Towards conditional RNAi : shape and sequence transduction with small conditional RNAs

RNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement “conditional RNAi.” Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.

To implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.

We demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.

Characterization of the neural mechanisms supporting the implementation of cognitive control in human decision making

Humans are particularly adept at modifying their behavior in accordance with changing environmental demands. Through various mechanisms of cognitive control, individuals are able to tailor actions to fit complex short- and long-term goals. The research described in this thesis uses functional magnetic resonance imaging to characterize the neural correlates of cognitive control at two levels of complexity: response inhibition and self-control in intertemporal choice. First, we examined changes in neural response associated with increased experience and skill in response inhibition; successful response inhibition was associated with decreased neural response over time in the right ventrolateral prefrontal cortex, a region widely implicated in cognitive control, providing evidence for increased neural efficiency with learned automaticity. We also examined a more abstract form of cognitive control using intertemporal choice. In two experiments, we identified putative neural substrates for individual differences in temporal discounting, or the tendency to prefer immediate to delayed rewards. Using dynamic causal models, we characterized the neural circuit between ventromedial prefrontal cortex, an area involved in valuation, and dorsolateral prefrontal cortex, a region implicated in self-control in intertemporal and dietary choice, and found that connectivity from dorsolateral prefrontal cortex to ventromedial prefrontal cortex increases at the time of choice, particularly when delayed rewards are chosen. Moreover, estimates of the strength of connectivity predicted out-of-sample individual rates of temporal discounting, suggesting a neurocomputational mechanism for variation in the ability to delay gratification. Next, we interrogated the hypothesis that individual differences in temporal discounting are in part explained by the ability to imagine future reward outcomes. Using a novel paradigm, we imaged neural response during the imagining of primary rewards, and identified negative correlations between activity in regions associated the processing of both real and imagined rewards (lateral orbitofrontal cortex and ventromedial prefrontal cortex, respectively) and the individual temporal discounting parameters estimated in the previous experiment. These data suggest that individuals who are better able to represent reward outcomes neurally are less susceptible to temporal discounting. Together, these findings provide further insight into role of the prefrontal cortex in implementing cognitive control, and propose neurobiological substrates for individual variation.

Protein-protein recognition: The neonatal Fe receptor and immunoglobulin G

The neonatal Fe receptor (FeRn) binds the Fe portion of immunoglobulin G (IgG) at the acidic pH of endosomes or the gut and releases IgG at the alkaline pH of blood. FeRn is responsible for the maternofetal transfer of IgG and for rescuing endocytosed IgG from a default degradative pathway. We investigated how FeRn interacts with IgG by constructing a heterodimeric form of the Fe (hdFc) that contains one FeRn binding site. This molecule was used to characterize the interaction between one FeRn molecule and one Fe and to determine under what conditions FeRn forms a dimer. The hdFc binds one FeRn molecule at pH 6.0 with a K_d of 80 nM. In solution and with FeRn anchored to solid supports, the heterodimeric Fe does not induce a dimer of FeRn molecules. FcRnhdFc complex crystals were obtained and the complex structure was solved to 2.8 Å resolution. Analysis of this structure refined the understanding of the mechanism of the pH-dependent binding, shed light on the role played by carbohydrates in the Fe binding, and provided insights on how to design therapeutic IgG antibodies with longer serum half-lives. The FcRn-hdFc complex in the crystal did not contain the FeRn dimer. To characterize the tendency of FeRn to form a dimer in a membrane we analyzed the tendency of the hdFc to induce cross-phosphorylation of FeRn-tyrosine kinase chimeras. We also constructed FeRn-cyan and FeRn-yellow fluorescent proteins and have analyzed the tendency of these molecules to exhibit fluorescence resonance energy transfer. As of now, neither of these analyses have lead to conclusive results. In the process of acquiring the context to appreciate the structure of the FcRn-hdFc interface, we developed a study of 171 other nonobligate protein-protein interfaces that includes an original principal component analysis of the quantifiable aspects of these interfaces.

Structural geology of the region between Pacoima and Little Tujunga Canyons, San Gabriel Mountains, California

An area of about 25 square miles in the western part of the San Gabriel Mountains was mapped on a scale of 1000 feet to the inch. Special attention was given to the structural geology, particularly the relations between the different systems of faults, of which the San Gabriel fault system and the Sierra Madre fault system are the most important ones. The present distribution and relations of the rocks suggests that the southern block has tilted northward against a more stable mass of old rocks which was raised up during a Pliocene or post-Pliocene orogeny. It is suggested that this northward tilting of the block resulted in the group of thrust faults which comprise the Sierra Madre fault system. It is show that this hypothesis fits the present distribution of the rocks and occupies a logical place in the geologic history of the region as well or better than any other hypothesis previously offered to explain the geology of the region.

Crystals that count! Physical principles and experimental investigations of DNA tile self-assembly

Algorithmic DNA tiles systems are fascinating. From a theoretical perspective, they can result in simple systems that assemble themselves into beautiful, complex structures through fundamental interactions and logical rules. As an experimental technique, they provide a promising method for programmably assembling complex, precise crystals that can grow to considerable size while retaining nanoscale resolution. In the journey from theoretical abstractions to experimental demonstrations, however, lie numerous challenges and complications.

In this thesis, to examine these challenges, we consider the physical principles behind DNA tile self-assembly. We survey recent progress in experimental algorithmic self-assembly, and explain the simple physical models behind this progress. Using direct observation of individual tile attachments and detachments with an atomic force microscope, we test some of the fundamental assumptions of the widely-used kinetic Tile Assembly Model, obtaining results that fit the model to within error. We then depart from the simplest form of that model, examining the effects of DNA sticky end sequence energetics on tile system behavior. We develop theoretical models, sequence assignment algorithms, and a software package, StickyDesign, for sticky end sequence design.

As a demonstration of a specific tile system, we design a binary counting ribbon that can accurately count from a programmable starting value and stop growing after overflowing, resulting in a single system that can construct ribbons of precise and programmable length. In the process of designing the system, we explain numerous considerations that provide insight into more general tile system design, particularly with regards to tile concentrations, facet nucleation, the construction of finite assemblies, and design beyond the abstract Tile Assembly Model.

Finally, we present our crystals that count: experimental results with our binary counting system that represent a significant improvement in the accuracy of experimental algorithmic self-assembly, including crystals that count perfectly with 5 bits from 0 to 31. We show some preliminary experimental results on the construction of our capping system to stop growth after counters overflow, and offer some speculation on potential future directions of the field.

Oxygen isotope ratios in coexisting minerals of regionally metamorphosed rocks

The O¹⁸/O¹⁶ ratios of coexisting minerals from a number of regionally metamorphosed rocks have been measured, using a bromine pentafluoride extraction-technique. Listed in order of their increasing tendency to concentrate O¹⁸, the minerals analyzed are magnetite, ilmenite, chlorite, biotite, garnet, hornblende, kyanite, muscovite, feldspar, and quartz. The only anomalous sequence detected occurs in a xenolith of schist, in which quartz, muscovite, biotite, and ilmenite, but not garnet, have undergone isotopic exchange with surrounding trondjemite.

With few exceptions, quartz-magnetite and quartz-ilmenite fractionations decrease with increasing metamorphic grade determined by mineral paragenesis and spatial distribution. This consistency does not apply to quartz-magnetite and quartz-ilmenite fractionations obtained from rocks in which petrographic evidence of retrogradation is present.

Whereas measured isotopic fractionations among quartz, garnet, ilmenite, and magnetite are approximately related to metamorphic grade, fractionations between these minerals and biotite or muscovite show poor correlation with grade. Variations in muscovite-biotite fractionations are relatively small. These observations are interpreted to mean that muscovite and biotite are affected by retrograde re-equilibration to a greater extent than the anhydrous minerals analyzed.

Measured quartz-ilmenite fractionations range from 12 permil in the biotite zone of central Vermont to 6.5 permil in the sillimanite-orthoclase zone of southeastern Connecticut. Analyses of natural assemblages from the kyanite and sillimanite zones suggest that equilibrium quartz-ilmenite fractionations are approximately 8 percent smaller than corresponding quartz-magnetite fractionations. Employing the quartz-magnetite geothermometer calibrated by O'Neil and Clayton (1964), a temperature of 560°C was obtained for kyanite-bearing schists from Addison County, Vermont. Extending the calibration to quartz-ilmenite fractionations, a temperature of 600°C was obtained for kyanite-schists from Shoshone County, Idaho. At these temperatures kyanite is stable only at pressures exceeding 11 kbars (Bell, 1963), corresponding to lithostatic loads of over 40 km.

Proton-Coupled Reduction of N2 Facilitated by Molecular Fe Complexes

The activation of Fe-coordinated N₂ via the formal addition of hydrogen atom equivalents is explored in this thesis. These reactions may occur in nitrogenase enzymes during the biological conversion of N₂ to NH₃. To understand these reactions, the N₂ reactivity of a series of molecular Fe(N₂) platforms is investigated. A trigonal pyramidal, carbon-ligated Fe^I complex was prepared that displays a similar geometry to that of the resting state 'belt' Fe atoms of nitrogenase. Upon reduction, this species was shown to coordinate N₂, concomitant with significant weakening of the C-Fe interaction. This hemilability of the axial ligand may play a critical role in mediating the interconversion of Fe(N_xH_y) species during N₂ conversion to NH₃. In fact, a trigonal pyramidal borane-ligated Fe complex was shown to catalyze this transformation, generating up to 8.49 equivalents of NH₃. To shed light on the mechanistic details of this reaction, protonation of a borane-ligated Fe(N₂) complex was investigated and found to give rise to a mixture of species that contains an iron hydrazido(2-) [Fe(NNH₂)] complex. The identification of this species is suggestive of an early N-N bond cleavage event en route to NH₃ production, but the highly-reactive nature of this complex frustrated direct attempts to probe this possibility. A structurally-analogous silyl-ligated Fe(N₂) complex was found to react productively with hydrogen atom equivalents, giving rise to an isolable Fe(NNH₂) species. Spectroscopic and crystallographic studies benefited from the enhanced stability of this complex relative to the borane analogue. One-electron reduction of this species initiates a spontaneous disproportionation reaction with an iron hydrazine [Fe(NH₂NH₂)] complex as the predominant reaction product. This transformation provides support for an Fe-mediated N₂ activation mechanism that proceeds via a late N-N bond cleavage. In hopes of gaining more fundamental insight into these reactions, a series of Fe(CN) complexes were prepared and reacted with hydrogen-atom equivalents. Significant quantities of CH₄ and NH₃ are generated in these reactions as a result of complete C-N bond activation. A series of Fe(CNH_x) were found to be exceptionally stable and may be intermediates in these reactions. The stability of these compounds permitted collection of thermodynamic parameters pertinent to the unique N-H bonds. This data is comparatively discussed with the theoretically-predicted data of the N₂-derived Fe(NNH_x) species. Exceptionally-weak N-H bond enthalpies are found for many of these compounds, and sheds light on their short-lived nature and tendency to evolve H₂. As a whole, these works both establish and provide a means to understand Fe-mediated N₂ activation via the addition of hydrogen atom equivalents.

Essays in revealed preference theory and behavioral economics

Time, 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.

Stationary Eddies and Zonal Variations of the Global Hydrological Cycle in a Changing Climate

This thesis advances our physical understanding of the sensitivity of the hydrological cycle to global warming. Specifically, it focuses on changes in the longitudinal (zonal) variation of precipitation minus evaporation (P - E), which is predominantly controlled by planetary-scale stationary eddies. By studying idealized general circulation model (GCM) experiments with zonally varying boundary conditions, this thesis examines the mechanisms controlling the strength of stationary-eddy circulations and their role in the hydrological cycle. The overarching goal of this research is to understand the cause of changes in regional P - E with global warming. An understanding of such changes can be useful for impact studies focusing on water availability, ecosystem management, and flood risk.

Based on a moisture-budget analysis of ERA-Interim data, we establish an approximation for zonally anomalous P - E in terms of surface moisture content and stationary-eddy vertical motion in the lower troposphere. Part of the success of this approximation comes from our finding that transient-eddy moisture fluxes partially cancel the effect of stationary-eddy moisture advection, allowing divergent circulations to dominate the moisture budget. The lower-tropospheric vertical motion is related to horizontal motion in stationary eddies by Sverdrup and Ekman balance. These moisture- and vorticity-budget balances also hold in idealized and comprehensive GCM simulations across a range of climates.

By examining climate changes in the idealized and comprehensive GCM simulations, we are able to show the utility of the vertical motion P - E approximation for splitting changes in zonally anomalous P - E into thermodynamic and dynamic components. Shifts in divergent stationary-eddy circulations dominate changes in zonally anomalous P - E. This limits the local utility of the "wet gets wetter, dry gets drier” idea, where existing P - E patterns are amplified with warming by the increase in atmospheric moisture content, with atmospheric circulations held fixed. The increase in atmospheric moisture content manifests instead in an increase in the amplitude of the zonally anomalous hydrological cycle as measured by the zonal variance of P - E. However, dynamic changes, particularly the slowdown of divergent stationary-eddy circulations, limit the strengthening of the zonally anomalous hydrological cycle. In certain idealized cases, dynamic changes are even strong enough to reverse the tendency towards "wet gets wetter, dry gets drier” with warming.

Motivated by the importance of stationary-eddy vertical velocities in the moisture budget analysis, we examine controls on the amplitude of stationary eddies across a wide range of climates in an idealized GCM with simple topographic and ocean-heating zonal asymmetries. An analysis of the thermodynamic equation in the vicinity of topographic forcing reveals the importance of on-slope surface winds, the midlatitude isentropic slope, and latent heating in setting the amplitude of stationary waves. The response of stationary eddies to climate change is determined primarily by the strength of zonal surface winds hitting the mountain. The sensitivity of stationary-eddies to this surface forcing increases with climate change as the slope of midlatitude isentropes decreases. However, latent heating also plays an important role in damping the stationary-eddy response, and this damping becomes stronger with warming as the atmospheric moisture content increases. We find that the response of tropical overturning circulations forced by ocean heat-flux convergence is described by changes in the vertical structure of moist static energy and deep convection. This is used to derive simple scalings for the Walker circulation strength that capture the monotonic decrease with warming found in our idealized simulations.

Through the work of this thesis, the advances made in understanding the amplitude of stationary-waves in a changing climate can be directly applied to better understand and predict changes in the zonally anomalous hydrological cycle.

Conditioned responses in the reticular formation

Unit activity was recorded from the midbrain and pons of 40 freely moving rats in an appetitive classical conditioning situation. Responses to auditory stimuli were observed from 100 units before and during a conditioning procedure in which presentation of food occurred 1 sec after the onset of the auditory stimulus. Conditioned unit responses (i.e., spike rate accelerations or decelerations) were considered to be positive when 1) no similar responses appeared prior to conditioning, and 2) latencies were equal to or less than those of sensory responses derived from the inferior colliculus. Such short latency conditioned unit responses were recorded from 11 probes located in the mid-lateral pert of the ventral region of the brain stem. This region was differentiated from paramedian, far lateral and dorsal parts of the brain stem reticular formation. Conditioned unit responses of considerably longer latencies were recorded from 76 probe located in these other regions. Among the longer latency responses interesting differences appeared in experiments conducted after the first conditioning series was completed. With additional training, units in the "reticular activating system" of midbrain and pons tended to yield stabilized responses in the early portion of the CS-US interval closely related in time to the orientation responses evoked by the CS. In contrast, the responses of units in the limbic midbrain tended to stabilize in the later part of the CS-US interval closely related in time to preparatory responses tied to the US. During extinction when the auditory stimulus was no longer followed by presentation of food, many of the responses were reduced to their pre-conditioning levels. However, there was a tendency for units which had displayed short latency responses on the first conditioning day to be more resistant to extinction than units which had displayed longer latency conditioned responses. The data were interpreted as indicating a local correlate of learning in the reticular formation of midbrain end pons and a separation of the midbrain system into at least two areas: 1) the classical "reticular activating system" related to orienting reactions, and 2) the limbic midbrain areas related to drives and rewards. Because the ventral and mid-lateral area with very short latency conditioned responses was not clearly tied to either of these; it was considered as possibly representing a third division.