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Towards in situ Single Cell Systems Biology
Get PDFSystems-level studies of biological systems rely on observations taken at a resolution lower than
the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled
measurements of the transcriptomes in single cells excised from their environment, but it remains a
daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In
this thesis I develop methods for the routine, quantitative in situ measurement of gene expression
using fluorescence microscopy.
The number of molecular species that can be measured simultaneously by fluorescence microscopy
is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally
limited to the simultaneous measurement of only five labeled biomolecules at a time. The
two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent
strategies for overcoming this limitation to monitor expression of many genes in a single cell.
Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial
labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual
mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically
increases the optical space in a cell, allowing a large numbers of barcodes to be visualized
simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium
stress response. The second method, sequential barcoding, reads out a temporal barcode through
multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of
sequential barcoding increases exponentially with the number of rounds of hybridization, allowing
over a hundred genes to be profiled in only a few rounds of hybridization.
The utility of sequential barcoding was further demonstrated by adapting this method to study
gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of
auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An
amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction),
was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated
in combination with light sheet microscopy and background reducing tissue clearing technology,
enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact
mammalian tissue.
The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed
transcriptional observations in any tissue of interest. These technologies will serve as a
general platform for future transcriptomic studies of complex tissues.</p
Evolution of Developmental Gene Regulatory Networks in Echinoids
Get PDFDevelopmental gene regulatory networks (dGRNs) are assemblages of regulatory genes that direct embryonic development of animal body plans and their morpho-logical structures. dGRNs exhibit recursively-wired circuitry that is encoded in the genome and executed during development. Alteration to the regulatory architecture of dGRNs causes variation in developmental programs both during the development of an individual organism and during the evolution of an individual lineage. The ex-planatory power of these networks is best exemplified by the global dGRN directing early development of the euechinoid sea urchin Strongylocentrotus purpuratus. This network consists of numerous regulatory genes engaging in hundreds of genomic regulatory transactions that collectively direct the delineation of early embryonic domains and the specification of cell lineages. Research on closely-related euechi-noid sea urchins, e.g. Lytechinus variegatus and Paracentrotus lividus, has revealed marked conservation of dGRN architecture in echinoid development, suggesting little appreciable alteration has occurred since their divergence in evolution at least 90 million years ago (mya).
We sought to test whether this observation extends to all sea urchins (echinoids) and undertook a systematic analysis of over 50 regulatory genes in the cidaroid sea urchin Eucidaris tribuloides, surveing their regulatory activity and function in a sea urchin that diverged from euechinoid sea urchins at least 268 mya. Our results revealed extensive alterations have occurred to all levels of echinoid dGRN archi-tecture since the cidaroid-euechinoid divergence. Alterations to mesodermal sub-circuits were particularly striking, including functional di˙erences in specification of non-skeletogenic mesenchyme (NSM), skeletogenic mesenchyme (SM), and en-domesodermal segregation. Specification of endomesodermal embryonic domains revealed that, while their underlying network circuitry had clearly diverged, regu-latory states established in pregastrular embryos of these two groups are strikingly similar. Analyses of E. tribuloides specification leading to the estab-lishment of dorsal-ventral (aboral-oral) larval polarity indicated that regulation of regulatory genes expressed in mesodermal embryonic domains had incurred significantly more alterations than those expressed in endodermal and ectodermal domains. Taken together, this study highlights the ability of dGRN architecture to buffer extensive alterations in the evolution and early development of echinoids and adds further support to the notion that alterations can occur at all levels of dGRN architecture and all stages of embryonic development.</p
Quantum of Vision
Get PDFVisual inputs to artificial and biological visual systems are often quantized: cameras accumulate photons from the visual world, and the brain receives action potentials from visual sensory neurons. Collecting more information quanta leads to a longer acquisition time and better performance. In many visual tasks, collecting a small number of quanta is sufficient to solve the task well. The ability to determine the right number of quanta is pivotal in situations where visual information is costly to obtain, such as photon-starved or time-critical environments. In these situations, conventional vision systems that always collect a fixed and large amount of information are infeasible. I develop a framework that judiciously determines the number of information quanta to observe based on the cost of observation and the requirement for accuracy. The framework implements the optimal speed versus accuracy tradeoff when two assumptions are met, namely that the task is fully specified probabilistically and constant over time. I also extend the framework to address scenarios that violate the assumptions. I deploy the framework to three recognition tasks: visual search (where both assumptions are satisfied), scotopic visual recognition (where the model is not specified), and visual discrimination with unknown stimulus onset (where the model is dynamic over time). Scotopic classification experiments suggest that the framework leads to dramatic improvement in photon-efficiency compared to conventional computer vision algorithms. Human psychophysics experiments confirmed that the framework provides a parsimonious and versatile explanation for human behavior under time pressure in both static and dynamic environments
Some Advances in Computational Geophysics: Seismic Wave and Inverse Geodynamic Modeling
Get PDFIn this thesis, I develop computational methods that link theory with geophysical observations, with one part devoted to the development of forward models of seismic wave propagation through the mantle and core, and a second part devoted to the inversion of viscous flow in the mantle.
First order seismic structure of the earth has been well described radially since the PREM model was introduced. With the help of seismic tomography methods, many large-scale heterogeneous structures have become well imaged. Based on this progress, the information in seismic waveforms, which provides extra constraints, is becoming more important in determination of the detailed structure within the earth's interior. However, 3-D modeling of seismic wave propagation remains computationally expensive, especially at high frequency, because the computing cost scales with fourth power of frequency. Thus 2-D modeling is often used, and in many cases is sufficient for the problem. To use 2-D modeling in global seismology, several issues need to be considered: how to handle the differences in geometric spreading between 2-D and 3-D modeling, how to incorporate earthquake sources into a 2-D code, and how to handle the spherical geometry of the earth. In the first part of my thesis, we solve all three problems, using a 2-D staggered finite difference method with a post-processing step. The post-processing automatically corrects the geometric spreading difference between 2-D and 3-D wave propagation; the earthquake sources are added to the 2-D finite difference simulation using a momentum source and transparent box approaches; the earth-flattening is discussed, especially for the density transformation. Benchmarks of the new method against with 1-D and 3-D code demonstrates the the accuracy of the method.
We then use the new code in a study of the interface between outer and inner core. Inner Core Boundary (ICB) is thought to be crucial in estimating the energy released in generating the geomagnetic field. One direct constraint on ICB properties is using reflected P wave from ICB, the PKiKP phase. Due to its small amplitude, near distance PKiKP is seldom observed. However, we find several events beneath Central American as having good set of PKiKP recordings from the USArray seismic network, as well as other core phases like P wave reflection with Core Mantle Boundary (CMB). The amplitude of the phases display large scatters across the stations, which are potentially caused by many factors, including site effects of the stations, upper mantle inhomogeneity, or a bumpy structure along either the CMB or ICB. After comparing amplitude ratio of between PKiKP and PcP phase, analyzing how this ratio changes for different nearby events, and computing forward models using our new method that investigate different factors influence the PKiKP phases, we attribute a stacked amplitude pattern as caused by ICB structure, in which PKiKP phase amplitude rapidly changes within a small range. Finally, we model this observed seismic pattern as a small dome-like anomalies above ICB, where the material changes from that of the outer core to that of inner core gradually.
The final part of my thesis is on a geodynamic inversion problem for mantle convection. Mantle convection is an important process that determines plate motions and subduction. Numerous forward models indicate that the constitute relation (viscosity law) is of key importance for mantle convection. Despite substantial effort attempting to determine the viscosity structure of the mantle, either through forward and inverse geophysical models or through laboratory work, many first order questions remain. We assume the realistic viscosity structure, which is temperature and strain-rate dependent, can be parameterized using a set of scalar parameters. Given this set of viscosity parameters and an initial temperature, the mantle evolves following a set of partial differential equations (PDEs). Our goal with the inverse problem is to recover the viscosity parameters and initial temperature by fitting the observational data, which here includes plate motion history and the present day temperature distribution of the mantle. We formulate this inversion problem following a PDE constrained optimization framework. We first define the cost function we want to minimize; then, the derivative of the cost function with respect to viscosity parameters and initial temperature is calculated following the discrete adjoint equations; finally, a gradient-based optimization method, limited memory Broyden-Fletcher-Goldfarb-Shanno (LBFGS) approach is used to find the minimum. To accelerate the optimization process, we modified the traditional LBFGS by adding a preconditioner, and achieve a more rapid convergence. To test our method, we use two synthetic cases: a sinking cylinder within a viscous layer and a realistic subduction model. We find that in the initial temperature-only inversion, the initial temperature can be recovered well; in the joint inversion of initial temperature and viscosity parameters, the temperature, as well as effective viscosity, can also be recovered reasonably, but there are trade offs between viscosity parameters. Presumably, the trade off in viscosity parameters is related to the ill-posedness of the problem.</p
SteelConverter and Caltech VirtualShaker: Rapid Nonlinear Cloud-Based Structural Model Conversion and Analysis
Get PDFSTEEL, the Caltech created nonlinear large displacement analysis software, is currently used by a large number of researchers at Caltech. However, due to its complexity, lack of visualization tools (such as pre- and post-processing capabilities) rapid creation and analysis of models using this software was difficult. SteelConverter was created as a means to facilitate model creation through the use of the industry standard finite element solver ETABS. This software allows users to create models in ETABS and intelligently convert model information such as geometry, loading, releases, fixity, etc., into a format that STEEL understands. Models that would take several days to create and verify now take several hours or less. The productivity of the researcher as well as the level of confidence in the model being analyzed is greatly increased.
It has always been a major goal of Caltech to spread the knowledge created here to other universities. However, due to the complexity of STEEL it was difficult for researchers or engineers from other universities to conduct analyses. While SteelConverter did help researchers at Caltech improve their research, sending SteelConverter and its documentation to other universities was less than ideal. Issues of version control, individual computer requirements, and the difficulty of releasing updates made a more centralized solution preferred. This is where the idea for Caltech VirtualShaker was born. Through the creation of a centralized website where users could log in, submit, analyze, and process models in the cloud, all of the major concerns associated with the utilization of SteelConverter were eliminated. Caltech VirtualShaker allows users to create profiles where defaults associated with their most commonly run models are saved, and allows them to submit multiple jobs to an online virtual server to be analyzed and post-processed. The creation of this website not only allowed for more rapid distribution of this tool, but also created a means for engineers and researchers with no access to powerful computer clusters to run computationally intensive analyses without the excessive cost of building and maintaining a computer cluster.
In order to increase confidence in the use of STEEL as an analysis system, as well as verify the conversion tools, a series of comparisons were done between STEEL and ETABS. Six models of increasing complexity, ranging from a cantilever column to a twenty-story moment frame, were analyzed to determine the ability of STEEL to accurately calculate basic model properties such as elastic stiffness and damping through a free vibration analysis as well as more complex structural properties such as overall structural capacity through a pushover analysis. These analyses showed a very strong agreement between the two softwares on every aspect of each analysis. However, these analyses also showed the ability of the STEEL analysis algorithm to converge at significantly larger drifts than ETABS when using the more computationally expensive and structurally realistic fiber hinges. Following the ETABS analysis, it was decided to repeat the comparisons in a software more capable of conducting highly nonlinear analysis, called Perform. These analyses again showed a very strong agreement between the two softwares in every aspect of each analysis through instability. However, due to some limitations in Perform, free vibration analyses for the three story one bay chevron brace frame, two bay chevron brace frame, and twenty story moment frame could not be conducted. With the current trend towards ultimate capacity analysis, the ability to use fiber based models allows engineers to gain a better understanding of a building’s behavior under these extreme load scenarios.
Following this, a final study was done on Hall’s U20 structure [1] where the structure was analyzed in all three softwares and their results compared. The pushover curves from each software were compared and the differences caused by variations in software implementation explained. From this, conclusions can be drawn on the effectiveness of each analysis tool when attempting to analyze structures through the point of geometric instability. The analyses show that while ETABS was capable of accurately determining the elastic stiffness of the model, following the onset of inelastic behavior the analysis tool failed to converge. However, for the small number of time steps the ETABS analysis was converging, its results exactly matched those of STEEL, leading to the conclusion that ETABS is not an appropriate analysis package for analyzing a structure through the point of collapse when using fiber elements throughout the model. The analyses also showed that while Perform was capable of calculating the response of the structure accurately, restrictions in the material model resulted in a pushover curve that did not match that of STEEL exactly, particularly post collapse. However, such problems could be alleviated by choosing a more simplistic material model. </p
Incremental Control Synthesis for Robotics in the Presence of Temporal Logic Specifications
Get PDFThis thesis presents methods for incrementally constructing controllers in the presence of uncertainty and nonlinear dynamics. The basic setting is motion planning subject to temporal logic specifications. Broadly, two categories of problems are treated. The first is reactive formal synthesis when so-called discrete abstractions are available. The fragment of linear-time temporal logic (LTL) known as GR(1) is used to express assumptions about an adversarial environment and requirements of the controller. Two problems of changes to a specification are posed that concern the two major aspects of GR(1): safety and liveness. Algorithms providing incremental updates to strategies are presented as solutions. In support of these, an annotation of strategies is developed that facilitates repeated modifications. A variety of properties are proven about it, including necessity of existence and sufficiency for a strategy to be winning. The second category of problems considered is non-reactive (open-loop) synthesis in the absence of a discrete abstraction. Instead, the presented stochastic optimization methods directly construct a control input sequence that achieves low cost and satisfies a LTL formula. Several relaxations are considered as heuristics to address the rarity of sampling trajectories that satisfy an LTL formula and demonstrated to improve convergence rates for Dubins car and single-integrators subject to a recurrence task
Essays in Revealed Preference Theory and Behavioral Economics
Get PDFTime, risk, and attention are all integral to economic decision making. The aim of this work is to understand those key components of decision making using a variety of approaches: providing axiomatic characterizations to investigate time discounting, generating measures of visual attention to infer consumers' intentions, and examining data from unique field settings.
Chapter 2, co-authored with Federico Echenique and Kota Saito, presents the first revealed-preference characterizations of exponentially-discounted utility model and its generalizations. My characterizations provide non-parametric revealed-preference tests. I apply the tests to data from a recent experiment, and find that the axiomatization delivers new insights on a dataset that had been analyzed by traditional parametric methods.
Chapter 3, co-authored with Min Jeong Kang and Colin Camerer, investigates whether "pre-choice" measures of visual attention improve in prediction of consumers' purchase intentions. We measure participants' visual attention using eyetracking or mousetracking while they make hypothetical as well as real purchase decisions. I find that different patterns of visual attention are associated with hypothetical and real decisions. I then demonstrate that including information on visual attention improves prediction of purchase decisions when attention is measured with mousetracking.
Chapter 4 investigates individuals' attitudes towards risk in a high-stakes environment using data from a TV game show, Jeopardy!. I first quantify players' subjective beliefs about answering questions correctly. Using those beliefs in estimation, I find that the representative player is risk averse. I then find that trailing players tend to wager more than "folk" strategies that are known among the community of contestants and fans, and this tendency is related to their confidence. I also find gender differences: male players take more risk than female players, and even more so when they are competing against two other male players.
Chapter 5, co-authored with Colin Camerer, investigates the dynamics of the favorite-longshot bias (FLB) using data on horse race betting from an online exchange that allows bettors to trade "in-play." I find that probabilistic forecasts implied by market prices before start of the races are well-calibrated, but the degree of FLB increases significantly as the events approach toward the end
Chemical and Electrochemical Behavior of Graphene-Covered Silicon Photoanodes
Get PDFThis dissertation describes efforts over the last five years to develop protective layers for semiconductor photoelectrodes based on monolayer or few-layer graphene sheets. Graphene is an attractive candidate for a protective layer because of its known chemical inertness, transparency, ease of deposition, and limited number of electronic states. Monolayer graphene was found to effectively inhibit loss of photocurrent over 1000 seconds at n-Si/aqueous electrolyte interfaces that exhibit total loss over photocurrent over 100 seconds. Further, the presence of graphene was found to effect only partial Fermi level pinning at the Si/graphene interface with respect to a range of nonaqueous electrolytes. Fluorination of graphene was found to extend the stability imparted on n-Si by the monolayer sheet in aqueous Fe(CN)63-/4- electrolyte to over 100,000 seconds. It was demonstrated that the stability of the photocurrent of n-Si/fluorinated graphene/aqueous electrolyte interfaces relative to n-Si/aqueous electrolyte interfaces is likely attributable to the inhibition of oxidation of the silicon surface.
This dissertation also relates efforts to describe and define terminology relevant to the field of photoelectrochemistry and solar fuels production. Terminology describing varying interfaces employed in electrochemical solar fuels devices are defined, and the research challenges associated with each are discussed. Methods for determining the efficiency of varying photoelectrochemical and solar-fuel-producing cells from the current-voltage behavior of the individual components of such a device without requiring the device be constructed are described, and a range of commonly employed performance metrics are explored. </p
Holistic Design In High-Speed Optical Interconnects
Get PDFIntegrated circuit scaling has enabled a huge growth in processing capability, which necessitates a corresponding increase in inter-chip communication bandwidth. As bandwidth requirements for chip-to-chip interconnection scale, deficiencies of electrical channels become more apparent. Optical links present a viable alternative due to their low frequency-dependent loss and higher bandwidth density in the form of wavelength division multiplexing. As integrated photonics and bonding technologies are maturing, commercialization of hybrid-integrated optical links are becoming a reality. Increasing silicon integration leads to better performance in optical links but necessitates a corresponding co-design strategy in both electronics and photonics. In this light, holistic design of high-speed optical links with an in-depth understanding of photonics and state-of-the-art electronics brings their performance to unprecedented levels. This thesis presents developments in high-speed optical links by co-designing and co-integrating the primary elements of an optical link: receiver, transmitter, and clocking.
In the first part of this thesis a 3D-integrated CMOS/Silicon-photonic receiver will be presented. The electronic chip features a novel design that employs a low-bandwidth TIA front-end, double-sampling and equalization through dynamic offset modulation. Measured results show -14.9dBm of sensitivity and energy efficiency of 170fJ/b at 25Gb/s. The same receiver front-end is also used to implement source-synchronous 4-channel WDM-based parallel optical receiver. Quadrature ILO-based clocking is employed for synchronization and a novel frequency-tracking method that exploits the dynamics of IL in a quadrature ring oscillator to increase the effective locking range. An adaptive body-biasing circuit is designed to maintain the per-bit-energy consumption constant across wide data-rates. The prototype measurements indicate a record-low power consumption of 153fJ/b at 32Gb/s. The receiver sensitivity is measured to be -8.8dBm at 32Gb/s.
Next, on the optical transmitter side, three new techniques will be presented. First one is a differential ring modulator that breaks the optical bandwidth/quality factor trade-off known to limit the speed of high-Q ring modulators. This structure maintains a constant energy in the ring to avoid pattern-dependent power droop. As a first proof of concept, a prototype has been fabricated and measured up to 10Gb/s. The second technique is thermal stabilization of micro-ring resonator modulators through direct measurement of temperature using a monolithic PTAT temperature sensor. The measured temperature is used in a feedback loop to adjust the thermal tuner of the ring. A prototype is fabricated and a closed-loop feedback system is demonstrated to operate at 20Gb/s in the presence of temperature fluctuations. The third technique is a switched-capacitor based pre-emphasis technique designed to extend the inherently low bandwidth of carrier injection micro-ring modulators. A measured prototype of the optical transmitter achieves energy efficiency of 342fJ/bit at 10Gb/s and the wavelength stabilization circuit based on the monolithic PTAT sensor consumes 0.29mW.
Lastly, a first-order frequency synthesizer that is suitable for high-speed on-chip clock generation will be discussed. The proposed design features an architecture combining an LC quadrature VCO, two sample-and-holds, a PI, digital coarse-tuning, and rotational frequency detection for fine-tuning. In addition to an electrical reference clock, as an extra feature, the prototype chip is capable of receiving a low jitter optical reference clock generated by a high-repetition-rate mode-locked laser. The output clock at 8GHz has an integrated RMS jitter of 490fs, peak-to-peak periodic jitter of 2.06ps, and total RMS jitter of 680fs. The reference spurs are measured to be –64.3dB below the carrier frequency. At 8GHz the system consumes 2.49mW from a 1V supply.</p
