Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents

Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.

In addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.

An investigation of detached shock waves

This investigation demonstrates an application of a flexible wall nozzle for testing in a supersonic wind tunnel. It is conservative to say that the versatility of this nozzle is such that it warrants the expenditure of time to carefully engineer a nozzle and incorporate it in the wind tunnel as a permanent part of the system. The gradients in the test section were kept within one percent of the calibrated Mach number, however, the gradients occurring over the bodies tested were only ± 0.2 percent in Mach number.

The conditions existing on a finite cone with a vertex angle of 75° were investigated by considering the pressure distribution on the cone and the shape of the shock wave. The pressure distribution on the surface of the 75° cone when based on upstream conditions does not show any discontinuities at the theoretical attachment Mach number.

Both the angle of the shock wave and the pressure distribution of the 75° cone are in very close agreement with the theoretical values given in the Kopal report, (Ref. 3).

The location of the intersection of the sonic line with the surface of the cone and with the shock wave are given for the cone. The blocking characteristics of the GALCIT supersonic wind tunnel were investigated with a series of 60° cones.

A report on increasing the thrust of a turbojet engine by combustion of fuel in the tail pipe

The purpose of this work was to develop a means of increasing the thrust of a turbojet engine by burning kerosene in the tail pipe.

A combustion system was developed which gave the following results:
(l) Maximum thrust increase using a G.E. I-14 engine was 64 per cent over straight tail pipe thrust corresponding to 42 per cent increase over the normal engine thrust. This increase was accomplished at an engine rpm of 12,000.
(2) Increase of maximum thrust obtained was 51 per cent over the straight tail pipe thrust corresponding to 23 per cent over the normal engine thrust. This increase was accomplished at an engine rpm of l6,000.
(3) For the thrust increases mentioned in (1) and (2) above, increases of Specific Fuel Consumption were 66 per cent and 76 per cent respectively over normal engine SFC.

Cavity Optomechanics at Millikelvin Temperatures

The field of cavity optomechanics, which concerns the coupling of a mechanical object's motion to the electromagnetic field of a high finesse cavity, allows for exquisitely sensitive measurements of mechanical motion, from large-scale gravitational wave detection to microscale accelerometers. Moreover, it provides a potential means to control and engineer the state of a macroscopic mechanical object at the quantum level, provided one can realize sufficiently strong interaction strengths relative to the ambient thermal noise. Recent experiments utilizing the optomechanical interaction to cool mechanical resonators to their motional quantum ground state allow for a variety of quantum engineering applications, including preparation of non-classical mechanical states and coherent optical to microwave conversion. Optomechanical crystals (OMCs), in which bandgaps for both optical and mechanical waves can be introduced through patterning of a material, provide one particularly attractive means for realizing strong interactions between high-frequency mechanical resonators and near-infrared light. Beyond the usual paradigm of cavity optomechanics involving isolated single mechanical elements, OMCs can also be fashioned into planar circuits for photons and phonons, and arrays of optomechanical elements can be interconnected via optical and acoustic waveguides. Such coupled OMC arrays have been proposed as a way to realize quantum optomechanical memories, nanomechanical circuits for continuous variable quantum information processing and phononic quantum networks, and as a platform for engineering and studying quantum many-body physics of optomechanical meta-materials.

However, while ground state occupancies (that is, average phonon occupancies less than one) have been achieved in OMC cavities utilizing laser cooling techniques, parasitic absorption and the concomitant degradation of the mechanical quality factor fundamentally limit this approach. On the other hand, the high mechanical frequency of these systems allows for the possibility of using a dilution refrigerator to simultaneously achieve low thermal occupancy and long mechanical coherence time by passively cooling the device to the millikelvin regime. This thesis describes efforts to realize the measurement of OMC cavities inside a dilution refrigerator, including the development of fridge-compatible optical coupling schemes and the characterization of the heating dynamics of the mechanical resonator at sub-kelvin temperatures.

We will begin by summarizing the theoretical framework used to describe cavity optomechanical systems, as well as a handful of the quantum applications envisioned for such devices. Then, we will present background on the design of the nanobeam OMC cavities used for this work, along with details of the design and characterization of tapered fiber couplers for optical coupling inside the fridge. Finally, we will present measurements of the devices at fridge base temperatures of T_f = 10 mK, using both heterodyne spectroscopy and time-resolved sideband photon counting, as well as detailed analysis of the prospects for future quantum applications based on the observed optically-induced heating.

Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.

In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.

Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for "tuning" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious "leak" pathways that compete with desired pathways.

We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.

Signatures of Topological Superconductors

Topological superconductors are particularly interesting in light of the active ongoing experimental efforts for realizing exotic physics such as Majorana zero modes. These systems have excitations with non-Abelian exchange statistics, which provides a path towards topological quantum information processing. Intrinsic topological superconductors are quite rare in nature. However, one can engineer topological superconductivity by inducing effective p-wave pairing in materials which can be grown in the laboratory. One possibility is to induce the proximity effect in topological insulators; another is to use hybrid structures of superconductors and semiconductors.

The proposal of interfacing s-wave superconductors with quantum spin Hall systems provides a promising route to engineered topological superconductivity. Given the exciting recent progress on the fabrication side, identifying experiments that definitively expose the topological superconducting phase (and clearly distinguish it from a trivial state) raises an increasingly important problem. With this goal in mind, we proposed a detection scheme to get an unambiguous signature of topological superconductivity, even in the presence of ordinarily detrimental effects such as thermal fluctuations and quasiparticle poisoning. We considered a Josephson junction built on top of a quantum spin Hall material. This system allows the proximity effect to turn edge states in effective topological superconductors. Such a setup is promising because experimentalists have demonstrated that supercurrents indeed flow through quantum spin Hall edges. To demonstrate the topological nature of the superconducting quantum spin Hall edges, theorists have proposed examining the periodicity of Josephson currents respect to the phase across a Josephson junction. The periodicity of tunneling currents of ground states in a topological superconductor Josephson junction is double that of a conventional Josephson junction. In practice, this modification of periodicity is extremely difficult to observe because noise sources, such as quasiparticle poisoning, wash out the signature of topological superconductors. For this reason, We propose a new, relatively simple DC measurement that can compellingly reveal topological superconductivity in such quantum spin Hall/superconductor heterostructures. More specifically, We develop a general framework for capturing the junction's current-voltage characteristics as a function of applied magnetic flux. Our analysis reveals sharp signatures of topological superconductivity in the field-dependent critical current. These signatures include the presence of multiple critical currents and a non-vanishing critical current for all magnetic field strengths as a reliable identification scheme for topological superconductivity.

This system becomes more interesting as interactions between electrons are involved. By modeling edge states as a Luttinger liquid, we find conductance provides universal signatures to distinguish between normal and topological superconductors. More specifically, we use renormalization group methods to extract universal transport characteristics of superconductor/quantum spin Hall heterostructures where the native edge states serve as a lead. Interestingly, arbitrarily weak interactions induce qualitative changes in the behavior relative to the free-fermion limit, leading to a sharp dichotomy in conductance for the trivial (narrow superconductor) and topological (wide superconductor) cases. Furthermore, we find that strong interactions can in principle induce parafermion excitations at a superconductor/quantum spin Hall junction.

As we identify the existence of topological superconductor, we can take a step further. One can use topological superconductor for realizing Majorana modes by breaking time reversal symmetry. An advantage of 2D topological insulator is that networks required for braiding Majoranas along the edge channels can be obtained by adjoining 2D topological insulator to form corner junctions. Physically cutting quantum wells for this purpose, however, presents technical challenges. For this reason, I propose a more accessible means of forming networks that rely on dynamically manipulating the location of edge states inside of a single 2D topological insulator sheet. In particular, I show that edge states can effectively be dragged into the system's interior by gating a region near the edge into a metallic regime and then removing the resulting gapless carriers via proximity-induced superconductivity. This method allows one to construct rather general quasi-1D networks along which Majorana modes can be exchanged by electrostatic means.

Apart from 2D topological insulators, Majorana fermions can also be generated in other more accessible materials such as semiconductors. Following up on a suggestion by experimentalist Charlie Marcus, I proposed a novel geometry to create Majorana fermions by placing a 2D electron gas in proximity to an interdigitated superconductor-ferromagnet structure. This architecture evades several manufacturing challenges by allowing single-side fabrication and widening the class of 2D electron gas that may be used, such as the surface states of bulk semiconductors. Furthermore, it naturally allows one to trap and manipulate Majorana fermions through the application of currents. Thus, this structure may lead to the development of a circuit that enables fully electrical manipulation of topologically-protected quantum memory. To reveal these exotic Majorana zero modes, I also proposed an interference scheme to detect Majorana fermions that is broadly applicable to any 2D topological superconductor platform.

San Gabriel Valley sewerage system

The disposal of sewage is the most important item in public sanitation. It is the most important present day problem in every city whether large or small. The direct cause of the majority of epidemics is the contamination of the water supply of the city by the excreta of man or animal. Public health varies directly as public sanitation, and if the public sanitation be good, the liability of sickness caused by contamination of the water supply is greatly lessened. When a city outgrows its sewerage system the public health becomes endangered. There are two causes for the increased amount of sewerage, increase in population and increase in industrial and manufacturing wastes. The main problem in this connection is the ultimate disposal of the matter which reaches the sewers.

Analysis of a concrete arch highway bridge reinforced with structural steel

An article in the Engineering News-Record for March 30, 1923, describes a new concrete arch bridge across the Connecticut River between Springfield and West Springfield, Mass.

A study of the durability of concrete in sea water

The durability of concrete in sea water has been a very widely debated question from the time that disintegration of concrete in sea water first became evident. Numerous investigations and reports have been made of isolated structures in various parts of the country and from these, hasty conclusions have been reached which engineers, situated where different conditions prevail, have not been warranted in accepting. A few engineers have made investigations of a much wider scope and consequently their conclusions should be given greater weight.

Mean-speed measurements in two-dimensional, incompressible, fully-developed turbulent channel flow

Mean velocity profiles were measured in the 5” x 60” wind channel of the turbulence laboratory at the GALCIT, by the use of a hot-wire anemometer. The repeatability of results was established, and the accuracy of the instrumentation estimated. Scatter of experimental results is a little, if any, beyond this limit, although some effects might be expected to arise from variations in atmospheric humidity, no account of this factor having been taken in the present work. Also, slight unsteadiness in flow conditions will be responsible for some scatter.

Irregularities of a hot-wire in close proximity to a solid boundary at low speeds were observed, as have already been found by others.

That Kármán’s logarithmic law holds reasonably well over the main part of a fully developed turbulent flow was checked, the equation u/u_t = 6.0 + 6.25 log₁₀ y^ut/v being obtained, and, as has been previously the case, the experimental points do not quite form one straight line in the region where viscosity effects are small. The values of the constants for this law for the best over-all agreement were determined and compared with those obtained by others.

The range of Reynolds numbers used (based on half-width of channel) was from 20,000 to 60,000.

Design of an oxygen flow meter for hospital application

A general description of the need for hospital flow meters is given along with an analysis of some common flow measurement methods.

The design criteria, establishment of the basic configuration of the instrument, and the evolution of the final design are presented in detail. The ability of the magnetic crossover mechanism to extract the square root of an input is explained, and design curves are presented. The action of the flow totalizer is described in relation to the rest of the instrument. A complete set of manufacturing drawings for the instrument and its tooling is included in the thesis.

In conclusion, an evaluation of the completed instrument is made, and improvements and modifications are indicated. Mention is made of the adaptability of the magnetic crossover mechanism to other instrumentation.

An investigation of a method of underwater propulsion by direct gas injection

This report presents the results of an investigation of a method of underwater propulsion. The propelling system utilizes the energy of a small mass of expanding gas to accelerate the flow of a large mass of water through an open ended duct of proper shape and dimensions to obtain a resultant thrust. The investigation was limited to making a large number of runs on a hydroduct of arbitrary design, varying between wide limits the water flow and gas flow through the device, and measuring the net thrust caused by the introduction and expansion of the gas.

In comparison with the effective exhaust velocity of about 6,000 feet per second observed in rocket motors, this hydroduct model attained a maximum effective exhaust velocity of more than 27,000 feet per second, using nitrogen gas. Using hydrogen gas, effective exhaust velocities of 146,000 feet per second were obtained. Further investigation should prove this method of propulsion not only to be practical but very efficient.

This investigation was conducted at Project No. 1, Guggenheim Aeronautical Laboratory, California Institute of Technology, Pasadena, California.

Pulsed neutron measurements in two adjacent finite media

The pulsed neutron technique has been used to investigate the decay of thermal neutrons in two adjacent water-borated water finite media. Experiments were performed with a 6x6x6 inches cubic assembly divided in two halves by a thin membrane and filled with pure distilled water on one side and borated water on the other side.

The fundamental decay constant was measured versus the boric acid concentration in the poisoned medium. The experimental results showed good agreement with the predictions of the time dependent diffusion model. It was assumed that the addition of boric acid increases the absorption cross section of the poisoned medium without affecting its diffusion properties: In these conditions, space-energy separability and the concept of an “effective” buckling as derived from diffusion theory were introduced. Their validity was supported by the experimental results.

Measurements were performed with the absorption cross section of the poisoned medium increasing gradually up to 16 times its initial value. Extensive use of the IBM 7090-7094 Computing facility was made to analyze properly the decay data (Frantic Code). Attention was given to the count loss correction scheme and the handling of the statistics involved. Fitting of the experimental results into the analytical form predicted by the diffusion model led to

Ʃ_av = 4721 sec^-1 (±150)

D_o = 35972 cm²sec^-1 (±800) for water at 21˚C

C (given) = 3420 cm⁴sec^-1

These values, when compared with published data, show that the diffusion model is adequate in describing the experiment.

An in vitro biomolecular breadboard for prototyping synthetic biological circuits

Biomolecular circuit engineering is critical for implementing complex functions in vivo, and is a baseline method in the synthetic biology space. However, current methods for conducting biomolecular circuit engineering are time-consuming and tedious. A complete design-build-test cycle typically takes weeks' to months' time due to the lack of an intermediary between design ex vivo and testing in vivo. In this work, we explore the development and application of a "biomolecular breadboard" composed of an in-vitro transcription-translation (TX-TL) lysate to rapidly speed up the engineering design-build-test cycle. We first developed protocols for creating and using lysates for conducting biological circuit design. By doing so we simplified the existing technology to an affordable ($0.03/uL) and easy to use three-tube reagent system. We then developed tools to accelerate circuit design by allowing for linear DNA use in lieu of plasmid DNA, and by utilizing principles of modular assembly. This allowed the design-build-test cycle to be reduced to under a business day. We then characterized protein degradation dynamics in the breadboard to aid to implementing complex circuits. Finally, we demonstrated that the breadboard could be applied to engineer complex synthetic circuits in vitro and in vivo. Specifically, we utilized our understanding of linear DNA prototyping, modular assembly, and protein degradation dynamics to characterize the repressilator oscillator and to prototype novel three- and five-node negative feedback oscillators both in vitro and in vivo. We therefore believe the biomolecular breadboard has wide application for acting as an intermediary for biological circuit engineering.