Browsing by Author "Luger, Karolin, committee member"
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Item Open Access A new dawn for Aurora B regulation: shining light on multiple discrete populations of Aurora B kinase at centromeres and kinetochores(Colorado State University. Libraries, 2020) Broad, Amanda J., author; DeLuca, Jennifer G., advisor; Luger, Karolin, committee member; Markus, Steven, committee member; Yao, Tingting, committee member; Amberg, Greg, committee memberCell division is a fundamental biological process that is essential for all eukaryotes to divide the replicated genome with high fidelity into individual daughter cells. Improper segregation of replicated DNA results in chromosome instability, a characteristic that is deleterious to most cells. Critical to the proper segregation of mitotic chromosomes is attachment to spindle microtubules, which are dynamic cytoskeleton filaments that drive the movement of chromosomes during mitosis. A complex network of proteins, collectively called the kinetochore, mediates microtubule attachments to chromosomes. Kinetochores are recruited to individual chromosomes through a specialized heterochromatin domain known as the centromere. Centromeric heterochromatin is comprised of both canonical, H3-containing nucleosomes as well as nucleosomes that contain the histone H3 variant CENP-A. Centromeres serve as a central point of organization in mitotic cells, recruiting both structural and regulatory kinetochore proteins to chromosomes. This extensive protein/DNA network ensures the accurate segregation of chromosomes by regulation of proper kinetochore-microtubule attachments in mitosis. Kinetochore-microtubule interactions are regulated by Aurora B kinase, which phosphorylates outer kinetochore substrates to promote release of erroneous attachments. Although Aurora B kinase substrates at the kinetochore are defined, little is known about how Aurora B is recruited to and evicted from kinetochores, in early and late mitosis, respectively, to regulate these essential interactions. We set out to determine how Aurora B kinase is regulated during mitosis. We found that, contrary to the current model, Aurora B kinase and the Chromosomal Passenger Complex are recruited to distinct regions within the centromere and kinetochore. Furthermore, we found that accumulation of Aurora B kinase at centromeres is independent from Aurora B localization and activity at outer kinetochores. These results lead us to hypothesize a new model for Aurora B kinase regulation. In the direct recruitment model, a population of the kinase is recruited directly to kinetochores in early mitosis, then as mitosis progresses and kinetochore-microtubule attachments are stabilized, architectural changes within the kinetochore result in the eviction of outer-kinetochore localized Aurora B kinase and the stabilization of kinetochore-microtubule attachments.Item Open Access A surface protease of Lyme disease bacteria degrades host extracellular matrix components and induces inflammatory cytokines in vitro(Colorado State University. Libraries, 2012) Russell, Theresa Michelle Tidd, author; Bamburg, James R., advisor; Johnson, Barbara J. B., advisor; Luger, Karolin, committee member; Cohen, Robert E., committee member; Gentry-Weeks, Claudia, committee memberFor nearly two decades, the paradigm in Lyme disease research has been that Borrelia burgdorferi does not produce proteases capable of damaging host molecules. Lyme disease has been considered, therefore, to be the consequence of an exuberant inflammatory response to infecting bacteria. This prevailing concept, however, has created a conundrum for the field. The bacterial burden in infected tissue is low, but the degree of inflammation is remarkable and seemingly out of proportion to this burden. The studies described in this dissertation provide evidence that, contrary to current thinking, B. burgdorferi does possess a protease that degrades numerous molecules of the host extracellular matrix (ECM). In addition to destabilization of the ECM which would be expected to benefit the organism, characterization of this proteolytic activity demonstrates that ECM fragments are produced that are known to be pro-inflammatory. These bioactive fragments may amplify the inflammatory processes triggered by the presence of the bacteria itself. When this hypothesis was tested directly by exposing chondrocytes to the borrelial protease in vitro, inflammatory cytokines and chemokines that are hallmarks of Lyme disease were induced. The studies herein suggest a new model for the pathogenesis of Lyme disease and offer an explanation for the paradox of debilitating inflammatory disease in the presence of few infecting organisms. Lastly, in contrast to current serology-based Lyme disease diagnostic tests, the activity of this protease in vitro may generate diagnostic biomarkers enabling detection of active B. burgdorferi infection.Item Open Access Activation of gene expression in yeast(Colorado State University. Libraries, 2010) Lee, Sarah Kathleen, author; Stargell, Laurie Ann, 1963-, advisor; Luger, Karolin, committee member; Nerger, Janice Lee, 1960-, committee member; Nyborg, Jennifer Kay, committee member; Paule, Marvin R., 1943-, committee member; Ross, Eric D., committee memberTranscription is the generation of RNA from the DNA template, and is the fundamental aspect of gene expression. As such, the initiation of transcription at genes that are transcribed by RNA polymerase II (RNAPII) is a major control point in gene expression. Organisms across the evolutionary spectrum possess genes whose transcription is regulated after recruitment of RNAPII to the promoter, or postrecruitment. This regulatory strategy has been observed in bacteria, yeast, worms, flies, and humans. Therefore, postrecruitment regulation is a conserved strategy for controlling gene expression. Genome-wide studies in Drosophila and humans demonstrate that a significant portion of these genomes are postrecruitment regulated. Recent studies in humans indicate two biologically important activators (p53 and c-myc) are involved in releasing paused polymerases from promoter DNA1,2. These regulators of cell growth and differentiation are both implicated in carcinogenesis. Thus, further understanding how activators regulate the transition from an inactive to active polymerase will prove crucial in our understanding of transcriptional regulation and human diseases. Coactivators are conserved, multiprotein complexes involved in regulating the transcription process at most genes. Yet, virtually nothing is known about the role of coactivators at postrecruitment regulated genes in yeast. The work presented in this dissertation details the identification of postrecruitment functions of two coactivators, the Mediator and SAGA complexes. My studies reveal that coactivators act as intermediaries with activator proteins to stimulate transcription after the recruitment of RNAPII to the promoter. Further, this work demonstrates that this conserved class of factors plays a role in postrecruitment regulation, a previously unappreciated aspect of coactivator function. Analysis of Mediator function at the postrecruitment regulated CYC1 gene revealed a functional submodule of the Mediator complex that is required for triggering the preloaded polymerase at the CYC1 promoter into an active polymerase. This requirement exists even when two different activator proteins control CYC1 expression, Hap2/3/4/5 and Yap1. Strikingly, this submodule is not required for activation of a recruitment regulated Yap1-dependent gene, GTT2. The Yap1 activator controls the expression of a number of genes during oxidative stress in yeast. Oxidative stress is a damaging condition that haunts all aerobic organisms, and is linked to many human ailments. Yeast respond to this biological assault with a rapid activation of many genes. My investigation of Yap1-dependent transcription demonstrated that postrecruitment regulation is more prevalent in yeast than previously thought. Analysis of SAGA function at Yap1-dependent genes revealed that Yap1 utilizes SAGA during oxidative stress. Despite a common reliance on the SAGA coactivator for expression, each gene has different specific SAGA requirements. This demonstrates an important role for the SAGA coactivator during the important biological response to oxidative stress, and the complexity inherent in transcriptional regulation. In sum, my findings illustrate the mechanisms of activated transcription yeast utilize in response to important biological stimuli. This work significantly advances our understanding of the regulation of transcription after RNAPII arrives at the promoter. It also reveals the novel role that coactivators play in stimulating transcription at the group of genes that are regulated in this fashion.Item Open Access Characterization of poliovirus 2CATPase bound to bilayer nanodiscs and involvement of the poliovirus 3Dpol thumb α-helix in determining poly(A) tail length(Colorado State University. Libraries, 2013) Springer, Courtney Lee, author; Peersen, Olve B., advisor; Ho, P. Shing, committee member; Luger, Karolin, committee member; Kennan, Alan, committee memberPoliovirus (PV) is a small non-enveloped picornavirus with a ≈7.5 kb long single-stranded, positive-sense RNA genome. Upon infection, the RNA is translated to generate a ≈250 kDa polyprotein that is subsequently cleaved into about a dozen fully processed proteins and several functional intermediates. PV replication occurs in large membrane associated complexes involving the "non-structural" P2 and P3 region proteins and two of these proteins, 2CATPase and 3Dpol, are the subjects of this dissertation. Part I of this work is focused on the 2C protein, an AAA+ family ATPase that plays a key role in host cell membrane rearrangements and virion assembly, but the membrane binding characteristics of 2C and its polyprotein precursors have made it difficult to elucidate their exact roles in virus replication. In this work I show that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C and the precursor 2BC and 2BC3AB polyproteins in a membrane bound form. Biochemical analysis shows that the proteins are highly active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Notably, the ATPase activity of the core 2C domain is stimulated ≈60-fold as compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. This data leads to a model wherein the viral replication complex can be assembled with a minimally active form of 2C that then becomes fully activated upon proteolytic cleavage from the adjacent 2B viroporin domain. In Part II of this dissertation, I focus on the role of the viral RNA polymerase, 3Dpol, in maintaining the ≈20-150 nucleotides long 3' poly(A) tail of the viral genome. The length of the tail is important for viral replication and initiation of (-)-strand synthesis, but the means by which the RNA is polyadenylated and how poly(A) tail length is regulated is not well understood. We have identified several mutations in an α-helix of the 3Dpol thumb domain that directly impact poly(A) tail length. Here, I tested the impact of these mutations on reiterative transcription of poly(A), poly(U), and poly(C) templates as well as characterized their effect on 3Dpol initiation, stability, elongation rate, and fidelity. I found that mutations in the thumb have the greatest impact on elongation complex stability and that 3Dpol is able to reiteratively transcribe homopolymeric poly(U) and poly(A), but not poly(C) RNA templates. Interestingly, distinct poly(A) and poly(U) transcripts are generated from 10 nucleotide homopolymers that are 1, 7, or 8 nucleotides longer than the template. Based on these findings, we propose a poly(A) slippage model in which the elongation complex stalls at the end of the homopolymer stretch in the absence of additional nucleotides to promote a single nucleotide slippage. This is followed by a slow structural rearrangement in which 3Dpol slips back to the 3' end of the homopolymer sequence, where it is able to re-transcribe starting from the fifth poly(U) in the template.Item Open Access In vivo regulation of chromatin dynamics by Saccharomyces cerevisiae histone chaperone Nap1(Colorado State University. Libraries, 2011) Barker, Kristi Leigh, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Wilusz, Carol J., committee memberEukaryotic cells must organize massive amounts of DNA into the nucleus. In order to accomplish this, the DNA must be compacted into a highly ordered structure known as chromatin. The basic, repeating unit of chromatin is the nucleosome, which consists of two copies of each histone (H2A, H2B, H3 and H4) and organizes 147 base pairs of DNA. Due to its highly compact nature, nucleosomes must be removed during gene expression in order for the transcription machinery to access the DNA. Shuttling of nucleosomes on and off DNA is mediated by a group of proteins known as histone chaperones. Importantly, histone chaperones interact with another family of chromatin remodeling complexes known as histone acetyltransferases (HATs). Acetylation of histones is correlated with the active transcription of genes. The work presented here explores the dynamics and kinetics of histone H3 occupancy and acetylation of histone H3-K9 and H3-K14 in a wild-type strain and strains deleted for three known histone chaperones (Nap1, Vps75 and Asf1) of the yeast Saccharomyces cerevisiae at the well characterized galactose inducible genes. This data offers insight into the epigenetic regulation of chromatin as well as possible mechanisms for the histone chaperones surveyed.Item Open Access Mitomycin alkaloids: synthetic studies(Colorado State University. Libraries, 2009) Gubler, Daniel Alan, author; Williams, Robert M., advisor; Rovis, Tomislav, committee member; Kennan, Alan J., committee member; Elliott, C. Michael, committee member; Luger, Karolin, committee memberDocumented herein are efforts towards the first asymmetric total synthesis of the mitomycin family of natural products. Methods have been developed that efficiently construct eight-membered ring precursors of the natural products. Additionally, a tetracyclic mitosane compound containing all the core features of the mitomycins except the C9a methyl aminal has been constructed. The above-mentioned synthetic efforts help set the stage for future completion of the asymmetric total synthesis of this family of compounds. The studies mentioned herein, while not totally successful, shed new light on the reactivity of the mitomycins as well as the remarkable electronic effect of the electron-rich arene ring.Item Open Access Nucleosomal array condensation: new insights into an old "tail"(Colorado State University. Libraries, 2011) Sorensen, Troy C., author; Hansen, Jeffrey C., advisor; Stargell, Laurie, committee member; Luger, Karolin, committee member; Bailey, Susan, committee memberThe DNA present within the nucleus of each human somatic cell, when extended end to end, would span a distance of about one meter. The first level of compaction critical to fitting the entire genome into the nucleus is the nucleosome, consisting of 147 base pairs of DNA wrapped 1.7 times around an octameric structure composed of the four core histones H2A, H2B, H3 and H4. Nucleosomes separated by up to 80 base pairs of linker DNA called nucleosomal arrays compact the DNA further through short range intra-array and long range inter-array contacts that generate different levels of higher order condensed structures. This dissertation investigates the involvement of the core histone "tail" domains as well as the influence of the H3 centromeric variant CENP-A in nucleosomal array condensation events. In vitro, 12-mer nucleosomal arrays condense intra- and inter-molecularly through nucleosome-nucleosome interactions driven primarily by the core histone tail. This dissertation details the contributions and the molecular determinants of the histone tail domains to the condensation processes. Importantly, we found that the H3 and H4 tail domains were the largest contributors to array condensation. The mode of action used by the H4 tail domain in intra- and intermolecular condensation centered on the following determinants: 1) position of the H4 tail, 2) amino acid composition, 3) positive charge density and 4) tail domain length. Importantly, the primary sequence of the H4 tail was found to not be an important molecular determinant. To date no study has been performed to determine short-range compaction between "bulk" H3 containing and H3 centromeric specific variant, CENP-A chromatin. 12-mer nucleosomal arrays containing either H3 or CENP-A histones were reconstituted and tested for their ability to fold intra-molecularly. Major finding include that CENP-A containing nucleosomal arrays assemble in the same stepwise manner as conical arrays and were always more compact than H3 containing arrays at every salt concentration tested. The increased compaction was found to be in part due to a lysine to arginine mutation at position 49 of CENP-A.Item Open Access Semiparametric regression in the presence of complex variance structures arising from small angle x-ray scattering data(Colorado State University. Libraries, 2014) Bugbee, Bruce D., author; Breidt, F. Jay, advisor; Estep, Don, advisor; Meyer, Mary, committee member; Hoeting, Jennifer, committee member; Luger, Karolin, committee memberAn ongoing problem in structural biology is how best to infer structural information for complex, biological macromolecules from indirect observational data. Molecular shape dictates functionality but is not always directly observable. There exists a wide class of experimental methods whose data can be used for indirectly inferring molecular shape features with varying degrees of resolution. Of these methods, small angle X-ray scattering (SAXS) is desirable due to low requirements on the sample of interest. However, SAXS data suffers numerous statistical problems that require the development of novel methodologies. A primary concern is the impact of radially reducing two-dimensional sensor data to a series of smooth mean and variance curves. Additionally, pronounced heteroskedasticity is often observed near sensor boundaries. The work presented here focuses on developing general model frameworks and implementation methods appropriate for SAXS data. Semiparametric regression refers to models that combine known parametric structures with flexible nonparametric components. Three semiparametric regression model frameworks that are well-suited for handling smooth data are presented. The first model introduced is the standard semiparametric regression model, described as a mixed model with low rank penalized splines as random effects. The second model extends the first to the case of heteroskedastic errors, which violate standard model assumptions. The latent variance function in the model is estimated through an additional semiparametric regression, allowing for appropriate uncertainty estimation at the mean level. The final model considers a data structure unique to SAXS experiments. This model incorporates both radial mean and radial variance data in hopes to better infer three-dimensional shape properties and understand experimental effects by including all available data. Each of the three model frameworks is structured hierarchically. Bayesian inference is appealing in this context, as it provides efficient and generalized modeling frameworks in a unified way. The main statistical contributions of this thesis are from the specific methods developed to address the computational challenges of Bayesian inference for these models. The contributions include new Markov Chain Monte Carlo (MCMC) procedures for numerical approximation of posterior distributions and novel variational approximations that are extremely fast and accurate. For the heteroskedastic semiparametric case, known form posterior conditionals are available for all model parameters save for the regression coefficients controlling the latent model variance function. A novel implementation of a multivariate delayed rejection adaptive Metropolis (DRAM) procedure is used to sample from this posterior conditional distribution. The joint model for radial mean and radial variance data is shown to be of comparable structure to the heteroskedastic case and the new DRAM methodology is extended to handle this case. Simulation studies of all three methods are provided, showing that these models provide accurate fits of observed data and latent variance functions. The demands of scientific data processing in the context of SAXS, where large data sets are rapidly attained, lead to consideration of fast approximations as alternatives to MCMC. {Variational approximations} or {Variational Bayes} describes a class of approximation methods where the posterior distribution of the parameters is approximated by minimizing the Kullback-Leibler divergence between the true posterior and a class of distributions under mild structural constraints. Variational approximations have been shown to be good approximations of true posteriors in many cases. A novel variational approximation for the general heteroskedastic semiparametric regression model is derived here. Simulation studies are provided demonstrating fit and coverage properties comparable to the DRAM results at a fraction of the computational cost. A variational approximation for the joint model of radial mean and variance data is also provided but is shown to suffer from poor performance due to high correlation across a subset of regression parameters. The heteroskedastic semiparametric regression framework has some strong structural relationships with a distinct, important problem: spatially adaptive smoothing. A noisy function with different amounts of smoothness over its domain may be systematically under-smoothed or over-smoothed if the smoothing is not spatially adaptive. A novel variational approximation is derived for the problem of spatially adaptive penalized spline regression, and shown to have excellent performance. This approximation method is shown to be able to fit highly oscillatory data while not requiring the traditional tuning and computational resources of standard MCMC implementations. Potential scientific contribution of the statistical methodology developed here are illuminated with SAXS data examples. Analysis of SAXS data typically has two primary concerns: description of experimental effects and estimation of physical shape parameters. Formal statistical procedures for testing the effect of sample concentration and exposure time are presented as alternatives to current methods, in which data sets are evaluated subjectively and often combined in ad hoc ways. Additionally, estimation procedures for the scattering intensity at zero angle, known to be proportional to molecular weight, and the radius of gyration are described along with appropriate measures of uncertainty. Finally, a brief example of the joint radial mean and variance method is provided. Guidelines for extending the models presented here to more complex SAXS problems are also given.Item Open Access Spn1, a highly conserved and essential node of RNA polymerase II dependent functions(Colorado State University. Libraries, 2011) Almeida, Adam Raymond, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Woody, Robert, committee member; Suchman, Erica, committee memberA multitude of proteins are responsible for regulating the activity of RNA Polymerase II (Pol II) in the nucleus of a eukaryotic cell. Two types of themes are used by these proteins to control transcription: recruitment-regulation and postrecruitment-regulation. The main difference between the two is the rate-limiting step for producing transcript. This rate-limiting step for the first mechanism is the recruitment of Pol II to the promoter. For the second mechanism, Pol II constitutively occupies the promoter, is "poised", and an unknown rate-limiting postrecruitment step prevents transcription from commencing. The highly conserved and essential transcription factor Spn1 was identified as a protein that functions postrecruitment of Pol II and has been characterized for having a direct role at regulating the poised CYC1 gene in Saccharyomyces cerevisiae. This activity has been determined from mutations made within the most conserved portion of Spn1 made up of a highly folded central domain. Little is known about the functions of the N-and C-terminal regions flanking this central domain, which is the focus of the work done here. Genetic characterization indicates that these regions have physiologically relevant and important functions within the cell outside of optimum growth conditions, but do not involve significant regulation of the CYC1 gene. A broader approach of experimentation is likely required to understand all of the Spn1 protein's functions regarding transcription. This led to the observation that Spn1 is able to bind to nucleosomes in vitro and that this interaction is dependent on the N-and C-terminal regions of the protein. The possibility that Spn1 could affect nucleosome dynamics in the cell is consistent with the physical and genetic interactions observed between Spn1 and the Spt6 and Swi/Snf histone chaperone and chromatin remodeling complexes. This result will provide several new avenues for future Spn1 research. A genomic ChIP-chip experiment performed by two independent groups revealed that Spn1 is recruited to a majority of the genes in the yeast genome. Evidence indicates that there are multiple, evolutionarily conserved pathways within the cell that are responsible for determining the rate at which an organism will age that include: ribosome biogenesis, protein translation, mitochondrial activity and function, heterochromatic stability, maintenance of the genome, and apoptosis. The possibility that Spn1 regulates the genes involved in these pathways is highly suggestive that this protein could be an aging factor within the cell. Chronological aging assays revealed that the removal of the N-and C-terminal regions of the Spn1 protein dramatically increase the lifespan of the BY4741 strain of yeast. These results further verify the physiological importance of this protein and the need for further Spn1 research.Item Open Access Spn1, a multifunctional player in the chromatin context(Colorado State University. Libraries, 2016) Li, Sha, author; Stargell, Laurie, advisor; Argueso, J. Lucas, committee member; Hansen, Jeffrey, committee member; Luger, Karolin, committee member; Yao, Tingting, committee memberSpn1 was initially identified as a transcription factor that copurified with Spt6. Spn1 functions in transcription initiation and elongation, mRNA processing and export, histone modification, as well as in heterochromatic silencing. Our recent study demonstrated that Spn1 could bind histones and assemble nucleosomes in vitro. Therefore, Spn1 is a new member of the histone chaperone family. Here we found that Spt6 regulates Spn1-nucleosome interaction and conversely, Spn1 regulates Spt6-H2A-H2B interaction. Co-regulation between Spn1 and Spt6 enables them to be independent histone chaperones in nucleosome assembly. In addition, abrogation of Spn1-Spt6 interaction does not generate cryptic transcripts at certain genes. Furthermore, we identified a new interaction between Spn1 and the histone chaperone Nap1. Spn1, Nap1 and histones can form a large complex. We also found Spt6 could compete Nap1 for Spn1 binding, therefore disrupting Spn1-Nap1 interaction and releasing Nap1. In sum, Spn1 plays a multifunctional role in the chromatin context via dynamic interactions with its binding partners.Item Open Access Statistical innovations for estimating shape characteristics of biological macromolecules in solution using small-angle x-ray scattering data(Colorado State University. Libraries, 2016) Alsaker, Cody, author; Breidt, F. Jay, advisor; Estep, Don, committee member; Kokoszka, Piotr, committee member; Luger, Karolin, committee memberSmall-angle X-ray scattering (SAXS) is a technique that yields low-resolution images of biological macromolecules by exposing a solution containing the molecule to a powerful X-ray beam. The beam scatters when it interacts with the molecule. The intensity of the scattered beam is recorded on a detector plate at various scattering angles, and contains information on structural characteristics of the molecule in solution. In particular, the radius of gyration (Rg) for a molecule, which is a measure of the spread of its mass, can be estimated from the lowest scattering angles of SAXS data using a regression technique known as Guinier analysis. The analysis requires specification of a range or “window” of scattering angles over which the regression relationship holds. We have thus developed methodology and supporting asymptotic theory for selection of an optimal window, minimum mean square error estimation of the radius of gyration, and estimation of its variance. The theory and methodology are developed using a local polynomial model with autoregressive errors. Simulation studies confirm the quality of the asymptotic approximations and the superior performance of the proposed methodology relative to the accepted standard. We show that the algorithm is applicable to data acquired from proteins, nucleic acids and their complexes, and we demonstrate with examples that the algorithm improves the ability to test biological hypotheses. The radius of gyration is a normalized second moment of the pairwise distance distribution p(r), which describes the relative frequency of inter-atomic distances in the structure of the molecule. By extending the theory to fourth moments, we show that a new parameter ψ can be calculated theoretically from p(r) and estimated from experimental SAXS data, using a method that extends Guinier's Rg estimation procedure. This new parameter yields an enhanced ability to use intensity data to distinguish between two molecules with different but similar Rg values. Analysis of existing structures in the protein data bank (PDB) shows that the theoretical ψ values relate closely to the aspect ratio of a molecular structure. The combined values for Rg and ψ acquired from experimental data provide estimates for the dimensions and associated uncertainties for a standard geometric shape, representing the particle in solution. We have chosen the cylinder as the standard shape and show that a simple, automated procedure gives a cylindrical estimate of a particle of interest. The cylindrical estimate in turn yields a good first approximation to the maximum inter-atomic distance in a molecule, Dmax, an important parameter in shape reconstruction. As with estimation of Rg, estimation of ψ requires specification of a window of angles over which to conduct the higher-order Guinier analysis. We again employ a local polynomial model with autoregressive errors to derive methodology and supporting asymptotic theory for selection of an optimal window, minimum mean square error estimation of the aspect ratio, and estimation of its variance. Recent advances in SAXS data collection and more comprehensive data comparisons have resulted in a great need for automated scripts that analyze SAXS data. Our procedures to estimate Rg and ψ can be automated easily and can thus be used for large suites of SAXS data under various experimental conditions, in an objective and reproducible manner. The new methods are applied to 357 SAXS intensity curves arising from a study on the wild type nucleosome core particle and its mutants and their behavior under different experimental conditions. The resulting Rg2 values constitute a dataset which is then analyzed to account for the complex dependence structure induced by the experimental protocols. The analysis yields powerful scientific inferences and insight into better design of SAXS experiments. Finally, we consider a measurement error problem relevant to the estimation of the radius of gyration. In a SAXS experiment, it is standard to obtain intensity curves at different concentrations of the molecule in solution. Concentration-by-angle interactions may be present in such data, and analysis is complicated by the fact that actual concentration levels are unknown, but are measured with some error. We therefore propose a model and estimation procedure that allows estimation of true concentration ratios and concentration-by-angle interactions, without requiring any information about concentration other than that contained in the SAXS data.Item Open Access Steady-state and time-resolved spectroscopy to probe the effects of confinement on Cy3 and the dynamics of AOT/iso-octane reverse micelles(Colorado State University. Libraries, 2010) McPhee, Jeffrey, author; Van Orden, Alan K., advisor; Levinger, Nancy E., committee member; Barisas, B. George, committee member; Prieto, Amy L., committee member; Luger, Karolin, committee memberThis dissertation describes the use of steady-state and time-resolved spectroscopy to probe the effects of localized confinement on the water soluble dye Cyanine-3 (Cy3) and the dynamics of intermicellar interactions using fluorescence correlation spectroscopy (FCS). The first set of experiments presents a wide range of steady-state and time-resolved spectroscopy data which indicate that the Cy3 molecules form H-aggregates at concentrations so dilute (nM) that this behavior is not observed in bulk aqueous solution. This unique behavior allowed for a series of FCS and dynamic light scattering measurements to be performed on the same system. These results indicate the formation of a transient reverse micelle dimer, whose lifetime has been identified to be on the order of 15 μs. Furthermore, preliminary experiments are presented on the same reverse micelle system containing the Rhodamine 6G and the results are consistent with those obtained for Cy3 in the reverse micelles. Lastly, fluorescence resonance energy transfer within the reverse micelles was investigated using Cy3 and Cy5. The preliminary results suggest that FRET may be occurring within this extremely confined environment. The work as a whole provides insight into the nature of confinement as well as the dynamics occurring within the world of reverse micelles.Item Open Access Synthetic studies of ecteinascidin 743 and fennebricin B(Colorado State University. Libraries, 2017) Le, Vy Hoang, author; Williams, Robert M., advisor; Kennan, Alan J., committee member; Finke, Richard G., committee member; Luger, Karolin, committee memberEcteinascidin 743 (Et-743, trabectidin or Yondelis®) possesses an impressive antitumor activity that it was approved for treatments of several cancer types worldwide. Since this natural product only presents as a trace amount in the nature, the main supply of this drug for research and commercial use is from laboratory synthesis. Many syntheses of Et-743 have been reported including three total syntheses, two formal syntheses and two semisyntheses. The biological activities of fennebricin B were unknown due to the scarcity of this natural product. However, fennebricin B share a common pentacyclic core with Et-743, thus may also possess interesting biological activities. Our group completed our formal synthesis of the natural product in 2008, featuring the Pictet-Spengler reaction to construct the pentacyclic core of Et-743. Our work, however, also produced both desired and undesired pentacycle without selectivity. We herein described an improved formal synthesis of Et-743 employing bromine auxiliary to generate the pentacylic core of Et-743 with the desired regioisomer as the only product. This approach was also utilized in the synthetic studies toward the total synthesis of fennebricin B.Item Open Access TATA binding protein dynamics within the cellular chromatin landscape(Colorado State University. Libraries, 2013) Yearling, Marie N., author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Nyborg, Jennifer K., committee member; Yao, Tingting, committee member; Slayden, Richard A., committee memberRNA polymerase II (RNAPII) is a twelve subunit enzyme that catalyzes messenger (mRNA) in eukaryotic organisms. A number of essential transcription factors associate with RNAPII to form the pre-initiation complex (PIC) at gene promoter regions. TATA binding protein (TBP) is one member of the transcription machinery indispensable for transcription. At some genes, the formation of the PIC correlates strongly with the transcription output (Ptashne, 2005). These genes have a low occupancy of TBP and other PIC components prior to activation. Upon activation, these factors assemble onto the promoter and transcriptional output increases. Genes that become active upon PIC formation are termed recruitment regulated because their transcription is regulated at the level of recruitment of the PIC to the promoter. While recruitment of the PIC is required for transcription, in many cases promoter-occupancy is not correlated with transcription output. Post-recruitment gene regulation has been conserved across evolution from prokaryotes to humans (Choy et al., 1997; Guenther et al., 2007). At these genes, TBP and RNAPII and other transcription-related factors occupy the promoter region regardless of whether transcription is occurring. Upon gene activation, the occupancy increases only slightly when compared to the increase in transcript level. These genes are described as being poised. At poised genes, these transcription proteins constitutively occupy the promoter region, but it is unknown if the promoter interaction is stable or dynamic. One principal objective of my work was to investigate TBP-promoter dynamics at the poised CYC1 gene in yeast. Due to the genetic and biochemical amenability of the yeast system, studies of the transition of poised CYC1 gene to the active form have provided key insights into the sophisticated molecular requirements involved in this post-recruitment process. To describe the dynamics of the transcription complex bound at the CYC1 promoter I developed a TBP exchange assay. The results suggest that the TBP within the RNAPII transcription complex exists in a relatively stable configuration at the poised gene prior to activation. Upon induction, TBP-promoter dynamics increased at the CYC1 gene promoter. Rapid exchange during activated transcription was also observed at other genes, including at recruitment regulated gene promoters. Overall, we found rapid TBP-promoter exchange to be associated with active transcription. From my findings I propose a model where frequently clearing the promoter offers a functional advantage to support activated transcription.Item Open Access The chromatin binding factor Spn1 contributes to genome instability in Saccharomyces cerevisiae(Colorado State University. Libraries, 2018) Thurston, Alison K., author; Stargell, Laurie, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey, committee member; Luger, Karolin, committee memberMaintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a "healthy level" of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle.Item Open Access The influence of H2AX and γH2AX on chromatin condensation(Colorado State University. Libraries, 2010) Norskog, Sarah Samaya, author; Hansen, Jeffrey, advisor; Luger, Karolin, committee member; Zabel, Mark, committee memberChromatin composition and structure are essential for the condensation of the genome and the regulation of a wide range of cellular activities. Chromatin condensation is thought to be controlled predominantly through interactions mediated by the unstructured amino terminal domains of the core histones H4, H3, H2A and H2B. In addition to the amino terminal domain, histone H2A contains an unstructured carboxyl terminal domain. Multiple H2A variants, many differing from major type H2A in this C-terminal domain sequence, have been identified. The most studied of variant is H2AX, which contains a conserved serine residue that becomes phosphorylated following double strand DNA breakage (yH2AX). Although the phosphorylation of the H2AX has been identified as a key step in major genomic activities, the basic mechanism by which it functions remains controversial. Here, I have determined the structural role of H2AX and yH2AX using in vitro assays which utilize defined nucleosomal arrays. H2AX and yH2AX alter chromatin folding under high salt concentrations but show no discernable differences in low concentrations of salt or under conditions which favor oligomerization. The phosphorylation of H2AX does not alter the folding or oligomerization relative to the unphosphorylated form, indicating yH2AX more likely functions as a signaling and recruitment motif rather than as a chromatin secondary structure remodeling factor.Item Open Access The spliceosome recycling factor, SART3, regulates H2B deubiquitination by Usp15(Colorado State University. Libraries, 2014) Long, Lindsey J., author; Yao, Tingting, advisor; DeLuca, Jennifer, committee member; Luger, Karolin, committee member; Wilusz, Jeffrey, committee memberIn eukaryotes plasticity of chromatin architecture is paramount to allow proper regulation of processes such as transcription regulation, DNA repair, and DNA replication. Modulation of chromatin dynamics is primarily achieved via signaling to chromatin modifiers and remodelers though a complex code of histone post-translational modifications (PTMs). These PTMs include methylation, acetylation, phosphorylation, and ubiquitination. In comparison to other histone PTMs, attachment of the 8.5 kDa ubiquitin (Ub) protein stands out due to its considerable size. The majority of histone monoubiquitination occurs on histones H2A and H2B (at lysine residues 119 and 120, respectively), and these modifications have roles in the regulation of many cellular processes including transcription, pre-mRNA processing, and DNA damage repair. To uncover the mechanisms underlying various functions associated with ubiquitinated histones, we generated non-hydrolyzable Ub-histone mimics and assembled them into H2A/H2B dimers or nucleosomes. Quantitative mass spectrometry was employed to identify proteins that bound to unmodified or modified histone dimers and mononucleosomes. We also found that, within the context of a mononucleosome, Ub, when attached to H2B, partially obscures the H2A/H2B acidic patch. Among the proteins that were identified, a deubiquitinating enzyme (DUB), Usp15, exhibited high affinity and specificity towards ubiquitinated histone dimers. Further characterization demonstrated that Usp15 is a bona fide histone DUB and preferentially deubiquitinates Ub-containing histone octamers versus Ub-containing mononucleosomes. Usp15 associates with the U4/U6 spliceosome recycling factor, SART3, which we found also bound to histones. These interactions result in more efficient histone deubiquitination by Usp15. In cells, depletion of SART3 results in elevated ubH2B levels that we show is due to a decreased rate in H2B deubiquitination. These observations indicate SART3 may play a role in regulating ubH2B dynamics as a possible mechanism by which regulate alternative splicing and transcription. Depletion of SART3 also alters transcriptional and alternative splicing patterns. By chromatin immunoprecipitation, we confirmed that SART3 localizes to at least a subset of genes whose transcription decreased upon SART3 depletion. Future studies will be designed to elucidate the mechanism by which Usp15, SART3, and ubH2B work together to regulate transcription and alternative splicing.