Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

Fiber-optic integration and efficient detection schemes for optomechanical resonators

With the advent of the laser in the year 1960, the field of optics experienced a renaissance from what was considered to be a dull, solved subject to an active area of development, with applications and discoveries which are yet to be exhausted 55 years later. Light is now nearly ubiquitous not only in cutting-edge research in physics, chemistry, and biology, but also in modern technology and infrastructure. One quality of light, that of the imparted radiation pressure force upon reflection from an object, has attracted intense interest from researchers seeking to precisely monitor and control the motional degrees of freedom of an object using light. These optomechanical interactions have inspired myriad proposals, ranging from quantum memories and transducers in quantum information networks to precision metrology of classical forces. Alongside advances in micro- and nano-fabrication, the burgeoning field of optomechanics has yielded a class of highly engineered systems designed to produce strong interactions between light and motion.

Optomechanical crystals are one such system in which the patterning of periodic holes in thin dielectric films traps both light and sound waves to a micro-scale volume. These devices feature strong radiation pressure coupling between high-quality optical cavity modes and internal nanomechanical resonances. Whether for applications in the quantum or classical domain, the utility of optomechanical crystals hinges on the degree to which light radiating from the device, having interacted with mechanical motion, can be collected and detected in an experimental apparatus consisting of conventional optical components such as lenses and optical fibers. While several efficient methods of optical coupling exist to meet this task, most are unsuitable for the cryogenic or vacuum integration required for many applications. The first portion of this dissertation will detail the development of robust and efficient methods of optically coupling optomechanical resonators to optical fibers, with an emphasis on fabrication processes and optical characterization.

I will then proceed to describe a few experiments enabled by the fiber couplers. The first studies the performance of an optomechanical resonator as a precise sensor for continuous position measurement. The sensitivity of the measurement, limited by the detection efficiency of intracavity photons, is compared to the standard quantum limit imposed by the quantum properties of the laser probe light. The added noise of the measurement is seen to fall within a factor of 3 of the standard quantum limit, representing an order of magnitude improvement over previous experiments utilizing optomechanical crystals, and matching the performance of similar measurements in the microwave domain.

The next experiment uses single photon counting to detect individual phonon emission and absorption events within the nanomechanical oscillator. The scattering of laser light from mechanical motion produces correlated photon-phonon pairs, and detection of the emitted photon corresponds to an effective phonon counting scheme. In the process of scattering, the coherence properties of the mechanical oscillation are mapped onto the reflected light. Intensity interferometry of the reflected light then allows measurement of the temporal coherence of the acoustic field. These correlations are measured for a range of experimental conditions, including the optomechanical amplification of the mechanics to a self-oscillation regime, and comparisons are drawn to a laser system for phonons. Finally, prospects for using phonon counting and intensity interferometry to produce non-classical mechanical states are detailed following recent proposals in literature.

Studies of bacteriophage T4 tail fibers and tail fiber genes

The distal half of the bacteriophage T4 tail fiber interacts with the surface of the bacterium during adsorption. The largest polypeptide in this half fiber is the product of gene 37 (P37). During assembly of the tail fiber, P37 interacts with the product of gene 38 (P38). These two gene products are incompatible with the corresponding gene products from the related phage T2. T2 P37 does not interact with T4 P38 and T2 P38 does not interact with T4 P37. Crosses between T2 and T4 phages mutant in genes 37 and 38 have shown that the carboxyl end of P37 interacts with P38 and with the bacterial surface. In the corresponding region of gene 37 and in gene 38 there is no recombination between T2 and T4. In the rest of gene 37 there are two small regions with relatively high recombination and a region of low recombination.

When T2/T4 heteroduplex DNA molecules are examined in the electron microscope four nonhomologous loops appear in the region of genes 37 and 38. Heteroduplexes between hybrid phages which have part of gene 37 from T4 and part from T2 have roughly located gene 37 mutations in the heteroduplex pattern. For a more precise location of the , mutations a physical map of gene 37 was constructed by determining the molecular weights of amber polypeptide fragments on polyacrylamide gels in the presence of sodium dodecyl sulfate. When the physical and heteroduplex maps are aligned, the regions of low recombination correspond to regions of nonhomology between T2 and T4. Regions with relatively high recombination are homologous.

The molecular weight of T2 P37 is about 13,000 greater than that of T4 P37. Analysis of hybrid phage has shown that this molecular weight difference is all at the carboxyl end of P37.

An antiserum has been prepared which is specific for the distal half fiber of T4. Tests of the ability of gene 37 hybrids to block this antiserum show that there are at least 4 subclasses of antigen specified by different parts of P37.

Observations in the electron microscope of the tailfiber - anti- body complexes formed by the gene 37 hybrids and the specific anti- serum have shown that P37 is oriented linearly in the distal half fiber with its N-terminus near the joint between the two half fibers and its C-terminus near the tip of the fiber. These observations lead to a simple model for the structure of the distal half fiber.

The high recombination in T4 gene 34 was also investigated. A comparison of genetic and physical maps of gene 34 showed that there is a gradient of increasing recombination near one end of the gene.

Distributed Optimal Control of Cyber-Physical Systems: Controller Synthesis, Architecture Design and System Identification

The centralized paradigm of a single controller and a single plant upon which modern control theory is built is no longer applicable to modern cyber-physical systems of interest, such as the power-grid, software defined networks or automated highways systems, as these are all large-scale and spatially distributed. Both the scale and the distributed nature of these systems has motivated the decentralization of control schemes into local sub-controllers that measure, exchange and act on locally available subsets of the globally available system information. This decentralization of control logic leads to different decision makers acting on asymmetric information sets, introduces the need for coordination between them, and perhaps not surprisingly makes the resulting optimal control problem much harder to solve. In fact, shortly after such questions were posed, it was realized that seemingly simple decentralized optimal control problems are computationally intractable to solve, with the Wistenhausen counterexample being a famous instance of this phenomenon. Spurred on by this perhaps discouraging result, a concerted 40 year effort to identify tractable classes of distributed optimal control problems culminated in the notion of quadratic invariance, which loosely states that if sub-controllers can exchange information with each other at least as quickly as the effect of their control actions propagates through the plant, then the resulting distributed optimal control problem admits a convex formulation.

The identification of quadratic invariance as an appropriate means of "convexifying" distributed optimal control problems led to a renewed enthusiasm in the controller synthesis community, resulting in a rich set of results over the past decade. The contributions of this thesis can be seen as being a part of this broader family of results, with a particular focus on closing the gap between theory and practice by relaxing or removing assumptions made in the traditional distributed optimal control framework. Our contributions are to the foundational theory of distributed optimal control, and fall under three broad categories, namely controller synthesis, architecture design and system identification.

We begin by providing two novel controller synthesis algorithms. The first is a solution to the distributed H-infinity optimal control problem subject to delay constraints, and provides the only known exact characterization of delay-constrained distributed controllers satisfying an H-infinity norm bound. The second is an explicit dynamic programming solution to a two player LQR state-feedback problem with varying delays. Accommodating varying delays represents an important first step in combining distributed optimal control theory with the area of Networked Control Systems that considers lossy channels in the feedback loop. Our next set of results are concerned with controller architecture design. When designing controllers for large-scale systems, the architectural aspects of the controller such as the placement of actuators, sensors, and the communication links between them can no longer be taken as given -- indeed the task of designing this architecture is now as important as the design of the control laws themselves. To address this task, we formulate the Regularization for Design (RFD) framework, which is a unifying computationally tractable approach, based on the model matching framework and atomic norm regularization, for the simultaneous co-design of a structured optimal controller and the architecture needed to implement it. Our final result is a contribution to distributed system identification. Traditional system identification techniques such as subspace identification are not computationally scalable, and destroy rather than leverage any a priori information about the system's interconnection structure. We argue that in the context of system identification, an essential building block of any scalable algorithm is the ability to estimate local dynamics within a large interconnected system. To that end we propose a promising heuristic for identifying the dynamics of a subsystem that is still connected to a large system. We exploit the fact that the transfer function of the local dynamics is low-order, but full-rank, while the transfer function of the global dynamics is high-order, but low-rank, to formulate this separation task as a nuclear norm minimization problem. Finally, we conclude with a brief discussion of future research directions, with a particular emphasis on how to incorporate the results of this thesis, and those of optimal control theory in general, into a broader theory of dynamics, control and optimization in layered architectures.