Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.

Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural α-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.

In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased β-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.

Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and α4β4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the α4β4 receptor.

Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the α7 nAChR. Additionally, binding interactions to the complementary face of the α7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.

Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.

Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.

Geological evolution of two crustal scale shear zones. Part I. The Rand thrust complex, northwestern Mojave Desert, California. Part II. The Magdalena metamorphic core complex, north central Sonora, Mexico

The geology and structure of two crustal scale shear zones were studied to understand the partitioning of strain within intracontinental orogenic belts. Movement histories and regional tectonic implications are deduced from observational data. The two widely separated study areas bear the imprint of intense Late Mesozoic through Middle Cenozoic tectonic activity. A regional transition from Late Cretaceous-Early Tertiary plutonism, metamorphism, and shortening strain to Middle Tertiary extension and magmatism is preserved in each area, with contrasting environments and mechanisms. Compressional phases of this tectonic history are better displayed in the Rand Mountains, whereas younger extensional structures dominate rock fabrics in the Magdalena area.

In the northwestern Mojave desert, the Rand Thrust Complex reveals a stack of four distinctive tectonic plates offset along the Garlock Fault. The lowermost plate, Rand Schist, is composed of greenschist facies metagraywacke, metachert, and metabasalt. Rand Schist is structurally overlain by Johannesburg Gneiss (= garnet-amphibolite grade orthogneisses, marbles and quartzites), which in turn is overlain by a Late Cretaceous hornblende-biotite granodiorite. Biotite granite forms the fourth and highest plate. Initial assembly of the tectonic stack involved a Late Cretaceous? south or southwest vergent overthrusting event in which Johannesburg Gneiss was imbricated and attenuated between Rand Schist and hornblende-biotite granodiorite. Thrusting postdated metamorphism and deformation of the lower two plates in separate environments. A post-kinematic stock, the Late Cretaceous Randsburg Granodiorite, intrudes deep levels of the complex and contains xenoliths of both Rand Schist and mylonitized Johannesburg? gneiss. Minimum shortening implied by the map patterns is 20 kilometers.

Some low angle faults of the Rand Thrust Complex formed or were reactivated between Late Cretaceous and Early Miocene time. South-southwest directed mylonites derived from Johannesburg Gneiss are commonly overprinted by less penetrative north-northeast vergent structures. Available kinematic information at shallower structural levels indicates that late disturbance(s) culminated in northward transport of the uppermost plate. Persistence of brittle fabrics along certain structural horizons suggests a possible association of late movement(s) with regionally known detachment faults. The four plates were juxtaposed and significant intraplate movements had ceased prior to Early Miocene emplacement of rhyolite porphyry dikes.

In the Magdalena region of north central Sonora, components of a pre-Middle Cretaceous stratigraphy are used as strain markers in tracking the evolution of a long lived orogenic belt. Important elements of the tectonic history include: (1) Compression during the Late Cretaceous and Early Tertiary, accompanied by plutonism, metamorphism, and ductile strain at depth, and thrust driven? syntectonic sedimentation at the surface. (2) Middle Tertiary transition to crustal extension, initially recorded by intrusion of leucogranites, inflation of the previously shortened middle and upper crustal section, and surface volcanism. (3) Gravity induced development of a normal sense ductile shear zone at mid crustal levels, with eventual detachment and southwestward displacement of the upper crustal stratigraphy by Early Miocene time.

Elucidation of the metamorphic core complex evolution just described was facilitated by fortuitous preservation of a unique assemblage of rocks and structures. The "type" stratigraphy utilized for regional correlation and strain analysis includes a Jurassic volcanic arc assemblage overlain by an Upper Jurassic-Lower Cretaceous quartz pebble conglomerate, in turn overlain by marine strata with fossiliferous Aptian-Albian limestones. The Jurassic strata, comprised of (a) rhyolite porphyries interstratified with quartz arenites, (b) rhyolite cobble conglomerate, and (c) intrusive granite porphyries, are known to rest on Precambrian basement north and east of the study area. The quartz pebble conglomerate is correlated with the Glance Conglomerate of southeastern Arizona and northeastern Sonora. The marine sequence represents part of an isolated arm? of the Bisbee Basin.

Crosscutting structural relationships between the pre-Middle Cretaceous supracrustal section, younger plutons, and deformational fabrics allow the tectonic sequence to be determined. Earliest phases of a Late Cretaceous-Early Tertiary orogeny are marked by emplacement of the 78 ± 3 Ma Guacomea Granodiorite (U/Pb zircon, Anderson et al., 1980) as a sill into deep levels of the layered Jurassic series. Subsequent regional metamorphism and ductile strain is recorded by a penetrative schistosity and lineation, and east-west trending folds. These fabrics are intruded by post-kinematic Early Tertiary? two mica granites. At shallower crustal levels, the orogeny is represented by north directed thrust faulting, formation of a large intermontane basin, and development of a pronounced unconformity. A second important phase of ductile strain followed Middle Tertiary? emplacement of leucogranites as sills and northwest trending dikes into intermediate levels of the deformed section (surficial volcanism was also active during this transitional period to regional extension). Gravitational instabilities resulting from crustal swelling via intrusion and thermal expansion led to development of a ductile shear zone within the stratigraphic horizon occupied by a laterally extensive leucogranite sill. With continued extension, upper crustal brittle normal faults (detachment faults) enhanced the uplift and tectonic denudation of this mylonite zone, ultimately resulting in southwestward displacement of the upper crustal stratigraphy.

Strains associated with the two ductile deformation events have been successfully partitioned through a multifaceted analysis. R_f/Ø measurements on various markers from the "type" stratigraphy allow a gradient representing cumulative strain since Middle Cretaceous time to be determined. From this gradient, noncoaxial strains accrued since emplacement of the leucogranites may be removed. Irrotational components of the postleucogranite strain are measured from quartz grain shapes in deformed granites; rotational components (shear strains) are determined from S-C fabrics and from restoration of rotated dike and vein networks. Structural observations and strain data are compatable with a deformation path of: (1) coaxial strain (pure shear?), followed by (2) injection of leucogranites as dikes (perpendicular to the minimum principle stress) and sills (parallel to the minimum principle stress), then (3) southwest directed simple shear. Modeling the late strain gradient as a simple shear zone permits a minimum displacement of 10 kilometers on the Magdalena mylonite zone/detachment fault system. Removal of the Middle Tertiary noncoaxial strains yields a residual (or pre-existing) strain gradient representative of the Late Cretaceous-Early Tertiary deformation. Several partially destrained cross sections, restored to the time of leucogranite emplacement, illustrate the idea that the upper plate of the core complex bas been detached from a region of significant topographic relief. 50% to 100% bulk extension across a 50 kilometer wide corridor is demonstrated.

Late Cenozoic tectonics of the Magdalena region are dominated by Basin and Range style faulting. Northeast and north-northwest trending high angle normal faults have interacted to extend the crust in an east-west direction. Net extension for this period is minor (10% to 15%) in comparison to the Middle Tertiary detachment related extensional episode.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

A multi-scale approach to shaping carbon nanotube structures for hollow microneedles

The concept of a carbon nanotube microneedle array is explored in this thesis from multiple perspectives including microneedle fabrication, physical aspects of transdermal delivery, and in vivo transdermal drug delivery experiments. Starting with standard techniques in carbon nanotube (CNT) fabrication, including catalyst patterning and chemical vapor deposition, vertically-aligned carbon nanotubes are utilized as a scaffold to define the shape of the hollow microneedle. Passive, scalable techniques based on capillary action and unique photolithographic methods are utilized to produce a CNT-polymer composite microneedle. Specific examples of CNT-polyimide and CNT-epoxy microneedles are investigated. Further analysis of the transport properties of polymer resins reveals general requirements for applying arbitrary polymers to the fabrication process.

The bottom-up fabrication approach embodied by vertically-aligned carbon nanotubes allows for more direct construction of complex high-aspect ratio features than standard top-down fabrication approaches, making microneedles an ideal application for CNTs. However, current vertically-aligned CNT fabrication techniques only allow for the production of extruded geometries with a constant cross-sectional area, such as cylinders. To rectify this limitation, isotropic oxygen etching is introduced as a novel fabrication technique to create true 3D CNT geometry. Oxygen etching is utilized to create a conical geometry from a cylindrical CNT structure as well as create complex shape transformations in other CNT geometries.

CNT-polymer composite microneedles are anchored onto a common polymer base less than 50 µm thick, which allows for the microneedles to be incorporated into multiple drug delivery platforms, including modified hypodermic syringes and silicone skin patches. Cylindrical microneedles are fabricated with 100 µm outer diameter and height of 200-250 µm with a central cavity, or lumen, diameter of 30 µm to facilitate liquid drug flow. In vitro delivery experiments in swine skin demonstrate the ability of the microneedles to successfully penetrate the skin and deliver aqueous solutions.

An in vivo study was performed to assess the ability of the CNT-polymer microneedles to deliver drugs transdermally. CNT-polymer microneedles are attached to a hand actuated silicone skin patch that holds a liquid reservoir of drugs. Fentanyl, a potent analgesic, was administered to New Zealand White Rabbits through 3 routes of delivery: topical patch, CNT-polymer microneedles, and subcutaneous hypodermic injection. Results demonstrate that the CNT-polymer microneedles have a similar onset of action as the topical patch. CNT-polymer microneedles were also vetted as a painless delivery approach compared to hypodermic injection. Comparative analysis with contemporary microneedle designs demonstrates that the delivery achieved through CNT-polymer microneedles is akin to current hollow microneedle architectures. The inherent advantage of applying a bottom-up fabrication approach alongside similar delivery performance to contemporary microneedle designs demonstrates that the CNT-polymer composite microneedle is a viable architecture in the emerging field of painless transdermal delivery.

From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Aerodynamics of vertical-axis wind turbines in full-scale and laboratory-scale experiments

Within a wind farm, multiple turbine wakes can interact and have a substantial effect on the overall power production. This makes an understanding of the wake recovery process critically important to optimizing wind farm efficiency. Vertical-axis wind turbines (VAWTs) exhibit features that are amenable to dramatically improving this efficiency. However, the physics of the flow around VAWTs is not well understood, especially as it pertains to wake interactions, and it is the goal of this thesis to partially fill this void. This objective is approached from two broadly different perspectives: a low-order view of wind farm aerodynamics, and a detailed experimental analysis of the VAWT wake.

One of the contributions of this thesis is the development of a semi-empirical model of wind farm aerodynamics, known as the LRB model, that is able to predict turbine array configurations to leading order accuracy. Another contribution is the characterization of the VAWT wake as a function of turbine solidity. It was found that three distinct regions of flow exist in the VAWT wake: (1) the near wake, where periodic blade shedding of vorticity dominates; (2) a transition region, where growth of a shear-layer instability occurs; (3) the far wake, where bluff-body oscillations dominate. The wake transition can be predicted using a new parameter, the dynamic solidity, which establishes a quantitative connection between the wake of a VAWT and that of a circular cylinder. The results provide insight into the mechanism of the VAWT wake recovery and the potential means to control it.

Non-Linear Scale Interactions in a Forced Turbulent Boundary Layer

This thesis explores the dynamics of scale interactions in a turbulent boundary layer through a forcing-response type experimental study. An emphasis is placed on the analysis of triadic wavenumber interactions since the governing Navier-Stokes equations for the flow necessitate a direct coupling between triadically consist scales. Two sets of experiments were performed in which deterministic disturbances were introduced into the flow using a spatially-impulsive dynamic wall perturbation. Hotwire anemometry was employed to measure the downstream turbulent velocity and study the flow response to the external forcing. In the first set of experiments, which were based on a recent investigation of dynamic forcing effects in a turbulent boundary layer, a 2D (spanwise constant) spatio-temporal normal mode was excited in the flow; the streamwise length and time scales of the synthetic mode roughly correspond to the very-large-scale-motions (VLSM) found naturally in canonical flows. Correlation studies between the large- and small-scale velocity signals reveal an alteration of the natural phase relations between scales by the synthetic mode. In particular, a strong phase-locking or organizing effect is seen on directly coupled small-scales through triadic interactions. Having characterized the bulk influence of a single energetic mode on the flow dynamics, a second set of experiments aimed at isolating specific triadic interactions was performed. Two distinct 2D large-scale normal modes were excited in the flow, and the response at the corresponding sum and difference wavenumbers was isolated from the turbulent signals. Results from this experiment serve as an unique demonstration of direct non-linear interactions in a fully turbulent wall-bounded flow, and allow for examination of phase relationships involving specific interacting scales. A direct connection is also made to the Navier-Stokes resolvent operator framework developed in recent literature. Results and analysis from the present work offer insights into the dynamical structure of wall turbulence, and have interesting implications for design of practical turbulence manipulation or control strategies.

Small scale turbulence in high Karlovitz number premixed flames

The purpose of this thesis is to characterize the behavior of the smallest turbulent scales in high Karlovitz number (Ka) premixed flames. These scales are particularly important in the two-way coupling between turbulence and chemistry and better understanding of these scales will support future modeling efforts using large eddy simulations (LES). The smallest turbulent scales are studied by considering the vorticity vector, ω, and its transport equation.

Due to the complexity of turbulent combustion introduced by the wide range of length and time scales, the two-dimensional vortex-flame interaction is first studied as a simplified test case. Numerical and analytical techniques are used to discern the dominate transport terms and their effects on vorticity based on the initial size and strength of the vortex. This description of the effects of the flame on a vortex provides a foundation for investigating vorticity in turbulent combustion.

Subsequently, enstrophy, ω² = ω • ω, and its transport equation are investigated in premixed turbulent combustion. For this purpose, a series of direct numerical simulations (DNS) of premixed n-heptane/air flames are performed, the conditions of which span a wide range of unburnt Karlovitz numbers and turbulent Reynolds numbers. Theoretical scaling analysis along with the DNS results support that, at high Karlovitz number, enstrophy transport is controlled by the viscous dissipation and vortex stretching/production terms. As a result, vorticity scales throughout the flame with the inverse of the Kolmogorov time scale, τ_η, just as in homogeneous isotropic turbulence. As τ_η is only a function of the viscosity and dissipation rate, this supports the validity of Kolmogorov’s first similarity hypothesis for sufficiently high Ka numbers (Ka ≳ 100). These conclusions are in contrast to low Karlovitz number behavior, where dilatation and baroclinic torque have a significant impact on vorticity within the flame. Results are unaffected by the transport model, chemical model, turbulent Reynolds number, and lastly the physical configuration.

Next, the isotropy of vorticity is assessed. It is found that given a sufficiently large value of the Karlovitz number (Ka ≳ 100) the vorticity is isotropic. At lower Karlovitz numbers, anisotropy develops due to the effects of the flame on the vortex stretching/production term. In this case, the local dynamics of vorticity in the strain-rate tensor, S, eigenframe are altered by the flame. At sufficiently high Karlovitz numbers, the dynamics of vorticity in this eigenframe resemble that of homogeneous isotropic turbulence.

Combined, the results of this thesis support that both the magnitude and orientation of vorticity resemble the behavior of homogeneous isotropic turbulence, given a sufficiently high Karlovitz number (Ka ≳ 100). This supports the validity of Kolmogorov’s first similarity hypothesis and the hypothesis of local isotropy under these condition. However, dramatically different behavior is found at lower Karlovitz numbers. These conclusions provides/suggests directions for modeling high Karlovitz number premixed flames using LES. With more accurate models, the design of aircraft combustors and other combustion based devices may better mitigate the detrimental effects of combustion, from reducing CO₂ and soot production to increasing engine efficiency.