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Ferrous Iron Sensing and Responding in Pseudomonas aeruginosa
Get PDFControlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator’s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa’s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future
The Neurocomputational Basis of Self-Control Success and Failure
Get PDFIndividuals often have difficulty delaying gratification – that is, forging smaller sooner rewards in favor of larger rewards delivered at a delay. Common examples of this deficit in self-control are difficulties in saving for retirement, going to the gym, or eating healthy foods. Despite an extensive literature on the neural substrates of decision-making, relatively little is still known about the sources of underlying individual variation in the ability to successfully execute self-control. This manuscript presents three studies examining the sources of individual differences in delay of gratification, with the goal of obtaining a more complete understanding of the neural mechanisms underlying choice. The main question this thesis addresses is: what features of the brain’s decision process allow individuals to down-regulate the appeal of smaller sooner rewards, in order to forgo them in favor of greater future reward?
In the first study, I present a novel method of measuring decision process dynamics, in which we harness the power of fine temporal resolution in recording computer mouse movements in dietary choices. We find that up to 39% of individual variation in dietary self-control can be explained by differences in the speed with which the decision-making circuitry processes basic attributes, such as tastiness, versus more complex, abstract, attributes, such as healthfulness.
In the second study, we extend this novel approach to a classic experimental economics paradigm, intertemporal choice. We found large individual variance in the speeds with which immediate and delayed reward values were processed. We found that about 25% of the individual differences can be explained by differences in the speed at which delayed rewards are processed. We also found that the relative speed at which immediate and delayed rewards are processed fluctuated across trials: subjects processed delayed rewards faster than immediate rewards when they made patient choices, but the order of processing speeds was reversed during impulsive choices. Together, these results show that a sizable fraction of variation in the ability to postpone gratification might be attributable to variables that affect the speed at which different types of rewards are processed, and not to differences on deep preference parameters like the temporal discount rate used by the brain’s valuation systems.
Across the lifespan, self-control improves in many choice domains. The third and final study capitalizes on this phenomenon of behavioral change with age to investigate the neural mechanisms underlying improvements in self-control. I use functional magnetic resonance imaging (fMRI) to examine the neural correlates of changes in discounting of future monetary rewards across the lifespan from adolescents (13 years old) to seniors (70 years old). We find that neural response to value in reward-related striatal brain regions dramatically decrease with age. In contrast, we find that the left dorsolateral prefrontal cortex, often found to be related to successful self-control, increases its functional connectivity to key valuation, reward, and future-thinking brain regions with age during very tempting trials. These results suggest a mechanism through which increased self-control is improved.
Taken together, these studies argue that individual features of the decision process have a large influence on the overall ability to exert self-control in both dietary and monetary choice domains. Specifically, we find that the speed with which abstract future attributes such as health information, relative to more concrete attributes such as taste, are processed have a large influence on individual self-control ability. We also find that decreased reward sensitivity, paired with increased effective connectivity between control and valuation regions specifically when control is required most, allow for increased ability to delay gratification with age.</p
Development and Function of Sleep Regulatory Circuits in Zebrafish
Get PDFSleep is widely accepted as an essential behavior for optimum mental and physical health, yet the genetic and neural circuits that govern sleep remain poorly understood. In this thesis, I briefly introduce the behavioral criteria that define sleep, currently known sleep regulatory mechanisms, and the distinct advantages of the zebrafish, Danio rerio, as a simple animal model for studying sleep. I then investigate two factors previously implicated in sleep behavior: epidermal growth factor receptor and hypocretin. First, I show that epidermal growth factor receptor signaling is both necessary and sufficient for normal sleep behavior in zebrafish, just as it is in invertebrates. This demonstrates that sleep regulatory mechanisms can be conserved over large evolutionary distances, and is the first genetic study showing that the epidermal growth factor receptor signaling is necessary for normal sleep behavior in a vertebrate. Second, I capitalize upon the rapid external development of zebrafish embryos to screen for developmental factors that specify hypocretin neurons, which are known to promote arousal and consolidate sleep/wake bouts. I identify the LIM homeobox 9 transcription factor as necessary for hypocretin neuronal development in zebrafish and sufficient to specify additional hypocretin neurons in both zebrafish and mice. This is the first time any factor has been shown to induce hypocretin neurons in vivo and may be an important step towards curing narcolepsy, a debilitating sleep disorder caused by the selective loss of hypocretin neurons. These studies deepen our understanding of how sleep is regulated at a genetic and cellular level and underscore the potential for zebrafish to make future contributions to sleep research
Manipulating Selectivity and Reactivity in Palladium-Catalyzed Oxidation Reactions
Get PDFSince the initial discovery of the Wacker process over half a century ago, the Wacker oxidation has become a premier reaction for the oxidation of terminal alkenes to methyl ketones. This thesis describes strategies for manipulating selectivity and reactivity in Wacker-type oxidations to provide synthetically useful transformations.
Chapter 2 describes how nitrite co-catalysts can be exploited in Wacker oxidations to reverse their typically high Markovnikov selectivity. Using these aerobic oxidation conditions, alkenes can be oxidized to aldehydes in high yield and selectivity. Preliminary mechanistic experiments are presented that are consistent with oxygen atom transfer from the nitrite catalyst to the substrate. The influence of proximal functionality on the new reaction is explored, yielding both synthetically useful transformations and further mechanistic insight.
Chapter 3 investigates how minor modifications to the nitrite-modified Wacker can interrupt the Wacker oxidation pathway, providing dioxygenated products using molecular oxygen as the terminal oxidant. A variety of functional groups are tolerated and high yields of 1,2-diacetoxylated products are obtained with a range of substrates. Mechanistic experiments are presented that demonstrate the kinetic competency of nitrogen dioxide to mediate the reaction and probe the nature of the reductive elimination event.
Chapter 4 details the development of a highly active Wacker-type oxidation capable of efficiently oxidizing internal alkenes, which are unreactive under
classical conditions. Under these simple and mild reaction conditions, a wide range of functional groups are tolerated and molecular oxygen can be employed as the terminal oxidant. Furthermore, the regioselectivity in unsymmetrical internal alkenes is investigated.
Chapter 5 explores the origins of innate regioselectivity in Wacker oxidations. Systematic investigations of both internal and terminal alkenes illustrate that inductive effects are sufficient to dramatically influence Wacker regioselectivity. These observations lead to the development of a simple set of reactions conditions that strongly enforces Markovnikov's rule, even with substrates that provide mixtures of aldehydes and ketones under classical conditions.</p
Surface Activity and Bulk Defect Chemistry of Solid Oxide Fuel Cell Cathodes
Get PDFIn the first half of this thesis, a new robotic instrument called a scanning impedance probe is presented that can acquire electrochemical impedance spectra in automated fashion from hundreds of thin film microelectrodes with systematically varied properties. Results from this instrument are presented for three catalyst compositions that are commonly considered for use in state-of-the-art solid oxide fuel cell cathodes. For (La0.8Sr0.2)0.95MnO3+δ (LSM), the impedance spectra are well fit by a through-the-film reaction pathway. Transport rates are extracted, and the surface activity towards oxygen reduction is found to be correlated with the number of exposed grain boundary sites, suggesting that grain boundaries are more surface-active than grains. For La0.5Sr0.5CoO3-δ (LSC), the surface activity degrades ~50x initially and then stabilizes at a comparable activity to that of previously measured Ba0.5Sr0.5Co0.8Fe0.2O3-δ films. For Sr0.06Nb0.06Bi1.87O3 (SNB), an example of a doped bismuth oxide, the activity of the metal-SNB boundary is measured.
In the second half of this thesis, SrCo0.9Nb0.1O3-δ is selected as a case study of perovskites containing Sr and Co, which are the most active oxygen reduction catalysts known. Several bulk properties are measured, and synchrotron data are presented that provide strong evidence of substantial cobalt-oxygen covalency at high temperatures. This covalent bonding may be the underlying source of the high surface activity.</p
Narrow-Linewidth Si/III-V Lasers: a Study of Laser Dynamics and Nonlinear Effects
Get PDFNarrow-linewidth lasers play an important role in a wide variety of applications, from sensing and spectroscopy to optical communication and on-chip clocks. Current narrow-linewidth systems are usually implemented in doped fibers and are big, expensive, and power-hungry. Semiconductor lasers compete favorably in size, cost, and power consumption, but their linewidth is historically limited to the sub-MHz regime. However, it has been recently demonstrated that a new design paradigm, in which the optical energy is stored away from the active region in a composite high-Q resonator, has the potential to dramatically improve the coherence of the laser.
This work explores this design paradigm, as applied on the hybrid Si/III-V platform. It demonstrates a record sub-KHz white-noise-floor linewidth. It further shows, both theoretically and experimentally, that this strategy practically eliminates Henry’s linewidth enhancement by positioning a damped relaxation resonance at frequencies as low as 70 MHz, yielding truly quantum limited devices at frequencies of interest.
In addition to this empirical contribution, this work explores the limits of performance of this platform. Here, the effect of two-photon-absorption and free-carrier-absorption are analyzed, using modified rate equations and Langevin force approach. The analysis predicts that as the intra-cavity field intensity builds up in the high-Q resonator, non-linear effects cause a new domain of performance-limiting factors. Steady-state behavior, laser dynamics, and frequency noise performance are examined in the context of this unique platform, pointing at the importance of nonlinear effects.
This work offers a theoretical model predicting laser performance in light of nonlinear effects, obtaining a good agreement with experimental results from fabricated high-Q Si/III-V lasers. In addition to demonstrating unprecedented semiconductor laser performance, this work establishes a first attempt to predict and demonstrate the key impact of nonlinear effects on silicon-based lasers.</p
Behavioral and fMRI Measures of Crossmodal Plasticity Induced by Auditory Sensory Substitution
Get PDFThirty nine million people are blind worldwide. Sensory Substitution (SS) attempts to aid the blind by translating images into sound and thereby restoring visual function. Previous studies have found that training on SS generates crossmodal neural changes allowing for activation in early visual regions in response to SS sounds. Unfortunately, training on auditory sensory substitution to become proficient at basic visual tasks takes 1 week to 3 months and even then is slow, inaccurate, and attention-intensive. In this thesis it was studied if SS interpretation could be performed by entirely naive users automatically, and if the crossmodal plasticity engendered through training could be engaged automatically. In contrast to the top-down SS interpretation, we have found that SS interpretation can be effortless and automatic in entirely naive individuals when crossmodally intuitive stimuli that contain crossmodal mappings are used. Crossmodal mappings are pre-existing associations in all individuals of images and sounds that were found to be used for entirely naive interpretation of SS. This result indicates that SS could potentially be made more useful to the blind with appropriate training and translation algorithms. We also studied if the crossmodal plasticity generated by SS training can also be activated automatically in trained blind and sighted device users. We found that crossmodal plasticity engendered through a week of training could be triggered automatically by SS stimuli. This indicates that crossmodal plasticity does not require an attention-intensive task be used and therefore is not entirely top-down cognitive. It might be possible to tap into this automatic processing in visual cortex of SS stimuli to make SS interpretation less effortful and more perceptual following the appropriate training. Overall, this thesis attempts to use SS to understand crossmodal neural processing and plasticity, and to through this broadened knowledge restore some visual function to the entirely blind
How Behavioral Economics Can Shape Firm Strategy and Public Policy: Lessons from the Field and Laboratory
Get PDFIncentives are not always economic or monetary in nature. Individuals are often influenced by socially-based incentives centered on how he or she wants to be perceived in a social setting, such as the desire to publicly adhere to a norm of fairness. Likewise, individuals can also be influenced by cognitively-based incentives centered on self-perception and self-attribution, such as the desire to convince oneself that he or she is altruistic. Behavioral economists have incorporated some of these concepts into standard economic models of decision-making, but there is much we still do not understand about the role of these psychology-based incentives in organizational strategy and public policy contexts.
This thesis examines the effects of several social and cognitive incentives across different settings. In Chapter I, I use a laboratory experiment to examine why individuals feel the need to reciprocate to gifts and favors, even when those gifts are from businesses looking to take advantage of our tendency to reciprocate. Specifically, I demonstrate that individuals reciprocate simply to the intent to give a gift or favor, regardless of the ulterior motives or actual utility resulting from the favor. In Chapter II, I use observational data to investigate how pharmaceutical firms exploit social incentives that invoke reciprocity in order to influence how physicians prescribe. I concurrently examine how regulators also use social incentives, but as a way to protect consumers from these manipulative marketing strategies. In Chapter III, I collaborate with a non-profit public radio station to test the interaction between psychological and economic incentives in a fundraising context. In particular, Chapter III uses a field experiment to show that economic incentives in a fundraising campaign can reduce donation rates by detracting attention from the psychological reasons for donating, and thereby inducing a different mindset in donors.
This thesis builds upon the field of behavioral economics in two ways. First, it uses experimental methods to extend our theoretical understanding of non-monetary, psychological incentives, including some of the mechanisms that drive reciprocity, social image, and motivation crowding out. Second, the thesis applies this knowledge toward understanding the effects of several commonly used marketing campaigns and regulatory policies.</p
Acceleration Sensing, Feedback Cooling, and Nonlinear Dynamics with Nanoscale Cavity-Optomechanical Devices
Get PDFLight has long been used for the precise measurement of moving bodies, but the burgeoning field of optomechanics is concerned with the interaction of light and matter in a regime where the typically weak radiation pressure force of light is able to push back on the moving object. This field began with the realization in the late 1960's that the momentum imparted by a recoiling photon on a mirror would place fundamental limits on the smallest measurable displacement of that mirror. This coupling between the frequency of light and the motion of a mechanical object does much more than simply add noise, however. It has been used to cool objects to their quantum ground state, demonstrate electromagnetically-induced-transparency, and modify the damping and spring constant of the resonator. Amazingly, these radiation pressure effects have now been demonstrated in systems ranging 18 orders of magnitude in mass (kg to fg).
In this work we will focus on three diverse experiments in three different optomechanical devices which span the fields of inertial sensors, closed-loop feedback, and nonlinear dynamics. The mechanical elements presented cover 6 orders of magnitude in mass (ng to fg), but they all employ nano-scale photonic crystals to trap light and resonantly enhance the light-matter interaction. In the first experiment we take advantage of the sub-femtometer displacement resolution of our photonic crystals to demonstrate a sensitive chip-scale optical accelerometer with a kHz-frequency mechanical resonator. This sensor has a noise density of approximately 10 micro-g/rt-Hz over a useable bandwidth of approximately 20 kHz and we demonstrate at least 50 dB of linear dynamic sensor range. We also discuss methods to further improve performance of this device by a factor of 10.
In the second experiment, we used a closed-loop measurement and feedback system to damp and cool a room-temperature MHz-frequency mechanical oscillator from a phonon occupation of 6.5 million down to just 66. At the time of the experiment, this represented a world-record result for the laser cooling of a macroscopic mechanical element without the aid of cryogenic pre-cooling. Furthermore, this closed-loop damping yields a high-resolution force sensor with a practical bandwidth of 200 kHZ and the method has applications to other optomechanical sensors.
The final experiment contains results from a GHz-frequency mechanical resonator in a regime where the nonlinearity of the radiation-pressure interaction dominates the system dynamics. In this device we show self-oscillations of the mechanical element that are driven by multi-photon-phonon scattering. Control of the system allows us to initialize the mechanical oscillator into a stable high-amplitude attractor which would otherwise be inaccessible. To provide context, we begin this work by first presenting an intuitive overview of optomechanical systems and then providing an extended discussion of the principles underlying the design and fabrication of our optomechanical devices.</p
Computationally Guided Monomerization of Red Fluorescent Proteins of the Class Anthozoa
Get PDFRed fluorescent proteins (RFPs) have attracted significant engineering focus because of the promise of near infrared fluorescent proteins, whose light penetrates biological tissue, and which would allow imaging inside of vertebrate animals. The RFP landscape, which numbers ~200 members, is mostly populated by engineered variants of four native RFPs, leaving the vast majority of native RFP biodiversity untouched. This is largely due to the fact that native RFPs are obligate tetramers, limiting their usefulness as fusion proteins. Monomerization has imposed critical costs on these evolved tetramers, however, as it has invariably led to loss of brightness, and often to many other adverse effects on the fluorescent properties of the derived monomeric variants. Here we have attempted to understand why monomerization has taken such a large toll on Anthozoa class RFPs, and to outline a clear strategy for their monomerization. We begin with a structural study of the far-red fluorescence of AQ143, one of the furthest red emitting RFPs. We then try to separate the problem of stable and bright fluorescence from the design of a soluble monomeric β-barrel surface by engineering a hybrid protein (DsRmCh) with an oligomeric parent that had been previously monomerized, DsRed, and a pre-stabilized monomeric core from mCherry. This allows us to use computational design to successfully design a stable, soluble, fluorescent monomer. Next we took HcRed, which is a previously unmonomerized RFP that has far-red fluorescence (λemission = 633 nm) and attempted to monomerize it making use of lessons learned from DsRmCh. We engineered two monomeric proteins by pre-stabilizing HcRed’s core, then monomerizing in stages, making use of computational design and directed evolution techniques such as error-prone mutagenesis and DNA shuffling. We call these proteins mGinger0.1 (λem = 637 nm / Φ = 0.02) and mGinger0.2 (λem = 631 nm Φ = 0.04). They are the furthest red first generation monomeric RFPs ever developed, are significantly thermostabilized, and add diversity to a small field of far-red monomeric FPs. We anticipate that the techniques we describe will be facilitate future RFP monomerization, and that further core optimization of the mGingers may allow significant improvements in brightness