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Descriptive Set Theory and Dynamics of Countable Groups
Get PDFThis thesis comprises four papers.
1. We show that for any Polish group G and any countable normal subgroup Γ ⊳ G, the coset equivalence relation G/Γ is a hyperfinite Borel equivalence relation. In particular, the outer automorphism group of any countable group is hyperfinite.
2. Given a countable Borel equivalence relation E and a countable group G, we study the problem of when a Borel action of G on X/E can be lifted to a Borel action of G on X.
3. Let Γ be a countable group. A classical theorem of Thorisson states that if X is a standard Borel Γ-space and µ and ν are Borel probability measures on X which agree on every Γ-invariant subset, then µ and ν are equidecomposable,
i.e., there are Borel measures (µγ)γϵΓ on X such that µ = Σγµγ and ν = Σγγµγ. We establish a generalization of this result to cardinal algebras.
4. Let R be a ring equipped with a proper norm. We show that under suitable conditions on R, there is a natural basis under continuous linear injection for the set of Polish R-modules which are not countably generated. When R is a division ring, this basis can be taken to be a singleton.</p
Structure and Function of the Human Nuclear Pore Complex
Get PDFThe nuclear pore complex (NPC) mediates the selective transport of macromolecules between the nucleus and the cytoplasm of the eukaryotic cell. In humans, the NPC is a ~110 MDa assembly of ~1,000 proteins, termed nucleoporins, which establishes a ~50 nm wide central transport channel that traverses both membranes of the nuclear envelope. In the central transport channel, natively unfolded nucleoporin regions rich in repeated phenylalanine-glycine (FG) motifs form a size-selective barrier that limits the diffusion of macromolecules. Active cargo transport is facilitated by dedicated mobile transport factors that exchange through the FG barrier and couple binding and unbinding of cargo to GTP hydrolysis by the small GTPase Ran. By contrast, nuclear export of mature mRNA is driven by ATP-dependent remodeling of messenger ribonucleoprotein (mRNP) at the NPC's cytoplasmic face. Whereas the mechanisms of the mobile Ran dependent transport machinery have been conceptually rationalized, the NPC's role in facilitating and modulating nucleocytoplasmic transport, including mRNA export, remains poorly understood. Addressing gaps in the understanding of the NPC's structure and function, this thesis presents comprehensive interdisciplinary analyses of the NPC's inner ring, linker scaffold, and cytoplasmic face architectures. First, we determined the structure, stoichiometry, and location of the channel nucleoporin heterotrimer that provides the bulk of the FG repeats in the central transport channel. Second, we elucidated the molecular details and topology of the network of linker nucleoporins previously shown to assemble folded scaffold nucleoporins into protomeric subcomplexes of the inner ring. Third, we characterized the composition, stoichiometry, and attachment mechanism of cytoplasmic filament nucleoporins, which present manifold Ran- and RNA-binding domains on the cytoplasmic face of the NPC. The resulting near atomic structure of the human NPC provides a rich foundation for rationalizing the dilation and constriction of the central transport channel, the nucleocytoplasmic transport of integral membrane proteins, and the spatial arrangement of cytoplasmic filament nucleoporins that are involved in mRNA export and etiologies of several human diseases.</p
Rheological Characterization of Polymer Additives for Mist Control and Drag Reduction
Get PDFLong flexible polymers in solution at low concentrations strongly change the extensional properties of fluids due to chain stretching that resists flow, while their compact conformation in shear has weak effects. This dramatic difference between their effects on extension and shear is desirable in a variety of applications--controlling drop size in sprayed mists, reducing drag in turbulent flow, and preventing rebound in drop impact. Traditional long covalent polymers, however, are not practical in many applications because they undergo mechanical degradation, i.e. chain scission, under strong flow conditions. Megasupramolecular polymer systems, consisting of long end-associative telechelic polymers that assemble in solutions into multi-million molecular weight supramolecules, meet this practical need. Through association, they act like traditional covalently-bonded polymers in extension, while reversibly dissociating under the strong flows that cause scission for those long polymers.
This thesis examines the interplay of flow and degradation that imposes an upper-bound on useful lengths of invididual end-associative chains (how long is too long) (Chapters 2 and 3); the quiescent coil size that affects the onset of stretching in fluids of interest (water and polyalphaolefin lubricant) (Chapters 2 and 4);
rheological approachs to detect variations in the degree of end-functionalization that affect formation of ultra-long supramolecules (Chapter 5); and the changes to turbulent flow when long polymers are present at low concentration (Chapter 2). Ultimately, the audience who might enjoy this thesis is limited by barriers of rheological jargon. In the pursuit of broader rheological and overall scientific understanding, I describe evidence-based pedagogical techniques and my approach to implementing them in chemical engineering and polymer physics classrooms (Chapter 6).</p
Strategies and Tools for Machine Learning-Assisted Protein Engineering
Get PDFProteins perform critical roles in a growing list of human-devised applications, and as demands for new applications arise, new proteins must be engineered to meet them. Machine learning-assisted protein engineering (MLPE) has recently arisen as a new philosophy of protein engineering, promising to overcome many of the limitations of existing engineering strategies. Despite its promise, however, as a relatively new approach to protein engineering, MLPE faces many challenges that hinder its routine application. This thesis is focused on addressing a number of them. Chapter 1 provides a theoretical overview of protein engineering, introduces the core steps of a typical MLPE pipeline, and discusses the challenges that currently hinder MLPE’s advancement. This chapter is written to be accessible to all members of the highly multidisciplinary audience that either use or develop MLPE tools, in turn providing a resource that eliminates the steep barrier to entry that can hinder broader participation in the field. Chapter 2 provides a solution to the challenge of applying MLPE to proteins whose fitness landscapes are dominated by “holes” (protein variants with zero or extremely low fitness). Using my development of the strategy “focused training machine learning-assisted directed evolution (ftMLDE)” as an example, I demonstrate how auxiliary information from protein sequence and structure can be used to navigate landscapes despite holes, in turn dramatically improving the efficiency of MLPE. Chapter 3 explores strategies for reducing the amount of sequence-fitness data needed for building MLPE models. Specifically, I detail the motivation behind and development of a new model designed to augment limited protein sequence-fitness datasets with information extracted from raw protein sequence and structure data. Finally, chapter 4 introduces “every variant sequencing” (evSeq), a collection of tools and protocols that enables extremely low-cost, routine collection of large protein sequence-fitness datasets. Not only does this technology drastically improve the financial feasibility of numerous MLPE applications, but it also potentiates the construction of a massive database of diverse protein sequence-fitness data, the likes of which would revolutionize our ability to engineer proteins with data-driven methods. Overall, the work described in this thesis advances both our understanding of MLPE and our ability to engineer proteins using it
Creating ARCHER: A 3D Hopping Robot with Flywheels for Attitude Control
Get PDFThe field of robotic hopping began over 40 years ago, when it was first shown that robust hopping could be achieved on real hardware. In the years since then, it's become clear that hopping requires high performance and precision from its actuation and planning, due to its extreme interactions with the environment occurring over periodic, yet very short durations of time. Despite being of lower dimensionality than many other legged robots, hoppers are very underactuated, which only adds to the difficulty of planning motions quickly for real-time needs.
The studies of robotic hopping presented in this thesis start with a look into two different actuation styles for creating vertical periodic motion: a compress-release mechanism and a moving-mass mechanism. The dynamics of each were examined from the perspective of stability and robustness to uncertainties in the model and measurements. The compress-release hopper (CRH) was found to be very stable, simple to control, and robust to all uncertainties, but inherently had some inefficiencies due to the requirement of holding compression during portions of the aerial phase. The moving-mass hopper (MMH) required optimization to generate the proper cyclic motions as well as closed-loop control to make them stable. Furthermore, the original configuration of the MMH was also less energetically efficient and robust to uncertainty than the CRH.
In an effort to improve the efficiency of the MMH, a second-generation robot was designed using the principle of parallel elasticity. This involved placing a second spring in parallel to the actuator which would naturally guide the motion of the moving-mass into an optimal path, eliminating a significant portion of actuation effort and improving the overall efficiency. An added benefit of this change was that the robot no longer required closed loop control to create stable hopping. This new robot was built and tested in the lab showing a dramatic improvement over the previous design. The principle of controlling the compliance in the actuator for efficient motion was then taken one step further by creating custom, nonlinear stiffness springs which would provide a more ideal trajectory of motion. This process utilized a design-in-the-loop optimization strategy that would both design these springs as well as the motions of the moving-mass to yield better actuation efficiency. A set of these springs was created and attached to the second-gen MMH, replacing the lower spring, and tested in the lab. These springs did slightly improve the efficiency of the robot, but were restricted by the material selection of the springs due to manufacturing limitations.
Moving into the realm of 3-Dimensional hopping, a final robot was designed and built: ARCHER. Unlike traditional hopping robots which use a torso with very large inertia to control the leg motion and balance, ARCHER uses a set of three flywheels. The goal of this robot was twofold: to study the feasibility of using flywheels alone to control attitude, and to take advantage of the principle of decoupled systems. By using strategically placed flywheels, the dynamics of the leg and the attitude subsystems were decoupled, meaning their actuation did not have a direct influence on each other. This allows for simpler motion planning and control. The culmination of this thesis was running experiments with this robot, showing its initial performance and ability to hop with separate controllers for each subsystem.</p
Controlling the Female Body: Obsession and Loss of Autonomy in Lolita and "Berenice"
No full text[Introduction] Why are we so obsessed with the female body? From high school dress codes to impossible beauty standards in the media, society polices and sexualizes young women's bodies. Nabokov's Lolita, which follows the charming Humbert Humbert and his horrifying relationship with adolescent Lolita, explores this hyperfixation of female bodies, particularly young female bodies. Edgar Allan Poe clearly inspires Nabokov, from the confusing foreward, reminiscent of Poe's novel, The Narrative of Arthur Gordon Pym of Nantucket, to the character of Annabel Lee, the titular subject of one of Poe's most famous poems. Poe's short story "Berenice" introduces the idea of monomania and explores themes of bodily autonomy from a physical and a mental perspective. "Berenice" also explores how control and obsession over the female body can make a narrator unreliable. Nabokov extracts these ideas from Poe, expands on them, and explores how Humbert controls Lolita and strips her of her identity. This loss of bodily autonomy and agency is jarring to read, despite Nabokov's stylistic beauty, and encourages the reader to examine how media and society treat young women.</p
Interface Optimization for Improved Photovoltaic Devices
Get PDFThe wide band gaps and superior conductivity of ZnSₓSe₁₋ₓ semiconductors to amorphous Si suggest an alternative carrier-selective contact in silicon heterojunction solar cells. Electron-selective ZnSₓSe₁₋ₓ front contacts on p-type c-Si solar cells are explored by simulating in Sentaurus TCAD a large design parameter space informed by experimentally determined optoelectronic properties. Comparable performance to experimental and simulated p-SHJ reference devices is shown, with a champion simulated device efficiency of 20.8%. X-ray photoelectron spectroscopy is used to measure band offsets at interfaces for the aforementioned ZnSₓSe₁₋ₓ-c-Si photovoltaic devices as well as various carrier-selective contacts and passivation layers for GaAs photovoltaic devices.</p
Formation, Abundance, and Evolution of Molecular Products in α-Pinene and β-Pinene Secondary Organic Aerosol
Get PDFThe atmospheric oxidation of α-pinene and β-pinene (C10H16), emitted in appreciable quantities from forested regions (~85 Tg y–1), contributes significantly to the global burden of secondary organic aerosol (SOA), a substantial component (15–80% by mass) of atmospheric fine particulate matter (PM2.5), which exerts large but uncertain effects on climate as well as adverse impacts on air quality and human health. Deciphering the molecular composition, and in turn formation and aging mechanisms, of α-pinene and β-pinene SOA is essential to reducing uncertainty in assessment of their environmental and health impacts. However, molecular characterization of α-pinene and β-pinene SOA is significantly hindered by their chemical complexity. In this work, we constrain the formation, abundance, and evolution of molecular products in SOA derived from ozonolysis and photooxidation of α-pinene and β-pinene using a combination of laboratory experiments, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), and organic synthesis. Through detailed MS/MS analysis, coupled with 13C isotopic labeling and OH scavenging, we identify a suite of dimeric compounds (C15–19H24–32O5–11) formed from synergistic O3 + OH oxidation of β-pinene (i.e., accretion of O3- and OH-derived products/intermediates). Informed by these structural analyses, together with 18O isotopic labeling and H/D exchange, we synthesize the first authentic standards of several major dimer esters identified in SOA from ozonolysis of α-pinene and β-pinene and elucidate their formation mechanism from targeted environmental chamber experiments. Additionally, we synthesize a series of pinene-derived carboxylic acid and dimer ester homologues and find that the ESI efficiencies of the dimer esters are 19–36 times higher than that of commercial cis-pinonic acid, a common quantification surrogate. Finally, we investigate the aqueous (photo)chemistry (kinetics, products, and mechanisms) of the carboxylic acid and dimer ester homologues at cloudwater-relevant concentrations as a function of pH.</p
Nanoparticle Technologies to Cure and Prevent Infectious Diseases
Get PDFDespite almost 40 years of intensive research, there is still no curative treatment for HIV-1/AIDS. Anti-retroviral therapy (ART) prolongs the life expectancy of HIV-1-infected individuals but is associated with side effects, and multiple drugs need to be given in combination to prevent the development of viral resistance. In addition, treatment must continue for the lifetime of the individual due to the existence of a long-lived latent proviral reservoir. While a "sterilizing" cure remains difficult to achieve due to difficulties associated with identifying and clearing latently-infected cells, recent research has focused on designing a "functional" cure, i.e., a therapeutic strategy that enables long-term suppression of HIV-1 replication and remission of symptoms in the absence of ART. The work presented here describes a new therapeutic direction for the development of a functional cure against HIV-1. This approach is based on the hypothesis that HIV-1 is unable to escape from a nanoparticle (NP)-based decoy that presents clusters of the HIV-1 receptor CD4, because CD4-NPs mimic viral target cells more accurately than soluble CD4-based inhibitors and permit high-avidity interactions with trimeric HIV-1 Env proteins. We demonstrate that CD4-NPs are >10,000-fold more potent than soluble CD4 (sCD4) and prevent viral escape in vitro. AAV-mediated delivery of self-assembling CD4-NPs produced stable CD4-NP serum concentrations in mice that were almost 1,000-fold higher than concentrations required to neutralize HIV-1 in vitro, suggesting that these concentrations could be therapeutic. Viral challenge studies in non-human primates are underway to evaluate the potential of this therapeutic strategy.
As an alternative approach to generate decoys against HIV-1, we generated engineered red blood cells (RBCs) that expressed viral receptors and potently inhibited HIV-1 infection of target cells in vitro. Because RBCs do not contain nuclei or functional organelles required for protein translation, infection of engineered RBCs represents a dead-end for a lentivirus such as HIV-1, which must integrate into the host cell genome as part of its lifecycle. We generated stable erythroid progenitor cell lines that continuously produced HIV-1 receptor-expressing RBCs that could be administered to HIV-1-infected individuals. As RBCs vastly outnumber CD4+ T-cells, HIV-1’s main target cells, and have extended lifetimes, only a fraction of an individual’s RBCs would need to be replaced with the engineered RBC viral traps in order to suppress HIV-1 infection in vivo.
My work on CD4-NP therapeutics against HIV-1 also led to the invention and development of the EBR NP technology that is ideally suited for vaccine design applications. This technology can be used to modify any type of membrane protein to self-assemble into enveloped virus-like NPs without the need for additional proteins. EBR NP assembly is induced by inserting a short amino acid sequence into the cytoplasmic tail of the membrane protein, which was designed to recruit host proteins from the endosomal sorting complex required for transport (ESCRT) pathway. We applied this technology to design protein NP-based vaccines against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which elicited potent serum neutralizing antibody responses in mice. The EBR NP technology is also ideally suited for the development of hybrid vaccine approaches that allow genetic encoding of protein-based NPs, thereby combining attributes of mRNA and protein-based NP vaccines. Pilot studies demonstrated that mRNA and DNA vaccines encoding the self-assembling SARS-CoV-2 spike-EBR construct elicited ~10-fold higher neutralizing antibody responses than mRNA and DNA vaccines encoding the unmodified spike protein. This hybrid approach has the potential to substantially enhance the potency of mRNA vaccines and could become a leading vaccine platform technology. Future applications for the EBR NP technology are discussed, including the development of a universal coronavirus vaccine to prevent future pandemics, and engineering EBR NPs to mRNA vaccines or therapeutic cargoes for efficient and targeted delivery.</p
Serine Integrase-Based Event Recording in E. coli
Get PDFDNA is a unique molecule that has evolved to serve as the genetic material for life. It seems straightforward to consider this molecule not only as a wonder of the natural world but as a tool for information storage and retrieval. Bacteria have evolved to conserve DNA, but bacteriophages have evolved to specifically integrate their genomes using integrases. In response to viruses, bacteria have evolved the RNA-guided nuclease Cas9 to destroy viral DNA before it can be integrated. The fruits of these evolutionary pressures prove useful to the researcher interested in easily editing DNA. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a synthetic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered "ink" plasmids, which correspond to two orthogonal small molecule inducers whose presence or absence as a function of time can be recorded with this system. Integrase sites present on these plasmids are found to not participate in intramolecular "deletion" reactions if closer than 100 bp. Guide RNAs overlapping integrase attachment sites are found to effectively block integrase activity at those sites if the overlap is equal to 9 or 19 base pairs. Other overlap values, including forward or reverse binding result in ineffective integrase activity repression. We develop 8 orthogonal guide RNA sequences capable of binding to and repressing integrase activity at the attP site. Plasmid multimers are sequenced using Oxford Nanopore sequencing and found to follow population-level predictions of event record identity. Single DNA states are found insufficient for identifying past history of events; an ensemble of DNA states at the population level must be used. A modular modeling framework is developed (Global enumeration) to describe this system, and integrated with the existing chemical reaction network creation automation software BioCRNpyler. The modeling framework developed here automatically creates chemical reaction networks based on typical linear DNA-based synthetic biology "genetic constructs" and predicts transcripts and proteins produced based on simple transcription/translation rules. Integrase-based recombination events can also be predicted in a recursive way.</p