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A Case for Ecclesiastical Minting of Anglo-Viking Coins
Get PDFWithin a few decades of the first Viking conquests of England, strong currency systems developed in Viking controlled parts of England. This preceded minting and the use of coins as currency in other Viking territories by about one hundred years. Herein, a case is made that the early development of currency in Anglo-Viking England is poorly explained by secular minting by Viking authorities but is well explained by ecclesiastical minting. The implications of ecclesiastical minting are discussed in the context of other sources of information on the church in early Anglo-Viking England
Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair
Get PDFDNA-mediated charge transport (DNA CT) is well established in both ground and excited state systems. Although theoretical models are still being developed, it is clear that the integrity of the extended π-stack of the aromatic heterocycles, the nucleic acid bases, plays a critical role. Electron donors and acceptors must be electronically well coupled into the π-stack, typically via intercalation. Perturbations that distort the π-stack, such as single-base mismatches, abasic sites, base lesions, and protein binding that kinks the double helix, attenuate DNA CT dramatically.
This thesis encompasses work that first aims to understand how DNA duplex structure informs characteristics of DNA CT and then continues to develop an understanding of the role these structural features play in biological systems. To contextualize these advancements, this first chapter outlines foundational work that has shown ways that DNA structure influences its ability to conduct charge.
Next, experiments were conducted on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. These results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.
This exquisite spin selectivity begged the question: how might biology take advantage of such a spin filter? Photolyase and cryptochromes both have been shown to exhibit magnetosensitive chemistry nearby a DNA binding pocket, and photolyase had previously been shown capable of DNA CT. Thus, electrochemical studies were conducted to monitor the repair of cyclobutane pyrimidine dimer lesions by E coli photolyase and truncated A Thaliana Cryptochrome 1 with an applied magnetic field. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 Gauss, though spin selective DNA CT is not involved. These data illustrate how cyclobutane dimer repair could be used in a biological compass that is informed by the angles of Earth’s magnetic field.
Next DNA-mediated electrochemistry and atomic force microscopy studies were used to describe a role for redox active [4Fe4S] clusters in DNA-mediated charge transport signaling. DNA-modified electrochemistry shows that the [4Fe4S] cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III, a base excision repair enzyme, cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. These data are then described using an equilibrium model which elucidates fundamental characteristics of this redox chemistry that allow DNA CT to coordinate the activities of DNA repair enzymes across the genome.
The importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process is then further explored. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.</p
Engineering Biosynthetic Pathways in Cell-Free Systems for Sustainability and Chemical Innovation
Get PDFThis work presents the cell-free transcription-translation (TX-TL) system as a research and development platform for renewable synthesis and molecular discovery. TX-TL is easy to use and provides a biomolecular breadboard for the rapid prototyping and engineering of biosynthetic pathways. This work has validated the capabilities of the cell-free TX-TL system for simultaneous protein expression and chemical synthesis. Specifically, this work shows that TX-TL supports the conversion of intermediates from carbohydrate metabolism and amino acids into valuable compounds. Metabolic flux through cofactor dependent pathways confirms that active cofactor metabolism is occurring in TX-TL. This work has also demonstrated the industrial relevance of TX-TL through exploring design space of a biosynthetic pathway for improved product yield and expanding substrate scope of another biosynthetic pathway.
Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. In this work, we have verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as the pathway bottleneck. We demonstrate the reliability of using linear DNA in TX-TL as a tool for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Pathway constructs of varying pathway enzyme expression levels are tested in TX-TL and in vivo to identify correlations between the two systems, and we find that the production of BDO is correlated to the expression of enzyme ald in both systems. The use of TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Different pathway combinations are also tested in TX-TL for its application in pathway ranking. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.
TX-TL simulates a customizable cellular environment that can be controlled by manipulating pH, changing cellular components, or adding exogenous substrates. By adding linear DNA encoding individual enzymes of the violacein pathway and tryptophan analogs in TX-TL reactions, we have discovered new violacein analogs. TX-TL enables rapid production of natural product analogs with diverse substitution, which allows small-scale biosynthesis of potential drug candidates and offers a new platform for drug discovery. This work also presents TX-TL as a platform for protein engineering. Residues targeted for site-saturated mutagenesis were identified with protein-ligand docking. Linear DNAs of individual enzyme mutants were added into TX-TL reactions to screen for improved enzyme variant. Screening result indicates vioE mutant Y17H reduces byproduct formation and redirects metabolic flux towards target metabolites. Protein engineering for improved enzyme activity can further expand the substrate scope of a natural product pathway and result with more natural product analogs that can be applied for medical applications.
This work demonstrates that the cell-free TX-TL system can become a valuable tool that complements the process of engineering biosynthesis in the whole cell in vivo system or the purified protein in vitro system. Future engineering and development of the TX-TL system can further expand the chemical space for biosynthesis.</p
Analysis and Characterization of Titan Aerosol Simulants by Mass Spectrometry
Get PDFSaturn’s large moon of Titan is unique as the only solid body in the outer solar system to possess a dense atmosphere, which is comprised of mostly nitrogen and methane. Atmospheric chemistry on this moon creates aerosols comprised of carbon, hydrogen, and nitrogen. Laboratory based synthesis of simulated Titan aerosols, also called tholins, led to molecules with rich chemical diversity, functionalities, and molecular weights. While this diversity has intriguing implications for Titan, it complicates the analysis and characterization of these simulants. This thesis discusses three different mass spectrometry methodologies, prioritizing a combination of mission applicability, broad spectrum characterization, and specific functional group identification. Chapters 2 and 3 utilize a home-built Direct Analysis in Real Time (DART) ionization source, termed EZ-DART, to test its suitability for the analysis of these simulants and the source’s mission applicability. Chapter 2 focuses on the development and characterization of the EZ-DART source. Chapter 3 presents the analysis of Titan aerosol simulants, produced in a new and unique way, by EZ-DART, allowing for the identification of various compounds of astrobiological significance. Solid phase microextraction gas chromatography mass spectrometry (SPME-GC-MS) is utilized in Chapter 4 with different Titan aerosol simulants, identifying multiple compounds of significance to Titan and astrobiology. The versatility allowed by SPME increases the applicability of GC for future lander missions. While not mission applicable, Chapter 5 shows the breadth of primary amines in Titan aerosol simulants through the use of supramolecular complexation to 18-crown-6 ether. This technique not only enabled the unambiguous identification of primary amines, but also allowed for the structural characterization of some components. The range of methods and identified compounds discussed within this thesis demonstrates not only the rich chemistry of these Titan aerosol simulants, but also introduces intriguing possibilities for Titan’s atmospheric chemistry and presents potential significance to astrobiology
Coherent Structures, their Interactions, and their Effects on Passive Scalar Transport and Aero-Optic Distortion in a Turbulent Boundary Layer
Get PDFThis thesis focused on the characterization of coherent structures and their interactions in a turbulent boundary layer using data from particle image velocimetry (PIV) measurements performed at Caltech and from a direct numerical simulation (DNS) of Wu et al. (2017). Connections were identified between instantaneous and statistical descriptions of coherent velocity structures, through the analysis of representative models for their structures derived from the resolvent analysis of McKeon and Sharma (2010). The representative models were used in a novel conditional averaging technique to identify the average behavior of small scales about variations in the large-scale streamwise velocity field. Based upon the results of this analysis, a hypothesis for a scale interaction mechanism was proposed involving three-dimensional critical layers. The modeling and analysis methods were then applied to the aero-optic problem in which optical beams are observed to be distorted after passing through variable-density turbulent flows. Measurements using simultaneous PIV and an aero-optic sensor called a Malley probe (Malley, Sutton, and Kincheloe, 1992) were conducted in an incompressible, mildly-heated turbulent boundary layer with Prandtl number of 0.7. A conditional averaging analysis of the data identified that the nonlinear interaction of two scales was most correlated to the aero-optic distortion. The modeling of this interaction using resolvent modes led to new insights regarding the instantaneous relationship between the velocity and scalar fields over a range of Prandtl numbers.</p
Elucidating the Role of [4Fe4S] Clusters in DNA Replication and Repair Proteins
Get PDF[4Fe4S] clusters, redox cofactors, have been discovered in DNA processing enzymes ranging from bacterial base excision repair glycosylases to eukaryotic DNA polymerases. Bacterial repair proteins are activated toward redox activity when bound to DNA and can take advantage of DNA-mediated charge transport (DNA CT) to search the genome for lesions. DNA CT involves the rapid transport of charges through the π-stacked base pairs and is sharply attenuated in the presence of lesions, mismatches, or other stacking perturbations. Thus, [4Fe4S] repair proteins use this chemistry to rapidly redistribute to target lesions and communicate with one another over long distances.
The general function of [4Fe4S] clusters in bacterial DNA repair has received much attention, but previous efforts have left several critical questions unanswered. First, while the redox potential of these proteins is affected by DNA binding, the relative importance of the negatively-charged DNA, the protein environment surrounding the cluster, and solvent has remained unclear. Second, the importance of [4Fe4S] clusters and DNA CT to human disease has never been directly addressed. The biological consequences of this chemistry are certainly a pressing issue, as numerous disease-relevant mutations in the human homologues of well-studied repair proteins have been recorded. Finally, the existence of [4Fe4S] clusters in eukaryotic DNA replication proteins in general, and in the B-family DNA polymerases in particular, was entirely unexpected. The function of the [4Fe4S] cluster in replication proteins was far from obvious, and the functional differences from repair proteins made them difficult to explain even in the context of CT signaling. Herein, these questions have been addressed using a combination of electrochemical, spectroscopic, and biochemical approaches.
First, we describe the use of pyrolytic graphite edge electrodes (PGE) and S K-edge X-ray absorption spectroscopy (XAS) to address the influence of protein environment, DNA, and solvation on the [4Fe4S] cluster redox potential in the bacterial base excision repair glycosylases endonuclease III (EndoIII) and MutY. The PGE surface is rough and favorable for protein binding; electron transfer can be further enhanced in the presence of carbon nanotubes. Electrochemical signals for EndoIII and MutY in the absence of DNA are large and reproducible, and a potential shift upon DNA binding is observed. With respect to studying proteins in the absence of DNA, the PGE electrode represents a significant advance over previously used highly-oriented pyrolytic graphite (HOPG), which is hydrophobic and difficult to prepare. To test the effect of protein environment on redox potential, a series of EndoIII point mutants were prepared in which the charge within 5 Å of the cluster was reversed or added in. None of these mutations induced a significant shift in redox potential relative to wild type, arguing that DNA electrostatics are the dominant factor in potential modulation. In parallel, XAS studies were performed on EndoIII and MutY in the presence and absence of DNA, and in the presence and absence of solvent. Ligating cysteinyl thiols and inorganic S atoms in the [4Fe4S] cluster absorb at different intensities in XAS depending on solvent environment and local electrostatics; these changes, in turn, directly correlate to redox potential. By XAS, DNA was found to induce a significant shift in absorbance, and thus potential; the removal of solvent had a smaller effect. Together, these studies provide new approaches for the study of DNA-binding [4Fe4S] proteins and reveal the critical role of DNA in tuning the redox potential.
Second, we report on a novel mutation in human MUTYH identified from a colorectal cancer patient and confirmed to be pathological. MUTYH is responsible for repairing certain lesions induced by oxidative stress and is thus frequently implicated in cancer. This new variant, C306W, contains a mutation in one of the cysteines that ligates the [4Fe4S] cluster. Electrochemistry, activity and DNA binding assays, and spectroscopic analyses were performed for C306W alongside wild type MUTYH and two other disease-relevant mutants, Y179C and G396D, with an unaltered cluster environment. From this work, it is now clear that C306W can still bind a cluster, but it is susceptible to oxidative degradation to the [3Fe4S]+ state upon redox signaling in an aerobic environment. Consequently, enzymatic activity is very low, and DNA binding is poor. Overall, this represents the first complete characterization of the [4Fe4S] cluster in a human homologue of MutY, and the first demonstration of pathology resulting from a mutation that primarily affects the [4Fe4S] cluster.
Moving into DNA replication proteins, we report on the characterization of the [4Fe4S] cluster in yeast DNA polymerase (Pol) δ, the eukaryotic lagging strand polymerase. Pol δ shows reversible electrochemical signals at a midpoint potential indistinguishable from EndoIII under the same conditions, and EPR spectroscopy confirms use of the [4Fe4S]3+/2+ couple. The electrochemical signal is attenuated on DNA containing an abasic site or a CA mismatch, confirming that Pol δ is capable of DNA-mediated signaling. Bulk electrolysis and photooxidation were used to oxidize Pol δ under anaerobic conditions, and activity assays were carried out using oxidized or untreated protein. Oxidation stalls replication activity, while electrochemical reduction of oxidized samples restores activity to untreated levels. These results thus reveal that cluster oxidation serves as a reversible switch regulating Pol δ activity, suggesting an in vivo role in responding to replication stress, especially oxidative stress. In an effort to address these possibilities, we have carried out preliminary efforts in the characterization of two potentially CT-deficient mutants, W1053A and Y1078A. Both mutants were found to be too structurally unstable to proceed with in vivo experiments, but they can serve to guide future efforts in this direction.
Finally, a strategy to examine charge transport through RPA-bound single-stranded DNA is reported. RPA is the eukaryotic single-stranded binding protein and forms a protective coat around vulnerable unwound DNA at replication forks. Given the importance of redox signaling in replication proteins, we aimed to use photooxidation experiments to determine if CT through RPA is a viable pathway; if so, this would open up a large set of long-range transfer pathways to [4Fe4S] proteins in replication. These efforts are ongoing, but the experimental strategy and initial efforts are discussed.</p
Quantum Computation and Information Storage in Quantum Double Models
Get PDFThe results of this thesis concern the real-world realization of quantum computers, specifically how to build their "hard drives" or quantum memories. These are many-body quantum systems, and their building blocks are qubits, the same way bits are the building blocks of classical computers.
Quantum memories need to be robust against thermal noise, noise that would otherwise destroy the encoded information, similar to how strong magnetic field corrupts data classically stored in magnetic many-body systems (e.g., in hard drives). In this work I focus on a subset of many-body models, called quantum doubles, which, in addition to storing the information, could be used to perform the steps of the quantum computation, i.e., work as a "quantum processor".
In the first part of my thesis, I investigate how long a subset of quantum doubles (qudit surface codes) can retain the quantum information stored in them, referred to as their memory time. I prove an upper bound for this memory time, restricting the maximum possible performance of qudit surface codes.
Then, I analyze the structure of quantum doubles, and find two interesting properties. First, that the high-level description of doubles, utilizing only their quasi-particles to describe their states, disregards key components of their microscopic properties. In short, quasi-particles (anyons) of quantum doubles are not in a one-to-one correspondence with the energy eigenstates of their Hamiltonian. Second, by investigating phase transitions of a simple quantum double, D(S3), I map its phase diagram, and interpret the physical processes the theory undergoes through terms borrowed from the Landau theory of phase transitions.</p
Decoding the Regulatory Genome: Quantitative Analysis of Transcriptional Regulation in Escherichia coli
Get PDFOver the past decades DNA sequencing has become significantly cheaper and faster, which has enabled the accumulation of a huge amount of genomic data. However, much of this genomic data is illegible to us. For noncoding regions of the genome in particular, it is difficult to determine what role is played by specific DNA sequences. Here we focus on regions of DNA that play a role in transcriptional regulation. We develop models and techniques that allow us to discover new regulatory sequences and better understand how DNA sequence determines regulatory output.
We start by considering how quantitative models serve as a powerful tool for testing our understanding of biological systems. We apply a statistical mechanical framework that incorporates the Monod-Wyman-Changeux model to analyze the effects of allostery in simple repression, using the lac operon as a test case. By fitting our model to experimental data, we are able to determine the values of the unknown parameter values in our model. We then show that we can use the model to accurately predict the induction responses of an array of simple repression constructs with a variety of repressor copy numbers and repressor binding energies.
Next, we consider how the DNA sequence of a promoter region can provide details about how the promoter is regulated. We begin by describing an approach for discovering regulatory architectures for promoters whose regulation has not previously been studied. We focus on six promoters from E. coli including three well-studied promoters (rel, mar, and lac) to serve as test cases. We use the massively parallel reporter assay Sort-Seq to identify transcription factor binding sites with base-pair resolution, determine the regulatory role of each binding site, and infer energy matrices for each binding site. Then, we use DNA affinity chromatography and mass spectrometry to identify each transcription factor.
We conclude with an in vivo approach for analyzing the sequence-dependence of transcription factor binding energies. Again using Sort-Seq, we show that we can represent transcription factor binding sites using energy matrices in absolute energy units. We then show that these energy matrices can be used to accurately predict the binding energies of mutated binding sites. We provide several examples of how understanding the relationship between DNA sequence and transcription factor binding provides us with a foundation for addressing additional scientific topics, such as the co-evolution of transcription factors and their binding sites.</p
Noncommutative Biology: Sequential Regulation of Complex Networks and Connected Matter
Get PDFDuring animal development from zygote to adult, a limited set of regulatory molecules are autonomously deployed in the service of tissue-specific gene expression (reviewed in chapter 1). Inherent in the process is the tension that single cells sample heterogeneous expression states while robustly maintaining a collective final outcome. This thesis addresses theoretical issues that help resolve the paradox that one cell simultaneously contains the fate information of many.
Previous models of development have likened cell fate to minima on a smooth potential energy surface. Such static pictures can be misleading because they suggest the egg knows the path it will take to the adult before it divides even once. Recognition that the potential analogy is an oversimplification has led others to propose that the surface is actually nonsmooth. Chapter 2 reviews the theoretical basis for smooth potentials and resolves these problems by appealing to the tangent space of gene expression. It is then shown that if the potential difference is sufficient to characterize the difference between egg and adult, then the tangent space controls on gene expression are one-dimensional. Furthermore, a shortcoming of models ignoring the connectivity and common origin of dividing cells is that they erect artificial barriers between alternative fates. A fundamentally different picture is sketched wherein the difference between egg and adult is schematized as the shape of the locus of equipotential fates accessible at the same point in time. The conjugacy of space and time is invoked to explain how the requirement that each fate be on a line of equipotential is the same as requiring that each alternative fate move the same distance down the surface at each step. The developmental trajectory is deterministic but not known in advance because it needs to be ascertained at each step which way cells "turn" in order to maintain their equipotential relationship. Chapters 3 and 4 refine this sequential model of collective development with specific examples.
A simple solution to the problem of cell-type specific gene expression is combinatorial binding of transcription factors at promoters. It is shown in chapter 3 that such models result in substantial information bottlenecks, because all cell fate information is concentrated at the start. We explore a novel, noncommutative model of gene regulation—known as sequential logic—that spreads the information out over time. It is shown using time sequences of noncommutative controllers that targets which otherwise would have been activated together can be regulated independently. We derive scaling laws for two noncommutative models of regulation, motivated by phosphorylation/neural networks and chromosome folding, respectively, and show that they scale super-exponentially in the number of regulators. It is also shown that specificity in control is robust to loss of a regulator. Consequently, sequential logic overcomes the information bottleneck in complex problems and enables novel solutions through roundabout strategies. The theoretical results are connected to real biological networks demonstrating specificity in the context of promiscuity.
Noncommutative sequential logic has improved storage capacity, but it does not specify who or what supplies the sequences of input that determine cell fate. Chapter 4 offers a solution by way of the seemingly unrelated problem of looping in twisted strings. Cells and strings obey a set of common space-time constraints, ultimately due to the conservation of energy. It is argued that the most parsimonious allocation of energy from the straight to strained string is the one in which each segment sees the same share of the total. Planar looping is shown to be a consequence of the parsimony principle and the Euler-Poincaré equations for rotational motion in the presence an applied torque. We then solve the problem for the looping of a twisted string; with two strains, the Euler-Poincaré predict a different answer than the classical Frenet-Serret equations. Using the results of chapter 2, it is concluded that the Frenet-Serret curvatures assigned ahead of time are not guaranteed to generate space curves that conserve energy: the predicted string has localized strains the Euler-Poincaré solution lacks. Rotational dynamics of strings are connected to developing organisms by postulating conserved RNA polymerase as an analog of angular momentum, and transcriptional activity as energy. Alternative fates along a one-dimensional "string" of dividing cells are possible by finding the RNAP distribution that conserves transcriptional activity along a curve of constant developmental potential. Consequently, each alternative fate samples a different sequence of changes to the distribution as it follows a local gradient downhill from high to low developmental potential over time.
In conclusion, regulation in the tangent space of gene expression resolves the paradox that development has a unique solution specified in the DNA of the egg which cannot be determined with certainty until completion of the adult. Noncommutative sequential logic generates complexity that cannot be realized at the start, while interdependent cells (and strings) require time to ensure that each fate is at the same potential difference from a common ancestor. This fundamental reimagining of the Waddington framework can be tested using new multiplexed mRNA imaging technologies that preserve the spatial context of cells in developing tissue.</p
Development of Stereoselective Iridium-Catalyzed Allylic Alkylation Methods
Get PDFThe Stoltz group, and moreover the synthetic community at large, has long been interested in the development of methods for the synthesis of enantioenriched all-carbon quaternary stereocenters. Historically, our group’s interest has centered on palladium-catalyzed allylic alkylation, though recently effort has moved to include the study of iridium catalysts. This thesis presents four related projects, all unified by the use of enantioselective iridium-catalyzed allylic alkylation to construct highly-congested C–C bonds.
First, the development of the first diastereo-, enantio-, and regioselective iridium-catalyzed allylic alkylation reaction of prochiral enolates to form vicinal tertiary and all-carbon quaternary stereodyads with alkyl-substituted allylic electrophiles is described. Next, the first enantioselective iridium-catalyzed allylic alkylation reaction of a masked acyl cyanide (MAC) nucleophile is presented, representing a rare example of umpolung strategy in iridium-catalyzed allylic alkylation. Additionally, the application of a MAC reagent in the first highly enantioselective iridium-catalyzed allylic alkylation to provide access to products bearing an allylic all-carbon quaternary stereocenter is detailed. The use of the MAC nucleophile enables the one-pot preparation of α-quaternary carboxylic acids, esters, and amides with a high degree of enantioselectivity. Finally, the first enantioselective transition metal-catalyzed allylic alkylation providing access to acyclic products bearing vicinal all-carbon quaternary centers is presented.</p