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The Development of RNA-based Control Systems to Regulate Signaling and Dictate Cell Fate in a Model MAPK Pathway
Get PDFCells integrate extracellular information via native signaling pathways to spatially and temporally coordinate complex tasks such as development and the immune response. Cellular programming holds the potential of harnessing the sophisticated and complex biological processes of living cells for diverse applications. In the last decade, cellular reprogramming has emerged as a viable therapeutic strategy. In large, reprogramming strategies have relied on statically programmed levels of gene expression to alter cellular behaviors. To construct more sophisticated programs requires dynamic control of expression and strategies for the facile construction of complex control architectures. Additionally, the application of synthetic programs to the control of native regulatory pathways requires the development of tools for interfacing with these pathways, as well as the construction of stringent controllers. Further, control systems composed of modular and tunable elements will facilitate the expansion of synthetic circuitry to a wide array of natural networks with varying system properties.
Here we describe the development of RNA-based control systems to regulate signaling and dictate cell fate in a model mitogen-activated protein kinase (MAPK) pathway. We construct networks of RNA-based control systems that interface with the Saccharomyces cerevisiae mating pathway to dictate entry into one of three programmed alternative fates dependent on environmental stimuli. We present a readily translatable method for identifying control points within natural networks that enable the construction of a modular interface between synthetic circuitry and native networks. In building these networks, we demonstrate the rational tuning of circuit performance via the exchange of well-defined parts to compose networks capable of actuating changes in cellular behavior in response to environmental cues. Further, we construct network architectures which facilitate reduced interference from simultaneously integrated opposing programs and identified sensitive parameters for engineering robust circuit performance. Finally, we present the development of a novel RNA-based control element for the regulation of both synthetic and endogenous transcripts. This work provides a model for engineering systems that regulate signaling and direct cell fate which may be applied to additional decision-making pathways to advance tissue engineering strategies, treat diseases, and study the behavior of natural regulatory networks.</p
Biomimetic Accommodating Intraocular Lens
Get PDFThe crystalline lens allows the eye to focus on near and far objects. During the aging process, it loses its ability to focus and often becomes cloudy during cataract formation. At this point, traditional medical therapy replaces the lens with an artificial replacement lens. Although replacement lenses for the crystalline lens have been implanted since 1949 for cataract surgery, none of the FDA-approved lenses mimic the anatomy of the natural lens. Hence, they are not able to focus in a manner similar to the youthful lens. Instead, they function in a manner similar to the aged lens and only provide vision at a single distance or at a very limited range of focal distances. Patients with the newest implants are often obliged to use reading glasses when using near vision, or suffer from optical aberrations, halos, or glare. Therefore, there is a need to provide youthful vision after lens surgery in terms of focusing ability, accurate optical power, and sharp focus without distortion or optical aberrations.
This thesis presents an approach to restoring youthful vision after lens replacement. An intraocular lens (IOL) that can provide accurate visual acuity along with focusing ability is proposed. This IOL relies on the natural anatomy and physiology of the eye, and therefore is actuated in a manner identical to the natural lens. In addition, the lens has the capability for adjustment during or after implantation to provide high-acuity vision throughout life.
The natural anatomy and physiology of the eye is described, along with lens replacement surgery. A lens design is proposed to address the unmet need of lens-replacement patients. Specific care in the design is made for small surgical incisions, high visual acuity, adjustable acuity over years, and the ability to focus similar to the natural lens. Methods to test the IOL using human donor tissue are developed based upon prior experiments on the ex vivo natural lens. These tools are used to demonstrate efficacy of the newly developed accommodating intraocular lens.
To further demonstrate implant feasibility, materials and processes for building the lens are evaluated for biocompatibility, endurance, repeatable manufacture, and stability. The lens biomechanics are determined after developing an artificial anatomy testing setup inspired by the natural anatomy of the human focusing mechanism. Finally, based upon a mechanical and optical knowledge of the lens, several improved lens concepts are proposed and demonstrated for efficacy.</p
Numerical Simulations of Black-Hole Spacetimes
Get PDFThis thesis covers various aspects of the numerical simulation of black-hole spacetimes according to Einstein's general theory of relativity, using the Spectral Einstein Code developed by the Caltech-Cornell-CITA collaboration. The first topic is improvement of binary-black-hole initial data. One such issue is the construction of binary-black-hole initial data with nearly extremal spins that remain nearly constant during the initial relaxation in an evolution. Another concern is the inclusion of physically realistic tidal deformations of the black holes to reduce the high-frequency components of the spurious gravitational radiation content, and represents a first step in incorporating post-Newtonian results in constraint-satisfying initial data. The next topic is the evolution of black-hole binaries and the gravitational waves they emit. The first spectral simulation of two inspiralling black holes through merger and ringdown is presented, in which the black holes are nonspinning and have equal masses. This work is extended to perform the first spectral simulations of two inspiralling black holes with moderate spins and equal masses, including the merger and ringdown. Two configurations are considered, in which both spins are either anti-aligned or aligned with the orbital angular momentum. Highly accurate gravitational waveforms are computed for all these cases, and are used to calibrate waveforms in the effective-one-body model. The final topic is the behavior of quasilocal black-hole horizons in highly dynamical situations. Simulations of a rotating black hole that is distorted by a pulse of ingoing gravitational radiation are performed. Multiple marginally outer trapped surfaces are seen to appear and annihilate with each other during the evolution, and the world tubes they trace out are all dynamical horizons. The dynamical horizon and angular momentum flux laws are evaluated in this context, and the dynamical horizons are contrasted with the event horizon. The formation of multiple marginally outer trapped surfaces in the Vaidya spacetime is also treated
The Faint Intergalactic Redshifted Emission Balloon and the Cosmic Web Imager: Two Integral Field Spectrographs Designed to Study Emission from the Intergalactic Medium
Get PDFGas in the intergalactic medium serves as the fuel for galaxies. It carries signatures of galactic feedback, including matter and energy outflows. Understanding the morphology, thermodynamics, chemistry, and kinematics of this gas is key to understanding galaxy formation and evolution. The principal method of characterizing this gas has been the study of the Lyman α forest and associated metal systems. While this work has yielded deep insights into the nature of intergalactic matter, the scarcity of suitable background sources does allow for a full three-dimensional picture. Numerical simulations and theoretical work indicate that this gas produces faint and extended recombinant line emission. Its signatures in Lyα(1216 Å), OVI(1033 Å), CIV(1550 Å) are expected to be the strongest. Recent advances in technology and fresh ideas in instrumentation are allowing access to the predicted surface brightness of intergalactic emission. The Faint Intergalactic Redshifted Emission Balloon (FIREBall) and the Cosmic Web Imager (CWI) are two integral field spectrographs probing different redshift regimes, which have been designed for the specific purpose of detecting and mapping emission from the intergalactic medium. FIREBall, operating in the balloon ultraviolet window around 2000 Å, probes the redshift range 0.3 < z < 1, while CWI, a ground-based optical instrument, studies the Universe at 2.5 < z < 7.0. Both instruments collected their first science data in mid-2009. This manuscript discusses the science case for the spectrographs, focuses on their designs, construction, testing, first light, target selection, observations, data reduction, and analysis. Initial results are presented and discussed
Analysis, Design, and in vitro Implementation of Robust Biochemical Networks
Get PDFThe functionalities of every living organism are wired in the biochemical interactions among proteins, nucleic acids, and all the other molecules that constitute life's building blocks. Understanding the general design principles of this "hardware of life" is an exciting and challenging task for modern bioengineers. In this thesis, I focus on the topic of molecular network robustness: I investigate several design rules guaranteeing desired functionalities in specific systems, despite their components variability. Experimental verifications of such design schemes are carried out using \emph{in vitro} transcriptional circuits, a minimal analogue of cellular genetic networks.
The first problem I consider is flux control, which is a fundamental feature for the correct performance of biochemical systems. I describe a simple model problem where two reagents bind stoichiometrically to form an output product. In the absence of any regulation, imbalances in the reagent production rates can cause accumulation of unused molecules, and limit the output flow. To match the reagents' flux robustly with respect to the open loop rates, I propose the use of negative or positive feedback schemes that rely on competitive binding. Such schemes are modeled through ordinary differential equations and implemented using transcriptional circuits; data are presented showing the performance of the two approaches.
The second topic I examine is the functional robustness of interconnected networks. Molecular devices characterized in isolation may lose their properties once interconnected. This challenge is illustrated with a case study: a synthetic transcriptional clock is used to time conformational changes in a molecular nanomachine called DNA tweezers. Mass conservation introduces parasitic interactions that perturb the oscillator trajectories proportionally to the total amount of tweezers "load". To overcome this problem, we can use a transcriptional switch that acts as a buffer amplifier, achieving signal propagation and at the same time reducing the perturbations on the source of signal.
Finally, I describe a general class of control-theoretic methods to analyze structural robustness in natural biological systems. Using Lyapunov theory and set invariance, the stability properties of several well-known case studies are analytically demonstrated. The key feature of this analysis is its reliance on parameter-independent models, which only capture essential dynamic interactions between molecular species.</p
Sequential Processivity and CAND1 Regulate SCF Ubiquitin Ligases
Get PDFThe modular design of the multi-subunit SCF ubiquitin ligases allows for recognition of a diverse set of target proteins. However, the speed and complexity of the SCF ubiquitylation reaction have precluded direct experimental tests to understand how SCF complex formation is regulated and the pathway by which ubiquitin chains are generated. Herein we introduce new theoretical and experimental methodologies to address both limitations. First, a quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCFβ-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Second, we present a novel FRET assay that enables real-time measurements of binding dynamics of the SCFFbxw7 complex. We find that the Cul1-associated protein CAND1 is able to actively remove Fbxw7/Skp1 from Cul1/Rbx1 by changing the dissociation rate of the complex a million-fold, yet CAND1 does not affect the assembly rate of SCFFbxw7. This activity is abolished when Cul1 is neddylated. Experiments show that CAND1 accelerates the rate at which multiple SCF complexes can form. Thus, CAND1 appears to function as an exchange factor. Lastly, several measurements reveal an extra step in the ubiquitylation pathway for yeast SCF that implies a substrate induced conformational change exists for Fbox proteins. These results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and their regulation by CAND1. </p
Global Analysis of Dynamic Epigenetic Marking and Transcriptional Regulation Underlying T-Cell Lineage Commitment
Get PDFT-cell lineage specification and commitment success depends on precise temporal induction of T-lineage specific genes, as well as repression of lineage-inappropriate programs. After entry into the thymus, T-cell progenitors still retain inherited lineage plasticity, reflected by the mixed-lineage pattern of gene expression and the abilities to give rise to alternative lineages. Although Notch-Delta signaling is an essential force to trigger and sustain T-lineage differentiation, it does not appear to be the only requirement for this process. Successful commitment also depends on additional transcription factors, which often cooperatively interact with Notch-Delta signaling. However, the molecular mechanism by which pro-T cells are advanced to become committed T cells, in particular how the alternative lineage potentials are eliminated, is not fully understood. Using the genome-wide high-throughput sequencing, we track global shifts in gene expression pattern and transcriptional activity associated histone modifications in five successive stages of T-cell differentiation that span the commitment process. Our results show that T-lineage commitment is defined by the surprisingly complex downregulation of progenitor- and/or alternative lineage-associated programs, with relatively few regulatory genes are substantially upregulated. Rather than being silenced by a single global repression event, progenitor- and/or alternative lineage-associated genes are regulated by individual gene-specific mechanisms, indicated by the unsynchronized epigenetic transformations at discrete cis-elements of genes loci linked to progenitor and/or alternative lineage programs. We also investigate the genome-wide occupancies of PU.1 and GATA-3, two regulatory factors that have critical but complementary roles in early T-cell development. Binding sites choices of these two factors imply that transcriptional regulation by one particular factor is developmental context as well as dosage dependent. Furthermore, We combine this genome-wide approach with gene perturbation to study the function of Bcl11b, a transcription factor required for the completion of T-cell lineage commitment. Our analyses reveal that, in part through directly or indirectly regulation of Notch1 and GATA-3, Bcl11b mediates the modulation of T-cell lineage specification and commitment
Thermoelasticity of Hexagonal Close-Packed Iron from the Phonon Density of States
Get PDFIron is the main constituent in Earth’s core, along with ~5 to 10 wt% Ni and some light elements (e.g., H, C, O, Si, S). This thesis explores the vibrational thermodynamic and thermoelastic properties of pure hexagonal close-packed iron (ε-Fe), in an effort to improve our understanding of the properties of a significant fraction of this remote region of the deep Earth and in turn, better constrain its composition.
In order to access the vibrational properties of pure ε-Fe, we directly probed its total phonon density of states (DOS) by performing nuclear resonant inelastic x-ray scattering (NRIXS) and in situ x-ray diffraction (XRD) experiments at Sector 3-ID-B of the Advanced Photon Source (APS) at Argonne National Laboratory. NRIXS and in situ XRD were collected over the course of ~14 days at eleven compression points between 30 and 171 GPa, and at 300 K. Our in situ XRD measurements probed the sample volume at each compression point, and our long NRIXS data-collection times and high-energy resolution resulted in the highest statistical quality dataset of this type for ε-Fe to outer core pressures. Hydrostatic conditions were achieved in the sample chamber for our experiments at smaller compressions (P ≤ 69 GPa) via the loading of a neon pressure transmitting medium at the GeoSoilEnviroCARS (GSECARS) sector of the APS. For measurements made at P > 69 GPa, the sample was fully embedded in boron epoxy, which served as the pressure transmitting medium.
From each measured phonon DOS and thermodynamic definitions, we determined a wide range of vibrational thermodynamic and thermoelastic parameters, including the Lamb-Mössbauer factor; vibrational components of the specific heat capacity, free energy, entropy, internal energy, and kinetic energy; and the Debye sound velocity. Together with our in situ measured volumes, the shape of the total phonon DOS and these parameters gave rise to a number of important properties for ε-Fe at Earth’s core conditions.
For example, we determined the Debye sound velocity (vD) at each of our compression points from the low-energy region of the phonon DOS and our in situ measured volumes. In turn, vD is related to the compressional and shear sound velocities via our determined densities and the adiabatic bulk modulus. Our high-statistical quality dataset places a new tight constraint on the density dependence of ε-Fe’s sound velocities to outer core pressures. Via comparison with existing data for iron alloys, we investigate how nickel and candidate light elements for the core affect the thermoelastic properties of iron. In addition, we explore the effects of temperature on ε-Fe’s sound velocities by applying pressure- and temperature-dependent elastic moduli from theoretical calculations to a finite-strain model. Such models allow for direct comparisons with one-dimensional seismic models of Earth’s solid inner core (e.g., the Preliminary Reference Earth Model).
Next, the volume dependence of the vibrational free energy is directly related to the vibrational thermal pressure, which we combine with previously reported theoretical values for the electronic and anharmonic thermal pressures to find the total thermal pressure of ε-Fe. In addition, we found a steady increase in the Lamb-Mössbauer factor with compression, which suggests restricted thermal atomic motions at outer core pressures. This behavior is related to the high-pressure melting behavior of ε-Fe via Gilvarry’s reformulation of Lindemann’s melting criterion, which we used to obtain the shape of ε-Fe’s melting curve up to 171 GPa. By anchoring our melting curve shape with experimentally determined melting points and considering thermal pressure and anharmonic effects, we investigated ε-Fe’s melting temperature at the pressure of the inner–core boundary (ICB, P = 330 GPa), where Earth’s solid inner core and liquid outer core are in contact. Then, combining this temperature constraint with our thermal pressure, we determined the density of ε-Fe under ICB conditions, which offers information about the composition of Earth’s core via the seismically inferred density at the ICB.
In addition, the shape of the phonon DOS remained similar at all compression points, while the maximum (cutoff) energy increased regularly with decreasing volume. As a result, we were able to describe the volume dependence of ε-Fe’s total phonon DOS with a generalized scaling law and, in turn, constrain the ambient temperature vibrational Grüneisen parameter. We also used the volume dependence of our previously mentioned vD to determine the commonly discussed Debye Grüneisen parameter (γD), which we found to be ~10% smaller than our vibrational Grüneisen parameter at any given volume. Finally, applying our determined vibrational Grüneisen parameter to a Mie-Grüneisen type relationship and an approximate form of the empirical Lindemann melting criterion, we predict the vibrational thermal pressure and estimate the high-pressure melting behavior of ε-Fe at Earth’s core pressures, which can be directly compared with our previous results.
Finally, we use our measured vibrational kinetic energy and entropy to approximate ε-Fe’s vibrational thermodynamic properties to outer core pressures. In particular, the vibrational kinetic energy is related to the pressure- and temperature-dependent reduced isotopic partition function ratios (β-factors) of ε-Fe and in turn, provide information about the partitioning behavior of solid iron in equilibrium processes. In addition, the volume dependence of vibrational entropy is directly related to the product of ε-Fe’s vibrational component of the thermal expansion coefficient and the isothermal bulk modulus, which we find to be independent of pressure (volume) at 300 K. In turn, this product gives rise to the volume-dependent thermal expansion coefficient of ε-Fe at 300 K via established EOS parameters, and the vibrational Grüneisen parameter and temperature dependence of the vibrational thermal pressure via thermodynamic definition.</p
Degradations and Improvements in PEM Fuel Cell Materials: A Computational Study
Get PDFThe advantages of Proton Exchange Membrane (PEM) fuel cells include lower operating temperature than other fuel cells and size small enough to fit into a car. Improving the cost and durability of PEM fuel cell materials is a hot topic of research today.
The Nafion membrane and cathode catalysts are two areas where PEM fuel cells have issues of cost, durability, and efficiency. In order to improve these materials, researchers need a better understanding of the detailed mechanisms for basic operation and degradation. Computational quantum mechanics has improved in recent years to the point where it can provide accurate potential energy maps of reactions that are difficult to determine by laboratory experiments alone. With the basic understanding of mechanisms, experimentalists can make educated predictions of ways to improve fuel cell materials.
Experimental studies suggest that Nafion degradation is caused by generation of trace radical species (such as OH●, H●) when in the presence of H2, O2, and Pt. We use density functional theory (DFT) to construct the potential energy surfaces for various plausible reactions involving intermediates that might be formed when Nafion is exposed to H2 (or H+) and O2 in the presence of the Pt catalyst. We find that OH● can be generated in trace amounts on the Pt surface from HOOH and OOHad. Next, we look at various ways in which the OH● can attack the Nafion sidechains or endgroups on the backbone.
Researchers are looking for ways to replace the Pt cathode catalyst, due to the preciousness of Pt and the low efficiency of the oxygen reduction reaction (ORR) on Pt, among other things. Alloying Pt with non-precious Co greatly increases the ORR efficiency. However, Pt3Co was reported to not withstand long-cycle testing due to the migration of Co metals onto the catalyst surface and leaching of Co into the electrolyte. To overcome these challenges, we first study Pt3Co to find out what makes these alloys so special in improving fuel cell efficiency, as well as what causes degradation to occur. Then, we apply the principles we learned in proposing improved fuel cell alloy catalysts.</p
The Radio Variability of Gamma-Ray Blazars
Get PDFSince late 2007, we have regularly monitored over 1100 systematically selected blazars at 15 GHz using the Owens Valley Radio Observatory 40 m radio telescope. The number of sources in the program has grown to nearly 1600, including all the active galactic nuclei associated with Fermi Large Area Telescope (LAT) gamma-ray point source detections north of our declination limit of -20°. Here, we describe the first 42 months of this program, including the design and implementation of an automated data reduction pipeline and a MySQL database system for storing the reduced data and intermediate data products. Using the "intrinsic modulation index," a maximum-likelihood method, we estimate the variability amplitudes for 1413 sources from their radio light curves and compare the properties of physically defined subpopulations of the sample. We find that, among our preselected sample, gamma-ray-loud blazars detected by the LAT are significantly more variable at 15 GHz, attributable to a difference in variability between the gamma-ray-loud and gamma-ray-quiet flat spectrum radio quasars. The BL Lacertae objects in the samples do not show this division in variability amplitudes. In the first two years of our program, a 3σ-significant difference between variability amplitudes for sources at redshift z ≥ 1 and for sources at z < 1 was found. This difference is found no longer to be significant in the full 42-month data set, particularly after we apply an analysis method to account for the effect of cosmological time dilation