy. In addition, the synthesis of the proposed biosynthetic precursor to hapalindole K was accessed. Currently, this newly developed silyl strategy is being employed in accessing some of the more functionalized hapalindoles (such as K) as well as the complex ambiguine core.
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    Tuberculosis as a romantic trope and a narrative device in Elizabeth Gaskell's novel North and South
    (Colorado State University. Libraries, 2022) Roess, Deborah A., author; Tsang, Philip, advisor; Gollapudi, Alpana, committee member; Crans, Debbie C., committee member
    A conventional reading of North and South by Elizabeth Gaskell has generally established the novel as highly realistic and Gaskell as a keen observer. However, the novel is full of contradictions that make this reading of the novel problematic. Bessy Higgins' death from tuberculosis gives us some insight into relationships in the novel if one considers Bessy Higgins as a "character" developed using a romantic trope associated with having tuberculosis and then sacrificed for the happy ending in this romance, Margaret Hale's marriage to John Thornton. The trope used by Gaskell to describe Bessy Higgins' romanticized death from tuberculosis was familiar to Gaskell's readers. In Chapter 1, I describe the perception of tuberculosis in the 18th and 19th century as a romantic disease. This trope is used in, for example, Samuel Richardson's novel Clarissa where Clarissa's gradual dying from tuberculosis was apparently painless but provided Clarissa with the opportunity to prepare for the rewards of her virtue upon her death. Similarly, John Keats' death from tuberculosis, documented in his letters and the writings of his contemporaries, was treated as an almost predestined romantic death for a highly sensitive poet. In Chapter 2, I examine the function of Bessy's tuberculosis in the narrative in North and South. Gaskell uses Bessy's tuberculosis to achieve the marriage of Margaret Hale to John Thornton as well as a resolution of the conflicts between Thornton and his workers. Bessy Higgins' tuberculosis is pivotal to the novel's narrative. Gaskell uses what had become an old-fashioned trope by the mid-19th century to establish a relationship between Bessy Higgins and Margaret Hale. Using this trope, Gaskell first transforms Bessy Higgins, a mill worker of humble origins, into both a romantic and individualized character whose death bears greater similarity to the death of Margaret Hale's mother than to the deaths of the lower-class characters, e.g., Mr. and Mrs. Boucher and Leonards. In conferring ambiguity to Bessy's class using the romantic trope associated with tuberculosis in the middle and upper classes, subsequent events in the novel become possible. A softening of class barriers, however incomplete, allows Margaret Hale to become part of a community in Milton that includes the striking mill workers. Within this newly constructed community, Bessy Higgins' father, Nicholas Higgins, an important figure in the mill worker's strike, becomes someone more closely a peer to John Thornton, the mill owner, than one of Thornton's workers. Gaskell can then use the relationship between John Thornton and Nicholas Higgins, based on a greater sense of equality between the two men, to address labor relations between masters and their workers and, ultimately, resolve the labor disputes that threaten Thornton's prosperity. Making use of the genre rules for a romance, Margaret Hale's relationship with Bessy and the softening of class barriers allows Margaret to consider Thornton, the son of a bankrupt speculator, as a suitable marriage partner despite her perception of Thornton as being not quite a gentleman. Margaret is quite specific early in the novel about her qualifications for a suitor and rejects, over the course of the novel, other suitors including Thornton. It is only when Milton's workers and their masters can be seen as individuals, made possible for Margaret through her relationship with Bessy Higgins and subsequently with Bessy's father, that John Thornton can be transformed from a mill master to a more humanized figure. As Thornton's relations with his workers become more equitable, Thornton becomes a desirable marriage partner for Margaret. In Chapter 3, I address the problem of treating Gaskell as a keen observer by examining the discordance between Gaskell's portrayal of a romanticized death from tuberculosis by a mill worker in mid-century Milton with what is likely to have been her own experiences with tuberculosis in Manchester and with the medical community's contemporary perspective on the disease and its treatment. A brief review of English medical literature from 1835-1850 suggests that active treatment of tuberculosis was available and, in many cases, successful. Thus, Gaskell's use of a romantic trope to describe Bessy Higgins' disease discredits both Gaskell as a keen observer and the notion that literary portrayals can alter general perceptions of disease, neither of which are supported by the text of North and South.
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    Unlocking the potential of crosslinked polymers: from dynamic covalent bonds to recyclable thermosets
    (Colorado State University. Libraries, 2022) Clarke, Ryan William, author; Chen, Eugene Y.-X., advisor; Miyake, Garret M., committee member; Li, Yan V., committee member; Crans, Debbie C., committee member
    This dissertation describes the development of dynamic covalent chemistries and bond exchange principles in the context of crosslinked polymers demonstrating high performance properties and sustainable end-of-life avenues. Traditional crosslinked polymers, or thermosets, present a significant challenge in meeting the goals of a circular materials economy caused by a strict orthogonality between performance and recyclability. Covalent adaptable networks (CANs) are a developing class of responsive, crosslinked materials that mimic the performance qualities of static thermosets but access flow-state melt processability by triggering crosslink-bond exchange. Several impactful and fundamental advances to this topic and broader sustainable chemistry are discussed herein: demonstration of new dynamic covalent bonds, establishment of intra- and inter-domain exchange principles, orthogonal working/healing exchange conditions for classic thermoset behavior, and upcycling compatibilization of mixed-feed commodity plastics via dynamic crosslinking. The multidisciplinary knowledge developed herein spans the themes of polymer synthesis, catalysis, block copolymers, self-assembly, structure/property relationships, mechanism elucidation, sustainability, materials chemistry, and polymer physics. Chapter 1 introduces dynamic covalent bonds, sustainable thermoset strategies, and standing challenges impeding wide-scale adoption. Background is also supplied on relevant themes to the following chapters, including pathways for plastics recycling, block copolymer technology, and mechanical reprocessing. Chapter 2 describes the Lewis pair polymerization (LPP) of renewable indenone to a high Tg (> 300 oC), optically transparent bio-polymer. Special emphasis is made for the unique upcycling of polyindenone, as well as the Lewis pair polymerization mechanism and methodology which is critical for complex polymer synthesis in Chapters 3 & 4. Chapter 3 discloses the merging of A-B-A triblock copolymer self-assembled thermoplastic elastomer (TPE) structure with CAN dynamic bond exchange function to establish tethered domain-restriction as an effective strategy to lock out creep deformation during working conditions. We also define the fundamental principles governing inter- and intra-domain exchange and thermomechanical activation of newly established dynamic covalent bonds. Chapter 4 describes our compounded-sequence-control LPP technology applied to produce architecturally and topologically sophisticated cyclic and linear A-B-A-B multiblock copolymers in timely (< 10 min), scalable (~40 g), one-pot two-step processes. Structure/property relationships are meticulously investigated in solution, bulk, and film phase for differentiation of isomeric products. Chapter 5 reports a highly collaborative, multidisciplinary (molecular, material, and computational) study on bis(diazirine) crosslinker molecules with imbedded dynamic covalent bond functionality as universal crosslinking agents for upcycling commodity thermoplastics (polyethylene, etc.) to high-performance, recyclable thermosets. Demonstration of successful crosslinking by C-H insertion, improvements to material performance (tensile pulling, creep-recovery, and thermomechanical durability), and reprocessability are thoroughly discussed. Most critical, we establish the compatibilization of (otherwise immiscible) mixed plastics in dynamically crosslinked polymer blends and examine the phenomenon by molecular modeling and structural characterization. Chapter 6 offers conclusions, remaining challenges, and future opportunities.