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Adeno-Associated Viral Vectors for Gene Delivery to the Non-Human Primate Brain
Get PDFViral vectors are efficient gene-delivery carriers for somatic cell gene therapy, and replication-deficient vectors are actively being used in the clinic to replace and correct disease-causing genes and mutations. Mirroring their therapeutic effectiveness, viral vectors are also powerful in vivo gene-delivery tools in basic research. In the field of neuroscience, adeno-associated viruses (AAVs) in particular have emerged as a major workhorse that enable efficient in vivo expression of opsins for optogenetics, designer GPCRs for chemogenetics, and GCaMP for calcium imaging. Recently, AAV engineering efforts by our group and others have expanded the toolbox of AAV vectors to include capsid variants that traverse the blood-brain-barrier (BBB) in rodents. However, not all engineered AAV capsids translate from mice to non-human primates (NHPs). This is especially true for translation to the rhesus macaque (Macaca mulatta), an Old World primate that is the predominant NHP model and shares a more recent common ancestor with humans (~25 million years ago) compared to rodents (~75 million years ago) and New World primates, such as the common marmoset (Callithrix jacchus; ~35 million years ago). The primary contents of this dissertation will focus on the development of AAV.CAP-Mac as a vector for non-invasive gene-transfer to the NHP brain and demonstrations of its utility to interrogate neuronal morphology and physiology in the macaque central nervous system. We identified and selected CAP-Mac using a multi-species selection strategy in adult marmosets and infant macaques, where it demonstrated improved delivery efficiency compared to AAV9 and other engineered variants. In individual characterization, CAP-Mac was biased towards neurons in two infant Old World primate species, the rhesus macaque and the green monkey (Chlorocebus sabaeus). Given this neuronal tropism in Old World primates, we demonstrated how CAP-Mac can be readily used for non-invasive, Brainbow-like labeling of macaque neurons and calcium imaging of GCaMP ex vivo. In closing, we describe CAP-Mac tropism across multiple developmental states, species, and routes of administration. Additionally, we present preliminary data on “orphan capsids,” capsid variants that were engineered to be non-infective, but can be readily re-functionalized using known receptor-ligand pairs. Collectively, the work covered in this dissertation disseminates non-invasive, gene-delivery tools for NHP researchers, and lays the groundwork for further development of more specific and efficacious AAVs that access the NHP brain
Dynamics and Performance of Wind-Energy Systems in Unsteady Flow Conditions
Get PDFWind energy is poised to play a considerable role in the global transition to clean-energy technologies within the next few decades. Modern wind turbines, like aircraft and other aerodynamic structures, are typically designed with the assumption that the flows they encounter will be uniform and steady. However, atmospheric flows are highly unsteady, and systems operating within them must contend with gust disturbances that can lead to performance losses and structural damage. Therefore, the next generation of wind-energy systems requires physics-informed design principles that effectively account for and even leverage these unsteady flow phenomena for enhanced power generation, robustness, and operational longevity. Accordingly, this work details experimental and analytical efforts to characterize unsteady aerodynamics in wind-turbine contexts. First, the effects of unsteady streamwise motion on turbine performance are studied, as recent work has suggested that these dynamics may enable time-averaged efficiencies that exceed the steady-flow Betz limit on turbine efficiency. The power production of and flow around a periodically surging wind turbine are thus investigated using wind-tunnel experiments, which suggest that turbines in these flow conditions could leverage unsteady surge motions for power-extraction gains of up to 6.4% over the stationary case. Linearized and nonlinear dynamical models of the response of the turbine to these time-varying flows are derived and validated against the experimental data. These models are also coupled with a potential-flow model of the upstream induction zone of the turbine in order to predict temporal variations in the flow velocities and pressures in this region. Unsteady contributions to the time-averaged efficiency are also considered through theoretical potential-flow derivations. Additionally, a novel three-dimensional particle-tracking velocimetry approach using artificial snow as seeding particles is deployed to obtain volumetric flow measurements in the wakes of full-scale vertical-axis wind turbines in field conditions. These measurements yield insights into the effects of unsteady vortex dynamics on the structure of the near wake, with implications for the performance of turbines in wind-farm arrays. These investigations provide the analytical and experimental foundations for future studies of unsteady atmospheric flows, and will lead to the development of principles and techniques for wind-farm siting, control, and optimization
Inference of Global Methane Emissions from Oil and Gas Production
No full textAtmospheric methane plays a significant role in warming the climate. Characterizing its sources and sinks is important for future climate and air quality impacts. Global methane background trends suggest a sustained increase in emissions since 2007. There is no debate that reducing anthropogenic (human-driven) emissions can lead to short-term decreases in atmospheric methane, posing an attractive avenue towards mitigating climate change. Yet, effective policy to limit emissions from energy-related activities relies on accurate emission estimates, and historically, it has been challenging to diagnose both the magnitude and origin of methane leaks from a wide range of facilities and components across production, transmission, storage, and distribution systems. We present a novel Bayesian hierarchical model to improve methane emission estimates on global and regional scales from oil and gas processes. We also present methods to optimize time and cost of model simulations of certain trace gases, including several of which have important climate implications. Finally, we present our efforts in characterizing fossil methane from burgeoning oil production in Oklahoma and Texas using long term ground-based remote-sensing observations combined with Stochastic Time-Inverted Larangian Transport modeling.</p
Scalable Fabrication of Micro-Architected Water Filtering Membranes
Get PDFPolymer-based filtration devices are predominantly mass manufactured via mechanical spinning or electrospinning of heated polymer materials or fiberglass to create a randomly oriented fibrous network. This technique, while effective at producing materials necessary for traditional filtering applications, fails to afford control over morphology, both macro- and microscopically. The filtering material produced often relies exclusively on its randomly assembled porosity (and occasionally on its surface charge) to capture materials from filtered fluids but provides little means for targeted analyte capture without bulk surface coating or functionalization. This thesis seeks to demonstrate a unique approach to filtration membrane manufacture via a novel high-throughput holographic lithography and contact lithography process in the visible spectrum that utilizes a customized negative-tone photoresist inherently capable of localized surface modification.
This thesis first describes the development of a large-scale holographic lithography process, from conceptualization to implementation, and demonstrates its efficacy by examining produced materials. A phase metasurface mask is utilized to produce a periodic intensity distribution of incident photons. This mask is irradiated at 0.23-0.25 W via linear raster scanning of a 2.2 mm diameter 532 nm laser at 1.5 mm/s and a scan offset of 0.4 mm to produce a homogeneous exposure profile in visible-light sensitized SU-8 negative-tone photoresist. Subsequent photoresist development results in 30–40 µm-thick nano-architected sheets with 2.1 × 2.4 cm² lateral dimensions and ~500 nm-wide struts organized in layered 3D brick-and-mortar-like patterns to result in ~50–70% porosity. Scanning electron micrographs of cross-sectioned materials reveal how pattern morphology varies with cure depth, and furthermore how the lack of complete porosity disqualifies this material for application as a membrane filter.
This thesis subsequently focuses on the development of a novel glycidyl methacrylate (GMA)-based negative-tone photoresist for implementation in the previously described lithography system to produce materials more amenable to functional membrane filter production. GMA is polymerized with a photo-caged aminated monomer, 2-((((2-nitrobenzyl)oxy)carbonyl)amino)ethyl 2-methyloxirane-2-carboxylate (ONBAMA) via free radical polymerization (FRP) and atom-transfer radical polymerization (ATRP) to produce ~30 kDa statistical co-polymers at an 85:15 monomer ratio, respectively. These linear co-polymers are then mixed with a photoacid generator (PAG) to produce a 532 nm sensitized negative-tone photoresist. Pre- and post-exposure bake temperatures are selected via glass-transition temperature identification (~62 °C) with differential scanning calorimetry (DSC) experiments, and cure depth varying with optical exposure dose is examined via establishment of contrast curves. The photoresist is then utilized in the previously described lithography system to produce square arrays of ~25 um circular holes, and the resulting films are characterized via optical and scanning electron microscopy.
This thesis concludes with an examination of the poly(GMA-rand-ONBAMA) films implemented as water-permeable filtration membranes. Efficacy of surface functionalization and solution capture explored via amine deprotection and subsequent tagging with fluorescein isothiocyanate (FITC) dye. The presence and intensity uniformity of tagged samples are examined via confocal microscopy. Transmission of water is justified analytical examination and phenomenologically demonstrated via droplet loading of supported membranes with methylene blue-dyed water. Results are preliminary but indicate potential application of manufactured films as water filters.
In summary, this thesis provides a foundation for the development of nano- and micro-architected materials at large scale and details its implementation for the design and preliminary testing of a GMA-based photoresist for water filtering membrane manufacture. Future research on optimizing photoresist design for mechanical stability could enable utilization of similar membranes for protein capture from biological fluids for use in diagnostic tools and assay automation.</p
Studies of mRNA Expression and Degradation
Get PDFPart 1: Protein degradation coupled to Nonsense-mediated mRNA decay
Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. In part 1 of this thesis, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, but suggested protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provides a reference for the field to identify and characterize required factors.
Part 2: The Commons Cell Atlas
Current cell atlas projects aim to curate representative datasets, cell-types, and marker genes for tissues across an organism. Despite their ubiquity, atlas projects rely on duplicated and manual effort to curate marker genes and annotate cell-types. Importantly, the lack of data-compatible tools and a fixed representation of the atlas make their reanalysis near-impossible. To overcome these challenges, we present a collection of data, algorithms, and tools to automate cataloging and analyzing cell-types across all tissues in an organism. We leveraged this work to build a Human Commons Cell Atlas comprising 2.9 million cells across 27 tissues that can be easily updated and that is structured to facilitate custom analyses. To showcase the flexibility of the atlas, we demonstrate that it can be used for isoform analyses. In particular, we study cell-type specificity of isoforms of OAS1, which has recently been shown to offer SARS-CoV-2 protection in certain individuals that display higher expression of the p46 isoform. Using our Commons Cell Atlas, we localize the OAS1 p44b isoform to the testis, and find that it is specific to germ line cells. By virtue of enabling customized analyses via a modular and dynamic atlas structure, the Commons Cell Atlas should be useful for exploratory analyses that are intractable within the rigid framework of current gene-centric static atlases.</p
Assembly of Intermediate-Mass Black Holes Along Star Formation
Get PDFIntermediate-mass black holes (IMBHs) are poorly observed and not as well understood as stellar-mass black holes (BHs) and supermassive black holes (SMBHs). However, they can be important to complement the formation scenario of massive BHs other than stellar-mass ones and reconcile the existence of some high-energy sources in the Universe. The thesis studies the assembly of IMBHs in star-forming giant molecular clouds (GMCs) of ∼ 5 – 500 pc and ∼ 10⁴ – 10¹⁰ M_⊙, which are realistic environments for some scenarios of IMBH formation, including runaway collisions in dense star clusters and super-Eddington accretion onto ∼ 100 M_⊙ BH seeds like remnants of massive stars.
We first inspect the runaway-collision scenario where IMBHs form as remnants of “quasi-stars” after stellar collisions. Density profiles of young massive clusters can be important for this scenario but are missing observational hints. We measure density profiles of cluster populations in star-formation simulations in GMCs and conduct both analytic derivations and Monte-Carlo simulations to estimate the mass of the quasi-star in different clusters. The analytic expression is in approximate agreement with observations.
The following three chapters are about the super-Eddington accretion scenario. The first question to solve is the availability of super-Eddington accretion in turbulent and star-forming environments. We run simulations of BH accretion in GMCs with star formation based on FIRE-2 physics. We find that dense clumps generated by stellar feedback and turbulence can feed BHs at high accretion rates. We also conclude that GMCs with high surface densities are favored for super- or hyper-Eddington accretion, in which self-gravity dominates over stellar feedback.
After convincing the availability of super-Eddington accretion in dense GMCs, we study the self-regulation of BH accretion through its feedback. We construct a sub-grid model of BH accretion and feedback, including radiation, winds/jets, and relativistic diffusive cosmic rays. We find that super-Eddington accretion is still achievable with proper radiative feedback models but is challenged by BH mechanical feedback. We also quantify BH feedback effects and find that they can be analytically explained with momentum-driven arguments. Moreover, we study the effects of multiple sub-grid parameters and BH feedback’s impact on star formation in GMCs.
Finally, we study another mode of accretion due to steady gas inflow towards BHs. This complements the missing interaction between stars and BHs in previous studies. Along with star formation, star clusters form and merge hierarchically, creating deep potential wells to capture BHs. At the late stage of the simulation, a ∼ 10 pc disk structures form. The gas inflow rate can be ∼ 10 M_⊙/yr. We find a non-trivial strong toroidal magnetic field in the disk, which is thermally heated and ionized by feedback from stars.</p
Microbial Transformations of Sulfur: Environmental and (Paleo) Ecological Implications
Get PDFThis thesis is centered around the role that sulfur plays in the cycling of carbon and in microbial energetics. In the oceans, sulfate is the most important electron acceptor for the remineralization of organic matter after oxygen has been depleted, and sulfate reduction is particularly relevant in coastal environments and in marine and freshwater sediments. The opposite process, reduced sulfur oxidation, allows autotrophic microorganisms to fix carbon in environments where oxygen is scarce. Organic sulfur is also a relevant component of the sulfur cycle, since sulfur is the sixth most abundant element in biomass, it can protect organic matter from degradation, and it is composed of hundreds of molecules that are produced mainly by microorganisms, with potentially relevant ecological roles.
This work has been divided into two parts. In the first one, we attempt to expand our understanding on different aspects of the sulfur cycle. In Chapter 2, published in Limnology and Oceanography, we focus on dimethylsulfoniopropionate (DMSP), the most abundant organic sulfur compound in the oceans with roles of UV, cryo, and osmoprotection, and involved in the formation of sulfate aerosols. We propose a framework to differentiate between the microbial degradation pathways of DMSP based on the sulfur isotope fractionations imprinted by each one of them. In Chapter 3, we perform a survey of sulfate, sulfide, and reduced sulfur intermediates, as well as redox-sensitive elements, in porewaters of a ~40 cm core from the San Clemente Basin (California) and three 1.2-2 m cores near Cocos Ridge (Costa Rica). We correlate these concentrations with the sediment microbial community composition to unveil the specifics of organic matter and sulfur cycling at these localities. In Chapter 4, we explore the utility of sulfur isotope fractionations to characterize different pathways involved in microbial sulfur oxidation (MSO), and examine the role of nutrient limitation and growth rates on the magnitude of the fractionation.
In the second part of this thesis, we aim at understanding biomineralization by consortia between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which comprise more than 90% of the microbial biomass in deep sea sediments around hydrocarbon seeps. In Chapter 5 (in review at Proceedings of the National Academy of Sciences) we establish that modern ANME-SRB aggregates precipitate amorphous silica in undersaturated solutions in sediments and carbonates, often in the form of rims, which pinpoints to a potentially new microbial biomineralization mechanism. In Chapter 6, we posit the use of this proxy, together with distinctive spectral and isotopic signals, to find potential microfossils of ANME-SRB aggregates in the rock record of the Earth and other planetary bodies where methane seepage has occurred throughout geologic time. This suite of tools is used in conjunction to identify ANME-SRB aggregates in the Tepee Buttes (Colorado, 75 Mya) seep carbonates.</p
Good Expectations: The Paradox of Expecting Great Things
No full textIntroduction: Whether or not we are aware of it, the average person is making thousands of split-second predictions. This is true in both the short term — whether or not their morning coffee will be good, or if they’ll do well on their test — and the long term: whether or not they will be at their current job when they retire, or if they will find long-term happiness with their partner. What these predictions lead to are expectations. After making good coffee for several months, one will come to predict – and expect – that their morning coffee will be good. Similarly, after several years of a happy relationship, one will expect that the relationship will continue to bring them happiness, enough to commit to it for the rest of their life. When these expectations are met, we are content. But when we meet the unexpected, it really sucks! If a student expects to receive an A on a test and receives a surprise C, or if a worker is fired suddenly from their dream job, the results can be upsetting, impacting their short-term happiness on a range from disappointing to devastating. While it is true that over time, these perceived failures will stop affecting one's happiness so dramatically, they can be hard to cope with. So, how can we prevent it
Parametrically-Driven Nonlinear Optical Resonators and their Networks for Sensing and Computing
Get PDF New physics and novel applications in various fields ranging from biology, and spectroscopy, to manipulation of quantum systems are driven by the availability of coherent light sources including frequency combs in the visible and mid-infrared spectral regimes. Nonlinear optical systems, that are parametrically driven by technologically mature near-infrared lasers, are leveraged in this regard to access challenging wavelengths where conventional lasers may be unavailable. It is of paramount importance to miniaturize these systems and replace the traditional bulky setups thereby paving the way for a plethora of applications. Optical parametric oscillators are among the most prominent examples of such nonlinear systems and beyond their indispensable usage as light sources (both classical and quantum) their unique non-equilibrium dynamics can endow a wealth of functionalities absent in their linear counterparts. These properties can be engineered and utilized for realizing highly sensitive sensors as well as special-purpose computing hardware that may outperform conventional digital computers. A network of these coupled parametric oscillators can be made to interact leading to emergent behaviors that are not expected from the individual constituents.
In this work, we experimentally and theoretically study the dynamics of individual and coupled optical parametric oscillators towards sensing and computing applications. We explore a previously avoided regime of operation for generating ultra-short pulses from these parametrically driven nonlinear resonators that lead to extreme pulse compression. We engineer the nonlinear dynamics of these systems to realize all-optical spectral phase transitions (both first-order and second-order) that behave as highly-sensitive sensors. We show how these critical phenomena can be utilized to enhance the solution accuracy of physics-based solvers in finding optimum solutions to combinatorial optimization problems in the context of coherent Ising machines. We also realize optical parametric oscillators in integrated lithium-niobate nanophotonic platform and demonstrate a mid-infrared frequency comb source that is widely tunable over an octave accompanied by visible frequency comb generation. We develop a comprehensive description to investigate the noise properties of optical parametric oscillators that provide new insights into the phase noise behavior of optical parametric oscillators in their various operating regimes. Finally, we propose a system of parametrically driven resonators as a synthetic medium with highly reconfigurable interactions that can host a plethora of emergent phenomena ranging from topological behaviors to non-Hermitian dynamics. These networks of nonlinear resonators display intriguing dynamical properties in contrast to their static counterparts in condensed-matter physics with implications in quantum sensing and robust device functionality. </p
Synthesis of Cyclic Polymers by Ring Expansion and Ring Opening Metathesis Polymerization
Get PDFCyclic polymers are topologically interesting and envisioned as a lubricant material. However, scalable synthesis of pure cyclic polymers remains elusive. The most straightforward way is to recover a used catalyst after the synthesis of cyclic polymers and reuse it. Unfortunately, this is demanding because of the catalyst’s vulnerability and inseparability from polymers, which reduce the practicality of the process. In Chapter 1 we describe a continuous circular process, where polymerization, polymer separation, and catalyst recovery happen in situ, to dispense a pure cyclic polymer after bulk ring-expansion metathesis polymerization of cyclopentene. This process is enabled by introducing silica-supported ruthenium catalysts and newly designed glassware. Different depolymerization kinetics of the cyclic polymer from its linear analogue are also discussed. This process minimizes manual labour, maximizes the security of vulnerable catalysts and guarantees the purity of cyclic polymers, thereby showcasing a prototype of a scalable access to cyclic polymers with increased turnovers (≥415,000) of precious catalysts.
α-Oxygenated Z-olefins are ubiquitous in biologically active molecules and serve as versatile handles for organic synthesis, but their syntheses are often tedious and less selective. In Chapter 2 we report the efficient Z-selective metathesis of various terminal acrylates and allyl alcohols, which enables facile and selective construction of high value-added α-oxygenated Z-olefins from readily available feedstock chemicals. These challenging metathesis transformations are enabled by novel cyclometalated Ru-carbene-nitrate complexes bearing bulky-yet-flexible side arms, whose assembly was unlocked by new organometallic syntheses.
Efficient separation of macrocyclic polyolefins from reaction mixtures of ring-opening metathesis polymerization is crucial for their application in materials science, drug delivery, and for the mechanistic study of the reaction mechanism. In Chapter 3, we present a facile method for obtaining topologically pure macrocyclic fractions by modifying chain ends using enyne metathesis chemistry and introducing polar functional groups into linear polymer chains through the addition of polar monomers. Nonpolar cyclic polyolefins are then readily separated using silica gel chromatography. The purity of the cyclic fractions was verified using multiple techniques, including gel permeation chromatography, nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We investigate the reaction factors affecting the yield and molecular weight of macrocycles during ring-opening metathesis polymerization of cyclooctadiene and discuss macrocycle formation in the ring-opening metathesis polymerization of cyclooctene, cyclopentene, and norbornene. Our work offers crucial insights into the synthesis and separation of macrocycles.</p