Molecular mechanisms of interleukin-2 gene inducibility: developmental control and combinatorial action of transcription factors

Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon activation mediated by T-cell receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage commitment and is regulated during T cell development and differentiation. Understanding the molecular mechanism of how IL-2 gene inducibility is controlled at each transition and each differentiation process of T-cell development is to understand one aspect of T-cell development. In the present study, we first attempted to elucidate the molecular basis for the developmental changes of IL-2 gene inducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4- CD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a complete set of transcription factors can be induced at this early stage to activate IL-2 gene expression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is accompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding activities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage. Further, the loss of factor binding, especially AP-1, is attributable to the reduced ability to activate expression of three potential components of AP-1 and NF-AT, including c-Fos, FosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2 promoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the IL-2 promoter is occupied by sequence-specific transcription factors when all the transcription factors are available; in resting T cells or non-T cells, no specific protein-DNA interaction is observed when only a subset of factors are present in the nuclei. Purposefully reducing a particular set of factor binding activities in stimulated T cells using pharmacological agents cyclosporin A or forskolin also abolished all interactions. The results suggest that a combinatorial and coordinated protein-DNA interaction is required for IL-2 gene activation. The thymocyte experiments clearly illustrated that multiple transcription factors are regulated during intrathymic T-cell development, and this regulation in tum controls the inducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction stressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter and to initiate its transcription, and provided a molecular mechanism for changes in IL-2 gene inducibility in T cells undergoing integration of multiple environmental signals.

Nonlinear optics and wavelength translation via cavity-optomechanics

The field of cavity-optomechanics explores the interaction of light with sound in an ever increasing array of devices. This interaction allows the mechanical system to be both sensed and controlled by the optical system, opening up a wide variety of experiments including the cooling of the mechanical resonator to its quantum mechanical ground state and the squeezing of the optical field upon interaction with the mechanical resonator, to name two.

In this work we explore two very different systems with different types of optomechanical coupling. The first system consists of two microdisk optical resonators stacked on top of each other and separated by a very small slot. The interaction of the disks causes their optical resonance frequencies to be extremely sensitive to the gap between the disks. By careful control of the gap between the disks, the optomechanical coupling can be made to be quadratic to first order which is uncommon in optomechanical systems. With this quadratic coupling the light field is now sensitive to the energy of the mechanical resonator and can directly control the potential energy trapping the mechanical motion. This ability to directly control the spring constant without modifying the energy of the mechanical system, unlike in linear optomechanical coupling, is explored.

Next, the bulk of this thesis deals with a high mechanical frequency optomechanical crystal which is used to coherently convert photons between different frequencies. This is accomplished via the engineered linear optomechanical coupling in these devices. Both classical and quantum systems utilize the interaction of light and matter across a wide range of energies. These systems are often not naturally compatible with one another and require a means of converting photons of dissimilar wavelengths to combine and exploit their different strengths. Here we theoretically propose and experimentally demonstrate coherent wavelength conversion of optical photons using photon-phonon translation in a cavity-optomechanical system. For an engineered silicon optomechanical crystal nanocavity supporting a 4 GHz localized phonon mode, optical signals in a 1.5 MHz bandwidth are coherently converted over a 11.2 THz frequency span between one cavity mode at wavelength 1460 nm and a second cavity mode at 1545 nm with a 93% internal (2% external) peak efficiency. The thermal and quantum limiting noise involved in the conversion process is also analyzed and, in terms of an equivalent photon number signal level, are found to correspond to an internal noise level of only 6 and 4 times 10x^-3 quanta, respectively.

We begin by developing the requisite theoretical background to describe the system. A significant amount of time is then spent describing the fabrication of these silicon nanobeams, with an emphasis on understanding the specifics and motivation. The experimental demonstration of wavelength conversion is then described and analyzed. It is determined that the method of getting photons into the cavity and collected from the cavity is a fundamental limiting factor in the overall efficiency. Finally, a new coupling scheme is designed, fabricated, and tested that provides a means of coupling greater than 90% of photons into and out of the cavity, addressing one of the largest obstacles with the initial wavelength conversion experiment.

Flexible microimplants for in vivo sensing

The work in this thesis develops two types of microimplants for the application of cardiovascular in vivo biomedical sensing, one for short-term diagnosis and the other for long-term monitoring.

Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic plaques has remained an unmet clinical need. A stretchable impedance sensor manifested as a pair of quasi-concentric microelectrodes was developed to detect unstable intravascular. By integrating the impedance sensor with a cardiac catheter, high-resolution Electrochemical Impedance Spectroscopy (EIS) measurements can be conducted during cardiac catheterization. An inflatable silicone balloon is added to the sensor to secure a well-controlled contact with the plaque under test in vivo. By deploying the device to the explants of NZW rabbit aorta and live animals, distinct EIS measurements were observed for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells.

On the other hand, zebrafish (Danio rerio) is an emerging genetic model for heart regenerative medicine. In humans, myocardial infarction results in the irreversible loss of cardiomyocytes. Zebrafish hearts can fully regenerate after two months with 20% ventricular resection. Long-term electrocardiogram (ECG) recording can characterize the heart regeneration in a functional dimension. A flexible microelectrode membrane was developed to be percutaneously implanted onto a zebrafish heart and record epicardial ECG signals from specific regions on it. Region-specific aberrant cardiac signals were obtained from injured and regenerated hearts. Following that, in order to achieve continuous and wireless recording from non-sedated and non-restricted small animal models, a wireless ECG recording system was designed for the microelectrode membrane, prototyped on a printed circuit board and demonstrated on a one-day-old neonatal mouse. Furthermore, a flexible and compact parylene C printed circuit membrane was used as the integration platform for the wireless ECG recording electronics. A substantially miniature wireless ECG recording system was achieved.

Self-healing Techniques for RF and mm-Wave Transmitters and Receivers

With continuing advances in CMOS technology, feature sizes of modern Silicon chip-sets have gone down drastically over the past decade. In addition to desktops and laptop processors, a vast majority of these chips are also being deployed in mobile communication devices like smart-phones and tablets, where multiple radio-frequency integrated circuits (RFICs) must be integrated into one device to cater to a wide variety of applications such as Wi-Fi, Bluetooth, NFC, wireless charging, etc. While a small feature size enables higher integration levels leading to billions of transistors co-existing on a single chip, it also makes these Silicon ICs more susceptible to variations. A part of these variations can be attributed to the manufacturing process itself, particularly due to the stringent dimensional tolerances associated with the lithographic steps in modern processes. Additionally, RF or millimeter-wave communication chip-sets are subject to another type of variation caused by dynamic changes in the operating environment. Another bottleneck in the development of high performance RF/mm-wave Silicon ICs is the lack of accurate analog/high-frequency models in nanometer CMOS processes. This can be primarily attributed to the fact that most cutting edge processes are geared towards digital system implementation and as such there is little model-to-hardware correlation at RF frequencies.

All these issues have significantly degraded yield of high performance mm-wave and RF CMOS systems which often require multiple trial-and-error based Silicon validations, thereby incurring additional production costs. This dissertation proposes a low overhead technique which attempts to counter the detrimental effects of these variations, thereby improving both performance and yield of chips post fabrication in a systematic way. The key idea behind this approach is to dynamically sense the performance of the system, identify when a problem has occurred, and then actuate it back to its desired performance level through an intelligent on-chip optimization algorithm. We term this technique as self-healing drawing inspiration from nature's own way of healing the body against adverse environmental effects. To effectively demonstrate the efficacy of self-healing in CMOS systems, several representative examples are designed, fabricated, and measured against a variety of operating conditions.

We demonstrate a high-power mm-wave segmented power mixer array based transmitter architecture that is capable of generating high-speed and non-constant envelope modulations at higher efficiencies compared to existing conventional designs. We then incorporate several sensors and actuators into the design and demonstrate closed-loop healing against a wide variety of non-ideal operating conditions. We also demonstrate fully-integrated self-healing in the context of another mm-wave power amplifier, where measurements were performed across several chips, showing significant improvements in performance as well as reduced variability in the presence of process variations and load impedance mismatch, as well as catastrophic transistor failure. Finally, on the receiver side, a closed-loop self-healing phase synthesis scheme is demonstrated in conjunction with a wide-band voltage controlled oscillator to generate phase shifter local oscillator (LO) signals for a phased array receiver. The system is shown to heal against non-idealities in the LO signal generation and distribution, significantly reducing phase errors across a wide range of frequencies.

How resources control aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner in which food and females promotes aggression.

In the first chapter, we explore how food controls aggression. As in many other species, food promotes aggression in flies, but it is not clear whether food increases aggression per se, or whether aggression is a secondary consequence of increased social interactions caused by aggregation of flies on food. Furthermore, nothing is known about how animals evaluate the quality and quantity of food in the context of competition. We show that food promotes aggression independently of any effect to increase the frequency of contact between males. Food increases aggression but not courtship between males, suggesting that the effect of food on aggression is specific. Next, we show that flies tune the level of aggression according to absolute amount of food rather than other parameters, such as area or concentration of food. Sucrose, a sugar molecule present in many fruits, is sufficient to promote aggression, and detection of sugar via gustatory receptor neurons is necessary for food-promoted aggression. Furthermore, we show that while food is necessary for aggression, too much food decreases aggression. Finally, we show that flies exhibit strategies consistent with a territorial strategy. These data suggest that flies use sweet-sensing gustatory information to guide their decision to fight over a limited quantity of a food resource.

Following up on the findings of the first chapter, we asked how the presence of a conspecific female resource promotes male-male aggression. In the absence of food, group-housed male flies, who normally do not fight even in the presence of food, fight in the presence of females. Unlike food, the presence of females strongly influences proximity between flies. Nevertheless, as group-housed flies do not fight even when they are in small chambers, it is unlikely that the presence of female indirectly increases aggression by first increasing proximity. Unlike food, the presence of females also leads to large increases in locomotion and in male-female courtship behaviors, suggesting that females may influence aggression as well as general arousal. Female cuticular hydrocarbons are required for this effect, as females that do not produce CH pheromones are unable to promote male-male aggression. In particular, 7,11-HD––a female-specific cuticular hydrocarbon pheromone critical for male-female courtship––is sufficient to mediate this effect when it is perfumed onto pheromone-deficient females or males. Recent studies showed that ppk23⁺ GRNs label two population of GRNs, one of which detects male cuticular hydrocarbons and another labeled by ppk23 and ppk25, which detects female cuticular hydrocarbons. I show that in particular, both of these GRNs control aggression, presumably via detection of female or male pheromones. To further investigate the ways in which these two classes of GRNs control aggression, I developed new genetic tools to independently test the male- and female-sensing GRNs. I show that ppk25-LexA and ppk25-GAL80 faithfully recapitulate the expression pattern of ppk25-GAL4 and label a subset of ppk23⁺ GRNs. These tools can be used in future studies to dissect the respective functions of male-sensing and female-sensing GRNs in male social behaviors.

Finally, in the last chapter, I discuss quantitative approaches to describe how varying quantities of food and females could control the level of aggression. Flies show an inverse-U shaped aggressive response to varying quantities of food and a flat aggressive response to varying quantities of females. I show how two simple game theoretic models, “prisoner’s dilemma” and “coordination game” could be used to describe the level of aggression we observe. These results suggest that flies may use strategic decision-making, using simple comparisons of costs and benefits.

In conclusion, male-male aggression in Drosophila is controlled by simple gustatory cues from food and females, which are detected by gustatory receptor neurons. Different quantities of resource cues lead to different levels of aggression, and flies show putative territorial behavior, suggesting that fly aggression is a highly strategic adaptive behavior. How these resource cues are integrated with male pheromone cues and give rise to this complex behavior is an interesting subject, which should keep researchers busy in the coming years.

Dynamic characterization of micro-particle systems

Ordered granular systems have been a subject of active research for decades. Due to their rich dynamic response and nonlinearity, ordered granular systems have been suggested for several applications, such as solitary wave focusing, acoustic signals manipulation, and vibration absorption. Most of the fundamental research performed on ordered granular systems has focused on macro-scale examples. However, most engineering applications require these systems to operate at much smaller scales. Very little is known about the response of micro-scale granular systems, primarily because of the difficulties in realizing reliable and quantitative experiments, which originate from the discrete nature of granular materials and their highly nonlinear inter-particle contact forces.

In this work, we investigate the physics of ordered micro-granular systems by designing an innovative experimental platform that allows us to assemble, excite, and characterize ordered micro-granular systems. This new experimental platform employs a laser system to deliver impulses with controlled momentum and incorporates non-contact measurement apparatuses to detect the particles’ displacement and velocity. We demonstrated the capability of the laser system to excite systems of dry (stainless steel particles of radius 150 micrometers) and wet (silica particles of radius 3.69 micrometers, immersed in fluid) micro-particles, after which we analyzed the stress propagation through these systems.

We derived the equations of motion governing the dynamic response of dry and wet particles on a substrate, which we then validated in experiments. We then measured the losses in these systems and characterized the collision and friction between two micro-particles. We studied wave propagation in one-dimensional dry chains of micro-particles as well as in two-dimensional colloidal systems immersed in fluid. We investigated the influence of defects to wave propagation in the one-dimensional systems. Finally, we characterized the wave-attenuation and its relation to the viscosity of the surrounding fluid and performed computer simulations to establish a model that captures the observed response.

The findings of the study offer the first systematic experimental and numerical analysis of wave propagation through ordered systems of micro-particles. The experimental system designed in this work provides the necessary tools for further fundamental studies of wave propagation in both granular and colloidal systems.

An Optically recombined laser interferometer for gravitational wave detection

Kilometer scale interferometers for the detection of gravitational waves are currently under construction by the LIGO (Laser Interferometer Gravitational-wave Observatory) and VIRGO projects. These interferometers will consist of two Fabry-Perot cavities illuminated by a laser beam which is split in half by a beam splitter. A recycling mirror between the laser and the beam splitter will reflect the light returning from the beam splitter towards the laser back into the interferometer. The positions of the optical components in these interferometers must be controlled to a small fraction of a wavelength of the laser light. Schemes to extract signals necessary to control these optical components have been developed and demonstrated on the tabletop. In the large scale gravitational wave detectors the optical components must be suspended from vibration isolation platforms to achieve the necessary isolation from seismic motion. These suspended components present a new class of problems in controlling the interferometer, but also provide more exacting test of interferometer signal and noise models.

This thesis discusses the first operation of a suspended-mass Fabry-Perot-Michelson interferometer, in which signals carried by the optically recombined beams are used to detect and control all important mirror displacements. This interferometer uses an optical configuration and signal extraction scheme that is planned for the full scale LIGO interferometers with the simplification of the removal of the recycling mirror. A theoretical analysis of the performance that is expected from such an interferometer is presented and the experimental results are shown to be in generally good agreement.

Feedback communication using orthogonal signals

This research is concerned with block coding for a feedback communication system in which the forward and feedback channels are independently disturbed by additive white Gaussian noise and average power constrained. Two coding schemes are proposed in which the messages to be coded for transmission over the forward channel are realized as a set of orthogonal waveforms. A finite number of forward and feedback transmissions (iterations) per message is made. Information received over the feedback channel is used to modify the waveform transmitted on successive forward iterations in such a way that the expected value of forward signal energy is zero on all iterations after the first. Similarly, information is sent over the feedback channel in such a way that the expected value of feedback signal energy is also zero on all iterations after the first. These schemes are shown to achieve a lower probability of error than the best one-way coding scheme at all rates up to the forward channel capacity, provided only that the feedback channel capacity be greater than the forward channel capacity. These schemes make more efficient use of the available feedback power than existing feedback coding schemes, and therefore require less feedback power to achieve a given error performance.

The global optimization of phase-incoherent signals

The problem of global optimization of M phase-incoherent signals in N complex dimensions is formulated. Then, by using the geometric approach of Landau and Slepian, conditions for optimality are established for N = 2 and the optimal signal sets are determined for M = 2, 3, 4, 6, and 12.

The method is the following: The signals are assumed to be equally probable and to have equal energy, and thus are represented by points ṡ_i, i = 1, 2, …, M, on the unit sphere S₁ in C^N. If W_ik is the halfspace determined by ṡ_i and ṡ_k and containing ṡ_i, i.e. W_ik = {ṙϵC^N:| ≥ | ˂ṙ, ṡ_k˃|}, then the Ʀ_i = ∩/k≠i W_ik, i = 1, 2, …, M, the maximum likelihood decision regions, partition S₁. For additive complex Gaussian noise ṅ and a received signal ṙ = ṡ_ie^jϴ + ṅ, where ϴ is uniformly distributed over [0, 2π], the probability of correct decoding is P_C = 1/π^N ∞/ʃ/0 r^2N-1e^-(r2+1)U(r)dr, where U(r) = 1/M M/Ʃ/i=1 Ʀ_i ʃ/∩ S₁ I₀(2r | ˂ṡ, ṡ_i˃|)dσ(ṡ), and r = ǁṙǁ.

For N = 2, it is proved that U(r) ≤ ʃ/C_α I₀(2r|˂ṡ, ṡ_i˃|)dσ(ṡ) – 2K/M. h(1/2K [Mσ(C_α)-σ(S₁)]), where C_α = {ṡϵS₁:|˂ṡ, ṡ_i˃| ≥ α}, K is the total number of boundaries of the net on S₁ determined by the decision regions, and h is the strictly increasing strictly convex function of σ(C_α∩W), (where W is a halfspace not containing ṡ_i), given by h = ʃ/C_α∩W I₀ (2r|˂ṡ, ṡ_i˃|)dσ(ṡ). Conditions for equality are established and these give rise to the globally optimal signal sets for M = 2, 3, 4, 6, and 12.