The construction of carbon\u2013nitrogen (C\u2013N) bonds is pivotal to the development of pharmaceuticals, agrochemicals, and advanced materials. While traditional synthetic methods offer robust and efficient solutions, biocatalysis has emerged as a powerful, sustainable alternative for constructing these bonds with precise stereochemical control. Yet, the enzymatic repertoire for C\u2013N bond formation remains limited compared to its chemical counterpart. By leveraging directed evolution and enzyme promiscuity, we have traversed beyond natural enzymatic functions and explored the new-to-nature catalytic landscape of enzymes.
\r\n\r\nThis thesis outlines a suite of strategies that expand the enzymatic toolbox for stereoselective C\u2013N bond construction in two main avenues: the functionalization of more challenging C\u2013H bonds and the development of novel nitrene species. Chapter I reviews contemporary chemical and biocatalytic approaches, highlighting how directed evolution can transcend native enzymatic functions to unlock new reactivities.
\r\n\r\nChapter II presents engineered serine-ligated cytochrome P411 variants capable of catalyzing enantioselective propargylic C(sp3)\u2013H amination, granting streamlined access to chiral propargylamines. Chapter III addresses the long-standing challenge of differentiating minimally distinct alkyl groups, methyl and ethyl substituents, by evolving P411 enzymes that perform enantiospecific functionalization of tertiary C\u2013H bonds to construct methyl-ethyl stereocenters with high selectivity.
\r\n\r\n\r\nChapter IV expands the scope of enzymatic nitrene transfer, enabling intramolecular alkyl and aryl nitrene C\u2013H insertions to synthesize chiral pyrrolidines and methyl indolines. Subsequent biocatalytic derivatization of these products affords complex molecules bearing multiple chiral centers, showcasing the potential of biocatalysis to rapidly build molecular complexity. Chapter V introduces engineered protoglobins capable of mediating intermolecular methylnitrene transfer using a simple and stable N-methyl hydroxylamine recursor. This process uniquely achieves the stereoconvergent conversion of both (Z)- and (E)-silyl enol ether isomers, affording enantiopure N-methyl-a-aminoketones, which defies conventional limitations of enzyme specificity.
\r\n\r\nTogether, these efforts address critical gaps in biocatalytic C\u2013N bond formation, establishing broadly applicable platforms for sustainable and stereoselective synthesis of N-containing molecules. The strategies developed herein not only deepen our understanding of heme-dependent enzymes but also lay the foundation for future innovations in biocatalysis and synthetic biology.
", "doi": "10.7907/5pv2-nr39", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18409", "collection": "thesis", "collection_id": "18409", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03092026-205538974", "type": "thesis", "title": "Human-Scale Ultrasound, Thermoacoustic, and Photoacoustic Tomography", "author": [ { "family_name": "Garrett", "given_name": "David Christopher", "orcid": "0000-0002-9747-8494", "clpid": "Garrett-David-Christopher" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Hajimiri", "given_name": "Ali", "orcid": "0000-0001-6736-8019", "clpid": "Hajimiri-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Modern healthcare relies on imaging modalities that visualize internal anatomy and pathology. While X-ray computed tomography (CT) and magnetic resonance imaging (MRI) provide clinically useful imaging across many applications, they face significant barriers to more frequent use: ionizing radiation limits repeated CT scanning, and MRI\u2019s high cost and long acquisition times create access disparities. Conventional handheld ultrasonography enables rapid, low-cost imaging but remains limited by narrow fields of view, operator dependence, and challenging image interpretation. Photoacoustic tomography has emerged as a promising alternative that combines optical absorption contrast with acoustic detection, offering molecular specificity without ionizing radiation. However, conventional photoacoustic imaging remains limited to depths of several centimeters, inhibiting applications in deep-tissue imaging like gastrointestinal or whole-body assessment.
\r\n\r\nIn this thesis, we develop three approaches to extract clinically relevant information at human scales: ultrasound, thermoacoustic, and photoacoustic tomography. All three modalities leverage a custom 512-element, 60 cm diameter receiver array designed to detect acoustic signals across human-scale geometries. We validate these approaches through in vivo imaging, ex vivo tissue experiments, and phantom studies. First, we demonstrate ultrasound tomography of full human cross-sections in the abdomen and lower extremities, reconstructing backscatter contrast alongside quantitative maps of the speed of sound and attenuation coefficient. We show that ultrasound tomography enables visualization of features such as the liver, vasculature, muscle, and subcutaneous adipose across entire 2D human cross-sections. Second, we develop a thermoacoustic approach to guiding microwave ablation procedures. By modulating the microwave signal delivered through the probe, we record the generated thermoacoustic signals and use them to model the thermal dynamics during ablation. We show that this approach yields more accurate estimates of ablation zone geometry than standard look-up tables, which could allow for more precise ablation therapy. Third, we develop a method to extend the imaging depth of photoacoustic tomography using a wireless, ingestible capsule-based optical source. We demonstrate imaging depth up to 12 cm, which could open the door to photoacoustic imaging of regions like the gastrointestinal tract. Together, these approaches aim to expand the range of safe, informative, and accessible imaging modalities available to patients and clinicians.
", "doi": "10.7907/s8t2-qb10", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18381", "collection": "thesis", "collection_id": "18381", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02192026-220300004", "primary_object_url": { "basename": "Thesis_Xin_Tong_final_v2.pdf", "content": "final", "filesize": 38728537, "license": "other", "mime_type": "application/pdf", "url": "/18381/1/Thesis_Xin_Tong_final_v2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Advanced Imaging with Sound and Light: Photoacoustic Tomography and Quantum Microscopy", "author": [ { "family_name": "Tong", "given_name": "Xin", "orcid": "0000-0003-2002-5638", "clpid": "Tong-Xin" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Marandi", "given_name": "Alireza", "orcid": "0000-0002-0470-0050", "clpid": "Marandi-A" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Optical imaging enables visualization of biological structure and function but is fundamentally limited by several physical constraints. Spatial resolution is bounded by optical diffraction, depth penetration is curtailed by strong absorption and scattering in tissue, and image contrast-to-noise ratio is often restricted by photon shot noise in low-light conditions. This thesis advances two novel directions\u2014photoacoustic imaging and quantum imaging\u2014to address these limitations.
\r\n \r\nIn photoacoustic imaging, we design and optimize high-speed photoacoustic computed tomography systems that enable deep, volumetric visualization of vasculature. By incorporating time-gated reconstruction and image-enhancement algorithms, these systems support small-animal imaging of cardiac structure, liver morphology, and brain hemodynamics non-invasively. Building on these foundations, we explore non-invasive breast photoacoustic imaging with high spatiotemporal resolution. Through integration with learning-based feature extraction, classification, and segmentation pipelines, we demonstrate the feasibility of applying photoacoustic imaging in clinical workflows to aid the characterization of breast tissue.
\r\n \r\nIn quantum imaging, we develop two complementary architectures that extend the state of the art in opposite but synergistic directions. The scanning quantum microscope scales up existing quantum imaging approaches, achieving the largest resolvable pixel counts to date by combining entangled-photon illumination with efficient coincidence detection. This platform enables the first demonstration of whole-organism imaging and shows potential in remote sensing and sub-shot-noise imaging. In contrast, the widefield quantum microscope scales down quantum imaging to the microscopic regime, integrating single-photon\u2013sensitive cameras with a covariance-based coincidence estimation algorithm. This approach enables cellular-level imaging and demonstrates quantum-enhanced resolution beyond the classical diffraction limit, establishing a practical pathway for quantum microscopy in biological imaging.
\r\n \r\nAcross both research directions, this thesis advances system design and engineering, quantitative characterization, calibration, reconstruction, and image-enhancement methodologies. Together, these developments establish pathways from physical principles to practical imaging systems, spanning laboratory prototypes through preclinical and clinical applications in biomedical imaging.
", "doi": "10.7907/013f-vd31", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17642", "collection": "thesis", "collection_id": "17642", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08212025-162748066", "type": "thesis", "title": "SLIM: Stochastic Lineage-Based Iterative Minimization", "author": [ { "family_name": "Lipschitz", "given_name": "Mikel", "orcid": "0000-0002-5764-1648", "clpid": "Lipschitz-Mikel" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The bacterial genomes we encounter today have been shaped by billions of years of genome altering events which involve rewriting, addition, and removal of genomic elements. The resulting product is a complex network of interactions composed of elements which are defined by contextual necessity. The elucidation of a minimal set of elements, comprised of just those essential for sustaining life, has long been sought after. This set, or minimal genome, has been proposed to be a representation for the foundation of life itself. Genome minimization, the pursuit of this foundation, is a process by which genomic segments deemed unnecessary, or non-essential, in an environmental context dependent manner are identified and removed, leaving only DNA that provides the cell with the resources and processes it needs to stay alive and reproduce. Numerous genome minimization efforts have been undertaken previously. However, each of these studies has resulted in the generation of a single genome-reduced strain derived from a single wild-type bacteria in a single environment. While these methods have shown a great deal of promise in their ability to identify foundational genomic pieces in this extremely narrow context, they lack the throughput and generalizability to identify foundational pieces of all bacterial life.
\r\n \r\nBuilding upon prior genome minimization efforts, we developed SLIM (Stochastic Lineage-based Iterative Minimization), a modular genome reduction system designed for unbiased DNA removal, high-throughput parallelization and cross-species compatibility. In this study we utilize SLIM to generate a library of ten genome-reduced E. coli strains. We then rigorously interrogate the library to identify patterns in deleted segments. We assess the effects that these deletions have on remaining genomic components and explore how these effects can result in substantial fitness changes in different environments. Finally, we demonstrate the modularity of SLIM by generating two additional libraries of genome-reduced strains from two phylogenetically distinct parent bacteria, S. flexneri and P. putida. This work highlights the power and promise of generating diverse libraries of genome-reduced strains; substantially expanding the number of minimized genomes that can be achieved, while simultaneously reducing the time to generation.
", "doi": "10.7907/q80m-3t75", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17830", "collection": "thesis", "collection_id": "17830", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01212026-163903186", "type": "thesis", "title": "Polymer Mechanochemistry Using Ultrasound: From Fundamental Reactivity to Controlled Drug Delivery", "author": [ { "family_name": "Luo", "given_name": "Meng (Stella)", "orcid": "0000-0003-4003-7468", "clpid": "Luo-Meng-Stella" } ], "thesis_advisor": [ { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polymer mechanochemistry harnesses mechanical force to drive specific chemical transformations in stress-sensitive molecules known as mechanophores. Through judicious chemical design, these force-driven reactions have enabled functional polymeric materials capable of sensing, self-healing, and catalysis. Research in this field encompasses expanding the mechanophore repertoire, elucidating the fundamental principles governing mechanically induced reactivity, and translating force-responsive systems into practical applications.
\r\n \r\nThis thesis advances the field by contributing both fundamental insight and applied functionality in mechanophore activation under ultrasonication, with a particular focus on controlled drug release in biological environments. First, we develop an improved methodology for characterizing mechanophore reactivity, addressing sensitivity limitations in sonication experiments and, combined with computational modeling, revealing underlying principles that govern force-induced bond activation. Separately, we establish a synergistic platform that couples cargo-releasing mechanophores with biocompatible focused ultrasound, enabling controlled release of a fluorophore and a chemotherapeutic agent under physiological conditions. Finally, we demonstrate mechanochemically triggered drug delivery in vivo and validate this process using an inducible protein-expression system as a biological readout, achieving the first constructive modulation of cellular function enabled by covalent polymer mechanochemistry. Together, these studies deepen the fundamental understanding of mechanophore reactivity and illustrate the substantial biomedical potential of mechanochemical approaches using ultrasound activation.
", "doi": "10.7907/mnbj-xq97", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17804", "collection": "thesis", "collection_id": "17804", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12182025-233110599", "type": "thesis", "title": "Neural Coding of Fear: From Genes to Brain-Wide Dynamics", "author": [ { "family_name": "Cheung", "given_name": "Yuen Man Kathy", "orcid": "0009-0007-1204-2178", "clpid": "Cheung-Yuen-Man-Kathy" } ], "thesis_advisor": [ { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Adolphs", "given_name": "Ralph", "orcid": "0000-0002-8053-9692", "clpid": "Adolphs-R" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Fear is essential for survival. Predator threats, aggressive conspecifics, and environmental dangers elicit innate defensive responses that do not require learning. Although these responses are hardwired, animals display remarkable flexibility in selecting the most adaptive behaviors. The neural circuits that enable such dynamic representations of internal fear states remain poorly understood. The dorsomedial and central subdivisions of the ventromedial hypothalamus (VMHdm) integrate multisensory threat-related inputs and are both necessary and sufficient to drive defensive responses. In this thesis, I investigate how VMHdm neurons encode internal states associated with predator fear, across multiple biological scales\u2014from transcriptomic profile, single-cell neural dynamics to brain-wide activity patterns.
\r\n\r\nFirst, I characterized the transcriptomic profile of VMH neurons activated by predator exposure using activity-dependent single-cell RNA sequencing (act-seq). I then combined optogenetic perturbation of VMHdm with act-seq to identify state-dependent transcriptomic correlates in a downstream output, the periaqueductal gray (PAG). To investigate the real-time dynamics of these neurons during naturalistic predator encounters, I developed a novel behavioral paradigm and performed microendoscopic single-cell calcium imaging. Contrary to the long-held view of VMHdm as a uniformly threat-activated population, my analyses revealed functionally distinct clusters that encode not only threat, but also safety, arousal or novelty or neophobia, predator imminence, and anxiety. Moreover, I found that individual variation in behavioral defensiveness was correlated with VMHdm neural dynamics.
\r\n\r\nFinally, to assess how local hypothalamic activation influences global brain states, I combined VMHdm optogenetic stimulation with functional ultrasound imaging (fUSI), which permits high-resolution recording of brain-wide hemodynamics. This approach revealed the spatiotemporal propagation of neural activity from the hypothalamus to distributed brain regions. These findings demonstrate how a genetically defined hypothalamic subpopulation can engage a dynamic, brain-wide ensemble to orchestrate defensive responses.
\r\n \r\nTogether, these studies provide a multi-modal, multi-scale analysis of the innate fear state and its flexible representation via the hypothalamus, embedded within a dynamic global network of interacting regions. These findings offer insight into how internal states are encoded and broadcast, with potential implications for understanding the neural basis of human psychiatric disorders and the distributed computation of affective states.
", "doi": "10.7907/vcr9-gp26", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17796", "collection": "thesis", "collection_id": "17796", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12112025-115800167", "type": "thesis", "title": "Strategies and Tactics in Alkaloid Synthesis: Total Synthesis of Strempeliopidine via a Non-Directed Petasis Reaction and Progress Toward the Synthesis of Mitomycin B", "author": [ { "family_name": "Gonzalez", "given_name": "Kevin Jaime", "orcid": "0000-0002-4904-590X", "clpid": "Gonzalez-Kevin-Jaime" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Fu", "given_name": "Gregory C.", "orcid": "0000-0002-0927-680X", "clpid": "Fu-G-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes research toward total synthesis of bioactive alkaloids with complex architectures that have demanded for the invention of methodology. Chapter 1 comprehensively reviews the semi-, partial, and total synthesis of heterodimeric monoterpenoid bisindole alkaloid natural products. Chapter 2 describes the enantioselective total synthesis of the bisindole alkaloid strempeliopidine. A convergent strategy featuring a diastereoselective Petasis reaction enabled the synthesis of the natural product and several stereoisomeric analogs. Chapter 3 details the development of a non-directed Petasis reaction inspired by the key step in the synthesis of strempeliopidine. This methodology couples hydroxyindoles with trifluoroborate salts under mild conditions, thereby enabling the synthesis of non-natural heterodimeric bisindole alkaloids. Chapter 4 explores ongoing efforts toward the asymmetric total synthesis of mitomycin B. Chapter 5 covers the development of an enantioselective 1,3-dipolar cycloaddition for the construction of nitrogen-rich spirocycles. A chiral magnesium Lewis acid catalyst facilitated the asymmetric [3+2] cycloaddition between alpha-methylene lactams and diazoacetates or nitrile oxides.", "doi": "10.7907/hakn-7h25", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17694", "collection": "thesis", "collection_id": "17694", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09212025-192830865", "primary_object_url": { "basename": "20250920_PhD_thesis_draft_v21_submitted-version.pdf", "content": "final", "filesize": 33782320, "license": "other", "mime_type": "application/pdf", "url": "/17694/1/20250920_PhD_thesis_draft_v21_submitted-version.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Acoustically Targeted Gene Delivery for Non-Invasive Neuroengineering", "author": [ { "family_name": "Li", "given_name": "Hongyi Richard", "orcid": "0000-0001-6970-0230", "clpid": "Li-Hongyi-Richard" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Andersen", "given_name": "Richard A.", "orcid": "0000-0002-7947-0472", "clpid": "Andersen-R-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Tsao", "given_name": "Doris Y.", "orcid": "0000-0003-1083-1919", "clpid": "Tsao-D-Y" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Noninvasive, spatially targeted gene delivery to the brain holds tremendous promise for addressing some of the most pressing neurological and psychiatric conditions of our time, including Parkinson\u2019s disease, treatment-resistant epilepsy, obsessive-compulsive disorder, and addictions. While adeno-associated viruses (AAVs) are the leading vectors for gene therapy in the state of the art, their clinical translation is hindered by the need for invasive injections to achieve site-specific delivery in the brain. Over the past two decades, focused ultrasound blood-brain barrier opening (FUS-BBBO) has emerged as a compelling alternative \u2014 enabling targeted entry of biomolecules, nanoparticles, and even small viral vectors like AAVs from the bloodstream into the brain without surgical intervention. Yet, natural AAV serotypes have shown only modest success with this method, often displaying low transduction efficiency and undesirable off-target expression in peripheral organs.
\r\n\r\nTo overcome these limitations, we have developed a new framework for acoustically targeted gene delivery \u2014 a noninvasive, spatially and cell-type-specific approach for delivering genetic material to the brain. In this thesis, I will describe how we harnessed high-throughput in vivo directed evolution to engineer AAV variants optimized for neuronal transduction specifically at the site of ultrasound targeting. In rodent models, these newly evolved vectors demonstrate significantly improved performance \u2014 achieving efficient, localized gene delivery to neurons while minimizing peripheral expression. Building on these successes, we advanced the platform toward clinical relevance by extending our evolutionary screening to non-human primates (NHPs). This allowed us to identify AAV variants with enhanced translational potential and establish a strong foundation for future studies in human clinical trials.
\r\n\r\nIn the final part of this thesis, I will showcase how these engineered AAVs can be further empowered by combining them with acoustic reporter genes \u2014 specifically, gas vesicle (GV) proteins \u2014 enabling non-invasive imaging of molecular activity deep within the brain. Using this powerful platform, we have also developed a novel therapeutic strategy for treating opioid addiction, in which biomolecular ultrasound coalesces with chemogenetic neuromodulation. Taken together, I hope to convince you that the technique of ultrasound-based acoustically targeted gene delivery, paired with engineered delivery vectors, unlocks a new frontier in non-invasive neurotherapeutics and brings us one step closer to precise, personalized neuroengineering in interfacing the human brain.
", "doi": "10.7907/qbrx-4132", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17678", "collection": "thesis", "collection_id": "17678", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09132025-054711849", "primary_object_url": { "basename": "Shaker_Sammy_2026.pdf", "content": "final", "filesize": 307026680, "license": "other", "mime_type": "application/pdf", "url": "/17678/1/Shaker_Sammy_2026.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "3D Vat Photopolymerization of Microarchitected Magnetic Metal Alloys for Chemotherapy Capture Filters", "author": [ { "family_name": "Shaker", "given_name": "Sammy", "orcid": "0000-0003-1751-4908", "clpid": "Shaker-Sammy" } ], "thesis_advisor": [ { "family_name": "Greer", "given_name": "Julia R.", "orcid": "0000-0002-9675-1508", "clpid": "Greer-J-R" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Greer", "given_name": "Julia R.", "orcid": "0000-0002-9675-1508", "clpid": "Greer-J-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Primary liver cancer constitutes an object of concern for communities across the globe. With the majority of new diagnoses of the most common subtype of primary liver cancer, hepatocellular carcinoma, inoperable at diagnosis, the standard of care revolves around the use of liver-directed therapies to treat tumors. The most popular therapy is termed transarterial chemoembolization, wherein the unique anatomy of the liver is exploited to deliver chemotherapy and embolic agents selectively to a large tumor, leading to tumor cell death and improved survival for patients. However, the chemotherapeutics used in this procedure have toxic effects on organs outside of the liver, and are as such dose-restricted on the basis of these side effects. In order to increase the amount of drug used and thus increase the chance of tumor cell death, chemotherapy capture devices are necessary. While some materials have been developed for this application, these devices regularly suffer from such restrictions as passive capture mechanisms necessitating the design of devices with poor hemodynamics or the use of immunogenic materials such as heteroDNA. Magnetic nanoparticles conjugated to chemotherapeutics as well as magnetic nanoparticle chemotherapy capture agents, delivered with the chemotherapeutics, present a potential way out of this conundrum, but the application of these materials requires the design of magnetic capture devices with favorable hemodynamic and magnetic properties. Architected magnetic metal devices can potentially provide the sought after solution to these difficulties, but techniques to such devices with high spatial resolution are lacking. An additive manufacturing technique that can provide high spatial resolution utilizes vat photopolymerization in tandem with thermal processing to produce well-resolved metal lattices that can provide for this ongoing need.
\r\n\r\nThis thesis applies this technique to the synthesis of magnetic lattices for particle capture. Lattices are synthesized in iron, nickel-iron, and copper-nickel-iron compositions and characterized structurally and magnetically. Simulations of particle capture in these lattices under various conditions are performed. In addition, attempts at particle capture are described and methods of characterization of particle capture are discussed. This technique is also explored with regards to the synthesis of iron-nickel and iron-cobalt lattices and the characterization of the resultant products is discussed and evaluated. Finally, a modification to this technique is used to generate metal-carbon microcomposites with unusual magnetic properties when compared with their counterparts synthesized using the unmodified procedure.
", "doi": "10.7907/gc1t-5869", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18415", "collection": "thesis", "collection_id": "18415", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03122026-031420036", "type": "thesis", "title": "Expanding Enzyme Function Through Data-Guided Evolution", "author": [ { "family_name": "Lal", "given_name": "Ravi Goel", "orcid": "0000-0001-6943-4147", "clpid": "Lal-Ravi-Goel" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Through the process of evolution, Nature has optimized enzymes for the chemical transformations which drive biology. The use of enzymes for chemical synthesis is enticing as these privileged catalysts can facilitate reactions in a highly sustainable and selective manner. Directed evolution (DE) is a strategy for engineering proteins to improve a desired function. This approach has been demonstrated to be of great effect for the development of enzymatic activities which have never been naturally observed. These \u2018new-to-nature\u2019 chemistries not only showcase the power of DE to unlock novel functions, but also the potential of biocatalysis to deliver high-value chemical compounds. This thesis details the exploration of new-to-nature biocatalytic reactions of hemoproteins using traditional DE and novel machine learning-assisted directed evolution (MLDE) methods. Chapter I provides an overview of how DE has enabled new-to-nature-biocatalysis, and the challenges associated with developing this novel biocatalytic reactions. In Chapter II, a cytochrome P411 is found to catalyze a carbene-mediated [1,2]-Stevens rearrangement to furnish azetidines via ring expansion, and is engineered to deliver these products with high yield and enantioselectivity. The evolution of this activity is a demonstration of how established DE techniques can be utilized to arrive at enzymes capable of unprecedented chemistry. Chapter III describes efforts towards advancing this ring-expansion chemistry for the synthesis of proline analogs. Though this activity could not be found with existing sequence diversity, several engineering insights were made about protoglobins (small, thermostable hemoproteins). Chapters II and III both highlight challenges associated with DE. In Chapter IV, active-learning assisted directed evolution (ALDE) is introduced as a workflow which leverages MLDE in an iterative fashion to greatly accelerate DE efforts. Alongside simulations on combinatorially complete datasets, ALDE was validated in the wet lab by simultaneously evolving a protoglobin-based cyclopropanation for improved yield and stereoselectivity. Finally, Chapter V describes the use of ALDE to engineer protoglobins with active sites which have been optimized to catalyze a broad scope of nitrene and carbene transfer reactions. This demonstration of enzyme ensemble engineering displays the power of diversity-oriented evolution to provide broad solutions in biocatalysis.", "doi": "10.7907/5zaf-8w29", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18487", "collection": "thesis", "collection_id": "18487", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04142026-193956786", "type": "thesis", "title": "A Tale of Two Strategies: Evolving and Engineering Biology Across Scales", "author": [ { "family_name": "Zhou", "given_name": "Jie", "orcid": "0009-0008-2076-2279", "clpid": "Zhou-Jie" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Demirer", "given_name": "Gozde S.", "orcid": "0000-0002-3007-1489", "clpid": "Demirer-G\u00f6zde-S" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Engineering biology at the scale of pathways and genomes requires two core capabilities: the precision to install defined changes and the capacity to generate diversity across large genetic elements. In this thesis, we developed complementary strategies that together span this spectrum. First, we introduced SCOPE (Selective Conjugation-mediated Optimization and Passaging for Evolution), a conjugation-based evolution platform that enables continuous diversification and passaging of replicons ranging from 10 kb to over 500 kb. Extending this approach, SCOPE established genome-to-genome \u201chopping\u201d by evolving CRISPR associated transposase based insertion systems as integrated units, yielding variants with up to 30-fold increased integration efficiency across cargo sizes ranging from 10 kb to 1 Mb. These results illustrate that entire polygenic systems, including genome-editing machines, can be diversified and optimized in vivo. Second, we developed an optimized prime editing framework for E.coli that achieves editing efficiencies above 95% across diverse genomic loci and edit types. Additional intron-based strategies enabled context-dependent template restoration, and together these layers established robust precision editing across diverse loci. Building on this foundation, we created PEACE (Prime Editing Additive Conjugative Engineering), which couples prime editing and conjugative transfer with site-specific recombination to achieve scarless installation of megabase-scale DNA segments across genera. This provides a continuum of editing capabilities ranging from single-nucleotide manipulations to large-scale genomic integrations.
\r\n\r\nTogether, SCOPE and PEACE address two longstanding bottlenecks in microbial genome engineering: the inability to efficiently diversify large genetic elements, and the lack of scalable tools for precise genomic integration. The results presented here suggest that these need not remain fundamental limitations. As the complexity of engineering targets grows, from single enzymes to entire biosynthetic pathways to multi-gene regulatory networks, the field will increasingly require tools that operate at matching scales. This thesis provides a foundation for that shift, offering approaches that are not merely incremental improvements but qualitatively different in the size and complexity of genetic systems they can access.
", "doi": "10.7907/qrag-gm36", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17070", "collection": "thesis", "collection_id": "17070", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03182025-023751137", "type": "thesis", "title": "RNA-Mediated Toxicity In Neurodegeneration: The Mechanistic Role Of The C9ORF72 Repeat Expansion In ALS Molecular Pathogenesis", "author": [ { "family_name": "Bhattacharya", "given_name": "Paulomi", "clpid": "Bhattacharya-Paulomi" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ichida", "given_name": "Justin K.", "orcid": "0000-0002-8827-8087", "clpid": "Ichida-Justin-K" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The G4C2 hexanucleotide repeat expansion in the first intron of the C9ORF72 gene is the most common genetic mutation linked to ALS, accounting for ~40 percent of familial and 10 percent of sporadic cases. Yet, its functional contribution to molecular pathogenesis remains unknown. The prevailing model is that this expansion leads to transcription of a novel RNA (C9-repeat RNA) that leads to disease either through its RNA product or translation of dipeptide repeat proteins it encodes (\u201cgain-of-function\u201d). However, recent attempts to degrade the C9-repeat RNA in several major clinical trials have failed to show any improvement in C9-ALS patients, raising questions about what role, if any, the C9-repeat RNA plays in ALS pathogenesis. Here, we demonstrate that the C9-repeat RNA is not detectable in C9-ALS patient-derived iPSNs or postmortem brain tissue. We show that transcription of the C9ORF72 gene initiates downstream of the G4C2 repeat sequence with the repeat expansion residing at a promoter-proximal region and displaying chromatin signatures of an enhancer. Because this region is GC-rich and has been reported to be preferentially methylated in C9-ALS patients, we explored whether this repeat expansion might lead to reduced C9ORF72 gene expression. We show that the C9-repeat is associated with reduced allele-specific expression of the C9ORF72 gene, consistent with the GC-rich features of the repeat expansion and previous reports of preferential DNA methylation in C9-ALS patients. Taken together, our findings challenge the prevailing gain-of-function models in C9-ALS and instead suggest that the repeat expansion region may function as a regulatory element that silences C9ORF72 expression from the mutant allele.", "doi": "10.7907/2ywx-7a47", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17291", "collection": "thesis", "collection_id": "17291", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292025-004444146", "type": "thesis", "title": "Biomolecular Engineering of Gas Vesicles with Thiol Functionality", "author": [ { "family_name": "Schrunk", "given_name": "Erik Tao", "orcid": "0009-0003-2002-7411", "clpid": "Schrunk-Erik-Tao" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Therapies involving the administration of engineered cells, such as CAR-T cell therapy and the delivery of genetically modified gut microbes, have enjoyed clinical success and increasing interest in recent years. While these therapies continue to show great promise, the opacity of tissue precludes the use of light in the observation and potential manipulation of these engineered cells as they carry out their functions within the body. To access these cells non-invasively in deep tissue requires the use of imaging modalities that do not involve light, of which ultrasound (US) is especially appealing due to its relatively low cost, safety, and widespread availability. Engineered cells can exhibit US contrast by expressing gas vesicles (GVs), air-filled polymeric proteinaceous nanostructures; GVs have already been used as acoustic reporters for gut colonization, tumor cell activity, and more.
\r\n \r\nWhereas GVs are most notable for their acoustic properties, we set out to further expand the function of GVs by chemically modifying them at the genetic level. Our goals were twofold: we wished to equip the external, solution-facing side of GVs with a unique chemical handle; and we wished to hide a reactive group within the internal, air-facing side of GVs that could only be revealed when the GV structures are irreversibly collapsed. To accomplish both these goals, we chose to incorporate cysteine into the shell of GVs because cysteine\u2019s thiol side chain is chemically unique among all natural amino acids and because wild-type GVs do not contain cysteine in their shells. We set up a cysteine scanning mutant library of the GV shell protein, GvpA/GvpA1, and screened for cysteine-tolerant mutations in the gene. Through this process, we discovered cysteine substitutions that furnished thiol groups facing both the GV exterior and interior.
\r\n\r\nThe GV-exterior-facing cysteines were leveraged to develop a modified GvpA that contains the bioorthogonal six amino acid tetracysteine tag, or TC tag. The TC tag reacts with the membrane-permeable molecule FlAsH, which becomes fluorescent upon reaction. We used TC-tagged GvpA, or tcGvpA, to express GVs in HEK 293T cells, and used confocal microscopy of FlAsH to study those GVs. Notably, we only substituted a small percentage of GvpA to tcGvpA, leaving the rest of the GvpA as wild type; to our knowledge, this is the only report of a polymeric proteinaceous structure that employs this chimeric assembly approach being successfully expressed and labeled with FlAsH. The microscopy results from this study were used to generate three-dimensional renderings that provided insights into the size and positioning of GV clusters expressed within HEK 293T cells.
\r\n\r\nSecond, we identified several interior-facing cysteine mutants to the GV shell protein GvpA1, which we used to develop \u201cSonoCages\u201d: chemical entities whose reactivity is gated by US. We purified GVs with one mutation from our screen, V47C, and reacted them with monobromobimane (mBBr), a fluorogenic, thiol-reactive molecule. The mutant GVs only reacted with mBBr after treatment with US, which collapsed the GVs and exposed their hydrophobic interiors to the bulk solution. Thus, we had developed thiol-bearing SonoCages whose cysteines could only engage in reactions after US-mediated collapse of the GVs\u2014a process we call \u201csono-uncaging\u201d in analogy to photo-uncaging. We further demonstrated the utility of SonoCages by preparing a hydrogel containing SonoCages and mBBr and using US to create fluorescent patterns corresponding to regions of GV collapse.
\r\n \r\nThe work presented in this thesis not only demonstrates the functionalization of the GV interior and exterior, but also establishes a framework through which further modifications can be performed. Whereas we used cysteine as our reactive chemical of choice, other amino acids (including non-canonical amino acids) could be used to explore a much wider library of reactivities. The vast potential of GV chemical modification, along with the amazing results from the rest of the Shapiro Lab and in labs across the world, serves as a reminder that GVs and GV-based technologies are not just a bubble (pun intended)\u2014they are going to be around for a long time.
", "doi": "10.7907/49mr-3744", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16529", "collection": "thesis", "collection_id": "16529", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06292024-034255793", "primary_object_url": { "basename": "MacKrell_Elliot_2025_thesis.pdf", "content": "final", "filesize": 10855249, "license": "other", "mime_type": "application/pdf", "url": "/16529/13/MacKrell_Elliot_2025_thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Global Analysis of Protein Synthesis and Degradation in Escherichia coli", "author": [ { "family_name": "MacKrell", "given_name": "Elliot James", "orcid": "0009-0006-5619-8548", "clpid": "MacKrell-Elliot-James" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein synthesis and degradation shape the cellular proteome to drive homeostasis and physiological adaptation. Many fundamental aspects of protein regulation have been elucidated through investigation of the Gram-negative bacterium Escherichia coli, which remains a fruitful model organism for uncovering conserved regulatory mechanisms relevant to cell biology, biotechnology, and medicine. Here, we used bioorthogonal noncanonical amino acid tagging (BONCAT) for the time-resolved analysis of protein synthesis and degradation in this organism in several contexts. We profiled protein degradation on a proteome-wide scale in growing and growth-arrested cells, identifying instability in a diverse panel of regulators. Our identifications served as training data in the validation and deployment of a machine learning classifier of in vivo protein stability, which highlighted the role of active degradation in motility and surface adhesion. We then utilized an efficient system of active degradation in this organism to engineer the instability of the mutant methionyl-tRNA synthetase NLL-MetRS for the analysis of protein synthesis in transient physiological states. Destabilized NLL-MetRS variants exhibited half-lives on the order of hours, which improved the fidelity of metabolic labeling in growth-arrested cells. Additionally, we leveraged the sensitivity of BONCAT to investigate protein synthesis in growth-arrested cells expressing a well-studied but controversial member of the widespread toxin-antitoxin family, MazF. Our proteomic profiling suggests this toxin activates several endogenous stress response systems, most notably the cold shock response system. Taken together, these investigations highlight the advantage of time-resolved proteomics in characterizing proteome dynamics.", "doi": "10.7907/n97w-ch36", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16751", "collection": "thesis", "collection_id": "16751", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09222024-230441454", "primary_object_url": { "basename": "HeatherLukas_PhDThesis.pdf", "content": "final", "filesize": 48618608, "license": "other", "mime_type": "application/pdf", "url": "/16751/1/HeatherLukas_PhDThesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Engineering Bioaffinity Sensors toward Continuous Electrochemical Biosensing", "author": [ { "family_name": "Lukas", "given_name": "Heather Lauren", "orcid": "0000-0002-8160-9066", "clpid": "Lukas-Heather-Lauren" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The rise of wearable sensing through smartwatches and continuous glucose monitors has made health data more widely accessible. Advances in machine learning have also been pivotal in identifying personalized health insights from biometric data streams. However, continuous biochemical data has been limited in sensor design by the availability of oxidoreductases (e.g., glucose oxidase, lactate dehydrogenase) to a given target. The challenge in engineering diverse oxidoreductase enzymes has led to the exploration of other generalized approaches to continuous electrochemical biosensing. To meet this need, we have explored a variety of bioaffinity sensing schemes using broad bioreceptor classes including antibodies, nucleic acids, and periplasmic binding proteins. We present a case study in electrochemical sensor design utilizing high-affinity antibodies for the rapid diagnosis of COVID-19 disease states. We then investigate the potential of nucleic acid-based electrochemical sensors for continuous sensing with a focus on structure-switching nucleic acid aptamers. The utility of aptamer sensors is demonstrated in the development of a serotonin aptamer sensor embedded in an ingestible capsule for continuous biosensing in the gastrointestinal tract. Applying the principles of electrochemical aptamer-based sensing, we explored the development of an electrochemical protein-based sensor for nicotine, which exploits the hinge-like binding motion of periplasmic binding proteins while also capitalizing on decades of protein evolution and characterization research. With the goal of continuous, noninvasive biochemical sensing, we evaluate the design considerations and translatability of these sensors for wearable sweat analysis. These biosensing techniques may enable the future hardware necessary to expand accessible biomedical data for the next wave of personalized health monitoring.", "doi": "10.7907/2c89-k924", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17355", "collection": "thesis", "collection_id": "17355", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-051628277", "type": "thesis", "title": "Time-Resolved Proteomic Analysis in Zebrafish and Cultured Neurons Using Bioorthogonal Noncanonical Amino Acid Tagging", "author": [ { "family_name": "Miller", "given_name": "Sophie Eve", "orcid": "0000-0001-6805-7036", "clpid": "Miller-Sophie-Eve" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" }, { "family_name": "Chou", "given_name": "Tsui-Fen", "orcid": "0000-0003-2410-2186", "clpid": "Chou-Tsui-Fen" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "3MT Competition (Caltech)" }, { "literal": "div_chem" } ], "abstract": "Temporally and spatially controlled protein synthesis plays a critical role in orchestrating the molecular events underlying behaviors, stress adaptations, and therapeutic responses to drugs. However, traditional proteomic techniques often fail to capture the dynamic changes in protein expression essential for understanding transient biological phenomena. To overcome this limitation, the work presented in this thesis leverages bioorthogonal noncanonical amino acid tagging (BONCAT) coupled with mass spectrometry to perform time-resolved proteomic analyses in zebrafish larvae and cultured neurons.
\r\n\r\nChapter II details the development and validation of BONCAT proteomics in zebrafish, demonstrating that newly synthesized proteins from zebrafish larvae could be reliably labeled, enriched, and identified even over short labeling periods. Proof-of-concept experiments using heat shock revealed that BONCAT proteomics was able to detect changes in expression of proteins known to be induced by heat shock with greater sensitivity than conventional approaches using global proteomics. These results establish BONCAT as a powerful tool for investigating dynamic changes in protein synthesis in zebrafish. In Chapter III, we applied BONCAT to neuronal cultures to profile the proteomic changes induced by sub-anesthetic, antidepressant-relevant doses of ketamine. These studies uncovered rapid alterations in protein synthesis, identifying significantly differentially regulated proteins and pathways involved in synaptic plasticity, cytoskeletal remodeling, cellular signaling, metabolism, and RNA processing. This work provides novel molecular insights into ketamine\u2019s rapid-acting antidepressant effects and further illustrates the utility of BONCAT for capturing early, transient proteomic responses to drug treatment. Finally, in Chapter IV, we explore changes in protein expression in zebrafish larvae underlying circadian rhythms and in response to low-dose ketamine treatment. We observed interesting protein synthesis patterns in both biological contexts, but our findings lacked the statistical significance and reproducibility across experiments required to draw strong biological conclusions from our data. Although methodological refinements are required, our work underscores BONCAT\u2019s potential to elucidate transient proteomic shifts underlying behavioral phenomena and pharmacological interventions in zebrafish.
", "doi": "10.7907/vdgh-6c46", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17354", "collection": "thesis", "collection_id": "17354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-044636280", "primary_object_url": { "basename": "Caltech-Thesis-Shirin-Shivaei-final.pdf", "content": "final", "filesize": 19237378, "license": "other", "mime_type": "application/pdf", "url": "/17354/6/Caltech-Thesis-Shirin-Shivaei-final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "A Viral Toolkit for Ultrasound Imaging of Cellular Activity and Gene Expression", "author": [ { "family_name": "Shivaei", "given_name": "Shirin", "orcid": "0000-0002-6894-3289", "clpid": "Shivaei-Shirin" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Tanter", "given_name": "Mickael", "orcid": "0000-0001-7739-8051", "clpid": "Tanter-Mickael" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Observing and manipulating cell dynamics in living organisms is essential for understanding biological processes and intervening when they malfunction. However, the lack of non-invasive, non-ionizing, and cost-effective imaging technologies limits our ability to study these processes in their native context. To address this gap, we developed a toolkit for ultrasound imaging of acoustic reporter gene expression in mammalian tissues using virally-delivered gas vesicle (GV) genes. We demonstrate the versatility of this toolkit across multiple applications, including tracking engineered cell-based therapies and imaging activity-dependent gene expression in the brain.
\r\n\r\nTo track cell-based therapies, we developed lentiviral vectors encoding the eight genes necessary for GV expression, achieving robust ultrasound contrast in both cell lines and primary human T cells. By expressing GVs downstream of activity-dependent promoters, we monitor T cell activation in cytotoxic T cells engaged with tumor cells. In a mouse xenograft model, we then image the targeted accumulation and proliferation of GV-expressing T cells within tumors. These ultrasound measurements, which closely correlate with immunohistological analysis, provide real-time, in vivo insights into the spatial dynamics of therapeutic cells. This approach offers a powerful tool to accelerate the development and clinical translation of cell-based therapies.
\r\n\r\nWe extend this technology to the brain by engineering an AAV-based system for GV expression in primary neurons. Following intracranial injection of the GV-encoding AAVs in mice, we demonstrate longitudinal imaging of in situ gene expression in the brain over several weeks. Moreover, by using immediate early gene promoters to drive GV expression, we track changes in neuronal activity in the hippocampus during seizure episodes, enabling repeated, longitudinal imaging of brain function within the same animal. Collectively, these advancements establish a robust platform for ultrasound imaging of cellular activity and gene expression in opaque tissues, with applications ranging from cancer immunotherapy to neuroscience.
", "doi": "10.7907/r9gz-1482", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17203", "collection": "thesis", "collection_id": "17203", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052025-211734908", "type": "thesis", "title": "Enriching Architectures for Biosensing and Motor-Filament Systems Through the Programmability of DNA", "author": [ { "family_name": "Guareschi", "given_name": "Matteo Michele", "orcid": "0000-0002-5197-3158", "clpid": "Guareschi-Matteo-Michele" } ], "thesis_advisor": [ { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Since its inception, the field of DNA nanotechnology has focused on studying the fundamental behaviors and capabilities of engineered nucleic acids. A deep understanding of this toolkit has enabled advancements in several fields, for research tools and in translational applications. Together with its programmability and nanometric resolution, the great promise of DNA nanotechnology lies in the incorporation of structure and function in a single molecule. In this work, we show how these advantages can be leveraged to expand the capabilities of two different systems: a sensor for biomarkers and a motor-filament architecture. During our exploration, we also discover and work to overcome some of the less obvious limitations of the technology, shining light on more foundational questions.
\r\n\r\nWe demonstrate an electrochemical biosensor based on a DNA origami that can detect and quantify nucleic acids and proteins in a package easily adaptable to different analytes by simply replacing the binder molecules. Upon target binding, the structure undergoes a large conformational change, bringing a multitude of redox reporters to the electrode surface where an electric current can be measured. The high number of reporter molecules on a single detector results in improved signal gain per binding event, allowing for the detection of low analyte concentrations, while the conformational change yields an unprecedented gain between the off and on state. We demonstrate how the system can be readily adapted to different analyte molecules and reused over several cycles to analyze multiple samples. We then run simulations of the detector molecule to understand structural deformations intrinsic to this design, in order to optimize the number and placement of the redox reporters. We discover and investigate a phenomenon that causes significant curling of the DNA origami, possibly limiting the contribution of many of the reporter molecules. We explore experimental directions to mitigate the issue by changing the configuration of the redox molecules and by designing stiffer sensors.
\r\n\r\nWe then set out to integrate DNA origami-based nanostructures with an engineered dynein protein that can bind to and kick double-stranded DNA instead of tubulin. Motor-filament architectures have been studied as the main mechanism for cellular transport and as a system that can exhibit mesoscopic active matter behaviors in biology, but the relative difficulty of engineering microtubules has hindered the exploration of their properties. The high-resolution programmability of DNA nanostructures makes them prime candidates to overcome this obstacle and this study has been enabled by the recent development of new protein motors where the tubulin binding domain is replaced by a DNA binding domain. We first look at DNA nanotubes, structures that resemble microtubules, but that retain a level of programmability that is typical of DNA nanotechnology. By exploiting the DNA strand displacement technique, we incorporate machinery that enables new behaviors, with a focus on different ways to turn gliding on and off by stopping the DNA nanotubes.
\r\n\r\nWe then turn our focus to more complex gliders designed with DNA origami. We explore the space of DNA origami polymers in order to assemble superstructures that can be detected under light microscopy, encountering again issues of deformations due to the addition of overhangs. We then assess the gliding capabilities of DNA origami, designing ways to incorporate motor binding sequences on them, but we find that DNA origami sticks nonspecifically to the engineered dynein motors. After testing several different hypotheses, we gather evidence that this interaction might be caused by the large sequence variability of the scaffold strand in DNA origami, coupled with the recognition of spurious binding sequences by the motor proteins.
", "doi": "10.7907/fmhp-r892", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17435", "collection": "thesis", "collection_id": "17435", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092025-211524859", "primary_object_url": { "basename": "2025-Andrew-Lu-Caltech-PhD-Thesis.pdf", "content": "final", "filesize": 9969754, "license": "other", "mime_type": "application/pdf", "url": "/17435/21/2025-Andrew-Lu-Caltech-PhD-Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineered Protein Circuits for Cancer Therapy", "author": [ { "family_name": "Lu", "given_name": "Andrew C.", "orcid": "0000-0002-7594-6445", "clpid": "Lu-Andrew-C" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Dawson", "given_name": "David", "orcid": "0000-0002-0215-5861", "clpid": "Dawson, David" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Engineered protein circuits seek to treat cancer by directly rewiring oncogenic signaling to cell death. However, it has remained unclear what circuit designs could operate effectively, and what advantages protein circuits could provide compared to existing small molecule inhibitors. Here, we introduce Ras-targeting circuits that accurately discriminate and kill Ras-mutant cells, circumventing drug resistance mechanisms and suppressing cancer in vivo. These circuits combine three modules: a protease-based sensor that responds to a broad spectrum of clinically relevant Ras mutations, an optional protease amplifier, and protease-triggered cell death effectors of the apoptosis and pyroptosis cell death pathways. When delivered as mRNA in lipid nanoparticles (LNPs), the circuits were effective against Ras-mutant human cancer cell lines with minimal off-target killing of wild-type Ras cells. In immunocompetent mice bearing aggressive, multifocal Ras-driven liver tumors, systemically delivered mRNA-LNP circuits strongly reduced tumor burden. Further, therapeutic circuits provided more complete killing of Ras-mutant cancer cells than the Ras inhibitors Sotorasib and RMC-7977 and did not require oncogene addiction. They also exhibited increased potency against Sotorasib-resistant cells. These results establish a programmable mechanism for treating cancer and other human diseases.", "doi": "10.7907/kxvr-r291", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17117", "collection": "thesis", "collection_id": "17117", "cite_using_url": "https://resolver.caltech.edu/CaltechThesis:03312025-203601435", "primary_object_url": { "basename": "KatsuyaColon_Thesis_Final.pdf", "content": "final", "filesize": 13949697, "license": "other", "mime_type": "application/pdf", "url": "/17117/1/KatsuyaColon_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Situ Signal Amplification for Spatial Transcriptomics Using Programmable DNA Assemblies", "author": [ { "family_name": "Col\u00f3n", "given_name": "Katsuya Lex", "orcid": "0000-0002-7347-6128", "clpid": "Col\u00f3n-Katsuya-Lex" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Sequential Fluorescent In Situ Hybridization (seqFISH) has been an invaluable tool in imaging-based spatial transcriptomics, aiding researchers in elucidating spatially-resolved, gene expression patterns in intact tissues and cell culture models. However, methods that rely on smFISH, such as seqFISH, suffer from poor signal-to-noise ratio in certain tissue types or target RNA, require many fluorescently labeled RNA targeting probes which prohibits imaging of small RNA species, and exhibit poor sample throughput due to the need of high magnification objective or long exposure times. Herein, we develop solutions to these limitations by developing and utilizing a robust signal amplification strategy. While various amplification technologies exist, their limitations often hinder broad applicability. Moreover, we desire an amplification platform that is amenable to the denaturing wash conditions used in seqFISH. We will begin Chapter I by discussing the background, technical challenges, and utility of various in situ signal amplification technologies. Chapter II details the exploration and technical limitations of rolling circle amplification (RCA) and branched DNA (bDNA) assembly utilizing ssDNA padlock amplifier strands. Chapter III discusses the design and development of a novel amplification strategy called Signal amPlicAtion by Recursive Crosslinking (SPARC), which builds upon the knowledge gained from Chapter II. We highlight SPARC as a unique photochemical signal amplification method that iteratively deposits amplifier strands near the primary probe target for linear signal amplification. Then, the deposited amplifier strands act as a scaffold for branched DNA assembly, leading to an exponential signal amplification. Through each deposition and assembly step, amplifier strands are photo-crosslinked to the extracellular matrix, forming highly stable DNA nanostructures that can withstand harsh denaturing wash conditions. We demonstrate the utility of SPARC in amplifying signal of both single-molecule transcripts and proteins.", "doi": "10.7907/pp5f-pk64", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17112", "collection": "thesis", "collection_id": "17112", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03292025-043938908", "type": "thesis", "title": "Probing the Biological Interactions of a Therapeutic Small Peptide at the Tissue, Cellular, and Molecular Levels for Treating Retinal Diseases", "author": [ { "family_name": "Koo", "given_name": "Jin Mo", "orcid": "0009-0003-8088-8790", "clpid": "Koo-Jin-Mo" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the leading causes of blindness in developed countries, affecting the lives of millions and lowering their quality of life due to limited eyesight. Although these retinal diseases afflict a large portion of the senior population, the current standard of care, primarily antibody injection treatments, merely treats symptoms and fails to address the root causes of diseases.
\r\n\r\nA therapeutic hexapeptide, risuteganib (RSG), designed by Allegro Ophthalmics, LLC., has shown efficacy in treating both angiogenic and inflammatory retinopathies such as wet- and dry-AMD and diabetic macular edema (DME), a progressive form of DR. In the past, many studies sought to identify integrins that demonstrated specific binding affinities to RSG, based on the structural similarity of RSG to the well-known RGD motif. In contrast, Caltech decided to pursue unbiased research directions to unveil the mechanism of action (MOA) of RSG. Following studies from Caltech, UC Irvine, Johns Hopkins University, and Duke University revealed unanticipated features of RSG, suggesting that it has both anti-angiogenic and anti-inflammatory effects and that it can rescue compromised mitochondrial functions in cells under chemically induced oxidative stress. In this thesis, we conducted a series of investigations, progressing from the tissue level to the cellular level and then to the molecular level, to test a hypothesis that may unify these observations.
\r\n\r\nIn Chapter 2, we developed a peptide-directed fluorescent staining method to identify the binding location of RSG-dye conjugate using aged BALB/c to recapitulate features of age-related retinal diseases. We identified crucial parameters that visualized replicable, RSG-specific labeling at the aged RPE layer. Findings from Chapter 2 and past studies laid the groundwork for the multi-layered investigations on the MOA of RSG in subsequent chapters, hinting at the cell type of interest, the need for appropriate stress to tissue or cell, and the importance of limiting RSG probes below clinical dosage. In collaboration with the Kenney lab at UC Irvine, we tested the protective effects of RSG in differentiated ARPE-19 cell model in response to chemically activated hypoxia-inducible factor 1 (HIF-1) signaling pathway (Chapter 3). Sub-clinical dosage of RSG (30 \u00b5M) demonstrated protective effects against chemically elevated HIF-1\u03b1 leading to cell death and compromised mitochondrial membrane integrity. Further, RSG-dye conjugate localized in the mitochondria of differentiated ARPE-19 cells, where partial co-localization with mitochondrial protein, pyruvate dehydrogenase E1 \u03b1 subunit (PDHA1), was observed. In Chapter 4, we developed a luminescence-based in vitro assay to quantify the effect of RSG on the activity of a mitochondrial kinase, pyruvate dehydrogenase kinase 1 (PDHK1). PDHK1 interacts with pyruvate dehydrogenase complex (PDC) which contains PDHA1. PDHK/PDC interaction governs the pyruvate decarboxylation process, thereby serving as a dynamic metabolism switch. Upregulation of PDHK1 decreases PDC activity, which subsequently increases glycolytic flux as an adaptive response against hypoxia, temporarily alleviating oxygen deficiency at the cost of total ATP production per mol glucose. When such response is prolonged in the RPE cells, energy deficiency and compromised homeostasis lead to cell death of not only RPE cells but other crucial retinal cells including the photoreceptor cells. Here, tested RSG concentrations (3.125, 6.25, and 12.5 \u00b5M) did not demonstrate significant inhibition of PDHK1 activity; nevertheless, the devised in vitro assay laid the foundation for a reliable, quantitative assessment of RSG or other inhibitors\u2019 effects on the activity of PDHK1 and its isoforms. Chapter 5 summarized the findings in the previous chapters and suggested future studies that might aid in the continued search for the mechanism of action of RSG.
", "doi": "10.7907/kye5-xz39", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16963", "collection": "thesis", "collection_id": "16963", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01282025-190331921", "primary_object_url": { "basename": "20250131_ffl_thesis.pdf", "content": "final", "filesize": 1123988, "license": "other", "mime_type": "application/pdf", "url": "/16963/1/20250131_ffl_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "A Multispecies Perspective on the Evolution of Form Vision", "author": [ { "family_name": "Lanfranchi", "given_name": "Francesco (Frank)", "orcid": "0000-0001-8176-320X", "clpid": "Lanfranchi-Francesco" } ], "thesis_advisor": [ { "family_name": "Tsao", "given_name": "Doris Y.", "orcid": "0000-0003-1083-1919", "clpid": "Tsao-D-Y" } ], "thesis_committee": [ { "family_name": "Perona", "given_name": "Pietro", "orcid": "0000-0002-7583-5809", "clpid": "Perona-P" }, { "family_name": "Tsao", "given_name": "Doris Y.", "orcid": "0000-0003-1083-1919", "clpid": "Tsao-D-Y" }, { "family_name": "Andersen", "given_name": "Richard A.", "orcid": "0000-0002-7947-0472", "clpid": "Andersen-R-A" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Rutishauser", "given_name": "Ueli", "orcid": "0000-0002-9207-7069", "clpid": "Rutishauser-U" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "In the mammalian visual system, photons captured by the retina are transformed into meaningful internal percepts of surroundings through a hierarchy of interconnected visual areas. Understanding the representation of visual information at each node of the hierarchy has been a central quest of visual systems neuroscience over the past 50 years. The primate visual system, with its over two dozen distinct areas broadly organized into a dorsal stream for visuo-motor transformations and a ventral stream for object recognition, has served as the gold standard for studying the organization of the visual system. Recent advances in artificial neural networks modeled on the primate visual system for object recognition have prompted the question, is hierarchical representation necessary, and if so, can we observe it across all highly visual mammalian species? Hierarchical organization appears to be a key architectural principle of both artificial and biological networks, enabling stepwise construction of a structured and compact representation from raw sensory input. Here we present a series of efforts to determine the cortical organization and connectivity of the tree shrew visual system and directly compare to that of the primate. This cross-species study sheds light on the evolution and mechanisms of vision in a close relative of primates. Using high-density Neuropixels recordings, we demonstrate that the tree shrew ventral visual pathway exhibits primate-like hierarchical processing, with progressively larger receptive fields, increasing response latencies, and enhanced selectivity for complex stimuli along the visual pathway. Area V2 in the tree shrew performs key functions similar to those of the primate inferotemporal (IT) cortex. Specifically, V2 contains strongly face-selective cells, supports a complete representation of high-level object space, and achieves the most accurate object identity decoding and reconstruction among all tree shrew visual areas. Yet we also found significant differences from the canonical template for hierarchical organization observed in the primate, including maintenance of relatively small, focal receptive fields throughout the hierarchy, and better decoding of latent variables in late deep neural network (DNN) layers by area V2 compared to other areas.
\r\n\r\nThe hierarchical organization of the visual system describes the arrangement of areas but does not reveal how information flows between them. Understanding the type of processing carried out at each node raised the next question of whether information that is transmitted across nodes is differentiated between feedforward and feedback connections. To explore this, we combined electrical microstimulation and extracellular recordings to identify the directionality of projections which is applicable in various species. We used this technique to first study the connections between the first two nodes of the tree shrew cortical hierarchy, V1 and V2. We found that V2 feedback neurons carry a full visual representation on par to other V2 cells. These feedback neurons were distinct with regards to their spatial features, including distinct locations and sizes of their receptive fields. We also found that both feedforward and feedback V2 neurons were modulated by perceptual conflict arising when distinct textures were presented to each eye, suggesting they could refine V1 processing to perceptual inconsistencies.
\r\n\r\nThese studies provide insights into how the tree shrew visual system generates object representations through a hierarchy of interconnected nodes, employing strategies adapted to its cortical constraints. In addition, by combining electrical microstimulation with electrophysiology we set the foundation for cross-species studies to determine the role of feedforward and feedback processing along the visual hierarchy. Together, this work reveals conserved principles of visual processing across species while showcasing unique adaptations in the tree shrew, offering insights into the evolutionary origins and functional organization of the primate visual system.
", "doi": "10.7907/154t-x703", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17035", "collection": "thesis", "collection_id": "17035", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02282025-172449360", "primary_object_url": { "basename": "Dongkwan_Lee_Thesis_Final_Deposited.pdf", "content": "final", "filesize": 10500825, "license": "other", "mime_type": "application/pdf", "url": "/17035/6/Dongkwan_Lee_Thesis_Final_Deposited.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single- Molecule Imaging", "author": [ { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" } ], "thesis_advisor": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Advances in optical microscopy have revolutionized cell biology, transforming our understanding of cellular processes from static structural observations to dynamic temporal and spatial insights at the single-molecule level. While fluorescence imaging remains the gold standard due to its high sensitivity, specificity, and versatile toolbox, it faces significant limitations, particularly in imaging small molecules that are not inherently fluorescent. Attaching fluorescent tags to these molecules often disrupts their physicochemical properties, highlighting the need for minimally invasive and intrinsic-contrast-based approaches.
\r\n\r\nVibrational spectro-microscopy, which probes the intrinsic vibrational frequencies of chemical bonds, offers a promising solution. Stimulated Raman scattering (SRS) microscopy, a well-established vibrational imaging technique, enhances vibrational excitation by up 10\u2078-fold through stimulated emission amplification, enabling rapid, label-free imaging of biological samples with high specificity.
\r\n \r\nIn the first half of this thesis, we advance SRS microscopy to tackle specific biological challenges and explore new methodological possibilities. To visualize glycogen metabolism, we combined a stable isotope labeling strategy with SRS imaging, achieving high-specificity imaging of glycogen in live cells. This approach was further applied to metabolic phenotyping of patient-derived melanoma cell lines. Additionally, we investigated strategies to photoswitch electronic pre-resonance (epr) SRS probes, which are typically photostable. By inducing electronic transitions that modulate electronic-vibrational coupling, we developed the first genetically encodable photoswitchable epr-SRS probe using a near-infrared fluorescent protein, unlocking new possibilities in Raman imaging.
\r\n\r\nIn the second half of this thesis, we address the limitations of SRS microscopy by developing a novel bond-selective nonlinear optical microscopy technique called bond-selective fluorescence-detected infrared-excited (BonFIRE). BonFIRE introduces a vibration-state-mediated two-photon process as a new vibrational contrast mechanism, overcoming key limitations in sensitivity and speed associated with SRS. By combining the high sensitivity and specificity of fluorescence with the rich chemical information provided by IR absorption-based vibrational contrast, BonFIRE offers a powerful platform for multidimensional insights into biological systems. We envision BonFIRE as a tool to tackle unique challenges that current technologies cannot address, representing a significant step forward in understanding the complex processes that define life.
", "doi": "10.7907/8qqq-z286", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16175", "collection": "thesis", "collection_id": "16175", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09022023-064950039", "primary_object_url": { "basename": "WackelinThesis.pdf", "content": "final", "filesize": 9962421, "license": "other", "mime_type": "application/pdf", "url": "/16175/1/WackelinThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "New to Nature C\u2013C Bond Forming Cyclases: Pushing the Boundaries of Ring Forming Reactions", "author": [ { "family_name": "Wackelin", "given_name": "Daniel Joseph", "orcid": "0000-0001-8189-6985", "clpid": "Wackelin-Daniel-Joseph" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biocatalysts have shown themselves to be extremely powerful for the synthesis of pharmaceuticals, fragrances, and fine chemicals, providing products with high yields and selectivities. Recently, new-to-nature biocatalysis has received increased attention, allowing for the benefits of biocatalysis to be applied to reactions that were previously the sole domain of chemocatalysts. Engineers have begun to develop enzymes that catalyze new-to-nature C\u2013C bond forming cyclisation reactions, which are quite powerful due to their ability to build the carbon skeleton of molecules. Despite this, this class of enzymes is limited in scope. This thesis details the expansion of C\u2013C bond forming cyclases, including expanding the scope of cytochrome P411 cyclopropanation and an intramolecular C\u2013H functionalization strategy for the synthesis of diverse rings. Chapter 1 introduces biocatalysis and its recent applications, especially as they apply to new-to-nature C\u2013C bond forming cyclisation reactions. Chapter 2 shows the development of a cytochrome P411 that catalyzes the enantio- and diastero-specific synthesis of 1,2,3-polysubstituted cyclopropanes. Using directed evolution, this carbene transferase was evolved to react with internal alkenes and build two C\u2013C bonds, expanding the scope and specificity of cyclopropanation reactions. Chapter 3 describes the expansion of this biocatalytic system toward the synthesis of stereoconvergent products, enabling more efficient synthesis from non-diasteropure starting materials. Chapter 4 details the evolution of a cytochrome P411 to perform an intramolecular C\u2013H functionalization using diazo compounds, making a variety of differently sized rings with different molecular geometries. In summary, this work addresses the need for expansion of new-to-nature C\u2013C bond forming cyclisation reactions and provides a guide for expanding new-to-nature reactions to their full potential.
", "doi": "10.7907/26ag-8c56", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16405", "collection": "thesis", "collection_id": "16405", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212024-231923059", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 153588118, "license": "other", "mime_type": "application/pdf", "url": "/16405/1/THESIS.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Oxygen-Regulating MEMS Devices for Cell Transplantation to Cure Type 1 Diabetes", "author": [ { "family_name": "Shang", "given_name": "Kuang-Ming", "orcid": "0000-0001-5065-7607", "clpid": "Shang-Kuang-Ming" } ], "thesis_advisor": [ { "family_name": "Tai", "given_name": "Yu-Chong", "orcid": "0000-0001-8529-106X", "clpid": "Tai-Yu-Chong" }, { "family_name": "Komatsu", "given_name": "Hirotake", "orcid": "0000-0003-0876-4809", "clpid": "Komatsu-Hirotake" } ], "thesis_committee": [ { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" }, { "family_name": "Komatsu", "given_name": "Hirotake", "orcid": "0000-0003-0876-4809", "clpid": "Komatsu-Hirotake" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Tai", "given_name": "Yu-Chong", "orcid": "0000-0001-8529-106X", "clpid": "Tai-Yu-Chong" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Type 1 diabetes is an autoimmune disease in which immune cells specifically attack and destroy the insulin-producing beta cells in the pancreatic islets that regulate blood glucose levels. Traditionally managed with frequent injections of exogenous insulin, beta cell replacement therapy\u2014also known as islet transplantation\u2014has emerged as an alternative clinical option. Recently, the focus has shifted toward subcutaneous islet transplantation, offering a promising and minimally invasive therapy. However, the survival of transplanted islets has been shown to be significantly challenged by hypoxia-induced graft loss stemming from inadequate oxygen supply.
\r\n\r\nTo address this issue, we have developed innovative hollow mesh devices that regulate oxygen. These devices can either bring oxygen from the adjacent oxygen-rich tissue or draw additional oxygen from ambient air to improve oxygen delivery to the hypoxic microenvironment of islet grafts. Fabricated using MEMS techniques and biocompatible materials, these devices feature a network of unobstructed air-containing microchannels. Utilizing the property that oxygen diffuses 10,000 times faster in air than in interstitial fluids, these devices effectively overcome oxygen supply barriers when co-transplanted with islet grafts. By integrating these hollow meshes with the islet grafts, oxygen can be rapidly redistributed throughout the graft, establishing local oxygen balance and regulation. This approach significantly reduces hypoxia-induced graft loss and improves the efficacy of post-transplant blood glucose regulation in recipients.
\r\n\r\nIn this thesis, we first delved into the physiology of oxygen transport within an islet, establishing the critical oxygen threshold necessary for islet cell survival. We developed equivalent circuit models for oxygen diffusion and constructed oxygen-regulating hollow mesh MEMS devices based on these models. We investigated the effects of oxygenation through both computational models and benchtop experiments. Finally, using our device, we demonstrated enhanced survival of islet grafts in diabetic rodent models, successfully achieving a long-term cure for diabetes.
\r\n\r\nWith the preclinical success of this oxygen-regulating hollow mesh in mitigating cellular oxygen deficiency, we also explored and proposed future pathways toward clinical effectiveness. Our device holds significant therapeutic potential to revolutionize clinical outcomes in islet transplantation with the ultimate goal of curing type 1 diabetes.
", "doi": "10.7907/xf5z-0p34", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16384", "collection": "thesis", "collection_id": "16384", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152024-052419062", "type": "thesis", "title": "Acoustic Biosensors for Noninvasive Imaging of Molecular Processes", "author": [ { "family_name": "Jin", "given_name": "Zhiyang", "orcid": "0000-0002-4411-6991", "clpid": "Jin-Zhiyang" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Understanding biology in its native context has been a major scientific endeavor. Yet, it is challenging to visualize cellular dynamics at the molecular scale in the context of a living organism at the macroscopic scale. Ultrasound imaging represents a promising candidate to address this challenge, with its unique advantages of large imaging volume, deep penetration, and good spatiotemporal resolution. However, ultrasound was historically limited in retrieving molecular information that biology carries. Until very recently, the discovery of the first ultrasound-interacting biomolecules, gas vesicles (GVs), established a connection between connect cellular function and ultrasound signals, which later enabled ultrasound imaging of gene expression and thus the location of GV-expressing cells. Going beyond location tracking, this thesis describes the engineering of GV-based acoustic biosensors that made it possible to noninvasively image the dynamics of cellular signaling in living organisms.
\r\n \r\nGVs are genetically encoded intracellular air-filled \u201cballoons\u201d that are encapsulated by protein shells. The acoustic biosensor design leverages the GV surface protein GvpC, which controls GVs' ultrasound scattering by setting the stiffness of their protein shell. We developed the first acoustic biosensors by engineering GvpC to change its confirmation and thereby GVs\u2019 ultrasound contrast in response to the activity or concentration of specific molecules. Specifically, we first built the biosensors for three different types of enzymes and demonstrated noninvasive imaging of enzyme activity inside probiotic cells in the mouse colon in vivo. Next, we engineered the acoustic biosensors for calcium, a ubiquitous signaling molecule that is essential in many cellular processes (e.g., neural activity). With the first generation of this calcium sensor for ultrasound, we demonstrated imaging of receptor-specific calcium signaling deep inside the mouse brain through the intact skull noninvasively, which opened up the possibility of whole-brain neuroimaging that can lead to many breakthroughs in neuroscience. Last, we established a high-throughput engineering platform to develop all these GV-based imaging agents in a much shorter time frame. Collectively, this thesis presents the first demonstration of noninvasively imaging dynamic cellular signaling with acoustic biosensors and the feasibility of efficiently improving them for potential real-world applications with our engineering pipeline, opening up a new route towards understanding biology across scales.
", "doi": "10.7907/b51h-q307", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16342", "collection": "thesis", "collection_id": "16342", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03292024-213352165", "primary_object_url": { "basename": "Wang_thesis_final.pdf", "content": "final", "filesize": 12860605, "license": "other", "mime_type": "application/pdf", "url": "/16342/3/Wang_thesis_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Chemo-Selective Proteomics for Discovery of Polymicrobial Interactions", "author": [ { "family_name": "Wang", "given_name": "Grace Zimu", "orcid": "0000-0002-0938-304X", "clpid": "Wang-Grace-Zimu" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The future of microbiome research lies in our ability to engineer polymicrobial interactions toward improved host health outcomes, which requires a fundamental molecular understanding of how microbial species sense and respond to ecological competition. Chronic respiratory infection by polymicrobial communities is the leading cause of mortality and morbidity in people living with cystic fibrosis (CF). My thesis work adapts chemo-selective proteomics to dissect molecular mechanisms that drive interspecies dynamics between two notorious opportunistic pathogens dominating chronic CF infection, Pseudomonas aeruginosa and Staphylococcus aureus.
\r\n\r\nIn Chapter 1, I introduce bioorthogonal noncanonical amino acid tagging (BONCAT)-based comparative proteomics, focusing on time-resolved, cell-specific, and cellular state-selective proteomic applications in the dissection of complex microbial systems. In Chapter 2, I discuss a new usage of time-resolved BONCAT\r\nto monitor immediate competition-sensing responses in interbacterial warfare. While coinfection by the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. We\r\ndiscovered that P. aeruginosa senses S. aureus secreted cytotoxic peptides from a distance and preempts potential competition through activation of type six secretion system (T6SS). P. aeruginosa enhances such competition-sensing-induced antagonism through concomitant attraction toward S. aureus peptides, effectively reducing cellular distances between neighboring species and providing a competitive advantage. In Chapter 3, I discuss a new usage of cell-selective BONCAT to target protein synthesis analysis of the lowabundance organism, S. aureus, in a coculture environment predominated by P. aeruginosa. P. aeruginosa robustly outcompetes S. aureus, and conventional shotgun proteomics, which is biased toward highly abundant proteins on principle, could only identify and quantify less than 5% of total protein synthesis by S. aureus in coculture. We demonstrate that chemical enrichment affords a more than 12-fold increase in total protein abundances synthesized by S. aureus. About 50% of protein \u201chits\u201d with statistically significant changes in expression were not detected in pre-enrichment lysates, highlighting BONCAT as a powerful strategy that facilitates high-resolution proteomic analysis of low-abundance organisms in polymicrobial communities.
", "doi": "10.7907/1vw0-gt98", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16494", "collection": "thesis", "collection_id": "16494", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042024-004047037", "type": "thesis", "title": "Neuropsychiatric Drug Biosensors in Organelles, Cells, Biofluids, & Behaving Animals", "author": [ { "family_name": "Muthusamy", "given_name": "Anand Kumar", "orcid": "0000-0003-1041-914X", "clpid": "Muthusamy-Anand-Kumar" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biology is distinguished in its several levels of spatial organization\u2014from molecular to whole body\u2014that give rise to coherent, goal-related behaviors. Cutting across these layers are definable circuits, each with its own dynamics. These systems should be studied in their natural context to preserve their structure and function. However, quantitative measurements typically demand invasive apparatuses or infrequent, ex vivo measurements. The advent of genetically encoded fluorescent biosensors solves this problem by detecting molecules in situ, read by a microscope or implantable optical probe. These biosensors typically are fusions of a conformational switch and a fluorescent protein. A naturally occurring protein that binds the target of interest typically provides an initial scaffold. However, several molecules, particularly various human-made drugs, do not have similar naturally occurring cognate conformational switches in nature robust enough for this approach.
\r\n\r\nThis work develops and applies the first genetically encoded drug biosensors in cellular and behavioral assays addressing substance abuse disorders. We term these biosensors intensity-based drug-sensing fluorescent reporters or \u201ciDrugSnFRs.\u201d These biosensors are based on a choline-binding periplasmic binding protein (PBP), OpuBC, interrupting a circularly permuted green fluorescent protein (GFP). This work reports a method of optimizing this construct toward the detection of several classes of neural drugs, including nicotinic, SSRIs, ketamine family drugs, and opioids.
\r\n\r\nThe opportunity for continuous monitoring is particularly prominent in brain-body-behavior relationships. For example, a core tenet of behavioral neuropharmacology is the existence of some stereotyped relationship between the time course of a drug and behavioral outcomes such as opioid use disorder. Interindividual variability in pharmacokinetics (PK) complicates the problem of optimized opioid dosing, especially outside the clinic. The problem of personalizing pharmacokinetics is severe in substance use disorders: the patient must receive opioid levels that relieve pain, minimize tolerance and other side effects, and remain within a therapeutic window to maximize adherence. That ideal window is a \u201cmoving target\u201d due to tolerance, changes in metabolism, and stressors.
\r\n\r\nAs an end-to-end study with a preclinical model, the final chapter reports the development of iOpioidSnFRs and their application to the continuous monitoring of fentanyl alongside a computer vision routine to quantify behavior. The fentanyl sensor, iFentanylSnFR2.0, was expressed in the ventral tegmental area of mice and reported [fentanyl] vs. time. This recording is the longest continuous measurement of the brain [drug] alongside behavior (4 hours). We found a stereotypic, repetitive motor pattern that tracked the entire fentanyl time course (2-3 hours) despite variable PK across individuals. This result challenges current models of cellular desensitization and acute tolerance timescales. In a separate experiment, we investigated if this stereotypical pattern impaired mice in a survival task where mice forage for water through a labyrinth maze. Like in the open arena, mice in the maze exhibited circling/stalling for approximately 3 h, to the complete exclusion of successful foraging. Critically, this paradigm offers a normative definition of a deficit, as mice should have a baseline level of successful foraging to survive. We introduce this task to the substance use disorder field as an additional metric for the deficits caused by opioid administration.
\r\n\r\nFinally, this work demonstrates the utility of iOpioidSnFRs in diagnostic tests owing to their suitable aqueous solubility, dynamic range, sensitivity, selectivity, kinetics, and stability after lyophilization. Plate reader assays using iFentanylSnFR2.0, iS-methadoneSnFR, iTapentadolSnFR, and iLevorphanolSnFR provided quantitation across the pharmacologically relevant concentration ranges. These biosensors were also used in a simulated field test using readily available parts: dark box, blue LED strips, band pass filter, and a cellphone camera. This test could be used to determine the presence of a health hazard in the environment (e.g., fentanyl) or determine the exposure level in a person. These results encourage diagnostic and continuous monitoring approaches to personalizing opioid regimens.
", "doi": "10.7907/1043-8k76", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16473", "collection": "thesis", "collection_id": "16473", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-011201674", "primary_object_url": { "basename": "Mengtong Duan 2024 Jun 14.pdf", "content": "final", "filesize": 35875270, "license": "other", "mime_type": "application/pdf", "url": "/16473/1/Mengtong Duan 2024 Jun 14.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression", "author": [ { "family_name": "Duan", "given_name": "Mengtong (Tom)", "orcid": "0000-0002-1601-8876", "clpid": "Duan-Mengtong-Tom" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis presents a comprehensive exploration of the next generation of mammalian Acoustic Reporter Genes (mARGs), unveiling a novel approach for non-invasive, real-time imaging of cellular processes and gene expression within live animals1. Building on the foundational work of first-generation ARGs2,3, which introduced the groundbreaking concept of using gas vesicle (GV) genes as genetically encoded ultrasound contrast agents, this research tackles the inherent limitations of these pioneering systems. The first segment details the development and characterization of the second-generation mARGs which significantly improve upon their predecessors by offering robust expression without the need for monoclonal screening, dynamic non-destructive imaging capabilities, and customizable acoustic properties through gene and protein level modifications. This advancement not only enhances the utility of mARGs in biomedical imaging but also paves the way for their application in novel therapeutic monitoring strategies, as exemplified by real-time tracking of tumor development and ultrasound-guided tumor biopsies that leverage gene expression information.
\r\n\r\nFurther, the thesis delves into the structural, genetic, and biochemical principles underpinning GV assembly, addressing a critical knowledge gap that has persisted despite the utility of GVs in ultrasound imaging. Understanding these assembly mechanisms is crucial for the engineering of improved ARGs.
\r\n\r\nThe exploration then extends into innovative bioengineering methodologies, specifically Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER), a synthetic biology breakthrough enabling the expression of multiple proteins at precise stoichiometries from single, compact transcripts.4 SEMPER represents a strategic advancement in the field, facilitating efficient formation of multi-protein complexes, minimizing cellular toxicity, and broadening the scope of potential applications in genetic engineering, including the creation of enhanced cell lines and circuits for research and therapeutic purposes.
\r\n\r\nCollectively, this work not only advances our understanding of GV-based ultrasound imaging and gene expression tracking but also introduces versatile genetic tools for the manipulation of cellular machinery. These achievements mark significant strides in the fields of synthetic biology and molecular imaging, setting the stage for future innovations in non-invasive diagnostics, cellular therapy, and cancer monitoring research. Through the integration of improved acoustic reporter genes, insights into gas vesicle assembly, and the SEMPER method for gene expression, this thesis embodies a holistic approach to overcoming current challenges and unlocking new potentials in biomedical engineering and synthetic biology.
", "doi": "10.7907/avgk-yc71", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16324", "collection": "thesis", "collection_id": "16324", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03132024-155539035", "primary_object_url": { "basename": "JLee_Thesis_Final.pdf", "content": "final", "filesize": 14709267, "license": "other", "mime_type": "application/pdf", "url": "/16324/1/JLee_Thesis_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Ultrasound Control and Imaging of Cellular Immunotherapy", "author": [ { "family_name": "Lee", "given_name": "Justin", "orcid": "0000-0002-3657-4386", "clpid": "Lee-Justin" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Biomedical ultrasound-based therapeutics and diagnostics are becoming an increasingly important clinical tool. Techniques like focused ultrasound tissue heating and microbubble-enhanced ultrasound imaging have enabled new ways to noninvasively treat and detect diseases cost-effectively and safely. While these are great leaps forward in ultrasound technology, leveraging synthetic biology tools to engineer cells with the capabilities to interact with ultrasound in novel ways may enable even more avenues for ultrasound to address important clinical challenges.
\r\n\r\nIn this thesis, we explore the potential in engineering immune cells with various genetic elements which interact with either therapeutic or diagnostic ultrasound in novel ways. In Chapter 2, we engineer T-cells capable of sensing increases in temperature and responding by activating expression of therapeutic proteins to potentially increase safety of cell-based immunotherapies by controlling their spatiotemporal activation. In Chapters 3 and 4, we develop monocytes as ultrasound reporter cells for cancer detection by engineering them to express gas vesicles (GVs), a class of air-filled protein nanostructures natively found in certain aquatic microbes, which have been demonstrated to produce ultrasound contrast. We demonstrate the potential to confine GV expression to certain disease related signals to create ultrasound reporter cells. Together, these findings highlight the potential of engineering cells to activate in certain locations in response to ultrasound heating or serve as sentinel cells for disease detection.
", "doi": "10.7907/60sh-a389", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16289", "collection": "thesis", "collection_id": "16289", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02072024-100209417", "type": "thesis", "title": "Design and Implementation of a Microparticle Delivery Device for the Cornea", "author": [ { "family_name": "Ko", "given_name": "Dennis Lok", "orcid": "0000-0001-8700-0844", "clpid": "Ko-Dennis-Lok" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biolistic drug delivery offers an alternative path for delivering therapeutics into the cornea. Until now, none of the commercially available gene guns are suitable for clinical delivery of therapeutics due to tissue damage caused by high speed gas used to accelerate microparticles. Here, we demonstrated the use of a device that both eliminates the exit gas, only allowing high speed particles through, and one that works in a clinical setting.
\r\n\r\nMicroparticles ranging from 5 to 22 \u03bcm were accelerated and delivered into both the agarose gels and ex vivo corneas. In gels, we found that normalized penetration depth was proportional to particle diameter and density. As the standoff distance between the device and the target increased, more particles were left stranded at the surface, as their penetrating power decreased, and their dispersion from the center of mass on the target increased. The orifice size served to control both the number of particles and the amount of exit gas. Increasing the inlet pressure did not a show significant increase in the penetration depth of microparticles.
\r\n\r\nIn the cornea, we found that we were able to use our device to deliver particles into both the epithelium and the stroma, although only higher density particles were able to enter the stroma. There was little to no damage to the cornea due to particle delivery. If epithelial defects were detected in the cornea due to particle penetration, they were quickly resolved within 30 minutes. Our device demonstrated performance (penetration depth) comparable to previous biolistic delivery methods in the cornea, while also maintaining clinical relevance by eliminating exit gas flow.
", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16280", "collection": "thesis", "collection_id": "16280", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01162024-161326655", "primary_object_url": { "basename": "TDilanyan_Thesis.pdf", "content": "final", "filesize": 7266593, "license": "other", "mime_type": "application/pdf", "url": "/16280/4/TDilanyan_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Open-Source Custom Beads for Single-Cell Transcriptomics", "author": [ { "family_name": "Dilanyan", "given_name": "Taleen Gaied", "orcid": "0000-0002-3131-3259", "clpid": "Dilanyan-Taleen-Gaied" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Open-source single-cell genomics technologies have helped democratize single-cell genomics and expedite method development. Methods such as inDrops and Drop-seq for single-cell RNA-seq preceded popular technologies such as the 10x Genomics\u2019 Chromium platform, however despite initial enthusiasm for open-source methods, their popularity has waned. A major reason has been the lack of availability of low-cost, customizable beads, which are essential for microfluidics based single-cell RNA-seq. We address this challenge by introducing a new method for producing barcoded hydrogel beads for single-cell RNA-seq called HiPER (High-throughput PER-barcoded hydrogel beads) that allows for increasing the diversity of barcode sequences, reducing manufacturing cost, and that can be readily adapted to custom applications. HiPER barcodes are decoupled from the capture sequences and can therefore be configured to capture RNA, DNA, or tailored for specific-gene enrichment.", "doi": "10.7907/v52p-gf80", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16239", "collection": "thesis", "collection_id": "16239", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11062023-050222447", "type": "thesis", "title": "Wearable Sweat Sensors for Disease Monitoring and Management", "author": [ { "family_name": "Tu", "given_name": "Jiaobing", "orcid": "0000-0002-7653-6640", "clpid": "Tu-Jiaobing" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Dabiri", "given_name": "John O.", "orcid": "0000-0002-6722-9008", "clpid": "Dabiri-J-O" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "With the emphasis of healthcare shifting towards prevention and early detection of diseases and monitoring of chronic conditions, there is a growing need for hassle\u2010free telemedicine sensor technologies that can be seamlessly integrated into daily life. While significant progress has been made in the development of wearable sweat and salivary biosensors to meet this need for rapid, real-time collection of physiological information, the majority of current epidermal sensing systems are unable to detect trace-level disease-relevant biomarkers accurately in biofluids and cannot be mass produced. To meet this demand for low-cost, mass-producible mHealth devices for at-home settings, we developed several fully integrated laser-engraved graphene-based biosensors for the detection of low-concentration sweat and saliva analytes including hormones (cortisol) and proteins (C-reactive protein). Several graphene surface engineering strategies are investigated for the sensitive and selective detection of targets. System-level engineering and microfluidic designs are explored to achieve on-demand sweat induction and harvesting under sedentary settings and automated sweat and reagent routing and in situ signal correction and analysis for facile operation on the skin. The utility of these fully integrated flexible mHealth systems is evaluated through multiple human studies involving healthy and various patient subgroups towards stress assessment, as well as the monitoring and management of various chronic conditions including chronic obstructive pulmonary disease, heart failure, and inflammatory bowel diseases. These fully integrated mHealth devices demonstrate a technology that can be easily adapted to monitor a broad spectrum of disease-specific proteins, cytokines, and hormones, thus advancing future applications in personalized disease diagnosis, management, and prevention.", "doi": "10.7907/7jdg-z479", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16437", "collection": "thesis", "collection_id": "16437", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292024-221307093", "primary_object_url": { "basename": "BussMarjorie_thesis_v04.pdf", "content": "final", "filesize": 30293546, "license": "other", "mime_type": "application/pdf", "url": "/16437/1/BussMarjorie_thesis_v04.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Tools for Noninvasive Imaging and Control of Engineered Bacteria In Vivo", "author": [ { "family_name": "Buss", "given_name": "Marjorie Theresa", "orcid": "0000-0002-4266-9197", "clpid": "Buss-Marjorie-Theresa" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Genetically engineered bacteria are promising new cell-based diagnostic and therapeutic agents due to their ability to sense and respond to unique signals, access and interface with hard-to-reach areas of the body, and deliver therapeutics directly to these areas. However, currently tools to noninvasively monitor and control their activity in vivo are limited. Optical imaging methods, which are based on fluorescent and luminescent reporter genes, and optogenetics, which are based on light-activated proteins, are widely used in cell culture and rodent studies. However, these optical methods suffer from the poor penetration depth of light in tissue which limits their use in larger animals or humans. On the other hand, nuclear imaging methods such as PET and SPECT have good imaging depth but rely on radioactive tracers whose synthesis can be complex and exposes patients to radiation. Here I present tools for imaging and control of bacteria that based on non-ionizing forms of energy that easily penetrate tissue: sound waves and magnetic fields.
\r\n\r\nThe first two parts of my thesis focuses on imaging bacteria in vivo with ultrasound, which is a widely available imaging modality that does not use ionizing radiation and has tissue penetration depth of several centimeters. Bacteria can be imaged with ultrasound by expressing acoustic reporter genes (ARGs) which result in the production of gas vesicles (GVs), air-filled protein nanostructures that aquatic microbes use to regulate their buoyancy. However, the first-generation acoustic reporter genes expressed too poorly under in vivo conditions to enable ultrasound imaging of bacteria in therapeutically relevant contexts. Here, we present a new and improved ARG construct that produces high levels of robust gas vesicle expression in the probiotic bacterium E. coli Nissle (EcN), enabling ultrasound imaging of these cells with high sensitivity. This second-generation ARG construct, bARGSer, uses genes derived from Serratia sp. ATCC 39006 and was optimized for plasmid-based expression in EcN. We demonstrate that with bARGSer, we can visualize the spatial distribution of engineered EcN after they home to and colonize tumors upon systemic administration. We also demonstrate that the engineered EcN can be imaged with ultrasound when colonizing the gastrointestinal tract of mice after sensing dietary sugars as well as biomarkers of inflammation. By enabling monitoring of the precise spatial location of engineered probiotic bacteria inside the body, this technology could greatly improve the development and eventual clinical use of this emerging class of microbial cell-based theranostics.
\r\n\r\nThe last part of my thesis focuses on control of bacteria in vivo with magnetic fields. Many bacteria have limited ability to selectively colonize specific targeted regions of the GI tract due to a lack of external control over their location and persistence. Magnetic fields are well suited to provide such control due to their ability to freely penetrate biological tissues, but they are difficult to apply with enough strength to directly manipulate magnetically labeled cells within deep tissue or viscous environments such as in the GI tract. Here, we show that ingestible micron-sized magnetic particles, combined with an externally applied magnetic field, act as in vivo magnetic field gradient amplifiers, enabling the trapping and retention of orally administered probiotic E. coli within the mouse GI tract. This technology improves the ability of these probiotic agents to accumulate at specific locations and stably colonize without antibiotic treatment. By enhancing the ability of GI-targeted cellular agents to be at the right place at the right time, cellular localization assisted by magnetic particles (CLAMP) adds external physical control to an important emerging class of biotherapeutics.
", "doi": "10.7907/mvgg-ch02", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16255", "collection": "thesis", "collection_id": "16255", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052023-185529151", "primary_object_url": { "basename": "Thesis - Jihong Min.pdf", "content": "final", "filesize": 9341850, "license": "other", "mime_type": "application/pdf", "url": "/16255/11/Thesis - Jihong Min.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Innovations in Wireless Bioelectronics for Precision Medicine, from Sustainable Sweat Sensing to Ingestible Gut Monitoring", "author": [ { "family_name": "Min", "given_name": "Jihong", "orcid": "0000-0002-5788-1473", "clpid": "Min-Jihong" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Biofluids, constituting about 60% of the human body, serve as treasure troves of biomarkers such as metabolites and electrolytes, shedding light on individual health conditions. Although blood and urine tests have been routinely utilized, they are limited by their invasive and episodic nature. However, the promise of continuous and noninvasive access to other biofluids like sweat, GI fluids, and saliva paves the way for real-time, onsite health monitoring. This thesis delves into the untapped potential of wearable sensors and noninvasive biofluid analysis, emphasizing the importance of continuous and sustainable monitoring for predictive personal healthcare. Chapter 1 introduces the paradigm of biofluid sensing, focusing on sweat as a key candidate for personalized healthcare applications. Chapter 2 delves into the physiology of sweat glands, highlighting the composition of sweat and the mechanisms behind sweat extraction, either through natural exercise or iontophoretic stimulation. Chapter 3 embarks on the development of innovative sensors designed for detecting clinically pertinent biomarkers in sweat, a step forward in predictive health analytics. In Chapter 4, the spotlight is on system integration, as the study emphasizes the need for miniaturized and reliable wireless sensor devices that ensure minimal discomfort and maximum reliability. Chapters 5 and 6 delve into strategies for sustainably powering wearable devices from energy harvested from body motions and from ambient light, respectively. The final chapter, Chapter 7, extrapolates the aforementioned technologies for the realm of ingestible devices, adapting them for electrochemical sensing in alternate media, primarily gastrointestinal fluids. This allows for enhanced detection of gastrointestinal diseases and a deeper understanding of the intricate gut-brain axis. The ultimate vision of this research is to equip individuals with wearable and ingestible sensors that can seamlessly monitor a broad spectrum of clinically relevant biomarkers. This continuous monitoring, coupled with data analytics, will potentially catalyze a shift from reactive to predictive healthcare, ushering in an era of personalized therapeutic interventions. As wearable sweat and ingestible sensors become mainstream, a confluence of biosensing mechanisms, materials science, and flexible electronics is anticipated enable continuous and unobtrusive acquisition of clinically relevant biomarkers over prolonged periods and large populations, further refining the nexus between health monitoring and precision medicine.", "doi": "10.7907/kcm7-wz71", "publication_date": "2024-06-14", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16171", "collection": "thesis", "collection_id": "16171", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08312023-053642106", "type": "thesis", "title": "Stimulated Raman Scattering: a Biophysical Perspective for Imaging Cells and Tissues", "author": [ { "family_name": "Miao", "given_name": "Kun", "orcid": "0000-0001-6567-3650", "clpid": "Miao-Kun" } ], "thesis_advisor": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis explores the utilization of Stimulated Raman Scattering (SRS) microscopy as a novel imaging method in the biomedical field, aiming to overcome the limitations associated with traditional fluorescence-based techniques. Given the drawbacks of fluorescence imaging, such as photobleaching, auto-fluorescence, and the complexity of fluorophore labeling, SRS microscopy emerges as a promising solution. The optical imaging contrast in this method originates from bond vibrations of endogenous biomolecules. Grounded in the principle of Raman scattering, SRS amplifies weak spontaneous Raman transitions through stimulated emission, offering a target-specific, high-speed, and label-free imaging modality that can overcome the challenges of traditional bio-imaging techniques.
\r\n\r\nTo tackle the interference from fluorescent proteins when imaging small proteins of interest, we demonstrated a combination of SRS with selective deuterium labeling for visualizing polyQ aggregates in Huntington's disease. We targeted the C-D vibration on deuterated glutamines, which are metabolically enriched in the polyQ sequence. This allowed us to image Huntingtin aggregates without using fluorescent labels. Our method enables, for the first time, the quantification of protein concentrations and compositional analyses of polyQ and non-polyQ proteins within native Huntingtin aggregates. This novel perspective suggests that aggregates have distinct biophysical roles at different stages of aggregation.
\r\n\r\nIn addition to fluorescent proteins, immunofluorescence is the gold standard for visualizing the location and distribution of proteins within cells or tissues. However, the proper delivery of antibodies is slow and labor-intensive. To overcome this issue, we developed a novel method, Vibrational Imaging of Swelled Tissue and Analysis (VISTA), that combines SRS microscopy with sample expansion to enable label-free super-resolution volumetric imaging in tissues. We developed a unique fixation hydrogel chemistry to maximize protein retention, delipidation, and isotropic expansion in tissue samples. By targeting the bond vibrations from endogenous proteins, VISTA bypasses the limitations of antibody labeling and provides an efficient tool for high-throughput imaging that can be scaled to large-volume clinical samples. The addition of image segmentation methods to VISTA equips it with protein-level specificity similar to immunofluorescence. We further used this technique to study protein aggregates, such as amyloid-\u03b2 plaques in Alzheimer's disease, revealing intricate aggregate structures and polymorphisms absent in conventional fluorescence methods.
\r\n\r\nFinally, as fluorescent biosensors are indispensable tools for studying intracellular dynamics, we worked on extending the utility of SRS microscopy into the realm of sensing. We employed hydrogen-deuterium exchange on alkyne substrates to develop a Raman-based sensing strategy sensitive to subtle variations in local microenvironments. The rate of hydrogen-deuterium exchange changes under different conditions, and the resulting frequency shift from alkyne to deuterated alkyne is captured by SRS microscopy. This new platform enhances the study of chemical environments in various biological structures, marking a pivotal step in integrating imaging and sensing in biophysical research.
", "doi": "10.7907/ch88-9173", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16062", "collection": "thesis", "collection_id": "16062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-194724728", "type": "thesis", "title": "Stochastic Foundations for Single-Cell RNA Sequencing", "author": [ { "family_name": "Gorin", "given_name": "Gennady", "orcid": "0000-0001-6097-2029", "clpid": "Gorin-Gennady" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Single-cell RNA sequencing, which quantifies cell transcriptomes, has seen widespread adoption, accompanied by proliferation of analysis methods. However, there has been relatively little systematic investigation of its best practices and their underlying assumptions, leading to challenges and discrepancies in interpretation. I present a set of generic, principled strategies for modeling the biological and technical components of sequencing experiments and use case studies to motivate their application to sequencing data.
", "doi": "10.7907/jn6n-x368", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15009", "collection": "thesis", "collection_id": "15009", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08272022-063655097", "type": "thesis", "title": "Bioorthogonal Noncanonical Amino Acid Tagging for Understanding Bacterial Persistence", "author": [ { "family_name": "Liu", "given_name": "Xinyan", "orcid": "0000-0003-3258-5720", "clpid": "Liu-Xinyan" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of \u201cpersister\u201d cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. On the other hand, bacteria often have multiple molecular mechanisms to adapt to fluctuating environments. Understanding these mechanisms, and their redundancy, requires examinations in depth at the molecular level. This thesis describes a time- and cell state-selective proteome-labeling approach that enables researchers to investigate heterogeneous systems and molecular redundancy.
\r\n\r\nIn Chapter 1, we review the concept of bacterial persistence. The definition of bacterial persistence is introduced. Both the differences and connections between bacterial persistence and resistance are covered. In particular, we discuss research related to Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen found in many cystic fibrosis patients. State-of-the-art technologies to investigate bacterial persistence are discussed, and we conclude that advanced tools are needed to advance research on bacterial persistence further.
\r\n\r\nIn Chapter 2, we highlight the concept of bioorthogonal noncanonical amino acid tagging (BONCAT). BONCAT is a powerful tool developed in the Tirrell and Schuman laboratories allowing the incorporation of noncanonical amino acids (ncAA) into newly-synthesized proteins. We review established strategies for proteomics, especially cell-selective proteomics. We introduce the concept and mechanism of BONCAT and address the advantages of BONCAT in the investigation of phenotypic heterogeneity and bacterial persistence.
\r\n\r\nIn Chapter 3, we describe our work using BONCAT for understanding bacterial persistence. In particular, we investigated the process of persister resuscitation, as it is closely related to the reoccurrence of P. aeruginosa infections. The characteristics of the heterogeneity of persister cells during persister awakening were examined by survival assays and by ScanLag, an automated colony-based system allowing high-throughput acquisition of time-lapse images, quantification, and analysis of growth of bacterial colonies. Two BONCAT methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (Aha), which avoids extensive usage of antibiotic markers and allows direct integration with PA14 transposon insertion library, or with L-azidonorleucine (Anl), which has the advantage of specificity, as well as direct application in nutrition-rich medium. Through BONCAT enrichment experiments, we found proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were up-regulated in the regrowth phase. We further explored whether the up-regulation was a result of the modulation of HigB-HigA toxin-antitoxin system.
\r\n\r\nIn Chapter 4, we describe our work for understanding molecular redundancy. The chapter follows up on our observation of up-regulation of pyochelin-related proteins during persister regrowth. We discuss the hypothesis that pyochelin confers a growth advantage in persister cells subject to carbon-limited conditions. In addition, we discuss the potential role of Fur, a ferric uptake regulator, in bacterial persistence.
", "doi": "10.7907/q6bx-kt39", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15038", "collection": "thesis", "collection_id": "15038", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10032022-224522966", "type": "thesis", "title": "The Hemodynamics of Native and Surgical Aortic Valves with Regards to Wall Shear Stress and Residence Time", "author": [ { "family_name": "Rosakis", "given_name": "Alexandros Yiannis", "orcid": "0000-0001-9170-1002", "clpid": "Rosakis-Alexandros-Yiannis" } ], "thesis_advisor": [ { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "thesis_committee": [ { "family_name": "Ravichandran", "given_name": "Guruswami", "orcid": "0000-0002-2912-0001", "clpid": "Ravichandran-G" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Koochesfahani", "given_name": "Manoochehr", "orcid": "0000-0002-7001-8455", "clpid": "Koochesfahani-Manoochehr" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Cardiovascular diseases are the leading causes of illness and death all around the world. The third most common cardiovascular disease is aortic stenosis (AS). AS is most commonly characterized as a stiffening of the native trileaflet aortic valve, which impedes blood flow into the aorta and puts extra stress on the heart. The aorta is the main artery that supplies oxygenated blood to the body. AS has been widely studied in the past. However, there has been little work in understanding the complex effects that non uniform stiffening of the aortic valve can have on the hemodynamics inside the aorta.\r\n\r\nThe most effective treatment for AS is to replace the stiffened valve with a prosthetic valve. Care must be taken to ensure that the replacement actually performs better hemodynamically. A major metric for prosthetic valve performance is the transvalvular pressure drop which is a measure of how much pressure, and energy, is lost as the heart pumps blood through the valve. Generally speaking, larger valves exhibit a smaller pressure drop because they restrict the flow to a lesser degree. This phenomenon has led to a trend for surgeons to implant the largest prosthetic valve possible, and in some cases, to expanding the aorta to fit even larger valves. However, there has been relatively little work done on determining the effects of valve oversizing on the blood flow inside the Aorta. \r\n\r\nThe aims of this study were two-fold. First, a model of AS was tested inside an in vitro aortic simulator in order to identify how different individual leaflet stiffnesses would affect blood flow. Digital particle image velocimetry (DPIV) was used to measure velocity profiles inside a model aorta. The DPIV results were used to estimate the wall shear stress and blood residence time. Our analysis suggests that leaflet asymmetry greatly affects the amount of WSS by vectoring the systolic jet and that stiffened leaflets have an increased residence time. This study indicates that valve leaflets with different stiffness conditions can have a more significant impact on wall shear stress than stenosis caused by the uniform increase in all three leaflets (and the subsequent increased systolic velocity) alone. Second, the experimental apparatus was used to test different prosthetic valve sizes and valve mounting methods in order to identify how they affected residence time inside the sinus bulge. Dye residence experiments and DPIV were used to measure fluid stasis in several different combinations of prosthetic valve sizes, sinus sizes, and valve mounting methods. Our results indicate that valve to sinus sizing and mounting method is very important and can lead to greatly increased residence time and thrombosis risk. We have also identified a metric that can predict the threshold at which valves become oversized.", "doi": "10.7907/exsa-nm30", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15089", "collection": "thesis", "collection_id": "15089", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01192023-014456964", "primary_object_url": { "basename": "griggs_whitney_phd_thesis_v10-correctionp29-20240705.pdf", "content": "final", "filesize": 5444797, "license": "other", "mime_type": "application/pdf", "url": "/15089/14/griggs_whitney_phd_thesis_v10-correctionp29-20240705.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Listening to the Internal Representation of Actions Within the Posterior Parietal Cortex", "author": [ { "family_name": "Griggs", "given_name": "Whitney Scott", "orcid": "0000-0003-2941-6803", "clpid": "Griggs-Whitney-Scott" } ], "thesis_advisor": [ { "family_name": "Andersen", "given_name": "Richard A.", "orcid": "0000-0002-7947-0472", "clpid": "Andersen-R-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Andersen", "given_name": "Richard A.", "orcid": "0000-0002-7947-0472", "clpid": "Andersen-R-A" }, { "family_name": "Adolphs", "given_name": "Ralph", "orcid": "0000-0002-8053-9692", "clpid": "Adolphs-R" }, { "family_name": "O'Doherty", "given_name": "John P.", "orcid": "0000-0003-0016-3531", "clpid": "O'Doherty-J-P" }, { "family_name": "Rutishauser", "given_name": "Ueli", "orcid": "0000-0002-9207-7069", "clpid": "Rutishauser-U" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "More than 5.4 million people in the United States live with chronic paralysis and roughly 20 million people worldwide live with spinal cord injuries. Brain-machine interfaces (BMIs) can be transformative for these people, enabling them to control computers, robots, and more with only thought. State-of-the-art BMIs have already made this future a reality in limited clinical trials. However, these state-of-the-art BMIs have shortcomings that limit user adoption; high-performance BMIs currently require highly invasive electrodes above or in the brain; device degradation limits longevity to about 5 years; and their field of view is small, restricting the number, and type, of applications possible. This illustrates the need for a new generation of BMIs with a brain recording modality that is longer lasting, less invasive, and scalable to sense activity from large regions of the brain.
\r\n\r\nFunctional ultrasound imaging (fUSI) is a recently developed technique that meets these criteria. fUSI measures cerebral hemodynamics with exceptional spatiotemporal resolution (<100 \u00b5m; ~100 ms) and a large field of view (several cm)\u2014specifications ideally suited to recording detailed activity of entire cortical regions in parallel. In a series of novel results, we work towards developing the first high-performance ultrasonic BMI for human use. We first demonstrate that posterior parietal cortex (PPC), an area important for sensorimotor transformation, contains mesoscopic populations tuned to the intended movement direction. Using offline recorded data from several rhesus macaque monkeys, we can decode intended movement direction, task state, and expected action reward magnitude on a single trial basis. Having demonstrated that we could decode a variety of motor and cognitive variables using offline data, we developed a real-time, closed-loop ultrasonic BMI capable of decoding up to eight directions of intended movement with high accuracy. Finally, we began to translate these results into human applications and demonstrate the ability to measure changes in cerebral hemodynamics with high sensitivity through an acoustically transparent skull replacement in human subjects.
\r\n \r\nTaken together, our work is a novel characterization of how functional ultrasound neuroimaging may enable a new generation of BMIs. Additionally, this work reinforces the validity of fUSI as a robust and accessible neuroimaging technique for future neuroscience questions about mesoscopic populations and their interrelationships throughout the brain.
", "doi": "10.7907/0eae-hk18", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15093", "collection": "thesis", "collection_id": "15093", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01272023-184413283", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 16608292, "license": "other", "mime_type": "application/pdf", "url": "/15093/1/Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Towards Integrated Molecular Machines: Structural, Mechanical, and Computational Motifs", "author": [ { "family_name": "Sarraf", "given_name": "Namita", "orcid": "0000-0001-8692-7429", "clpid": "Sarraf-Namita" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The programmability of DNA has made it well-suited for building molecular machines, performing nanoscale self-assembly, and computing via biochemical circuits. In the last few decades, great strides have been made in characterizing the interactions between DNA molecules such that they can be predicted and engineered.
\r\n\r\nThe development of frameworks for those interactions has enabled the construction of more complex molecular systems that can execute specified programs. Such programs have included mechanical tasks, like walking and sorting cargo; assembly and reconfiguration of 2D and 3D shapes; and computation, like Boolean logic and pattern recognition.
\r\n\r\nHowever, the continuing development of more complex molecular programs relies upon expanding the modules available for molecular systems to use to execute them. Expanded functionality of mechanical, structural, and computation modules are required in order to build compound systems that can interact with the physical world, reconfigure, and analyze signals in a variety of interesting ways. In this dissertation, we will discuss our contributions to this effort, which include exploring a motif for molecular robotic behavior, characterizing tile-tile interactions, and developing new capabilities for bimolecular circuits.
\r\n\r\nWithin the framework of a maze-solving molecular robot, we aim to implement walking behavior on DNA origami that introduces a surface modification via a four-way strand displacement reaction. Surprisingly, our experiments suggest that the walking behavior is at least two orders of magnitude slower than expected. To understand why, we quantitatively explore to what extent the speed and completion level of the robot can be modulated by design considerations such as toehold lengths, track redundancy, and strand purity. Another factor affecting the reaction rate is the number of tethering points, and we demonstrate an order of magnitude speed up in the four-way strand displacement reaction when we remove one tethering point. The characterization of a surface-modifying four-way strand displacement reaction is a useful tool for the continued development of molecular robots with more complex functionality.
\r\n\r\nFree-floating DNA origami tiles, called invaders here, can swap out DNA origami tiles within larger assemblies via a technique called tile displacement, which has previously been demonstrated using single tile and dimer invaders with 4- and 9-tile arrays. We introduce initial structures and invading assemblies with more complex shapes. We explore the robustness of this reaction by testing a variety of edge configurations and comparing their reaction rates. We demonstrate tunable growth of one of the invaders, which can grow into polymers of arbitrary length or close into 3D structures. By a tile displacement reaction, we reconfigure the 3D structures into 2D. The invaders with complex shapes are able to reconfigure the original tile assembly at rates comparable to simpler tile displacement reactions, and two reconfiguration events can take place sequentially or simultaneously.
\r\n\r\nFinally, we build two new modules for use with biochemical circuits. The first, a loser-take-all circuit, yields binary outputs indicating which analog signal is the smallest among all inputs. We implement a signal reversal function that converts the smallest input to the largest output, which can then be composed with a previously developed winner-take-all function to achieve loser-take-all. By making concentration adjustments, we can mitigate biases in the circuit that are a result of sequence-dependent different in reaction rates. We experimentally demonstrate a three-input loser-take-all circuit with nine input combinations. With further development, this circuit could be used to implement the activation function in neural networks that perform pattern classification according to which memory an input pattern is least similar to.
\r\n\r\nThe second circuit processes information using temporary memory. We design and implement a circuit that outputs distinct logic decisions based on relative timing information of a pair inputs and their logic values. We show that we can mitigate crosstalk in the circuit by utilizing mismatches and adjusting toehold lengths. The circuit is able to display clear ON-OFF separation at time intervals as short as one minute between the two inputs arriving.
", "doi": "10.7907/cdwp-c709", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15238", "collection": "thesis", "collection_id": "15238", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312023-034316442", "type": "thesis", "title": "Low-Power and Miniaturized Medical Electronics for In-Vivo Localization and Tracking", "author": [ { "family_name": "Sharma", "given_name": "Saransh", "orcid": "0000-0002-5052-4932", "clpid": "Sharma-Saransh" } ], "thesis_advisor": [ { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "orcid": "0000-0003-3003-7042", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Marandi", "given_name": "Alireza", "orcid": "0000-0002-0470-0050", "clpid": "Marandi-A" }, { "family_name": "Traverso", "given_name": "Giovanni", "orcid": "0000-0001-7851-4077", "clpid": "Traverso-Giovanni" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Medical electronic devices are an integral part of the healthcare system today. Significant advances have been made over the past few decades to yield highly miniaturized and low-power medical devices that are suitable for implantable, ingestible, or wearable applications. A key feature of medical devices that is central to their use in many applications is the capability to locate them precisely inside the body, and quite a lot of research effort has been expended in this direction. Location sensing is crucial for several applications: tracking pills in the GI tract, navigation during precision surgeries, endovascular procedures, robotic and minimally invasive surgery, and targeted therapy. The current gold-standard solutions for these procedures include invasive techniques such as endoscopy, or procedures that require repeated use of potentially harmful X-ray radiation such as CT scans. These techniques also require repeated evaluation in a hospital setting and are not conducive for non-clinical environments. While there are several alternative non-ionizing methods for imaging and localization based on electromagnetic tracking, radio-frequency, ultrasound, and optical tracking, none of them are able to simultaneously achieve a high field-of-view of tracking, high spatiotemporal resolution, fully wireless operation and miniaturization of the sensing devices, and system scalability with the number of devices. In this dissertation, we present a radiation-free system for high-precision localization and tracking of miniaturized wireless devices in vivo, using harmless magnetic field gradients.
\r\n\r\nFirst, we demonstrate our system for precision surgery applications. We designed highly miniaturized, wireless and battery-less microdevices, capable of measuring and transmitting their local magnetic field. One such device can be attached to an implant inside the body and another to a surgical tool, such that both can simultaneously measure and communicate the magnetic field at their respective locations to an external receiver. The relative location of the two devices on a real-time display can enable precise surgical navigation without using X-ray fluoroscopy. The prototype device consists of a micro-chip fabricated in 65nm CMOS technology, a 3D magnetic sensor and an inductor-coil. The chip performs wireless power management, wireless bi-directional data-telemetry, and I2C communication with the sensor. Planar electromagnetic coils are designed for creating monotonically varying magnetic fields in the X, Y, and Z directions, resulting in field gradients that encode each spatial point with a unique magnetic field value. The concept of gradient-based spatial encoding is inspired by MRI. The system is tested in vitro to demonstrate a localization accuracy of <100\u00b5m in 3D, the highest reported to the best of our knowledge.
\r\n\r\nSecond, we demonstrate our system for localization and tracking of ingestible microdevices in the GI tract, which is valuable for the diagnosis and treatment of GI disorders. We designed highly miniaturized, low-power, and wireless ingestible devices to sense and transmit their local magnetic field as they travel through the GI tract. These devices consist of a 3D magnetic sensor, a Bluetooth microprocessor and a 2.4GHz Bluetooth antenna for wireless communication, all packaged into a 000-size capsule. The magnetic field sensed by the devices is created by using high-efficiency planar electromagnetic coils that encode each spatial point with a distinct magnetic field magnitude, allowing us to track the location of the devices unambiguously. The system functionality is demonstrated in vivo in large animals under different chronic conditions and disease models to show 3D localization and tracking in real time and in non-clinical settings, with mm-scale spatial resolution, and without using any X-ray radiation. This has the potential for significant clinical benefit for quantitative assessment of GI transit-time, motility disorders, constipation, incontinence, medication adherence monitoring, anatomic targeting for drug delivery, and targeted stimulation therapy.
\r\n\r\nThird, in order to further miniaturize the devices developed for the above two applications and to make them even more low-power, we present a monolithic 3D magnetic sensor in 65nm CMOS technology that measures <5mm\u00b2 in area and consumes 14.8\u00b5W in power while achieving <10\u03bcTrms noise. Our novel 3D magnetic sensor overcomes the challenges faced by traditional magnetic sensors by being fully CMOS compatible and achieving high sensitivity with only \u00b5W-level power, which is in sharp contrast with Hall and Fluxgate sensors. The sensor is comprised of three orthogonal and highly dense metal coils implemented in the 65nm node, which generate a voltage signal in response to AC magnetic fields by electromagnetic induction. The EMF voltage signal is processed by on-chip circuitry that performs low-noise amplification, filtering, peak detection, and 12-bit digitization. Though the sensor can be used for a variety of applications that require AC field sensing, it is particularly useful for biomedical applications\u2014tracking catheters and guidewires during endovascular procedures, minimally invasive surgeries, targeted radiotherapy, and for use as fiducial markers during preoperative planning. The proposed magnetic sensor is demonstrated for use in 3D tracking of catheters using the magnetic-field gradient-based spatial encoding scheme, and achieves 500\u00b5m of mean 3D localization accuracy.
", "doi": "10.7907/xrw0-k789", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15248", "collection": "thesis", "collection_id": "15248", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312023-220301021", "primary_object_url": { "basename": "ling_bill_thesis_final3.pdf", "content": "final", "filesize": 20171760, "license": "other", "mime_type": "application/pdf", "url": "/15248/1/ling_bill_thesis_final3.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Acoustic Biomolecules for Diagnostic Ultrasound Imaging", "author": [ { "family_name": "Ling", "given_name": "Bill", "orcid": "0000-0002-1276-7204", "clpid": "Ling-Bill" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Chan", "given_name": "Warren C. W.", "orcid": "0000-0001-5435-4785", "clpid": "Chan-Warren-C-W" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nanotechnology has enabled significant breakthroughs in the early detection and treatment of disease, but many of these advances rely on expensive and less-accessible imaging modalities. Ultrasound, on the other hand, is a noninvasive imaging modality that stands out for its universal availability, cost-effectiveness, and safety. However, harnessing the benefits of nanomaterials for ultrasound has been challenging due to the size and stability constraints of typical ultrasound contrast agents. Recently, an innovative solution has emerged in the form of gas vesicles (GVs), a class of air-filled protein nanostructures found in certain aquatic microbes. These promising next-generation ultrasound contrast agents offer a crucial bridge between nanotechnology and ultrasonography.
\r\n\r\nIn this thesis, we investigate the in vivo behavior of GVs, explore their potential applications as nanodiagnostic agents, and consider key factors for their future clinical deployment. In Chapter 2, we examine the interactions of GVs with blood components, focusing on imaging performance and immunogenicity. In Chapter 3, we show that intravenously injected GVs are cleared by liver-resident macrophages and subsequently undergo lysosomal degradation. We leverage this finding to develop an ultrasound-based method for visualizing cellular degradative processes and demonstrate its potential as a liver disease diagnostic. In Chapter 4, we introduce bicone GVs, the smallest known ultrasound contrast agent. We show that these sub-80 nm particles can penetrate tumors, deliver potent ultrasound-induced mechanical effects, and are readily engineered for molecular targeting, extended circulation time, and payload conjugation.
\r\n\r\nTogether, these findings highlight the tremendous potential of GVs as injectable nanomaterials for ultrasound imaging, laying the foundation for future studies to further refine the design and application of these agents.
", "doi": "10.7907/va8g-tb47", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14989", "collection": "thesis", "collection_id": "14989", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07272022-064642720", "type": "thesis", "title": "Applications of Genetically Engineered Bacillus subtilis in Biocatalysis and Functional Materials", "author": [ { "family_name": "Hui", "given_name": "Yue", "orcid": "0000-0002-0354-0382", "clpid": "Hui-Yue" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Bacillus subtilis is a gram-positive model bacterium that forms endospores as a response to nutrient limitation and other environmental stresses. The B. subtilis spore contains a dehydrated core, where the bacterial genome is safely stored, and multilayer proteinaceous coats, protecting the spore from various physical and chemical insults. Because of the outstanding resilience of the B. subtilis spore, it has attracted increasing interest for application in biotechnology. In this thesis, we demonstrate the utilization of genetically engineered B. subtilis cells and spores for heterologous protein display and functional material synthesis and characterization.
\r\n\r\nIn Chapter 1, we review the fundamentals of sporulation and germination in B. subtilis. We highlight notable biotechnological applications of native and engineered B. subtilis spores in recent years. We also discuss limitations associated with prior studies that inspire us to pursue the work in this thesis.
\r\n\r\nIn Chapter 2, we describe the T7 RNA polymerase (RNAP) enabled high density protein display on B. subtilis spores (TIED) method. The TIED constructs employ a coat protein promoter \u2013 PcotG, PcotV, or PcotZ \u2013 to drive the expression of the T7 RNAP. Target proteins are fused to the C-terminus of a spore crust protein \u2013 CotY or CotZ \u2013 and subjected to amplification by the T7 promoter. We prepare the endogenous constructs in which coat protein promoters directly regulate fusion protein expression for comparison with TIED. In addition, we develop a supplementary procedure to harvest spores before mother cell lysis, further improving the loading density of the target proteins. We verify the performance of the TIED architectures with a fluorescent reporter protein, mWasabi. Together with the early harvest protocol, the TIED method substantially enhances the total expression level and loading density of the crust-mWasabi fusion proteins relative to the endogenous expression system, as evidenced by bulk fluorescence measurements and microscopy.
\r\n\r\nIn Chapter 3, we implement the TIED architectures described in Chapter 2 for enzyme display on B. subtilis spores. We demonstrate the spore-based biocatalyst platform with three enzymes \u2013 lipase A and lipase B secreted by vegetative B. subtilis, and an engineered peroxidase, APEX2. We manifest that TIED enables massive accumulation of all three enzymes on the spore surface, with loading densities in the range of 106-107 enzymes per spore. Further, TIED-enzymes show comparable catalytic performance to the respective free-form enzymes, enhanced catalytic activity in methanol, and increased temperature stability. We conduct Michaelis-Menten studies to elucidate the kinetic characteristics of TIED-enzymes and their free form counterparts. Finally, we demonstrate that TIED-enzymes are not only recyclable, but also fully renewable after loss of activity through induction of germination and sporulation, demonstrating the potential for perpetual regeneration of the immobilized biocatalysts.
\r\n\r\nIn Chapter 4, we describe a new class of living composite materials (LCMs), in which genetically engineered B. subtilis cells and spores are effectively crosslinked into the surrounding polymeric scaffold. The resulting LCMs can be dried to yield portable materials. When re-immersed in aqueous media, entrapped cells and spores in previously- dried LCMs exhibit metabolic activity, including synthesis and secretion of recombinant proteins. Notably, we show that the scaffold based on photopolymerization of N-(hydroxymethyl) acrylamide (NHMAA) achieves effective cellular confinement, showing no evidence of cellular leakage over a period of 72 hours. We envision that the design principles elucidated in this work can provide a promising route to functional living materials engineered for biomedical and other applications.
", "doi": "10.7907/cdja-ck19", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16067", "collection": "thesis", "collection_id": "16067", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-223752519", "primary_object_url": { "basename": "AndrewFriedmanThesis.pdf", "content": "final", "filesize": 14702200, "license": "other", "mime_type": "application/pdf", "url": "/16067/1/AndrewFriedmanThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Scalable Fabrication of Micro-Architected Water Filtering Membranes", "author": [ { "family_name": "Friedman", "given_name": "Andrew Collin", "clpid": "Friedman-Andrew-Collin" } ], "thesis_advisor": [ { "family_name": "Greer", "given_name": "Julia R.", "orcid": "0000-0002-9675-1508", "clpid": "Greer-J-R" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Giapis", "given_name": "Konstantinos P.", "orcid": "0000-0002-7393-298X", "clpid": "Giapis-K-P" }, { "family_name": "Greer", "given_name": "Julia R.", "orcid": "0000-0002-9675-1508", "clpid": "Greer-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polymer-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.
\r\n\r\nThis 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\u201340 \u00b5m-thick nano-architected sheets with 2.1 \u00d7 2.4 cm\u00b2 lateral dimensions and ~500 nm-wide struts organized in layered 3D brick-and-mortar-like patterns to result in ~50\u201370% 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.
\r\n\r\nThis 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 \u00b0C) 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.
\r\n\r\nThis 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.
\r\n\r\nIn 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.
", "doi": "10.7907/rktj-2v55", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15113", "collection": "thesis", "collection_id": "15113", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03022023-175118888", "primary_object_url": { "basename": "Caltech_Thesis_KimGunho.pdf", "content": "final", "filesize": 34728792, "license": "other", "mime_type": "application/pdf", "url": "/15113/1/Caltech_Thesis_KimGunho.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Wave Propagation in Periodic Acoustic Metamaterials: from 1D to 3D", "author": [ { "family_name": "Kim", "given_name": "Gunho", "orcid": "0000-0003-1796-0908", "clpid": "Kim-Gunho" } ], "thesis_advisor": [ { "family_name": "Daraio", "given_name": "Chiara", "orcid": "0000-0001-5296-4440", "clpid": "Daraio-C" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Daraio", "given_name": "Chiara", "orcid": "0000-0001-5296-4440", "clpid": "Daraio-C" }, { "family_name": "Greer", "given_name": "Julia R.", "orcid": "0000-0002-9675-1508", "clpid": "Greer-J-R" }, { "family_name": "Asimaki", "given_name": "Domniki", "orcid": "0000-0002-3008-8088", "clpid": "Asimaki-D" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Wave propagation in periodic structures has been studied for centuries; for example, Newton derived the velocity of sound based on a linear lattice. Recently, advanced manufacturing techniques have led to the fabrication of geometrically complex architected materials with acoustic properties unattainable by their constituent materials. Such rationally designed structures are often called acoustic metamaterials and they can be engineered to transmit, block, amplify, or redirect acoustic waves. Subwave-length building blocks, typically periodic (but not necessarily so), can be assembled into effectively continuous materials to manipulate dispersive properties of vibrational waves in ways that differ substantially in conventional media. This thesis investigates rationally designed acoustic metamaterials, ranging from 1D to 3D, and how acoustic wave propagation can be controlled by these artificially structured composite materials for ultrasound-related biomedical applications.
\r\n\r\nI first explore 1D wave propagation in acoustic metamaterials to study the basic mechanics and relevant analysis skills. Bio-inspired helical mechanical metamaterials are designed and their normal modes are investigated. I demonstrate the ability to vary the acoustic properties of the helical metamaterials by perturbing the geometrical structure and mass distribution. By locally adding eccentric and denser elements in the unit cells, I change the moment of inertia of the system and introduce centro-asymmetry. This allows me to control the degree of mode coupling and the width of subwavelength band gaps in the dispersion relation, which are the product of enhanced local resonance hybridization.
\r\n\r\nThen I study 2D wave propagation in microlattice acoustic metamaterials for ultra- sound manipulation. When coupled with pressure waves in the surrounding fluid, the dynamic behavior of microlattices in the long wavelength limit can be explained in the context of Biot\u2019s theory of poroelasticity. I exploit elastoacoustic wave propagation within 3D-printed polymeric microlattices to design a gradient refractive index lens for underwater wave focusing. A modified Luneburg lens index profile adapted for ultrasonic wave lensing is demonstrated via the finite element method and underwater testing, showcasing a computationally efficient poroelasticity-based design approach that enables accelerated design of acoustic wave manipulation devices.
\r\n\r\nLastly, I show that tailorable 3D wave propagation can be achieved based on the findings from the previous chapters. Functional ultrasound imaging enables sensitive, high-resolution imaging of neural activity in freely behaving animals and human patients. However, the skull acts as an aberrant and absorbing layer for sound waves, leading to most functional ultrasound experiments being conducted after skull removal. A microscale 2-photon polymerization technique is adopted to fabricate a conformal acoustic window with a high stiffness-to-density ratio and sonotransparency. Long-term biocompatibility and lasting signal sensitivity are demonstrated over a long period of time (> 4 months) by conducting ultrasound imaging in mouse models implanted with the metamaterial skull prosthesis.
", "doi": "10.7907/dyq0-vm69", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15153", "collection": "thesis", "collection_id": "15153", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052023-192721059", "primary_object_url": { "basename": "Xiaozhe_Ding_Caltech_Thesis_final.pdf", "content": "final", "filesize": 23591445, "license": "other", "mime_type": "application/pdf", "url": "/15153/1/Xiaozhe_Ding_Caltech_Thesis_final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Computation-Aided Protein Engineering for Targeted Therapeutic Delivery", "author": [ { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "My Ph.D. projects centered on using computational structural biology tools to develop protein engineering methods for targeted therapeutic delivery, emphasizing delivering molecules to the brain. In this thesis, I focus on three main projects. First, utilizing computational structural biology techniques, I investigate the molecular mechanism that enables engineered adeno-associated viral (AAV) capsids to cross the blood-brain barrier (BBB). I develop a pipeline to model the vast and dynamic complex between engineered AAV capsids and their BBB receptors. I also apply a tool, recently developed by myself and discussed in Chapter 3, to distinguish capsids that bind to different receptors. The findings of this study can lead to novel approaches for developing chemicals and biologicals that can penetrate the human brain (Chapter 2). Second, I describe the development of Automated Pairwise Peptide-Receptor AnalysIs for Screening Engineered proteins (APPRAISE). This computational pipeline predicts the receptor binding propensity of engineered proteins based on competitive modeling and physics-grounded analysis. I show that APPRAISE is capable of distinguishing between receptor-dependent and receptor-independent adeno-associated viral vectors and ranking various engineered proteins, such as miniproteins binding to the SARS-CoV-2 spike and nanobodies binding to a G-protein-coupled receptor. A top performer in an in silico screening using APPRAISE was validated experimentally (Chapter 3). Third, I show an example to engineer a genetically encoded transmitter indicator (GETI), which may eventually be a cargo delivered to the brain. The GETI has a novel scaffold based on bacterial repressors, a class of transcriptional regulators that are critical for bacteria to respond to environmental chemicals. I repurposed an antibiotic-sensing repressor protein to bind a neurotransmitter, melatonin, using machine-learning-guided directed evolution. A melatonin indicator was then created by integrating the repurposed receptor with a fluorescent protein. This engineering platform may be adapted to create bio-orthogonal GETIs for various neurotransmitters (Chapter 4).", "doi": "10.7907/7n15-3076", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16115", "collection": "thesis", "collection_id": "16115", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06132023-010825748", "primary_object_url": { "basename": "Thesis-Gholamin Sharareh June14th 1.pdf", "content": "final", "filesize": 11640358, "license": "other", "mime_type": "application/pdf", "url": "/16115/3/Thesis-Gholamin Sharareh June14th 1.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Mechanism of Response and Resistance to CAR T Cell Therapies", "author": [ { "family_name": "Gholamin", "given_name": "Sharareh", "orcid": "0000-0001-7425-6074", "clpid": "Gholamin-Sharareh" } ], "thesis_advisor": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Brown", "given_name": "Christine", "clpid": "Brown-Christine" }, { "family_name": "Forman", "given_name": "Stephen", "clpid": "Forman-Stephen" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Brown", "given_name": "Christine", "clpid": "Brown-Christine" }, { "family_name": "Ribas", "given_name": "Antoni", "clpid": "Ribas-Antoni" }, { "family_name": "Forman", "given_name": "Stephen", "clpid": "Forman-Stephen" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "While chimeric antigen T (CAR) T cell therapy has shown remarkable success in leukemia, lymphoma, and multiple myeloma, its effectiveness in solid tumors including glioblastoma (GBM) remains limited. It is crucial to understand mechanisms that reduce the efficacy of CAR T cell therapies and develop strategies to prevent tumor resistance. In this study, we conjectured that alterations in tumor cell-intrinsic interferon (IFN) signaling pathways contribute to establishment of immunosuppressive tumor microenvironment in solid tumors, leading to resistance of solid tumor cells to CAR T cell-mediated killing. We established syngeneic IFN signaling-deficient tumor models for murine IL-13Ra2 targeted CAR T cell therapy and showed that these models modulate the tumor microenvironment (TME), leading to resistance to CAR T cell therapy. We identified variations in gene expression associated with IFN signaling components and cytokines between IFN signaling-deficient tumor cells and wild type (WT) tumor cells after CAR T cell treatment. Furthermore, single-cell RNA sequencing and mass cytometry analysis of the tumor immune cell infiltrates in IFN-signaling deficient tumors compared to WT controls identified the immune-mediated causal components for the resistance of Janus Kinase1 knockout (JAK1/KO) tumors to CAR T cell therapy. CAR T cell-treated IFN signaling-deficient tumors presented decreased T-cell transcripts, with decreased frequency of CD8-early active, CD8-naive like T cells. Conversely, there were more regulatory and follicular T cells, exhausted endogenous T cells , and exhausted CAR T cells in treated IFN signaling-deficient tumors compared to treated WT tumors. The analyses also showed the superior enrichment and crosstalk of genes that identified fibroblasts, neutrophils, and myeloid cells in IFN signaling-deficient tumors compared to those of WT tumors. Mass cytometry analysis on the immune cells infiltrates of JAK1/KO and WT tumors post CAR T cell treatment corroborated the results from gene expression analysis. The potential cause of immune suppressive crosstalk in IFN signaling-deficient tumor niches could be attributed to the varied enhancement of receptor-ligand interactions such as SPP1+ tumor-associated macrophages (TAMs) and CD44+ cancer-associated fibroblasts (CAFs), as well as SPP1+ TAMs and integrins present on other cell lineages. To overcome resistance to CAR T cell therapies, we employed two distinct actionable approaches: triggering the immune microenvironment and disrupting the extracellular matrix. Unconjugated interferon signaling gene-15 (ISG-15) enhanced CAR T cell efficacy in an INF-signaling deficient model, increasing the recruitment of endogenous T cells and reshaping the TME. Anti-SPP1 blocking antibody was used to prime the JAK1/KO tumors prior to the treatment with CAR T cell therapy potentially via enhancing the persistence and trafficking of CAR T cells in the TME.
\r\n\r\nWe next identified immune signatures of 32 GBM patients who had progressive disease after CAR T cell treatment compared to those who had relatively stable disease or showed improvement. We identified the presence of fibroblasts and SPP1+ APOE+ C1QA+ C1QC+ myeloid cells in GBM signatures that are associated with immune suppression and resistance to therapy. Patients with GBM who exhibited a relatively stable response to treatment and increased T cell recruitment had differential expression of interferon regulatory factors (IRFs) and ISGs compared to patients with less response to the treatment. Our findings uncover a correlation between tumor-intrinsic driver mutations, the composition of the TME, and the responsiveness of solid tumors to CAR T cell therapy, providing insights into potential approaches to address resistance in IFN non-responsive tumors.
", "doi": "10.7907/jf79-pv58", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15157", "collection": "thesis", "collection_id": "15157", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05092023-163511031", "primary_object_url": { "basename": "PhD_thesis_final.pdf", "content": "final", "filesize": 56663983, "license": "other", "mime_type": "application/pdf", "url": "/15157/5/PhD_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes", "author": [ { "family_name": "Dutka", "given_name": "Przemys\u0142aw", "orcid": "0000-0003-3819-1618", "clpid": "Dutka-Przemys\u0142aw" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cryo-electron tomography (cryo-ET) is a powerful method for investigating the 3D structure of intact cells, organelles, and complex protein macromolecules that cannot be crystallized or are too heterogenous for single-particle cryo-electron microscopy (cryo-EM). However, obtaining high- resolution cryo-ET structures for many biologically important targets is still a challenge. To address this challenge, cryo-ET can be combined with other methods, including X-ray crystallography, single-particle cryo-EM, structure predictions, cross-linking mass spectrometry, biochemistry, and evolutionary analysis to produce integrative models. Recently, with the development of AI-based tools such as AlphaFold2, structure prediction has played an increasingly important role in integrative modeling. The combination of cryo-ET and structure prediction in particular has provided unprecedented insights into the ultrastructure of cellular components. This thesis focuses on two bacterial multi-megadalton protein complexes which are difficult to study by classical structural biology approaches: gas vesicles (GVs) and the Legionella pneumophila Dot/Icm type IV secretion system (T4SS). GVs are gas-filled protein nanostructures that regulate the position of certain microorganisms in water and consequently their access to sunlight and nutrients. Here, we investigate the mechanical properties of GVs and reveal the molecular structure of GVs and its implication for the assembly mechanism. The Dot/Icm T4SS is a macromolecular complex formed by approximately 27 proteins, utilized by L. pneumophila to hijack the host cell's biology for its replication purposes. A nearly-complete integrative model of this complex provides crucial insights into its structural organization and its evolution from conjugation to secretion, as well as the transportation of substrates into the host cell.
", "doi": "10.7907/87jm-7v06", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15167", "collection": "thesis", "collection_id": "15167", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05132023-220300179", "primary_object_url": { "basename": "230520_Ifkovits_Zachary_Thesis.pdf", "content": "final", "filesize": 5054710, "license": "other", "mime_type": "application/pdf", "url": "/15167/1/230520_Ifkovits_Zachary_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Strategies for Enabling Stable and Efficient (Photo)Electrochemical Water Splitting", "author": [ { "family_name": "Ifkovits", "given_name": "Zachary Philip", "orcid": "0000-0003-2538-0794", "clpid": "Ifkovits-Zachary-Philip" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Flagan", "given_name": "Richard C.", "orcid": "0000-0001-5690-770X", "clpid": "Flagan-R-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The electrolysis of water splits H\u2082O into its constituent parts, generating H\u2082 fuel and O\u2082 as a by-product. Although electrolysis has been known since late 1700s and has a consistently expanding industrial capacity, several barriers still exist to its widespread utilization as a clean method of generating hydrogen for industrial uses or as a grid-scale energy storage chemical. Among these, the materials and costs constraints surrounding the use of precious metal catalysts and expenses associated with balance-of-system costs are of primary importance. In this thesis, the first point is addressed by utilizing earth-abundant catalysts for chemical, electrochemical, and photoelectrochemical water splitting reactions. Specifically, MnySb1-yOx catalysts were synthesized for use as both cerium-mediated chemical water oxidation catalysts and as electrochemical water oxidation catalysts, furthering steps towards removing Ir from industrial electrolysis devices. Addition of Sb was shown to stabilize reactive Mn centers in these configurations, offering enhanced stability over pure Mn oxide catalysts. Reduction of electrolyzer balance-of-system costs were addressed in this thesis through the integration of multiple components of a solar-powered electrolysis system into a single, integrated photoelectrochemical water splitting device. Specifically, electrodeposition conditions were shown to affect the spontaneous mesostructuring of Ni-P hydrogen evolution catalysts on silicon photocathodes, leading to enhanced transmission of light to the semiconductor substrate. Furthermore, Y\u2082SiO\u2085 protective layers were shown to mitigate the corrosion of Si photocathodes in alkaline environments, an electrochemical environment known to be destructive towards silicon.", "doi": "10.7907/jt8t-w739", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15194", "collection": "thesis", "collection_id": "15194", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05192023-001330664", "type": "thesis", "title": "Engineering of Second-Generation Acoustic Reporter Genes", "author": [ { "family_name": "Hurt", "given_name": "Robert Cooper", "orcid": "0000-0002-4347-6901", "clpid": "Hurt-Robert-Cooper" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 \u00b5m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.
\r\n\r\nThe goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the \u201cnature and nurture\u201d of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.
\r\n\r\nThis thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them\u2014one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.
\r\n\r\nThe approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.
", "doi": "10.7907/qs6v-5d67", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15215", "collection": "thesis", "collection_id": "15215", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272023-093432414", "primary_object_url": { "basename": "MPAbundo_05272023.pdf", "content": "final", "filesize": 11712317, "license": "other", "mime_type": "application/pdf", "url": "/15215/1/MPAbundo_05272023.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Ultrasound Controlled Drug Delivery by Acoustically Switchable Hydrogels", "author": [ { "family_name": "Abundo", "given_name": "Maria Paulene Bernal", "orcid": "0000-0002-5122-6937", "clpid": "Abundo-Maria-Paulene-Bernal" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Daraio", "given_name": "Chiara", "orcid": "0000-0001-5296-4440", "clpid": "Daraio-C" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Not only is ultrasound widely used as a diagnostic imaging modality, it can also be focused into deep tissues to perform non-invasive actuation of cells, implants and delivery vehicles and other biological targets. With the addition of gas vesicles (GV), generic hydrogel materials gain the ability to communicate with ultrasound, equipping them with in vivo tracking, targeting and actuation capabilities to safely transport biomolecular cargo. This is possible as GVs function simultaneously as ultrasound contrast agents and steric blockers that can be \"erased\" by an increase in ultrasound pressure to trigger a rapid outflow diffusion of the payload from within the material. We evaluate this concept through in vitro measurements of ultrasound-modulated diffusion and drug release and targeted in vivo release in the lower gastrointestinal tract. Then we demonstrate the use of orally administered hydrogel particles to deliver etanercept in the duodenum to treat gastrointestinal inflammation in a rat model of colitis. Finally, we explore new directions and applications of GV-hydrogel systems, showcasing their potential for deployment in a wide range of biomedical applications.
", "doi": "10.7907/5rr6-q625", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15216", "collection": "thesis", "collection_id": "15216", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272023-161541041", "primary_object_url": { "basename": "2023-05-26_Xiong_Thesis.pdf", "content": "final", "filesize": 68354418, "license": "other", "mime_type": "application/pdf", "url": "/15216/1/2023-05-26_Xiong_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Expanding the Toolbox for Thermal Control of E. coli: Cold-Activated Transcription with Applications in Temperature Self-Regulation", "author": [ { "family_name": "Xiong", "given_name": "Lealia Li", "orcid": "0000-0001-7636-5936", "clpid": "Xiong-Lealia-Li" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Temperature can be used to control engineered E. coli \u2014 for example, the living component of an engineered living material (ELM) - through the use of thermolabile transcription factors. Sharp induction of gene expression with heat has been established using these bacteria- and phage-derived proteins. Here, we expand the toolbox for thermal control of E. coli through both direct cold-induced gene expression and through the construction of genetic circuits to invert heat-induced gene expression.
\r\n\r\nWe accomplish direct induction at low temperatures through the use of temperature-sensitive mutants of Lambda repressor as transcriptional activators. In addition, we show that a temperature-sensitive mutant of Lambda repressor can serve as an activator and a repressor of different genes simultaneously in one genetic circuit, leading to opposite thermal responses and serving as a temperature switch.
\r\n\r\nNext, we demonstrate inversion of a temperature-sensitive repressor using a temperature insensitive repressor. We apply this multicomponent switch to engineer a temperature self-regulation circuit for E. coli-based ELMs. Seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment, because E. coli growth rate is impaired both below and above 37\u00b0C. Our construct enables E. coli to produce a light-absorptive pigment in response to environmental temperature below 36\u00b0C with the goal of allowing the cells to absorb sunlight and locally warm to their optimal growth temperature. We demonstrate the efficacy of our pigment temperature switch in a model flat ELM growing at 32\u00b0C and 42\u00b0C in a home-built illuminated growth chamber. Below 36\u00b0C, our engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented bacteria. On the other hand, above 36\u00b0C, they decrease in pigmentation, protecting their growth compared to bacteria with temperature- independent high pigmentation. Integrating our temperature homeostasis circuit into an ELM has the potential to improve ELM performance by optimizing growth and protein production in the face of seasonal temperature changes.
", "doi": "10.7907/5w59-0667", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14445", "collection": "thesis", "collection_id": "14445", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12102021-231944176", "primary_object_url": { "basename": "KjeldbjergCamilla2022Thesis.pdf", "content": "final", "filesize": 57009594, "license": "other", "mime_type": "application/pdf", "url": "/14445/1/KjeldbjergCamilla2022Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "The Effects of Confinement in Active Matter: the Casimir Effect, Partitioning, and Hindered Diffusion", "author": [ { "family_name": "Kjeldbjerg", "given_name": "Camilla Maria", "orcid": "0000-0003-2224-0534", "clpid": "Kjeldbjerg-Camilla-Maria" } ], "thesis_advisor": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Seinfeld", "given_name": "John H.", "orcid": "0000-0003-1344-4068", "clpid": "Seinfeld-J-H" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Active matter describes a class of materials for which constituent \"particles\" convert chemical energy into mechanical motion leading to self-propulsion (swimming). The origins of this swimming motion for both biological and synthetic constituents is a thriving area of research. However, here we focus on the physical properties and mechanics of the active matter systems. We model active particles using the active Brownian particle (ABP) model that is the simplest model that captures the essential physics, where a particle translates with a swim speed U0 in a direction q for a characteristic reorientation time \tτR; the average length they move between each reorientation is called the run, or persistence, length ℓ = U0τR. Owing to this persistent swimming, the ABPs distribute non-homogenously near surfaces, accumulating at no-flux boundaries leading to a concentration boundary layer near solid surfaces. Active particles often have an effective size—their run length—which can be much larger than their geometric size such that they experience confinement in geometries whose size is on the order of the run length. Active systems are inherently far from equilibrium, and we cannot appeal to properties of equilibrium thermodynamic such as the chemical potential to predict the partitioning. Fortunately, active particles are still subject to the laws of mechanics, and in this work, we present a simple macroscopic balance that allows one to predict behavior without detailed calculations. We predict the attractive force between two parallel plates in a reservoir (also called the Casimir effect) and find that the average concentration between the plates equals that in the bulk reservoir independent of the degree of confinement (ratio of run length to the spacing between the plates). We then examine the confinement effects in a channel geometry, where the behavior is fundamentally different, and the average concentration grows linearly with the degree of confinement. The understanding of these fundamental geometries motivated us to look into more complex geometries such as porous media. Based on dimensional analysis and our predictive model, we explain the transient behavior and steady-state partitioning of active particles between a fluid reservoir and a porous medium. Lastly, we discuss the hindered diffusion in periodic porous media and how the diffusion depends not only on the porosity of the medium but also on the degree of confinement. We believe that utilizing the insights in effects of confinement for these fundamental geometries and the porous media will be valuable in designing optimal structures for enhancing or isolating active particles.
", "doi": "10.7907/avfw-fh81", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14295", "collection": "thesis", "collection_id": "14295", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07062021-155047044", "type": "thesis", "title": "Engineering the Tryptophan Synthase \u03b2-Subunit for Synthesis of Noncanonical Amino Acids", "author": [ { "family_name": "Watkins-Dulaney", "given_name": "Ella Jenn\u00e1", "orcid": "0000-0002-0585-1598", "clpid": "Watkins-Dulaney-Ella-Jenn\u00e1" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Buller", "given_name": "Andrew R.", "orcid": "0000-0002-9635-4844", "clpid": "Buller-A-R" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The tryptophan synthase \u03b2-subunit (TrpB) naturally catalyzes a pyridoxal phosphate cofactor-mediated \u03b2-substitution reaction between indole and serine to form L-tryptophan. Almost half a century ago, it was realized that TrpB could accept nucleophiles other than indole to synthesize noncanonical amino acids (ncAAs), which are highly useful small-molecule building blocks that are found in many bioactive molecules. Since then, TrpB has been applied to synthesize a wide range of ncAAs. This thesis details the engineering of TrpB for synthesis of new and useful ncAAs and the application of TrpB as a model to study the principles that govern intra-protein interactions. Chapter I chronicles the history of tryptophan synthase, provides useful information about the enzyme\u2019s catalytic cycle, and describes how TrpB has been used to synthesize ncAAs in works preceding this thesis. Chapter II describes the evolution, application, and characterization of TrpB for the synthesis of a blue, fluorescent noncanonical amino acid \u03b2-(1-azulenyl)-L-alanine (AzAla). Chapter III details the engineering and mechanistic characterization of TrpB to asymmetrically catalyze C\u2013C bond formation with an entirely new class of nucleophile: ketones. Chapter IV describes the in vivo continuous evolution of TrpB which resulted in sequence-diverse TrpB orthologs that have been adapted to function at lower temperatures and display a range of substrate-selectivity profiles. Chapter V describes the development of a deep mutational scanning experiment of combinatorial site-saturation mutagenesis (SSM) libraries for generating a large dataset that maps enzyme sequence to function for the purpose of studying epistasis with machine learning. Overall, the work presented in this thesis expands the repertoire of ncAAs that can be synthesized by TrpB and demonstrates unique applications of TrpB as a model enzyme for continuous in vivo directed evolution and for generating a dataset that will be useful to the protein machine learning community.
", "doi": "10.7907/yekm-y267", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14617", "collection": "thesis", "collection_id": "14617", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252022-172145394", "type": "thesis", "title": "Mechanical Approach to Active Matter: Reverse Osmotic Effect and Motility-Induced Phase Separation", "author": [ { "family_name": "Row", "given_name": "Hyeongjoo", "orcid": "0000-0003-3623-512X", "clpid": "Row-Hyeongjoo" } ], "thesis_advisor": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The defining feature of active matter, self-propulsion requires constant consumption of energy to be maintained. As a result, active matter systems are inherently out of equilibrium and some principles that are accepted as common knowledge, particularly from thermodynamics, do not apply to the active matter systems. Arguably the most popular example is the motility-induced phase separation (MIPS) -- active matter can spontaneously phase separate into liquid-like dense phase and gas-like sparse phase even without any attractive interactions between the self-propelling constituents. In this thesis, I demonstrate the utility of a mechanical perspective in revealing and understanding the underlying physics of seemingly confounding behaviors of active matter systems. In Chapters 2 and 3, I consider the mechanics of a suspension of active colloidal particles when the transport properties (self-propelling speed and diffusivities) vary spatially. The mechanical analysis reveals the reverse-osmotic nature of active matter systems with a spatial variation in activity. I provide an explanation for why physical processes governed by the osmotic pressure of particles can appear in a reversed manner in active matter systems, e.g. a fluid can flow from regions of high concentration to low in a suspension of active colloids. In Chapter 4, I develop a mechanical theory of phase coexistence that applies to both equilibrium and nonequilibrium systems. By applying the mechanical theory to MIPS, I find phase coexistence conditions of the MIPS that allow a construction of a phase diagram, which excellently agrees with the results from computer simulations. The mechanical theory also allows access to the microscopic structure of phase interfaces. By investigating the interfacial structure, I discover interesting nonequilibrium interfacial behavior of the MIPS. I find that the width of the MIPS interface varies nonmonotically with the activity of particles and provide a mechanical explanation for the phenomena.", "doi": "10.7907/qef0-e420", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14391", "collection": "thesis", "collection_id": "14391", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10092021-015926914", "type": "thesis", "title": "Open-Circuit Stability and Integration of Silicon Electrodes for Solar Fuels Devices", "author": [ { "family_name": "Fu", "given_name": "Harold Jin", "orcid": "0000-0001-9738-209X", "clpid": "Fu-Harold-Jin" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Flagan", "given_name": "Richard C.", "orcid": "0000-0001-5690-770X", "clpid": "Flagan-R-C" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Two significant challenges that impede the realization of inexpensive, solar-driven water electrolysis involve the corrosion and integration of component materials. For instance, Si is a prominent light absorbing material that readily corrodes in alkaline electrolyte unless subjected to an oxidative potential. Although a protective coating can be applied to mitigate corrosion, the underlying semiconductor remains exposed to electrolyte at pinholes on the protective coating. Illumination slows the dissolution of Si photoanodes further by 2-3 orders of magnitude via oxidation to SiOx. However, Si is still susceptible to corrosion under nighttime conditions and device stability must be maintained regardless of diurnal patterns of sunlight. This thesis explores two approaches to drive Si passivation in the dark at open circuit. First, a protective electrolyte can be introduced to solution that acts as an oxidizing agent to Si. Secondly, a catalytic thin film like NiOx on Si can drive the electrode potential positive by catalyzing O2 in electrolyte. Applying either passivation strategy yielded extended stability of Si photoanodes subjected to simulated day/night cycling. In addition to corrosion, device performance is critically dependent on the integration of component materials. Efficient water splitting requires that at least two semiconductors be connected in series to drive the reaction, while lateral resistance losses in electrolyte preclude large (> cm2) planar photoelectrode areas. Si can be vertically arranged as high aspect ratio microwires that can be embedded in an ion exchange membrane. This assembly can be laminated to a tandem partner arranged in a similar configuration using an electrically conductive interlayer. This thesis additionally investigates the bulk and interfacial properties of Nafion-PEDOT:PSS composite films as a candidate material for this interlayer. After solvent treatment, the composite film exhibited percolation of electrically conductive PEDOT domains even at dilute PEDOT concentrations (~ 0.2 wt%). Despite the presence of an insulating Nafion-rich layer on the surface, the composite forms a low resistance contact to CH3-terminated p-Si, thereby making the composite a viable interlayer for use in a fully integrated, tandem water splitting device.
", "doi": "10.7907/pgx5-fj77", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14567", "collection": "thesis", "collection_id": "14567", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292022-213605156", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 12618424, "license": "other", "mime_type": "application/pdf", "url": "/14567/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Transport and Microrheology of Active Colloids", "author": [ { "family_name": "Peng", "given_name": "Zhiwei", "orcid": "0000-0002-9486-2837", "clpid": "Peng-Zhiwei" } ], "thesis_advisor": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Colonius", "given_name": "Tim", "orcid": "0000-0003-0326-3909", "clpid": "Colonius-T" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Active colloids are micron-sized particles that self-propel through viscous fluids by converting energy extracted from their environment into mechanical motion. The origin or mechanism of their locomotion can be either biological or synthetic ranging from motile bacteria to artificial phoretic particles. Owing to their ability to self-propel, active colloids are out of thermodynamic equilibrium and exhibit interesting macroscopic or collective dynamics. In particular, active colloids exhibit accumulation at confining boundaries, upstream swimming in Poiseuille flow, and a reduced or negative apparent shear viscosity. My work has been focused on a theoretical and computational understanding of the dynamics of active colloids under the influence of confinement and external fluid flows, which are ubiquitous in biological processes. I consider the transport of active colloids in channel flows, the microrheology of active colloids, and lastly I propose and study a vesicle propulsion system based on the learned principles.
\r\n\r\nA generalized Taylor dispersion theory is developed to study the transport of active colloids in channel flows. I show that the often-observed upstream swimming can be explained by the biased upstream reorientation due to the flow vorticity. The longitudinal dispersion of active colloids includes the classical shear-enhanced dispersion and an active swim diffusivity. Their coupling results in a non-monotonic variation of the dispersivity as a function of the flow speed. To understand the effect of particle shape on the transport of active colloids, a simulation algorithm is developed that is able to faithfully resolve the inelastic collision between an ellipsoidal particle and the channel walls. I show that the collision-induced rotation for active ellipsoids can suppress upstream swimming. I then investigate the particle-tracking microrheology of active colloids. I show that active colloids exhibit a swim-thinning microrheology and a negative microviscosity can be observed when certain hydrodynamic effects are considered. I show that the traditional constant-velocity probe model is not suitable for the quantification of fluctuations in the suspension. To resolve this difficulty, a generalized microrheology model that closely mimics the experimental setup is developed. I conclude by proposing a microscale propulsion system in which active colloids are encapsulated in a vesicle with a semi-permeable membrane that allows water to pass through. By maintaining an asymmetric number density distribution, I show that the vesicle can self-propel through the surrounding viscous fluid.
", "doi": "10.7907/wa00-y892", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:13838", "collection": "thesis", "collection_id": "13838", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07082020-113341068", "type": "thesis", "title": "Guiding Self-Organization in Active Matter with Spatiotemporal Boundary Conditions", "author": [ { "family_name": "Ross", "given_name": "Tyler David", "orcid": "0000-0002-7872-3992", "clpid": "Ross-Tyler-David" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "In this thesis, I demonstrate that self-organized structures and forces can be guided by modulating the interactions between force-generating molecules in space and time. The physics of self-organizing systems is an open frontier. We do not have a complete set of principles that can describe how a dynamic structure forms based on the non-equilibrium dynamics of its constituent components. Yet, living systems appear to depend on some set of rules of self-organization in order to reliably carry out their mechanical functions. Force-generating, active, molecules in the form of motor proteins and filamentous polymers are responsible for performing fundamental tasks in living matter, such as locomotion and division. While it is known that the regulation of motor-filament interactions is necessary to achieve the dynamic structures that drive movement and propagation, the role of spatial and temporal patterning in self-organizing systems has not been explored. I design a artificial system of purified molecules where the interactions between motors and filaments are toggled with light. By patterning molecular interactions in space and time, I show that it is possible to localize the formation of spherically symmetric asters, which can be moved, merged, and used to generate advective fluid flows. The ability to pattern molecular interactions in space and time offers a new perspective in the search for principles of active self-organization. Spatial and temporal control makes it possible to start distilling how the interactions between active molecules determine the mesoscopic behaviors of self-organized structures. These rules ultimately govern the physics of living matter and may eventually be harnessed to build new materials and cell-like machines.
", "doi": "10.7907/q85h-j730", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14174", "collection": "thesis", "collection_id": "14174", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262021-021059637", "primary_object_url": { "basename": "PDF.pdf", "content": "final", "filesize": 53443918, "license": "other", "mime_type": "application/pdf", "url": "/14174/8/PDF.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Biomolecular Tools for Noninvasive Imaging and Manipulation of Engineered Cells", "author": [ { "family_name": "Wu", "given_name": "Di", "orcid": "0000-0002-6848-668X", "clpid": "Wu-Di" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Tai", "given_name": "Yu-Chong", "orcid": "0000-0001-8529-106X", "clpid": "Tai-Yu-Chong" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Today\u2019s most advanced tools for imaging and controlling cellular function are based on fluorescent or light-controlled proteins, which have limited utility in large organisms or engineered living materials due to the scattering of photons. Deeply penetrant forms of energy such as magnetic fields and sound waves, while routinely used to monitor and treat diseases on the tissue and organism level, do not process the equivalent set of biomolecular tools for interfacing with biology on the molecular and cellular level. Emerging technologies discussed in this thesis aim to bridge this gap by harnessing biomolecules that have the appropriate physical properties to interact with sound waves or magnetic fields in such a way that enables the visualization and control of specific cells (Chapter 1). We describe two additions to the expanding toolkit for noninvasive imaging and control. In the first case, we show that gas vesicles, a class of hollow protein nanostructures naturally found in aquatic single-cell organisms, can be used as acoustic actuators to enable the control of cellular forces, movement, and patterning using ultrasound (Chapter 2). In the second case, we show that aquaporins, a class of membrane water channels, can be used to alter cellular permeability and serve as genetic reporters for magnetic resonance imaging (Chapter 3). These tools provide critical capabilities for interfacing with cellular function noninvasively and could open the door to applications in various research, biomedical, and industrial settings.", "doi": "10.7907/dt22-nv14", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14232", "collection": "thesis", "collection_id": "14232", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022021-043318684", "type": "thesis", "title": "Enhanced Noninvasive Imaging of Acoustic Biomolecules", "author": [ { "family_name": "Sawyer", "given_name": "Daniel Patrick", "orcid": "0000-0003-2926-191X", "clpid": "Sawyer-Daniel-Patrick" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The extensive scientific interest in cellular and biomolecular processes is due in large part to the importance of such processes deep inside living organisms, in the context of both health and disease. However, most methods for imaging cellular processes such as gene expression have relied on fluorescent proteins and other optical reporters that, while providing a direct optical readout of the biomolecular environment in cells readily exposed to light, have greatly limited performance in large animals due to the poor penetration of visible light beyond 1 mm of biological tissue. In contrast, ultrasound is widely used to noninvasively image tissue deep inside living organisms but has rarely been used to investigate cellular function due a lack of acoustic reporters whose production and properties are coupled to biomolecular events. Recently, the first acoustic reporter genes (ARGs) were developed for ultrasound imaging of a unique class of air-filled protein nanostructures known as gas vesicles, or GVs, which scatter sound waves when expressed in bacterial and mammalian cells. ARGs allow gene expression to be visualized with ultrasound similar to how green fluorescent protein (GFP) allowed gene expression to be visualized with light. However, ARGs will have limited utility in practical applications involving living organisms without ultrasound imaging methods providing the specificity to reliably distinguish GVs from surrounding tissue and the sensitivity to detect GVs at low concentrations.
\r\n\r\nIn this thesis, we present two novel ultrasound imaging methods that exploit the unique nonlinear physical properties of gas vesicles to enhance image quality in situations that pose challenges for conventional imaging methods. In Chapter 1, we address the problem of distinguishing GVs from tissue with cross-Amplitude Modulation (xAM), an ultrasound pulse sequence that uses X-waves to isolate the signal generated by reversible buckling of the GV shell while cancelling scattering and artifacts from tissue. In Chapter 2, we present an application of xAM to imaging of dynamic biomolecular processes. We show that, when GVs are engineered such that buckling is induced by enzyme activity, xAM can visualize enzymatic processes deep inside living animals. In Chapter 3, we address the problem of detecting very low concentrations of ARG-expressing cells with Burst Ultrasound Reconstructed with Signal Templates (BURST), an imaging method that exploits the strong, transient signals generated during sudden GV collapse under acoustic pressure by unmixing the temporal dynamics of such signals from background scattering. BURST imaging improves cellular sensitivity by more than 1000-fold and, in dilute cell suspensions, enables the detection of gene expression in individual bacteria and mammalian cells. In Chapter 4, we present an application of an early formulation of BURST to imaging gene expression in mammalian cells. We use this imaging method to visualize vascularization patterns in tumors containing mammalian cells expressing acoustic reporter genes.
", "doi": "10.7907/p52e-qv56", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14257", "collection": "thesis", "collection_id": "14257", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072021-211522720", "type": "thesis", "title": "Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices", "author": [ { "family_name": "Chong", "given_name": "Lucy Shin", "orcid": "0000-0002-5858-9984", "clpid": "Chong-Lucy-Shin" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Cell-specific targeting of therapeutics is a fundamental challenge in biomedicine. The use of engineered proteins that interact with one another as designed, synthetic circuits represents a promising solution to this challenge. These circuits can be constructed to directly sense endogenous cell signals, act on these signals to classify cellular state, and produce a specific response such as conditional triggering of cell death or targeted expression of a reporter. Synthetic protein circuits can also be delivered in mRNA vectors transiently to avoid permanent gene modification.
\r\n\r\nWe recently showed viral proteases can be engineered to regulate one another in a composable manner, permitting the construction of diverse protein-level circuits (Circuits of Hacked Orthogonal Modular Proteases). CHOMP could perform a wide range of computations including Boolean logic, analogue signal processing, and dynamic signal processing. Using this system we were also able to directly sense key cellular pathways and conditionally respond to trigger apoptosis in cancer-like cells. Further expansion of synthetic protein circuits to include nonlinear signal processing enables new system-level behaviors.
\r\n\r\nProtein-based circuits are compatible with innovative delivery methods including mRNA encapsulated in lipid-nanoparticle formulations and engineered viruses. As a proof of principle, we were able to develop a controllable, transient RNA-virus delivery system that allowed for targeted delivery to defined cell populations. This paradigm requires control over multiple aspects of the viral delivery system, including (1) production and release of viral particles, (2) target cell entry based on cell-surface proteins, (3) replication within the cell depending on intracellular proteins, and (4) drug-dependent elimination of the virus. Here, we integrate each of these distinct levels of control can into a single system based on the well-characterized negative stranded RNA virus. This RNA-virus platform will enable synthetic protein circuit delivery.
\r\n\r\nCombining viral engineering and protein circuit construction, the work described here suggests a roadmap towards \u201csmarter\u201d circuit-based therapies that can integrate multiple cues to maximize therapeutic specificity and establishes a role for post-translational circuits as future therapeutic devices.
", "doi": "10.7907/jdhe-by95", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14186", "collection": "thesis", "collection_id": "14186", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282021-020006678", "type": "thesis", "title": "Mechanism of Action of a Therapeutic Peptide, Risuteganib, Suggests that Supporting Mitochondrial Function Underlies its Clinical Efficacy in Treating Leading Causes of Blindness", "author": [ { "family_name": "Zhou", "given_name": "Dan", "orcid": "0000-0003-2367-2822", "clpid": "Zhou-Dan" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the leading causes of blindness in the developed world and on the rise globally due to the growth of an aging population and an increasing number of diabetics. Antibodies of vascular endothelial growth factor (VEGF), which target neovascularization in the advanced stages of both diseases, have been the main treatment for the past decade. However, anti-VEGF therapies suffer from short half-life and high cost inherent to antibodies, limiting the medical availability to a broader population.
\r\n\r\nTo fill the unmet medical need for treating retinal diseases, a novel therapeutic oligopeptide, risuteganib, is currently in Phase II clinical trials. Results from completed trials suggest that risuteganib has comparable drug efficacy to anti-VEGF therapies, long half-life, low cost, and absence of drug-related adverse events in several hundred patients enrolled in clinical trials for diabetic macular edema (DME) and dry AMD. Risuteganib was originally designed to target neovascularization, intending to inhibit integrin cell-surface receptors and thereby block adhesion and migration of abnormal blood vessel cells. Early in our study, we found experimental evidence contrary to this mechanism of action (MOA).
\r\n\r\nOur journey began with an unbiased search for the binding loci in retinal tissue, using peptide-directed fluorescent labeling. We found out that risuteganib specifically binds to a monolayer of cells, the retinal pigment epithelium (RPE), which has essential functions in maintaining the homeostasis of the retina, and its dysfunction is the hallmark for both blinding retinal diseases. In vitro study in an RPE cell model, ARPE19, showed that risuteganib protects cells against elevated oxidative stress that is associated with AMD and DR. This protective effect correlates with maintaining mitochondrial function. Further study of mitochondrial bioenergetics, in collaboration with Dr. Cris Kenney at UCI, revealed that risuteganib supports oxidative phosphorylation metabolism in the mitochondria.
\r\n\r\nBased on the chemical similarity of risuteganib with a natural product, we hypothesized that risuteganib may act through a mitochondrial enzyme, pyruvate dehydrogenase kinase (PDK), specifically PDK1 that is responsive to disease-related hypoxia-inducible factor 1 alpha (HIF-1\u03b1). Protein phosphorylation assay and enzymatic assay confirmed that risuteganib inhibits PDK1, as a result, reducing phosphorylation of an essential enzyme, pyruvate dehydrogenase (PDH). Leaving PDH in its unphosphorylated form allows its continued activity in oxidative phosphorylation metabolism, which offers a molecular explanation of the ability to support mitochondrial activity. This leads to our current hypothesis that risuteganib\u2019s mechanism of action (MOA) is through inhibition of PDK and protection of mitochondrial functions in RPE cells for treating retinal diseases.
\r\n\r\nProtecting mitochondrial functions may be beneficial to other cell types and in other diseases that subject cells to oxidative stress. As the mitochondria targeting is a potential therapy for diverse life-threatening diseases, including inflammatory disease, cardiovascular disease, and cancer, the present hypothesis invites us to expand our scope of view for this study to broader applications.
", "doi": "10.7907/9kqf-yr35", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14098", "collection": "thesis", "collection_id": "14098", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03032021-224823348", "primary_object_url": { "basename": "Caltech-Thesis-Mohamad-Final.pdf", "content": "final", "filesize": 7550092, "license": "other", "mime_type": "application/pdf", "url": "/14098/1/Caltech-Thesis-Mohamad-Final.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Thermal Bioswitches for Non-Invasive Control of Cellular Therapies", "author": [ { "family_name": "Abedi", "given_name": "Mohamad", "orcid": "0000-0001-9717-6288", "clpid": "Abedi-Mohamad" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Temperature is a unique input signal that could be used by engineered therapeutic cells to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, I present here a new class of thermal bioswitches that enables thermal control over bacterial and mammalian cells. For bacterial applications, we developed two new families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32\u201346 \u00b0C. We integrated these molecular bioswitches into thermal logic circuits and demonstrated their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.
\r\n\r\nTo apply this technology in a relevant clinical scenario, we sought to engineer microbial immunotherapies that can be thermally controlled with focused ultrasound. This technology was enabled by rapid advances in synthetic biology that are driving the development of genetically modified microbes as therapeutic agents for a multitude of human diseases, including cancer. In particular, the reduced immune surveillance within the core of some solid tumors creates an ideal environment for microbes to engraft and release therapeutic payloads. However, these therapeutic payloads could be harmful if released in healthy tissues where microbes tend to also engraft in smaller numbers. As described in Chapter 2, my colleagues and I introduced a temperature-actuated state switch that enables tight spatiotemporal control over the activity of therapeutic microbes when combined with focused ultrasound hyperthermia. Through a combination of rational design and high throughput screening, we optimized the behavior of this switch to minimize leakage and maximize inducibility. When tested in a clinically relevant in vivo model, engineered microbes, successfully switched states, and induced a marked suppression of tumor growth upon focal activation. This bioswitch provides a critical tool to attain selective and sustained activity of therapeutic microbes in vivo.
\r\n\r\nEncouraged by the successful development of thermally actuated circuits in microbes, we aimed to establish equivalent technologies for thermal control of human T cells. Genetically engineered T cells are actively being developed to perform a variety of therapeutic functions with great clinical promise. However, no robust mechanisms exist to externally control the activity of T cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. In Chapter 4, my colleagues and I tested the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T cells in the well-tolerated temperature range of 37\u221242 \u00b0C, and we introduced genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrated the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. Overall, the technologies developed here provide critical tools to direct control therapeutic cells after they have been deployed deep inside the body.
", "doi": "10.7907/z7ac-2g66", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14084", "collection": "thesis", "collection_id": "14084", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02192021-010538691", "primary_object_url": { "basename": "chour_william_2021_thesis.pdf", "content": "final", "filesize": 140220437, "license": "other", "mime_type": "application/pdf", "url": "/14084/1/chour_william_2021_thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Molecular Technologies for Antigen-Based Immunity", "author": [ { "family_name": "Chour", "given_name": "William", "orcid": "0000-0003-1817-0123", "clpid": "Chour-William" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The presence and proliferation antigen-specific T cells is a defining characteristic of an adaptive immune response against various disease types (autoimmune, cancer, and infectious). The use of Class I and Class II peptide-major histocompatibility complex (pMHC) reagents to identify such cells, however, is technically difficult and expensive, and it has been challenging to refine synthesis protocols for higher yield and more efficient assembly to accommodate large-scale applications. This achievement would enable high-throughput capture of corresponding T cell receptors (TCR), which may be further used in clinical applications such as adoptive cell transfer therapies. Overcoming this hurdle requires the development and integration of various molecular technologies and analytical methods.
\r\n\r\nToward this end, the bulk of my thesis work, covered in Chapter 2, introduces these developments in the context of pMHCs, where the three subunits of each reagent are covalent linked together and expressed as a single protein. These single-chain trimer (SCT) technologies primarily consist of traditional DNA cloning and protein production techniques which have been streamlined for applications requiring output on the scale of 102-103 of reagents. This chapter serves as the foundation for much of the methodology discussed throughout the rest of my thesis, and thus should serve as a reference point. The generated constructs are also functionally validated here, and potential future research directions are outlined.
\r\n\r\nIn Chapter 3, I explore the use of this technology in the context of COVID-19 to enumerate antigen specificity of the CD8+ T cell immune response. Class I SCTs were constructed to present peptides across several SARS-CoV-2 protein domains, using various HLA alleles to match haplotyped participant blood samples. These reagents were then used to capture SARS-CoV-2-specific T cells through flow and nanoparticle cytometry to demonstrate HLA-dependent, domain-dependent immune responses. Identified TCRs were cloned into T cells for confirmation of antigen specificity and functional cytotoxicity.
\r\n\r\nIn Chapters 4 and 5, I explore potential pMHC applications in cancer antigen contexts, covering both tumor-associated and tumor-specific antigens. Through various collaborations across the west coast (UCLA, Parker Institute, Fred Hutchinson Cancer Research Center), I make use of the SCT platform to showcase new assays to discover and rank key tumor targets (Chapter 4). Finally, Chapter 5 is a reproduction of our lab\u2019s published work concerning identification of antigen-specific CD8+ T cells from melanoma cancer patients.
\r\n\r\nIn summary, the adaptation of SCTs in a high-throughput format allows for the rapid enumeration of antigen-specific T-cell receptor sequences. As demonstrated in the contexts of COVID-19 and cancer, this SCT platform enables subsequent downstream applications, such as single-cell, antigen-specific immunophenotypic mapping/analysis and target discovery for personalized immunotherapies.
", "doi": "10.7907/z20t-nq62", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13824", "collection": "thesis", "collection_id": "13824", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06212020-111155395", "type": "thesis", "title": "Expanding the Scope of Metalloprotein Families and Substrate Classes in New-to-Nature Reactions", "author": [ { "family_name": "Knight", "given_name": "Anders Matthew", "orcid": "0000-0001-9665-8197", "clpid": "Knight-Anders-Matthew" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Heme proteins, in particular cytochromes P450, have been extensively used in biocatalytic applications due to their high degree of regio-, chemo-, and stereoselectivity in oxene-transfer reactions. In 2013, it was shown for the first time that engineered heme proteins can also catalyze analogous carbene- and nitrene-transfer reactions. Research in this field has since grown dramatically, with emphasis on developing new heme protein variants to increase the scope of biotransformations accessible through these new transfer reactions. This thesis details the expansion of these new-to-nature carbene and nitrene-transfer reactions to include new substrate classes previously unexplored with iron-porphyrin proteins, the use of non-heme metalloproteins for these transformations, and steps toward improving the robustness of the new-to-nature biocatalytic platform. Chapter 1 introduces the steps the field of biocatalysis has taken toward engineering enzymes with new catalytic functions and the process by which these activities are discovered and enhanced. Chapter 2 details the discovery and engineering of heme proteins which catalyze the stereodivergent cyclopropanation of unactivated and electron-deficient alkenes via carbene transfer, expanding the substrate classes beyond styrenyl alkenes. Chapter 3 shows the development of engineered variants of a heme protein (Rhodothermus marinus nitric oxide dioxygenase) for the diastereodivergent synthesis of cyclopropanes functionalized with a pinacolborane moiety, enabling product diversification through standard cross-coupling reactions. In Chapter 4, a collection of non-heme metalloproteins is curated, and a non-heme iron enzyme (Pseudomonas savastanoi ethylene-forming enzyme) is shown to be both amenable to directed evolution and non-native ligand substitution to enhance its nitrene-transfer activity. Chapter 5 describes the expansion of sequence space targeted for screening in the serine-ligated cytochrome P411 from Bacillus megaterium (P411BM3) biocatalytic platform to enhance the mutational robustness of these remarkable enzymes. Overall, this work provides a framework for bringing model new-to-nature reactions to their full potential in synthetic biocatalytic reactions.
", "doi": "10.7907/7qh5-5130", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13721", "collection": "thesis", "collection_id": "13721", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242020-045332969", "type": "thesis", "title": "Modifying Ultrasound Waveform Parameters to Control, Influence, or Disrupt Cells", "author": [ { "family_name": "Mittelstein", "given_name": "David Reza", "orcid": "0000-0001-8747-0483", "clpid": "Mittelstein-David-Reza" } ], "thesis_advisor": [ { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Colonius", "given_name": "Tim", "orcid": "0000-0003-0326-3909", "clpid": "Colonius-T" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ortiz", "given_name": "Michael", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Ultrasound can be focused into deep tissues with millimeter precision to perform non-invasive ablative therapy for diseases such as cancer. In most cases, this ablation uses high intensity ultrasound to deposit non-selective thermal or mechanical energy at the ultrasound focus, damaging both healthy bystander tissue and cancer cells. Here we describe an alternative low intensity pulsed ultrasound approach known as \u201concotripsy\u201d that leverages the distinct mechanical properties of neoplastic cells to achieve inherent cancer selectivity. We show that when applied at a specific frequency and pulse duration, focused ultrasound selectively disrupts a panel of breast, colon, and leukemia cancer cell models in suspension without significantly damaging healthy immune or red blood cells. Mechanistic experiments reveal that the formation of acoustic standing waves and the emergence of cell-seeded cavitation lead to cytoskeletal disruption, expression of apoptotic markers, and cell death. The inherent selectivity of this low intensity pulsed ultrasound approach offers a potentially safer and thus more broadly applicable alternative to non-selective high intensity ultrasound ablation.
\r\n\r\nIn this dissertation, I describe the oncotripsy theory in its initial formulation, the experimental validation and investigation of testable predictions from that theory, and the refinement of said theory with new experimental evidence. Throughout, I describe how careful modifications to the ultrasound waveform directly can significantly impact how the ultrasound bio-effects control, influence, or disrupt cells in a selective and controlled manner.
\r\n", "doi": "10.7907/71ak-w328", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13684", "collection": "thesis", "collection_id": "13684", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04272020-152058259", "primary_object_url": { "basename": "Manuel_Bedrossian_Caltech_PhD_Thesis.pdf", "content": "final", "filesize": 121568711, "license": "other", "mime_type": "application/pdf", "url": "/13684/8/Manuel_Bedrossian_Caltech_PhD_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "A Novel Digital Holographic Microscope (DHM) to Investigate and Characterize Microbial Motility in Extreme Aquatic Environments", "author": [ { "family_name": "Bedrossian", "given_name": "Manuel M.", "orcid": "0000-0003-2524-3765", "clpid": "Bedrossian-Manuel-M" } ], "thesis_advisor": [ { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Nadeau", "given_name": "Jay L.", "orcid": "0000-0001-5258-0076", "clpid": "Nadeau-Jay-L" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Recent shifts in the astrobiological community have prompted the development of methods for the direct search for extant life within our solar system. In order to look for life elsewhere in our solar system, it is important to also investigate the broad spectrum of extant life on Earth. Over millions of years of evolution, life has continually adapted such that an 'extreme' environment has become a relative term. What is considered extreme for one type of organism is home to another and vice versa. Furthermore, very little is known about the organisms that inhabit these extreme environments, and even less in known about their in situ behavior. Investigating various extreme environments around Earth in order to understand the in situ behavior of organisms that inhabit it will better inform the astrobiological community when planning future space missions for the direct search for extant life within our solar system. However, no suitable instrument exists to conduct these in situ field campaigns, while also being physically robust enough to withstand the rugged terrains that can be expected from extreme environments.
\r\n\r\nThis thesis describes the development of a novel off-axis digital holographic microscope (DHM) for the direct in situ observation of microscale organisms in extreme aquatic environments. The hardware developments of this instrument are introduced and validated experimentally as well as software developments including autonomous particle detection and tracking algorithms. This instrument is then used in novel laboratory experiments involving the development of optical phase contrast agents, as well as deployed to multiple field campaigns where off-axis DHM is used to observe the in situ behavior of microorgansisms in various extreme aquatic environments around North America.
", "doi": "10.7907/m3a3-4610", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13679", "collection": "thesis", "collection_id": "13679", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04222020-163929825", "type": "thesis", "title": "Acoustic Reporter Genes for Noninvasive Imaging of Cellular Function", "author": [ { "family_name": "Farhadi", "given_name": "Arash", "orcid": "0000-0001-9137-8559", "clpid": "Farhadi-Arash" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The study of cellular function within the context of intact living organisms is a grand challenge in biological research. Addressing this challenge requires imaging tools that can visualize cells inside the body. If successful, this would greatly increase our ability to study a battery of processes from brain development to tumorigenesis, to monitoring cell-based therapeutics. To date, most common methods for imaging cellular processes such as gene expression have relied on optical reporters, such as fluorescent or luminescent proteins, which provide high molecular precision for studies in petri dishes and transparent organisms, but have limited performance in large animals due to the poor penetration of light in biological tissue. Conversely, magnetic resonance imaging (MRI) and ultrasound can image tissues at depth with high spatial and temporal resolution, but they lack molecular reporters analogous to the green fluorescent protein (GFP). As a result, they have made limited impact on biological research. To address this, we focus on developing biomolecular reporters for MRI and ultrasound \u2014 based on a unique class of air-filled protein nanostructures called gas vesicles \u2014 using them to image the location and function of cells deep inside the body.
\r\n\r\nThis thesis begins with a brief review of genetically encoded materials for noninvasive imaging, highlighting key advances over the past two decades and providing context for the work below. We discuss the development of increasingly sophisticated tools starting from early efforts to engineer single molecule reporters to recent work on multi-component genetic machinery (including gas vesicles) with multi-modality capabilities. In Chapter 2, we present a platform for engineering the surface of gas vesicles to modulate their acoustic, surface charge, and molecular- targeting properties as injectable acoustic biomolecules. In Chapter 3, we present the recombinant expression of gas vesicles as injectable contrast agents in common lab strain bacteria to facilitate the genetic engineering of the entire gas vesicle gene cluster and to assist this technology\u2019s adoption by other (non-specialist) research groups. This work characterized the ultrasound and hyperpolarized 129Xenon-MRI contrast of gas vesicles as nanoscale contrast agents.
\r\n\r\nIn a parallel effort, we developed a hybrid gene cluster that when introduced to microbes enables the imaging of their gene expression using ultrasound. These bacterial acoustic reporter genes were used to image the location of probiotic cells inside the gastrointestinal tract of mice. However, the ability for these genes to be expressed in mammalian cells had not been demonstrated and presented a major challenge in synthetic biology. In Chapter 4, we addressed this by introducing the first mammalian acoustic reporter genes \u2014 a genetic program whose introduction to mammalian cells resulted in the expression of gas vesicles that can be visualized by ultrasound. These mammalian acoustic reporter genes will enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo.
\r\n\r\nIn Chapter 5, we explore a new paradigm in MRI by taking advantage of the acousto-magnetic property of gas vesicles. Here, we present background-free MRI to address a longstanding challenge in untangling the signal of exogenous contrast agents from the endogenous MRI contrast produced by biological tissues. Chapter 6 explores the optical properties of gas vesicles as genetically encodable phase contrast agents in digital holographic imaging. Chapter 7 is a brief discussion of the potential future directions for this work.
\r\n\r\nThe data presented in this thesis lays the ground for exciting new research on developing noninvasive biomolecular tools that will enable the discovery of novel biological processes.
", "doi": "10.7907/zght-4j47", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13639", "collection": "thesis", "collection_id": "13639", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02122020-151048251", "primary_object_url": { "basename": "Ng_Ryan_2020.pdf", "content": "final", "filesize": 20979784, "license": "other", "mime_type": "application/pdf", "url": "/13639/1/Ng_Ryan_2020.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Nanophotonic Phenomena in Dielectric Photonic Crystals", "author": [ { "family_name": "Ng", "given_name": "Ryan Cecil", "orcid": "0000-0002-0527-9130", "clpid": "Ng-Ryan-Cecil" } ], "thesis_advisor": [ { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "thesis_committee": [ { "family_name": "Faraon", "given_name": "Andrei", "clpid": "Faraon-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Brady", "given_name": "John F.", "clpid": "Brady-J-F" }, { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "Photonic crystals are periodic optical nanostructures with varying dielectric constant that allow light flow to be controlled and manipulated much in a similar way to electrons within a semiconductor crystal. These nanostructures tend to have a spatially varying refractive index on the order of the wavelength of light to be manipulated. 1D and 2D photonic crystals have already garnered significant attention in the realm of thin-film optics, while 3D photonic crystals have been thus far limited in application, due to difficulties in fabrication and a lack of available materials for fabrication.
\r\n\r\nIn this work, we first explore 1D and 2D photonic crystals based on the concept of a guided mode resonance, which manifests as a narrow near-unity resonance in reflection or transmission that arise from the coupling of an incident wave into a leaky waveguide mode via a grating vector that is subsequently re-radiated. Such a resonance is well-suited for multi- and hyper- spectral filtering applications in the infrared. We designed a platform consisting of amorphous Si arrays embedded in SiO2 in simulation and experiment for application as narrow stopband filters. We present the tunability of the spectral characteristics of the resonance in these arrays through variation of array geometric parameters in simulation and experiment. Guided mode resonance designs often consider only the case of an infinite array, where the leaky waveguide mode can propagate laterally for hundreds of periods, allowing for this mode to eventually scatter out of the array giving rise to the characteristic narrow near-unity rapid spectral variations of a GMR. With an insufficient number of periods, the quality factor and thus the optical filtering performance is greatly diminished. Thus, we further extend our analysis to compact periodic arrays of finite size, which are required for high spatial resolution snapshot imaging, and introduce array designs that operate under finite size limitations in the near-infrared.
\r\n\r\nWe then transition to 3D photonic crystals, exploring the use of an additive manufacturing process to directly fabricate nanocrystalline rutile TiO2 with ~100 nm resolution. Though TiO2 was chosen as the model material, the key to this work is that a similar process can be used to print many different materials, enabling future applications of 3D photonic crystals. The focus here is the additive manufacturing of high index materials such as TiO2, and its potential for photonic applications is demonstrated by characterizing the optical band gap of 3D PhC TiO2 structures printed with this method. We present a system where the ability to print high refractive index 3D photonic crystals would be useful, by studying 3D polymer-germanium core-shell structures that should exhibit all-angle negative refraction in the mid-infrared regime.
", "doi": "10.7907/ZP30-F550", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13601", "collection": "thesis", "collection_id": "13601", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092019-141850032", "type": "thesis", "title": "Visualizing Small Proteins with the cryoEM Platform and The Structure of the Vibrio cholerae Type IV Competence Pilus Secretin PilQ", "author": [ { "family_name": "Weaver", "given_name": "Sara Jean", "orcid": "0000-0001-7753-6215", "clpid": "Weaver-Sara-Jean" } ], "thesis_advisor": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Voorhees", "given_name": "Rebecca M", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 \u00c5 resolution overall, with 5-8 \u00c5 resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.
\r\n\r\nNatural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV competence pilus is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound Type IV competence pilus retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.0 \u00c5 and provide insight on the channel DNA may traverse through during uptake.
", "doi": "10.7907/9B9V-PK08", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13600", "collection": "thesis", "collection_id": "13600", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092019-113537728", "type": "thesis", "title": "Photoacoustic Tomography: From Bench to Bedside", "author": [ { "family_name": "Lin", "given_name": "Li", "orcid": "0000-0002-0517-8436", "clpid": "Lin-Li" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "thesis_committee": [ { "family_name": "Tai", "given_name": "Yu-Chong", "orcid": "0000-0001-8529-106X", "clpid": "Tai-Yu-Chong" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Photoacoustic imaging (PAI) is an emerging imaging modality that shows great potential for preclinical research and clinical practice. As a hybrid technique, PAI uniquely combines the advantages of optical excitation and of acoustic detection. Optical absorption provides a rich contrast mechanism from either endogenous chromophores or exogenous contrast agents. Because ultrasound scatters much less than light in tissue, PAI generates high-resolution images in both the optical ballistic and diffusive regimes, overcoming the limitations imposed by light scattering in deep biological tissues. PAI has led to a variety of exciting discoveries and applications from laboratory research to clinical patient care.
\r\n\r\nTo translate photoacoustic technology from the bench to the bedside, this thesis focuses on efforts to increase the imaging depth, provide clinically useful information (i.e., relevant imaging contrast), reduce system size, and improve system reliability. Assisted by powerful pulsed lasers and advanced data acquisition circuits, modern PAI has achieved applications such as functional imaging of the whole rat brain, revealing detailed angiography and functional connectivity at high spatiotemporal resolution. The advancement of deep imaging in small animal PAI has been transferred to human breast and brain imaging, showing early promise for clinical practice. To further extend the imaging depth and provide dielectric imaging contrast, microwave-based thermoacoustic tomography has been demonstrated in vivo. To map further physiological contrasts, spectroscopic PAI has been performed to image the oxygenation states of hemoglobin and myoglobin. In addition to the effort towards deep penetration and multiple contrasts, benchtop photoacoustic microscopy has been minimized to a handheld probe for human skin imaging. As a rapidly evolving imaging technology, PAI is being translated from the bench to the bedside and promises exciting and useful clinical applications.
\r\n", "doi": "10.7907/1DGY-T168", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11888", "collection": "thesis", "collection_id": "11888", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-135312842", "primary_object_url": { "basename": "Hanewich-Hollatz_Mikhail_2020.pdf", "content": "final", "filesize": 16901753, "license": "other", "mime_type": "application/pdf", "url": "/11888/2/Hanewich-Hollatz_Mikhail_2020.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology", "author": [ { "family_name": "Hanewich-Hollatz", "given_name": "Mikhail Henning", "orcid": "0000-0002-5369-3846", "clpid": "Hanewich-Hollatz-Mikhail-Henning" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. There are two possible logical directions for single-input cgRNAs: constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON\u2192OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF\u2192ON logic). Using an in vitro assay for cgRNA activity with synthetic trigger, in vitro transcribed cgRNA, and recombinant dCas9, we observe a conditional (ON\u2192OFF logic) response for a set of four allosteric constitutively active cgRNAs with a median \u22486% crosstalk between noncognate cgRNA/trigger pairs. Motivated by the observed lack of conditional response of this mechanism when ported to E. coli, we describe a systematic study of unstructured sequence inserts into the standard gRNA structure and report the conditional response of a set of 34 candidate cgRNAs in living cells. Molecular mechanisms for both ON\u2192OFF and OFF\u2192ON cgRNAs are demonstrated in E. coli. For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to produce an orthogonal library of cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of \u224815-fold for a library of three orthogonal ON\u2192OFF \"splinted switch\" cgRNA/trigger pairs, and \u22483-fold for a library of three orthogonal OFF\u2192ON \"toehold switch\" cgRNA/trigger pairs; the median crosstalk within each library is <2% and \u224820% for the two mechanisms, respectively. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for conditional gene regulation and synthetic biology.
", "doi": "10.7907/NS2B-DJ96", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11809", "collection": "thesis", "collection_id": "11809", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10072019-141728052", "primary_object_url": { "basename": "Davis_HunterC_2019_final.pdf", "content": "final", "filesize": 4540297, "license": "other", "mime_type": "application/pdf", "url": "/11809/1/Davis_HunterC_2019_final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanistic Insights for Magnetic Imaging and Control of Cellular Function", "author": [ { "family_name": "Davis", "given_name": "Hunter Cole Davis", "orcid": "0000-0003-1655-692X", "clpid": "Davis-Hunter-Cole-Davis" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Miller", "given_name": "Thomas F.", "orcid": "0000-0002-1882-5380", "clpid": "Miller-T-F" }, { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" }, { "family_name": "Cushing", "given_name": "Scott K.", "orcid": "0000-0003-3538-2259", "clpid": "Cushing-Scott-K" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The vast biomolecular toolkit for optical imaging and control of cellular function has revolutionized the study of in vitro samples and superficial tissues in living organisms but leaves deep tissue unexplored. To look deeper in tissue and observe system-level biological function in large organisms requires a modality that exploits a more penetrant form of energy than visible light. Magnetic imaging with MRI reveals the previously unseen, with endogenous tissue contrast and practically infinite penetration depth. While these clear advantages have made MRI a cornerstone of modern medical imaging, the sparse library of molecular agents for MRI have severely limited its utility for studies of cellular function in vivo. The development of new molecular agents for MRI has suffered from a lack of tools to study the connection between changes in the microscale cellular environment and the corresponding millimeter-scale MRI contrast. Bridging this gap requires revisiting the mechanistic underpinnings of MRI contrast, casting aside some of the simplifications that smooth over sub-voxel heterogeneity that is rich with information pertinent to the underlying cell state.
\r\n\r\nHere, we will demonstrate theoretical, computational, and experimental connections between subtle changes in microscale cellular environment and resultant MRI contrast. After reviewing some foundational principles of MRI physics in the first chapter, the second chapter of the thesis will explore computational models that have significantly enhanced the development of genetically encoded agents for MRI, including the first genetically encoded contrast agent for diffusion weighted imaging. By improving the efficacy of these genetically encoded agents, we unlock MRI reporter genes for in vivo studies of cellular dynamics much in the same way that the engineering of Green Fluorescent Protein has dramatically improved in vitro studies of cellular function.
\r\n\r\nIn the third chapter, we introduce our study that maps microscale magnetic fields in cells and tissues and connects those magnetic fields to MRI contrast. Such a connection has previously been experimentally intractable due to the lack of methods to resolve small magnetic perturbations with microscale resolution. To overcome this challenge, we leverage nitrogen vacancy diamond magnetometry to optically probe magnetic fields in cells with sub-micron resolution and nanotesla sensitivity, together with iterative localization of field sources and Monte Carlo simulation of nuclear spins to predict the corresponding MRI contrast. We demonstrate the utility of this technology in an in vitro model of macrophage iron uptake and histological samples from a mouse model of hepatic iron overload. In addition, we show that this technique can follow dynamic changes in the magnetic field occurring during contrast agent endocytosis by living cells. This approach bridges a fundamental gap between an MRI voxel and its microscopic constituents and provides a new capability for noninvasive imaging of opaque tissues.
\r\n\r\nIn the fourth chapter, we focus on the use of magnetic fields to perturb, rather than image, biological function. Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radiofrequency alternating magnetic fields have generated intense interest due to the potential utility of this phenomenon in non-invasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from classical heat transfer theory. We present an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radiofrequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface, nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems and can help direct future resources towards tractable avenues of magnetic control of cellular function.
", "doi": "10.7907/9QEJ-6H55", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13735", "collection": "thesis", "collection_id": "13735", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292020-131840076", "primary_object_url": { "basename": "200529_erik_jue_2020_thesis_final.pdf", "content": "final", "filesize": 13853909, "license": "other", "mime_type": "application/pdf", "url": "/13735/1/200529_erik_jue_2020_thesis_final.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Improved Tools for Point-of-Care Nucleic Acid Amplification Testing", "author": [ { "family_name": "Jue", "given_name": "Erik Bradley", "orcid": "0000-0001-7585-3794", "clpid": "Jue-Erik-Bradley" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "There is a critical need for improved diagnostic tools to detect infectious diseases, especially in low-resource regions. A sample-to-answer point-of-care nucleic acid amplification test (NAAT) would be incredibly valuable for many different applications (e.g. COVID-19, Chlamydia/Gonorrhoeae, Influenza, Ebola, Zika/Chikungunya/Dengue, etc.). However, sample preparation (purification of pure nucleic acids) is a challenging bottleneck. In Chapter 2, commercial NA extraction methods were studied and improved. In Chapter 3, commercial stocks of SARS-CoV-2 RNA used in FDA emergency-use authorizations were found to be inaccurate and were independently quantified using reverse transcription digital PCR. In Chapter 4, a 3D printed meter-mix device was developed for initial processing prior to the sample preparation device. In Chapter 5, a 3D printed sample-to-device interface was prototyped to facilitate loading multi-volume SlipChip devices with purified template mixed with LAMP reactants. In Chapters 6-7, advancements were made for image processing of commercial chips to study digital LAMP reactions. In Chapter 8, additional tools were developed towards sample-to-answer point-of-care NAAT including a sample preparation module, amplification module, cell-phone readout, and automated base station.", "doi": "10.7907/d6mf-5081", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13722", "collection": "thesis", "collection_id": "13722", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252020-134146453", "primary_object_url": { "basename": "Thesis Draft_v1.3.pdf", "content": "final", "filesize": 32451964, "license": "other", "mime_type": "application/pdf", "url": "/13722/12/Thesis Draft_v1.3.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Additive Manufacturing of 3D Nano-Architected Metals and Ceramics", "author": [ { "family_name": "Vyatskikh", "given_name": "Andrey", "orcid": "0000-0002-6917-6931", "clpid": "Vyatskikh-Andrey" } ], "thesis_advisor": [ { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Faber", "given_name": "Katherine T.", "clpid": "Faber-K-T" }, { "family_name": "Gao", "given_name": "Wei", "clpid": "Gao-Wei" }, { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "local_group": [ { "literal": "Resnick Sustainability Institute" }, { "literal": "Rosen Bioengineering Center" }, { "literal": "div_eng" } ], "abstract": "Additive manufacturing (AM) represents a set of manufacturing processes that create complex 3D parts out of polymers, metals, and ceramics. AM of metals and ceramics is widely used to produce parts for aerospace, automotive, and medical applications. At the micro- and nano-scales, AM is poised to become the enabling technology for efficient 3D microelectromechanical systems (MEMS), 3D micro-battery electrodes, 3D electrically small antennae, micro-optical components, and photonics. Today, the minimum feature size for most commercially available metal and ceramic AM is limited to ~20-50 \u03bcm. Currently, no established processes can reliably produce complex 3D metal and ceramic parts with sub-micron features.
\r\n\r\nIn this thesis, we first demonstrate a nanoscale metal AM process that can produce ~300 nm features out of nanocrystalline, nanoporous nickel using synthesized hybrid organic-inorganic materials, two-photon lithography, and pyrolysis. We study microstructure and mechanical properties of as-fabricated nickel architectures and compare their structural strength to established AM processes. We then show how this process can be extended to other metals and metalloids, including Mg, Ge, Si, and Ti.
\r\n\r\nThis study extends further into nanoscale AM of transparent, high refractive index materials for micro-optics and photonic crystals. We develop an AM process to 3D print fully dense nanocrystalline rutile titanium dioxide (TiO\u2082) with feature dimensions down to ~120 nm. We carefully study and model the relationship between feature dimensions and process parameters to achieve a <2% variation in critical dimensions. We then use this understanding of the process to fabricate and study 3D dielectric photonic crystals with a full photonic bandgap in the infrared.
\r\n\r\nFinally, a microscale AM process of titanium dioxide is demonstrated for photocatalytic water treatment. We show how synthesized hybrid organic-inorganic materials can be applied for stereolithography to print TiO\u2082 architectures with 100 \u03bcm features. We use the developed 3D printing process to investigate the effect of 3D architecture on the efficiency of photocatalytic water treatment.
\r\n\r\nThis work establishes a versatile and efficient pathway to create three-dimensional nano-architected metals and ceramics and to investigate their properties for applications in 3D MEMS, micro-optics, photonics, and photocatalysis.
\r\n", "doi": "10.7907/pdz2-dd59", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11525", "collection": "thesis", "collection_id": "11525", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202019-151055724", "primary_object_url": { "basename": "JChung_thesis_v5.pdf", "content": "final", "filesize": 35150878, "license": "other", "mime_type": "application/pdf", "url": "/11525/1/JChung_thesis_v5.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Computational Imaging: a Quest for the Perfect Image", "author": [ { "family_name": "Chung", "given_name": "Jaebum", "orcid": "0000-0003-3932-8428", "clpid": "Chung-Jaebum" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Lihong", "clpid": "Wang-Lihong" }, { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Hassibi", "given_name": "Babak", "clpid": "Hassibi-B" }, { "family_name": "Zheng", "given_name": "Guoan", "clpid": "Zheng-Guoan" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "A physical lens is limited in its ability to capture an image that is both high- resolution and wide-field due to aberrations even with a sophisticated lens design. This thesis explores computational methods that expand on the recently developed Fourier ptychographic microscopy (FPM) to overcome the physical limitations. New algorithms and imaging methods extend the computational aberration correction to more general imaging modalities including fluorescence microscopy and incoherent bright-field imaging so as to allow even a crude lens to perform like an ideal lens. This paradigm shift from the lens design to computational algorithms democratizes high-resolution imaging by making it easier to use and less complicated to build.", "doi": "10.7907/8W3A-HE02", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11337", "collection": "thesis", "collection_id": "11337", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01102019-190818557", "primary_object_url": { "basename": "Phototherapeutic Devices for the Treatment of Diabetic Retinopathy_Colin Andrew Cook_2019_Caltech.pdf", "content": "final", "filesize": 94243327, "license": "other", "mime_type": "application/pdf", "url": "/11337/1/Phototherapeutic Devices for the Treatment of Diabetic Retinopathy_Colin Andrew Cook_2019_Caltech.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Phototherapeutic Devices for the Treatment of Diabetic Retinopathy", "author": [ { "family_name": "Cook", "given_name": "Colin Andrew", "orcid": "0000-0002-6283-5105", "clpid": "Cook-Colin-Andrew" } ], "thesis_advisor": [ { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Gao", "given_name": "Wei", "clpid": "Gao-Wei" }, { "family_name": "Martinez-Camarillo", "given_name": "Juan Carlos", "clpid": "Martinez-Camarillo-Juan-Carlos" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Diabetic retinopathy is a microvascular disease of the retina and a leading cause of vision loss worldwide. In the non-proliferative phase, diabetes-induced degradation of the retinal blood supply leads to edema and progressive tissue hypoxia. In response, the retinal tissue expresses proangiogenic growth factors (e.g. vascular endothelial growth factor), which drive proliferation of aberrant blood vessels within the eye. These poorly formed vessels leak fluid and blood cells into the eye and grow into the vitreous, which puts traction on the retina and leads to detachment. Given the hypoxic etiology, retinal oxygen tension and metabolism have received considerable attention. Dark-adapted conditions drive the retina to a significantly lower oxygen tension compared to light- adapted conditions as rod cells consume more energy in order to boost sensitivity. While tolerable in the healthy retina, it has been hypothesized that increased nightly metabolism overwhelms the compromised oxygen supply in the diabetic retina, leading to hypoxia and pathological vascular endothelial growth factor expression.
\r\n\r\nThis thesis develops ocular devices that shine light onto the retina to modulate rod metabolism, reducing oxygen demand and mitigating nightly hypoxia. The phototherapeutic effect is characterized through mathematical modeling of retinal metabolism and in vivo testing. Implantable phototherapy devices are designed, fabricated, and evaluated. This thesis also develops overnight phototherapeutic contact lenses utilizing radioluminescence, chemiluminescence, and electroluminescence approaches. Phototherapy holds promise as a non-invasive, preventative therapy for the treatment of hypoxic retinal diseases such as diabetic retinopathy.
", "doi": "10.7907/62R5-RF78", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11716", "collection": "thesis", "collection_id": "11716", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-145818414", "primary_object_url": { "basename": "AEM_Blom_2019_0530_Full_Thesis.pdf", "content": "final", "filesize": 7524685, "license": "other", "mime_type": "application/pdf", "url": "/11716/1/AEM_Blom_2019_0530_Full_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Functional Evaluation and Development of Novel Agonists and Modulators of Neuronal Ion Channels", "author": [ { "family_name": "Blom", "given_name": "Antoinette Elisabeth Maria", "orcid": "0000-0002-7441-4893", "clpid": "Blom-Antoinette-Elisabeth-Maria" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes studies of activation of neuronal ion channels and evaluating new ligands to modulate this process. In chapter two, we expanded the binding model of cytisine to the \u03b14\u03b22 nicotinic acetylcholine receptor. We also determined how C(10)-modification of cytisine impacts the key binding interactions between cytisine and its binding site. To achieve this, we used non-canonical amino acid mutagenesis to probe the electrostatic binding interactions of a novel series of C(10)-cytisine derivatives. In order to perform similar studies in the \u03b13\u03b24 nAChR subtype, we describe the heterologous expression of mouse and human \u03b13\u03b24 nAChRs in Xenopus Laevis oocytes in appendix one. Chapter three describes the development and functional evaluation of a novel series of pyrrolidinoindolines for agonism and modulation of the GABAA receptor. Additionally, we performed mutagenesis studies to identify the binding site of these novel ligands. Appendix two describes a different screen for activation or modulation of GABAA receptors using a set of phenolic compounds implicated in Autism Spectrum Disorder. Chapter four shifts focus to voltage-gated ion channels: in this chapter, the ultimate goal was to photochemically control the activation of VGSCs and make progress towards developing a RubpyC17-based photoswitch that could be used in an artificial retina. To this end, we determined the functional effects of several ruthenium bipyridine analogs on voltage-gated sodium and potassium channels.
", "doi": "10.7907/RG56-G044", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11567", "collection": "thesis", "collection_id": "11567", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-112215583", "type": "thesis", "title": "Effects of Branching on Conformation, Crystallization, and Self-Assembly of Polymers", "author": [ { "family_name": "Kim", "given_name": "Joey Dongjin", "orcid": "0000-0002-3359-4875", "clpid": "Kim-Joey-Dongjin" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "clpid": "Brady-J-F" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The central feature of bottlebrush polymers is the stiffening of the main-chain (MC) due to side-chain side-chain (SC-SC) repulsion, amplified by densely grafting long SCs, particularly in good solvent conditions. The expectation of stiffening has led most prior studies to refer to bottlebrush polymers as \"worm-like,\" \"cylindrical\" or a \"self-avoiding walk (SAW) of superblobs\". However, there is no direct evidence of stiffening of the main-chain and measurements of the overall segment distribution of the whole molecule have failed to discriminate between competing models. Here, we provide a set of measurements of the main-chain conformation (neutron scattering in a solvent that is contrast matched to the side chains) together with the overall conformation of the bottlebrush as a whole (light, X-ray, and neutron scattering) under conditions that highlight SC-SC repulsion: the side-chains are relatively long compared to prior literature, the concentration of bottlebrushes is low, and the solvent quality is good. Surprisingly, the main-chain has a conformation that does not conform to any prior models: all three main-chain lengths examined showed a window of length scales in which the scattering power increased less than linearly with length scale. In particular, the MC conformation is not worm-like. Direct observation of the main-chain conformation and the overall conformation discriminates among models more powerfully than the overall conformation alone. Inspired by the Paturej-Rubinstein tension blob model, we examined a conceptual model in which tension of the MC accumulates with distance from the ends of the MC and found that it can capture the salient features of both the MC- and whole bottlebrush scattering more gradually than previous theoretical models predicted. The conceptual model also explains our observation of a substantial increase in anisometry with increasing MC length, opposite to a worm-like chain. The results indicate that synthetically accessible bottlebrushes are not fractals; they cannot have self-similar (fractal) conformation because each increase in main-chain length accesses greater side-chain crowding than any of its shorter siblings. We expanded the work to understand the behavior in \u03b8 conditions and shorter side-chains expected to have reduced tension as well as the behavior at different concentrations.
\r\n\r\nIn addition, we characterized the interplay of self-assembly and polymer crystallization through analysis of three representative bottlebrush copolymer systems. Our results revealed a surprising number of unexpected behaviors ranging from unexpected morphologies, control of thermal properties even to complete suppression of phase transitions, and control of the orientation of crystal stem with respect to the morphological interface, which highlights the potential of the bottlebrush architecture.
", "doi": "10.7907/8AFE-YC54", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11713", "collection": "thesis", "collection_id": "11713", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-131615199", "primary_object_url": { "basename": "mosesso_richard_2019_thesis_proofread.pdf", "content": "final", "filesize": 9464813, "license": "other", "mime_type": "application/pdf", "url": "/11713/1/mosesso_richard_2019_thesis_proofread.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanistic Investigations of Receptor Signaling via Canonical and Non-Canonical Amino Acid Mutagenesis", "author": [ { "family_name": "Mosesso", "given_name": "Richard Albert", "orcid": "0000-0003-0927-0843", "clpid": "Mosesso-Richard-Albert" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes investigations of the mechanisms by which pentameric ligand-gated ion channels (pLGICs) activate (\"gating\") using canonical and non-canonical amino acid mutagenesis. Chapter 1 provides an introduction to the systems being studied, their physiological roles, and the techniques that we have used to study them. Chapter 2 describes a series of experiments comparing the roles of amino acid residues proximal to the neurotransmitter binding site in the type 3 serotonin receptor (5-HT3R) to the aligning residues of the muscle-type nicotinic acetylcholine receptor (nAChR). The findings presented in Chapter 3 assess the functional roles of proline residues in the prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC). Chapter 4 describes an extensive investigation of salient details of 5-HT3R gating using canonical and non-canonical amino acid mutagenesis of amino acid residues at the interface of the extracellular domain and transmembrane domain of this receptor. Chapter 5 applies a photocrosslinking strategy employing the non-canonical amino acid p-azidophenylalanine to study dimerization and cofactor interactions of the estrogen receptor \u03b1.
", "doi": "10.7907/X18Z-XE16", "publication_date": "2019-06-14", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11501", "collection": "thesis", "collection_id": "11501", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072019-120355881", "primary_object_url": { "basename": "Caltech-Thesis-LeiLi_v12.pdf", "content": "final", "filesize": 7692212, "license": "other", "mime_type": "application/pdf", "url": "/11501/1/Caltech-Thesis-LeiLi_v12.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Multi-Contrast Photoacoustic Computed Tomography", "author": [ { "family_name": "Li", "given_name": "Lei", "orcid": "0000-0001-6164-2646", "clpid": "Li-Lei" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Tai", "given_name": "Yu-Chong", "orcid": "0000-0001-8529-106X", "clpid": "Tai-Yu-Chong" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1\u20132 mm) or a poor depth-to-resolution ratio (~3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. A stand-alone single-impulse photoacoustic computed tomography (PACT) system has been built, which successfully mitigates these limitations by integrating high spatiotemporal resolution, deep penetration, and full-view fidelity, as well as anatomical, dynamical, and functional contrasts. Based on hemoglobin absorption contrast, the whole-body dynamics and large scale brain functions of rodents have been imaged in real time. The absorption contrast between cytochrome and lipid has enabled PACT to resolve MRI-like whole brain structures. Taking advantage of the distinct absorption signature of melanin, unlabeled circulating melanoma cells have been tracked in real time in vivo.
\r\n\r\nAssisted by near-infrared dyes, the perfusion processes have been visualized in rodents. By localizing single-dyed droplets, the spatial resolution of PACT has been improved by six-fold in vivo. The migration of metallic-based microrobots toward the targeted regions in the intestines has been monitored in real time. Genetically encoded photochromic proteins benefit PACT in detection sensitivity and specificity. The unique photoswitching characteristics of different photochromic proteins allow quantitative multi-contrast imaging at depths. A split version of the photochromic protein has permitted PA detection of protein-protein interactions in deep-seated tumors. The photochromic behaviors have also been utilized to guide photons to form an optical focus inside live tissue. As a rapidly evolving imaging technique, PACT promises pre-clinical applications and clinical translation.
\r\n", "doi": "10.7907/FYGX-7M29", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11146", "collection": "thesis", "collection_id": "11146", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08142018-105206326", "type": "thesis", "title": "Conformations and Charge Fluctuations in Polyelectrolyte Solutions", "author": [ { "family_name": "Shen", "given_name": "Kevin", "orcid": "0000-0001-9715-7474", "clpid": "Shen-Kevin" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Miller", "given_name": "Thomas F.", "orcid": "0000-0002-1882-5380", "clpid": "Miller-T-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "From DNA and RNA encoding life to flocculation agents used in water remediation, charged polymers (polyelectrolytes) are prevalent in nearly all facets of our lives. The charged nature of polyelectrolytes has rendered them useful in many applications, from the stabilization of colloids to the formation of nanoparticles for drug or gene delivery. There are open questions regarding the factors that dictate polyelectrolyte stability, and electrostatic fluctuations, first elucidated by Debye and H\u00fcckel for simple electrolytes, are key to the thermodynamic description of such charged systems. Electrostatic fluctuations lead to ionic clouds around charges, leading to favorable energy decreases. While charge-fluctuations are well-described for simple electrolytes, the impact of polyelectrolyte (PE) charge connectivity on charge fluctuations is much less well understood: a huge number of degrees of freedom must be considered in order to describe the multicomponent nature of polyelectrolyte solutions and the large number of conformations the polyelectrolytes themselves can assume. Past theories have both under- and over-estimated the connectivity effects on electrostatic fluctuations, and do not give a clear picture of the transition from weak to strong electrostatic fluctuations.
\r\n\r\nMy work has focused on coming up with a theory that self-consistently accounts for the coupling of chain connectivity and electrostatic fluctuations, thus spanning electrostatic fluctuations from weak to intermediate fluctuation strengths. In particular, I present a novel renormalized Gaussian fluctuation (RGF) theory that identifies the renormalization of chain structure as a key physical consequence of intermediate-strength electrostatic fluctuations. The theory self-consistently couples chain structure with the thermodynamics, and mediates the transition from weak, linearized fluctuations to the onset of stronger fluctuation effects like ion pairing. While the onset of these different fluctuation effects has a clear sequence, they are all coupled and must be determined self-consistently. A key concept introduced by the theory is the notion of the polyelectrolyte self energy, which describes the electrostatic work required to charge the molecule in solution, and provides a useful perspective from which to understand and rationalize the effects of chain conformation on thermodynamic behavior. We use the theory to study the phase behavior of polyelectrolyte solutions and connect theory to experimental results.
", "doi": "10.7907/Y6VG-0297", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11698", "collection": "thesis", "collection_id": "11698", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062019-165907194", "primary_object_url": { "basename": "Caltech-Thesis-Anupama Lakshmanan_06062019.pdf", "content": "final", "filesize": 56790831, "license": "other", "mime_type": "application/pdf", "url": "/11698/1/Caltech-Thesis-Anupama Lakshmanan_06062019.pdf", "version": "v20.0.0" }, "type": "thesis", "title": "Engineering Acoustic Protein Nanostructures for Non-Invasive Molecular Imaging using Ultrasound", "author": [ { "family_name": "Lakshmanan", "given_name": "Anupama", "orcid": "0000-0002-6702-837X", "clpid": "Lakshmanan-Anupama" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Visualizing biomolecular and cellular processes in real time within deep tissues is fundamental to our understanding of the normal and pathological activity underlying health and disease. Ultrasound provides the ability to non-invasively image deep inside biological tissues with high spatial and temporal resolution. However, this technology has limited capacity to monitor molecular and cellular processes, due to the lack of appropriate intra-cellular and endogenously producible nanoscale contrast agents, which can directly couple sound waves to the activity or concentration of physiologically relevant molecules. This problem could in principle be solved by developing genetically encodable ultrasound sensors \u2013 biomolecules that can get illuminated in ultrasound imaging in response to specific cellular or molecular activity. This thesis describes the engineering and characterization of acoustic protein nanostructures called 'gas vesicles', or 'GVs', to accomplish this task.
\r\n\r\nGVs are protein-shelled gas-filled nanostructures produced by buoyant microbes, and were recently shown to be capable of scattering sound waves to produce ultrasound contrast. Owing to this property, they were initially conceptualized as a new class of ultrasound contrast agents. However, little was known about their tunability to enable molecular ultrasound imaging for a wide range of applications. In this thesis, we leveraged the genetic encodability of GVs to modify them at the level of their DNA sequence and constituent proteins, and thereby tune their mechanical, acoustic, surface and targeting properties. We accomplished this by establishing a facile and modular molecular engineering platform, to produce GVs that provide enhanced nonlinear signals for sensitive and specific detection in deep tissues, target specific cell types such as cancer and immune cells, and also provide distinct acoustic collapse spectra for multiplexed imaging. We then extended this platform to build GV-based biosensors that modulate their nonlinear ultrasound signals in response to changes in the activity or concentration of specific molecules in their environment. Specifically, we engineered acoustic sensors for three different types of enzymes and for calcium \u2013 whose activity or flux underlie a wide range of important cellular processes. Furthermore, we succeeded in transferring the genetic code of gas vesicles from their species of origin into a variety of other microbes that do not naturally produce them, in order to unlock their potential as ultrasound reporter genes. Our results establish GVs as reliable acoustic biomolecules, and thereby extend the capabilities of ultrasound for molecular and cellular imaging in a manner analogous to green fluorescent protein (GFP) and its derivatives in optical microscopy. When combined with the advantages of ultrasound for non-invasive imaging, this work facilitates novel technology to significantly enhance our understanding of molecular and cellular processes in basic biology, as well as enable improved diagnosis, monitoring and treatment of diseases.
", "doi": "10.7907/ASX5-KB62", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11230", "collection": "thesis", "collection_id": "11230", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10122018-165154970", "type": "thesis", "title": "From Single-Cell to Whole-Body: Developing a Molecular Neuroscience Toolkit", "author": [ { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-Nicholas-C" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Throughout my Ph.D. I have worked on technology development, at first to answer basic scientific questions and eventually for therapeutic applications. This technology development applied to a variety of fields, from neuroscience to development to gene therapy, and acted upon biological systems in a wide range of scale, from the single-cell monitoring to organism-wide gene-transfer. My graduate research began with the engineering of microbial rhodopsin spectral properties and fluorescence. By making use of their ability to absorb light and emit fluorescence in a voltage-dependent manner, I aimed to interrogate neuronal activity during behavior at the single-cell level. That line of research ended with publication of the voltage-sensor Archer, which I used to track activity of a single cell in vivo in awake, behaving worms. I then shifted from tracking activity at the single cell level, to visualizing entire organisms, by developing clearing techniques that enable a high-resolution, three-dimensional analysis of a diverse range of tissues. I began by optimizing tissue-clearing parameters for various tissue types and a wide variety of experimental needs. I then took that knowledge and applied it to visualizing and tracking the developing neural crest in cleared, whole-mount chicken embryos, discovering some unexpected derivates. Finally, I became interested not only in visualizing entire organisms, but in developing technologies to facilitate gene transfer throughout the body. The rapidly growing field of gene therapy is in constant need of new tools that target specific tissues, avoiding off-target effects. The end of my Ph.D. has been spent engineering viruses that can be delivered body-wide, but target only specific areas of therapeutic interest, like the brain and lungs.
", "doi": "10.7907/S28C-DJ17", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11693", "collection": "thesis", "collection_id": "11693", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-181520170", "primary_object_url": { "basename": "PhD_Thesis_Ramesh_Pradeep_Final.pdf", "content": "final", "filesize": 4837595, "license": "other", "mime_type": "application/pdf", "url": "/11693/1/PhD_Thesis_Ramesh_Pradeep_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Imaging and Control of Engineered Cells using Magnetic Fields", "author": [ { "family_name": "Ramesh", "given_name": "Pradeep", "orcid": "0000-0001-6243-8145", "clpid": "Ramesh-Pradeep" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in 'ultraparamagnetic' macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this 'ultraparamagnetic' concept.
\r\n\r\nIn addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest.
\r\n \r\nFinally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.
\r\n", "doi": "10.7907/KY00-7Y74", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11692", "collection": "thesis", "collection_id": "11692", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-172451535", "primary_object_url": { "basename": "Rusty_Thesis_Final.pdf", "content": "final", "filesize": 21140108, "license": "other", "mime_type": "application/pdf", "url": "/11692/1/Rusty_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Evolution and Characterization of Carbene Transferases for Cyclopropanation and Carbon\u2013Silicon Bond Formation", "author": [ { "family_name": "Lewis", "given_name": "Russell DeRieux", "orcid": "0000-0002-5776-7347", "clpid": "Lewis-Russell-DeRieux" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Heme proteins have recently been demonstrated to catalyze cyclopropanation reactions via a putative carbene transfer mechanism. Carbene transfer reactions are not known to occur in natural biological systems, but are highly useful synthetic reactions. There is growing interest in developing new \"carbene transferases\" that bring new chemical reactions into the realm of biology, and growing interest in engineering these enzymes for use in organic synthesis. Additionally, the mechanistic details of iron porphyrin-catalyzed carbene transfer reactions are largely unknown, especially with regards to how the enzyme environment influences the outcome of a carbene transfer reaction. This thesis details both the engineering of carbene transferases with novel catalytic capabilities and investigations into how these enzymes catalyze carbene transfer reactions. Chapter 1 introduces heme protein-catalyzed carbene transfer reactions and describes the directed evolution of new enzymes that allow access to a range of useful cyclopropane products. Chapter 2 describes the evolution of an enzyme that performs carbene transfer to silicon\u2013hydrogen bonds, resulting in a highly efficient and selective carbon\u2013silicon bond-forming enzyme, the first of its kind. Chapter 3 focuses on the characterization of a key reactive intermediate, the iron-porphyrin carbene, in the active site of the evolved carbon\u2013silicon bond-forming enzyme. This study provides an explanation of the remarkable enantioselectivity of the enzyme and provides a foundation from which to investigate the enzyme reaction mechanism. The mechanism of carbon\u2013 silicon bond formation is elucidated in Chapter 4, and the is then used to explain how the enzyme achieves chemoselectivity, which in turn guides the evolution of enzyme variants with altered chemoselectivity. Finally, two off-cycle catalytic pathways that cause inactivation of the carbene transferase are characterized, and methods to prevent and/or circumvent inactivation are investigated (Chapter 5). Overall, the work presented here expands the repertoire of enzyme-catalyzed reactions and facilitates the continuing development of new carbene transferases by developing our mechanistic understanding of this novel class of enzymes.
", "doi": "10.7907/RMEX-Q134", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11579", "collection": "thesis", "collection_id": "11579", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312019-045041264", "primary_object_url": { "basename": "Piraner_Dan_I_Thesis.pdf", "content": "final", "filesize": 7197165, "license": "other", "mime_type": "application/pdf", "url": "/11579/63/Piraner_Dan_I_Thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Tunable Thermal Bioswitches as a Control Modality for Next Generation Therapeutics", "author": [ { "family_name": "Piraner", "given_name": "Dan Ilya", "orcid": "0000-0003-3857-9487", "clpid": "Piraner-Dan-Ilya" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic biology is rapidly contributing to the field of therapeutic development to create increasingly potent agents for the treatment of a variety of diseases. These living \"designer therapeutics\" are capable of integrating multiple sensory inputs into decision making processes to unleash an array of powerful signaling and effector responses. Included in the great therapeutic potential of these agents, however, is a cognate risk of severe toxicity resulting from runaway on-target or erroneously induced off-target activity. The ability to remotely control engineered therapeutic cells after deployment into patient tissue would drastically reduce the potential dangers of such interventions. However, among existing biological control methods, systemic chemical administration typically lacks the spatial precision needed to modulate activity at specific anatomical locations, while optical approaches suffer from poor light penetration into biological tissue. On the other hand, temperature can be controlled both globally and locally \u2014 at depth \u2014 using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of the patient's condition. Overall, temperature is a versatile signal which can provide a handle to actuate a biological response for the control of therapeutic agents.
\r\n\r\nIn this thesis, a tunable and modular system is developed to respond to thermal perturbations in cellular environments and affect a biological response. At the core of this system is a pair of single-component thermosensing proteins whose dimerization is strongly and sharply coupled to their thermal environment. These domains are first utilized in their native context as negative regulators of transcription in prokaryotes, wherein they are integrated into genetic circuits to control expression of reporter genes. These gene circuits show strong and sharp thermal activation and can be utilized in multiplex to affect higher order logical operations. Cells imbued with these circuits demonstrate transcriptional activation upon global thermal elevation within the host animal within which they reside (fever) or upon a spatiotemporally localized temperature shift imparted by focused ultrasound hyperthermia. In subsequent work, one of these bioswitches is introduced into mammalian cells where it functions as a modular Protein-Protein Interaction (PPI) domain, conferring temperature-dependent protein localization.
\r\n\r\nThe work conducted in this thesis demonstrates the feasibility of utilizing temperature as a stimulus for biological activity. This technology can be harnessed to regulate therapeutically relevant processes in bacterial and mammalian cells such as transcriptional regulation and protein localization, and potentially broader protein function. The thermal bioswitches described herein could be utilized to engineer an array of research tools and biological therapies with actuation driven by spatiotemporally precise noninvasively applied stimuli or by real-time sensing of host conditions.
", "doi": "10.7907/DH10-6N61", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10399", "collection": "thesis", "collection_id": "10399", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08292017-182342238", "type": "thesis", "title": "Improving the Speed and Performance of Point-of-Care Diagnostics with Microfluidics", "author": [ { "family_name": "Schlappi", "given_name": "Travis Stratton", "orcid": "0000-0001-6132-6459", "clpid": "Schlappi-Travis-Stratton" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Microfluidic devices play an important role in improving global health because they reduce the study of biological phenomena into physiological scales and lay the foundation for point-of-care (POC) diagnostics. Health is improved and lives are saved because POC diagnostics can enable earlier diagnosis of diseases and therefore more effective treatment. Accurate and available diagnostics also prevent accelerated drug resistance that stems from overtreatment or mistreatment with antibiotics, which is projected to cause up to $100 trillion in lost economic output and 10 million deaths by 2050. This work details new diagnostic assays and theoretical analysis of microfluidic devices that can be implemented at the point-of-care to improve global health.", "doi": "10.7907/Z9K935Q6", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:11033", "collection": "thesis", "collection_id": "11033", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062018-180959061", "type": "thesis", "title": "Targeted Nanoparticle Delivery of Therapeutics Across the Blood-Brain and Blood-Tumor Barriers to Breast Cancer Brain Metastases", "author": [ { "family_name": "Wyatt", "given_name": "Emily Ann", "orcid": "0000-0002-7534-0582", "clpid": "Wyatt-Emily-Ann" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Brain metastases of human epidermal growth factor receptor 2 (HER2)-positive breast cancer are presenting an increasing problem in the clinic. While HER2-targeted therapies effectively control systemic disease, their efficacy against brain metastases is hindered by their inability to penetrate the blood-brain and blood-tumor barriers (BBB and BTB). One promising strategy to increase brain penetration of systemic therapeutics is to exploit endogenous transport systems at the BBB to shuttle drugs into the brain. Previous studies showed that gold nanoparticles designed to shed transferrin receptor (TfR)-targeting ligands under acidic conditions encountered during transcytosis of the BBB demonstrated increased accumulation in the brain. The focus of this work was to determine whether therapeutic, TfR-targeted nanoparticles using an improved acid-cleavable chemistry could be used to deliver therapeutically useful amounts of drug to the brain.
\r\n\r\nTo accomplish this goal, a new animal model of HER2-positive breast cancer brain metastasis was developed in an attempt to create a clinically representative, impermeable barrier to standard therapeutics. This new model establishes brain metastases by methods that more closely resemble the human disease, forming whole-body tumors that eventually metastasize to the brain. Brain metastases formed by this new methodology show no response to standard HER2-targeted agents, mimicking the clinical situation.
\r\n\r\nNext, efficacy and brain uptake of TfR-targeted, single-agent therapeutic nanoparticles were investigated in the newly developed model, as well as two common models from the literature. These nanoparticles show significant tumor growth delay and increased accumulation in both brain metastases and healthy brain tissue in all three models, highlighting their therapeutic potential. Additionally, non-BBB-penetrant small molecule and non-targeted nanoparticle therapeutics elicit a substantial antitumor response as well as brain tumor accumulation in the most commonly used literature model. In contrast, the new model and one gaining popularity in the literature provide for a more clinically relevant, impermeable barrier to non-BBB-penetrant agents, indicating that the method used to establish brain metastases can affect efficacy and brain uptake of therapeutics.
", "doi": "10.7907/5qpd-0736", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:11007", "collection": "thesis", "collection_id": "11007", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012018-165819361", "primary_object_url": { "basename": "thesis_maeda.pdf", "content": "final", "filesize": 16816964, "license": "other", "mime_type": "application/pdf", "url": "/11007/1/thesis_maeda.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Simulation, Experiments, and Modeling of Cloud Cavitation with Application to Burst Wave Lithotripsy", "author": [ { "family_name": "Maeda", "given_name": "Kazuki", "orcid": "0000-0002-5729-6194", "clpid": "Maeda-Kazuki" } ], "thesis_advisor": [ { "family_name": "Colonius", "given_name": "Tim", "orcid": "0000-0003-0326-3909", "clpid": "Colonius-T" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Blanquart", "given_name": "Guillaume", "orcid": "0000-0002-5074-9728", "clpid": "Blanquart-G" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Colonius", "given_name": "Tim", "orcid": "0000-0003-0326-3909", "clpid": "Colonius-T" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Modeling, numerical simulations, and experiments are used to investigate the dynamics of cavitation bubble clouds induced by strong ultrasound waves.
\r\n\r\nA major application of this work is burst wave lithotripsy (BWL), recently proposed method of lithotripsy that uses pulses (typically 10 wavelengths each) of highintensity, focused ultrasound at a frequency of O(100) kHz and an amplitude of O(1) MPa to break kidney stones. BWL is an alternative to standard shockwave lithotripsy (SWL), which uses much higher amplitude shock waves delivered at a typically much lower rate. In both SWL and BWL, the tensile component of the pressure can nucleate cavitation bubbles in the human body. For SWL, cavitation is a significant mechanism in stone communition, but also causes tissue injury. By contrast, little is yet known about cavitation in BWL.
\r\n\r\nTo investigate cloud cavitation in BWL, two numerical tools are developed: a model of ultrasound generation from a medical transducer, and a method of simulating clouds of cavitation bubbles in the focal region of the ultrasound. The numerical tools enable simulation of the cavitation growth and collapse of individual bubbles, their mutual interactions, and the resulting bubble-scattered acoustics. The numerics are implemented in a massively parallel framework to enable large-scale, three-dimensional simulations. Next, the numerical tools are applied to bubble clouds associated with BWL. Additionally, laboratory experiments are conducted in vitro in order to calibrate and validate the simulations. A major feature of the resulting bubble clouds is that the cloud size is similar to the ultrasound wavelength. This results in an anisotropic structure where the bubbles closest to the wave source grow to larger size and oscillate more rapidly. A new scaling parameter is introduced to characterize the nonlinear bubble cloud dynamics that generalizes the cloud interaction parameter of d'Agostino and Brennen (1989) defined for weak (linearized), bubble cloud dynamics excited uniformly by long-wavelength pressure waves. The mechanisms leading to the observed bubble dynamics are identified. The results further show that bubble clouds can scatter a large portion of incident ultrasound and consequently shield distal regions, including kidney stones, from irradiation. This energy shielding is quantified, and the simulations show that even a thin layer of bubbles can scatter up to 90% of the incident wave energy. A strong correlation is identified between the magnitude of energy shielding and the amplitude of the bubble-scattered acoustics. The correlation may be of use to control cavitation in the human body in real time by ultrasound monitoring for better outcomes of BWL.
", "doi": "10.7907/N7JK-F529", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10971", "collection": "thesis", "collection_id": "10971", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302018-095950094", "primary_object_url": { "basename": "Bedbrook_Caltech_Thesis_Revised_clean2.pdf", "content": "final", "filesize": 55577169, "license": "other", "mime_type": "application/pdf", "url": "/10971/1/Bedbrook_Caltech_Thesis_Revised_clean2.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineering Novel Rhodopsins for Neuroscience", "author": [ { "family_name": "Bedbrook", "given_name": "Claire Nicole", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-Nicole" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The overarching goal of my PhD research has been engineering proteins capable of controlling and reading out neural activity to advance neuroscience research. I engineered light-gated microbial rhodopsins, primarily focusing on the algal derived, light-gated channel, channelrhodopsin (ChR), which can be used to modulate neuronal activity with light. This work has required overcoming three major challenges. First, rhodopsins are trans-membrane proteins, which are inherently difficult to engineer because the sequence and structural determinants of membrane protein expression and plasma membrane localization are highly constrained and poorly understood (Chapter 3-5). Second, protein properties of interest for neuroscience applications are assayed using very low throughput patch-clamp electrophysiology preventing the use of high-throughput assays required for directed evolution experiments (Chapter 2, 5-6). And third, in vivo application of these improved tools require either retention or optimization of multiple protein properties in a single protein tool; for example, we must optimize expression and localization of these algal membrane proteins in mammalian cells while at the same time optimizing kinetic and functional properties (Chapter 5-6). These challenges restricted the field to low-throughput, conservative methods for discovery of improved ChRs, e.g., structure-guided mutagenesis and testing of natural ChR variants. I used an alternative approach: data-driven machine learning to model the fitness landscape of ChRs for different properties of interest and applying these models to select ChR sequences with optimal combinations of properties (Chapters 5-6). ChR variants identified from this work have unprecedented conductance properties and light sensitivity that could enable non-invasive activation of populations of cells throughout the nervous system. These ChRs have the potential to change how optogenetics experiments are done. This work is a convincing demonstration of the power of machine learning guided protein engineering for a class of proteins that present multiple engineering challenges. A component of the novel application of these new ChR tools relies on recent advances in gene delivery throughout the nervous system facilitated by engineered AAVs (Chapter 7). And finally, I developed a behavioral tracking system to monitor behavior and demonstrate sleep behavior in the jellyfish Cassiopea, the most primitive organism to have this behavior formally characterized (Chapter 8).
", "doi": "10.7907/7RA0-BC29", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10617", "collection": "thesis", "collection_id": "10617", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12242017-060345135", "primary_object_url": { "basename": "Maggi-2018.pdf", "content": "final", "filesize": 9300158, "license": "other", "mime_type": "application/pdf", "url": "/10617/1/Maggi-2018.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Three-Dimensional Nano-Architected Materials as Platforms for Designing Effective Bone Implants", "author": [ { "family_name": "Maggi", "given_name": "Alessandro", "clpid": "Maggi-Alessandro" } ], "thesis_advisor": [ { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "thesis_committee": [ { "family_name": "Ravichandran", "given_name": "Guruswami", "clpid": "Ravichandran-G" }, { "family_name": "Daraio", "given_name": "Chiara", "clpid": "Daraio-C" }, { "family_name": "Burdick", "given_name": "Joel Wakeman", "clpid": "Burdick-J-W" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Greer", "given_name": "Julia R.", "clpid": "Greer-J-R" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "GALCIT" }, { "literal": "div_eng" } ], "abstract": "The growing world population coupled with longer human life expectancy warrants the need for better medical implant development. Recent advances in lithographic techniques have opened the door to a variety of approaches to tackle the aforementioned issue. However, several scientific hurdles must be overcome before patients can use fully synthetic and effective implants.
\r\n\r\nIdentifying the optimal material, porosity, and mechanical properties of the scaffold to induce cell functionality are key obstacles. Limitations in established fabrication techniques have hindered the ability to fully understand cell behavior on 3D substrates. 3D printing is limited to feature sizes that are at least one order of magnitude larger than a single cell (~10\u03bcm); electrospinning is able to yield features that are on the same scale as cells, but its stochastic nature leads to scaffolds with poor mechanical properties; salt leeching doesn\u2019t allow for control of pore size and distribution which have detrimental effects on nutrient diffusion and cell ingrowth, thereby thwarting the formation of functional tissue.
\r\n\r\nMuch effort has been made to create a suitable platform for regenerating a relatively less complex organ, such as bone, yet the inability to fully understand cell mechanics on 3D scaffolds has curbed the fabrication of effective bone implants.
\r\n\r\nThe first part of this thesis focuses on the suitability of nanoarchitected materials as 3D platforms for bone-tissue growth. We employed two-photon lithography to create polymeric and hydroxyapatite-coated 3D nanolattices to explore scaffold biocompatibility and material effects on osteoblast attachment and growth. Our experiments showed that the unit cell geometry, tetrakaidekahedron, and size, 25\u03bcm, were adequate for cell attachment and infiltration, which are hallmark signs of biocompatibility. Our study also corroborated previous findings that mammalian cells respond differently to different materials that they come in contact with. To isolate structural effects, we fabricated nanolattices coated with a uniform 20nm-thick outermost layer of TiO2. These nanolattices, which had fixed porosity and unit cell size (25\u03bcm) while they varied in structural stiffness (~2-9MPa) were used to explore the influence of scaffold properties on the viability of osteoblasts in a microenvironment similar to that of natural bone. Upon growing osteogenic cells on the nanolattices, significant cell attachment and presence of various calcium phosphate species, which are commonly found in natural bone, were observed. These findings suggest that 3-dimensional nano-architected materials can be used as effective scaffolds for bone cell growth and proliferation.
\r\n\r\nThe second part of the thesis investigates the effects of nanolattice structural stiffness and loading conditions on osteoblast behavior. We fabricated nanolattices with stiffness ranging from ~0.7MPa to 100MPa. Experiments done by seeding osteoblast-like cells on these nanolattices revealed that both stress fiber concentration and bioapatite deposition were higher on the most compliant nanolattice, (0.7 MPa) by ~20% and ~40% respectively. These results provide insights into cell behavior in 3D microenvironments which can lead to a better understanding of stress shielding at the cellular level. Preventing stress shielding by creating scaffolds with structural stiffness and porosity that enhances osteoblasts activity could lead to the creation of effective implants with improved mechanical stability which ultimately improves osteointegration.
\r\n\r\nIn addition to investigating static cell-scaffold interactions we took advantage of the nanolattices tunability to study the effects of dynamic loading on cell behavior. Bone adaptation is driven by dynamic, rather than static loading, however there is still wide controversy on whether stress, strain or loading frequency plays the most significant role in bone remodeling, which drives bone healing.
\r\n\r\nIn order to understand cell sensitivity to varying loads, displacements and frequencies, we fabricated hollow TiO2 nanolattices with stiffness ranging from ~0.7-35MPa which were populated with osteoblast-like cells and subjected to cyclic compression to either a constant stress or strain. After seeding SAOS-2 cells on the nanolattices for 12 days different dynamic loading conditions were tested: (1) cyclic uniaxial compressions to strains ranging from ~0.3-2% strain were carried out to investigate the effects of strain magnitude on cell behavior. (2) Cyclic uniaxial compressions to stresses spanning from ~0.02-1MPa were performed to explore the role of stress magnitude on the cells\u2019 stress fibers formation. (3) The nanolattices were cyclically loaded at different frequencies, ~0.1-3Hz, while maintaining stress and strain constant, which provided insights into how loading frequency affects osteoblasts behavior.
\r\n\r\nCell activity, which was measured by monitoring f-actin and vinculin fluorescence intensity, revealed increased fluorescence in those cells that were mechanically stimulated as opposed to those that were statically grown on the nanolattices regardless of loading condition. Cell response was most drastically affected by varying the loading frequency. A ~30% increase in f-actin fluorescence was observed in the cells grown on the nanolattices that were loaded at ~3Hz compared to those that were grown on the nanolattices that were cyclically compressed at ~0.1Hz.
\r\n\r\nThe last part of this thesis is focused on developing a three-dimensional architected capacitor that could be used as a strain gauge to further our understanding of cell mechanics in 3D. We took advantage of the mechanical tunability of the nanolattices to fabricate a 3D parallel-plate capacitor with a basal capacitance of ~280fF and able to sense forces as low as ~30\u03bcN. This work points to nano-architected materials as promising candidates for ideal platforms to investigate more realistic cellular conditions which can immensely benefit the field of tissue engineering.
", "doi": "10.7907/Z947482K", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10411", "collection": "thesis", "collection_id": "10411", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09042017-140704721", "primary_object_url": { "basename": "Jungwoo_Kim_Thesis_Final.pdf", "content": "final", "filesize": 4993313, "license": "other", "mime_type": "application/pdf", "url": "/10411/1/Jungwoo_Kim_Thesis_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Microfluidic Analysis in Patient Biopsies: toward Precision Medicine for Glioblastoma Multiforme", "author": [ { "family_name": "Kim", "given_name": "Jungwoo", "orcid": "0000-0002-5215-2044", "clpid": "Kim-Jungwoo" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Although every individual has a unique biology, most medicine still relies on the one-size-fits-all approach, which often fails in the treatment of heterogeneous diseases like cancer. An emerging approach to disease treatment is precision medicine, in which a specific treatment is tailored for individual patients using their biological information, including their genome, phenome, and proteome. Two clinical actions are important for implementing precision medicine in cancer therapies: choosing the correct drugs via patient stratification and choosing a suitable drug dosage and duration via drug response monitoring.
\r\n\r\nAfter selecting the potential drug candidate, it is crucial to monitor tumor response to drug therapy because cancer is a dynamic disease that can develop drug resistance. Although non-invasive tumor imaging techniques such as magnetic resonance imaging, computed tomography, and positron emission tomography can assess physical size and metabolic activity of tumors, these techniques have poor time resolution and cannot capture the dynamic changes of bio-molecules implicated with drug resistance. Thus, to effectively monitor drug response, supplemental diagnostic or prognostic markers must be routinely measured from patient biopsies. Unfortunately, routine monitoring of multiple biomarkers from patient biopsies is impractical, as conventional analytical assays require large sample amounts (up to 100-1,000 mg of tissue or 10 mL of blood).
\r\n\r\nIn response to this challenge, this thesis describes the development of various microfluidic technologies that can perform multiplexed measurements (up to 20-plex) using minute amounts of sample (10,000-100,000 cells or 30\u00b5L of blood) in a miniaturized analytical platform (maximum 75 \u00d7 26 \u00d7 1 mm footprint). We applied these technologies for drug screening and drug response monitoring in glioblastoma multiforme, a highly lethal brain tumor, assaying two different types of patient biopsies: cancer cells and blood.
\r\n\r\nFirst, we developed an integrated microfluidics-chip/beta particle imaging system that can screen for effective therapies using small amounts of patient-derived cell lines. Since glioblastoma cells have abnormally high glycolytic activity, this was used as a read-out for drug response. Single cells were isolated in micro-traps, and their glycolytic activity was quantitated using a radioactive probe. This platform can assess potential drug targets directly from patient biopsies without administering drugs to the patient.
\r\n\r\nSecond, we developed an in vitro diagnostic test that can monitor tumor drug resistance by measuring up to 14 proteins in finger-prick volumes of blood. This test relies on microfluidics and microarray patterning of antibodies to carry out multiplexed sandwich-type immunofluorescence assays. Using this technology and conventional tumor imaging techniques, we linked proteomic signatures to tumor growth, establishing diagnostic and prognostic models in two clinical treatment cases of bevacizumab and buparlisib. Moreover, we adopted the multiplexed proteomic measurement platform to rapidly screen out small peptide binding agents that target an oncogenic protein in glioblastoma.
\r\n\r\nThe microfluidic tools developed here are sample-efficient and highly informative, and we propose that these techniques could enable routine evaluation of drug response in a precision medicine workflow.
\r\n", "doi": "10.7907/Z9639MX2", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10401", "collection": "thesis", "collection_id": "10401", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08302017-121452132", "type": "thesis", "title": "Engineering Biosynthetic Pathways in Cell-Free Systems for Sustainability and Chemical Innovation\r ", "author": [ { "family_name": "Wu", "given_name": "Yong Yi", "orcid": "0000-0002-5401-3662", "clpid": "Wu-Yong-Yi" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Culler", "given_name": "Stephanie J.", "clpid": "Culler-S-J" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This work presents the cell-free transcription-translation (TX-TL) system as a research and development platform for renewable synthesis and molecular discovery. TX-TL is easy to use and provides a biomolecular breadboard for the rapid prototyping and engineering of biosynthetic pathways. This work has validated the capabilities of the cell-free TX-TL system for simultaneous protein expression and chemical synthesis. Specifically, this work shows that TX-TL supports the conversion of intermediates from carbohydrate metabolism and amino acids into valuable compounds. Metabolic flux through cofactor dependent pathways confirms that active cofactor metabolism is occurring in TX-TL. This work has also demonstrated the industrial relevance of TX-TL through exploring design space of a biosynthetic pathway for improved product yield and expanding substrate scope of another biosynthetic pathway.
\r\n\r\nCurrent methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. In this work, we have verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as the pathway bottleneck. We demonstrate the reliability of using linear DNA in TX-TL as a tool for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Pathway constructs of varying pathway enzyme expression levels are tested in TX-TL and in vivo to identify correlations between the two systems, and we find that the production of BDO is correlated to the expression of enzyme ald in both systems. The use of TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Different pathway combinations are also tested in TX-TL for its application in pathway ranking. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.
\r\n\r\nTX-TL simulates a customizable cellular environment that can be controlled by manipulating pH, changing cellular components, or adding exogenous substrates. By adding linear DNA encoding individual enzymes of the violacein pathway and tryptophan analogs in TX-TL reactions, we have discovered new violacein analogs. TX-TL enables rapid production of natural product analogs with diverse substitution, which allows small-scale biosynthesis of potential drug candidates and offers a new platform for drug discovery. This work also presents TX-TL as a platform for protein engineering. Residues targeted for site-saturated mutagenesis were identified with protein-ligand docking. Linear DNAs of individual enzyme mutants were added into TX-TL reactions to screen for improved enzyme variant. Screening result indicates vioE mutant Y17H reduces byproduct formation and redirects metabolic flux towards target metabolites. Protein engineering for improved enzyme activity can further expand the substrate scope of a natural product pathway and result with more natural product analogs that can be applied for medical applications.
\r\n\r\nThis work demonstrates that the cell-free TX-TL system can become a valuable tool that complements the process of engineering biosynthesis in the whole cell in vivo system or the purified protein in vitro system. Future engineering and development of the TX-TL system can further expand the chemical space for biosynthesis.
\r\n", "doi": "10.7907/Z99W0CN1", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10172", "collection": "thesis", "collection_id": "10172", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142017-141527247", "type": "thesis", "title": "Forces, Stresses, and the (Thermo?) Dynamics of Active Matter: The Swim Pressure", "author": [ { "family_name": "Takatori", "given_name": "Sho C.", "orcid": "0000-0002-7839-3399", "clpid": "Takatori-Sho-C" } ], "thesis_advisor": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A core feature of many living systems is their ability to move, self-propel, and be active. From bird flocks to bacteria swarms, to even cytoskeletal networks, active matter systems exhibit collective and emergent dynamics owing to their constituents' ability to convert chemical fuel into mechanical activity. Active matter systems generate their own internal stress, which drives them far from equilibrium and thus frees them from conventional thermodynamic constraints, and by so doing they can control and direct their own behavior and that of their surrounding environment. This gives rise to fascinating behaviors such as spontaneous self-assembly and pattern formation, but also makes the theoretical understanding of their complex dynamical behaviors a challenging problem in the statistical physics of soft matter.
\r\n\r\nIn this thesis, I present a new principle that all active matter systems display---namely, through their self-motion they generate an intrinsic `swim pressure' that impacts their dynamic and collective behavior. I combine experimental and computational methods to demonstrate how intrinsic activity imparts new behaviors to soft materials that explain a variety of complex phenomena, including the collective motion of self-propelled particles and the complete loss of shear viscosity in fluid suspensions. These nonequilibrium phenomena are driven fundamentally by the active constituent's tendency to diffuse, undergo a random walk, and exert a mechanical force or a pressure on a confining wall. The swim pressure theory is conceptually similar to the kinetic theory of gases, where molecular collisions with the container walls exert a pressure, or to the Brownian osmotic pressure exerted by molecular or colloidal solutes in solution. In contrast to thermodynamic quantities such as the chemical potential and free energy, the mechanical pressure (or stress) is valid out of equilibrium because it comes directly from the micromechanical equations of motion. I apply this swim pressure framework in a broad context to interpret living matter as a material and understand its complex behavior using tools of hydrodynamics, kinetic theory, and nonequilibrium statistical mechanics. The present theory is applied to active systems that are driven by self-propulsion and motility, but there are other types of nonequilibrium driving work that may fit into this general theoretical framework, like driven autocatalytic reactions in electrochemical and biochemical systems.
", "doi": "10.7907/Z9RN35WF", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10232", "collection": "thesis", "collection_id": "10232", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312017-143935777", "primary_object_url": { "basename": "thesis_v1.pdf", "content": "final", "filesize": 191384516, "license": "other", "mime_type": "application/pdf", "url": "/10232/41/thesis_v1.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Localization and Stimulation Techniques for Implantable Medical Electronics", "author": [ { "family_name": "Monge Osorio", "given_name": "Manuel Alejandro", "orcid": "0000-0001-9799-0693", "clpid": "Monge-Osorio-Manuel-Alejandro" } ], "thesis_advisor": [ { "family_name": "Emami", "given_name": "Azita", "clpid": "Emami-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Emami", "given_name": "Azita", "clpid": "Emami-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Hajimiri", "given_name": "Ali", "clpid": "Hajimiri-A" }, { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" }, { "family_name": "Weinreb", "given_name": "Sander", "clpid": "Weinreb-S" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Implantable medical devices (IMDs) are emerging as one of the keystones of tomorrow\u2019s medical technology. Although they have enabled a revolution in medicine, from research to diagnosis to treatment, most of today\u2019s devices have critical limitations. They are bulky, have low resolution, and, in some cases, are limited to basic functionality. Miniaturization of IMDs will have an enormous impact not only on the technology itself and the medical procedures they enable, but also on the lives of patients, who will be more comfortable, have greater confidence in their medical treatments, and enjoy an overall improvement in their quality of life. The path towards miniaturized bioelectronic devices requires a reevaluation of existing paradigms to reach a seamless integration of electronics and biology. Miniaturization of medical electronics then involves an exploration of advanced integrated circuit processes and novel circuit and system level architectures. In this dissertation, we provide an overview of implantable medical devices and present novel circuit and system level techniques for the miniaturization of medical electronics.
\r\n\r\nThe function of wireless miniaturized medical devices such as capsule endoscopes, biosensors, and drug delivery systems depends critically on their location inside the body. However, existing electromagnetic, acoustic, and imaging-based methods for localizing and communicating with such devices with spatial selectivity are limited by the physical properties of tissue or imaging modality performance. In the first part of this dissertation, we introduce a new approach for microscale device localization by embodying the principles of nuclear magnetic resonance in a silicon integrated circuit. By analogy to the behavior of nuclear spins, we engineer miniaturized RF transmitters that encode their location in space by shifting their output frequency in proportion to the local magnetic field. The application of external field gradients then allows each device\u2019s location to be determined precisely from the frequency of its signal. We demonstrate the core capabilities of these devices, which we call addressable transmitters operated as magnetic spins (ATOMS), in an integrated circuit smaller than 0.7 mm^3, manufactured through a standard 180 nm complementary metal-oxide-semiconductor (CMOS) process. We show that ATOMS are capable of sub-millimeter localization in vitro and in vivo. As a technology that is inherently robust to tissue properties and scalable to multiple devices, ATOMS localization provides an enabling capability for the development of microscale devices to monitor and treat disease.
\r\n\r\nIn neuroprosthetics, retinal prostheses aim to restore vision in patients suffering from advanced stages of retinal degeneration (e.g., retinitis pigmentosa) by bypassing the damaged photoreceptors and directly stimulating the remaining healthy neurons. In the second part of this dissertation, we describe a fully intraocular self-calibrating epiretinal prosthesis that reduces area and power consumption, and increases the functionality and resolution of traditional implementations. We introduce a novel novel digital calibration technique that matches the biphasic stimulation currents of each channel independently while sharing the calibration circuitry among every 4 channels. The system-on-chip presents dual-band telemetry for power and data with on-chip rectifier and clock recovery. These techniques reduce the number of off-chip components and achieve a power conversion efficiency >80% and supporting data rates up to 20 Mb/s. The system occupies an area of 4.5 x 3.1 mm2 and is implemented in 65 nm CMOS . It features 512 independent channels with a pixel size of 0.0169 mm2 and arbitrary waveform generation per channel. The chip is integrated with flexible MEMS origami coils and parylene substrate to provide a fully intraocular implant.
", "doi": "10.7907/Z9P55KJ7", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10323", "collection": "thesis", "collection_id": "10323", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082017-194534497", "primary_object_url": { "basename": "Anu_Thubagere_BBE.pdf", "content": "final", "filesize": 18346331, "license": "other", "mime_type": "application/pdf", "url": "/10323/1/Anu_Thubagere_BBE.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Programming Complex Behavior in DNA-based Molecular Circuits and Robots", "author": [ { "family_name": "Thubagere Jagadeesh", "given_name": "Anupama", "clpid": "Thubagere-Jagadeesh-Anu" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "clpid": "Qian-Lulu" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Qian", "given_name": "Lulu", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Integrated electronic circuits, like those found in cellphones and computers, are ubiquitous in our information-driven society. The success of electronics has, in part, been due its modular architecture that enables individual components to be independently improved while the overall device functionality remains unchanged. Over the last two decades the emerging field of dynamic DNA nanotechnology has been trying to apply the underlying philosophy of electronics to biochemical circuits. DNA nanotechnology employs rationally designed DNA molecules as building blocks of biochemical circuits that can, in principle, enable powerful applications like diagnostics and therapeutics.
\r\n\r\nResearchers in the field of DNA nanotechnology have developed simple elements to construct biomolecular systems with desired functions. They have also developed molecular compilers for defining design principles. The cost of DNA synthesis has decreased by over three orders of magnitude in the past decade. This has lead to a non-trivial number of small scale circuits, like DNA-based logic gates and chemical oscillators, being implemented. However, the scalability of this approach has yet to be clearly demonstrated. n this thesis, we will discuss our main contributions to facilitating the advancement of DNA nanotechnology by developing systematic approaches for constructing modular DNA building blocks. These modules can be used to construct biochemical circuits and molecular robotic systems. The performance of the modules can be individually tuned and integrated into large-scale systems.
\r\n\r\nUsing automated circuit-design software and cheap unpurified DNA, we demonstrated the design and construction of a complex synthetic biochemical circuit consisting of 78 distinct DNA species. The circuit is capable of computing the transition rules of a cell updating its state based on its neighboring cells, defined in a classic computational model called cellular automata. Using a bottom-up approach, we first characterized the component necessary for basic Boolean logic computation. We then systematically integrated more circuit elements and eventually constructed the full circuit. By developing a systematic procedure for building DNA-based circuits using unpurified components, we significantly simplified the experimental procedure. By using unpurified DNA components, we reduced the cost and technical barrier for circuit construction, thus making the design and synthesis of complex DNA circuits accessible to even novice researchers.
\r\n\r\nNext we demonstrated a cargo sorting DNA nano-robot, using a simple algorithm and modular building blocks. The DNA robot has a leg and two foot domains for exploring a two-dimensional DNA origami surface, and an arm and hand domain for picking up randomly located cargos and dropping them off at their designated locations. It is completely autonomous and is programmed to perform a random walk without requiring an external energy source. Further, we demonstrated sorting multiple copies of two distinct cargo species on the same origami. Additionally, by compartmentalizing each sorting task on a single origami, we showed that two distinct sorting tasks can be implemented on different origami simultaneously in the same test tube. The recognition of a cargo is embedded in its destination, therefore it is possible to scale up the system simply by having multiple types of cargos. The same robot design can be used for performing multiple instances of distinct tasks in parallel. The different modules can be integrated to perform diverse functions, including applications in time-release targeted therapeutics.
", "doi": "10.7907/Z9WD3XMS", "publication_date": "2017-06-16", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9779", "collection": "thesis", "collection_id": "9779", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262016-210208370", "type": "thesis", "title": "Novel Parylene Filters for Biomedical Applications", "author": [ { "family_name": "Liu", "given_name": "Yang", "clpid": "Liu-Yang-Electrical-Engineering" } ], "thesis_advisor": [ { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" } ], "thesis_committee": [ { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Choo", "given_name": "Hyuck", "clpid": "Choo-Hyuck" }, { "family_name": "Emami", "given_name": "Azita", "clpid": "Emami-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Goldkorn", "given_name": "Amir", "clpid": "Goldkorn-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Medical engineering plays a more and more important role in driving the fundamental biology research moving forward. The work presented in this thesis targets at engineer smart parylene filters for various biomedical applications. Three novel parylene membranes are discussed. The first device is parylene magnesium-embedded filter for circulating tumor cells isolation. Circulating tumor cells (CTCs) are cells that slough off the edges of a primary tumor and are swept away by the bloodstream or lymphatic system into the vasculature. They constitute seeds for subsequent growth of additional tumors in vital distant organs, triggering a mechanism that is responsible for the vast majority of cancer-related deaths. Thus CTCs in peripheral blood have been investigated as a valuable biomarker for patients with various types of cancers. However, CTCs are difficult targets to probe owing to their extremely low concentration in peripheral blood. Although rare, CTCs represent a potential approach for the detection, characterization and monitoring of non-haematologic cancers. Therefore, CTCs capture from whole blood has been identified to be an unmet need for cancer research and effective cell separation methods are required to facilitate the study of CTCs. In this study, we developed a novel design applying a buried sacrificial Magnesium (Mg) layer underneath the original microfilter. After filtration, the filter was immersed in DMEM. When the thin-film Mg was dissolved, the cells were released and thus were ready for further biology analysis.
\r\n\r\nThe second device is parylene based microelectrode filter for single-islet electroisletogram. Other than direct insulin injection, one promising treatment for Type I diabetes is islet transplantation. However, one of the key lacking technologies of islet transplantation is high-throughput islet screening since each transplantation requires about one million islets. Islets, which are heterogeneous by nature, are currently screened as whole populations containing a range of functioning and dysfunctional characteristics. This work represents the first attempt to develop a MEMS technology for the screening of every single islet so as to guarantee no bad islet at all, which should improve results of islet transplant therapy. Here we report the first MEMS device designed for in vitro measuring of electroisletogram (EIG) of individual rat islets. Strong EIG signals in millivolt range are obtained. This work proves the feasibility of using MEMS and EIG for high-throughput screening, in contrast to patch-clamp measurements, of islets for transplantation to treat diabetes.
\r\n\r\nThe third device is parylene-on-PDMS membrane for vaccine production. A parylene-on-PDMS design is proposed to supply oxygen to CV-1 cells for vaccine production. Because the cells are seeded and attached right onto the surface of the device, extra oxygen is provided through permeation from the PDMS and thin parylene layers. The permeation is studied and cell growth experiments are performed to demonstrate the feasibility of the device. Compared to commercialized bioreactors, this novel design could have large cell density because oxygen are supplied locally and shear force is not a limiting factor any more.
\r\n\r\nBesides the three devices, parylene properties are also studied and a novel origami design is proposed, which can potentially increase the surface areas of the membranes by fold the 2D flat film into 3D structures. Details are discussed in the following chapters.
", "doi": "10.7907/Z97P8WDF", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9688", "collection": "thesis", "collection_id": "9688", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282016-051723211", "primary_object_url": { "basename": "Xiaoze_Thesis_Caltech_04282016.pdf", "content": "final", "filesize": 13112405, "license": "other", "mime_type": "application/pdf", "url": "/9688/1/Xiaoze_Thesis_Caltech_04282016.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Computational Microscopy: Breaking the Limit of Conventional Optics", "author": [ { "family_name": "Ou", "given_name": "Xiaoze", "orcid": "0000-0001-9918-0221", "clpid": "Ou-Xiaoze" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Computational imaging is flourishing thanks to the recent advancement in array photodetectors and image processing algorithms. This thesis presents Fourier ptychography, which is a computational imaging technique implemented in microscopy to break the limit of conventional optics. With the implementation of Fourier ptychography, the resolution of the imaging system can surpass the diffraction limit of the objective lens's numerical aperture; the quantitative phase information of a sample can be reconstructed from intensity-only measurements; and the aberration of a microscope system can be characterized and computationally corrected. This computational microscopy technique enhances the performance of conventional optical systems and expands the scope of their applications.", "doi": "10.7907/Z9M32SRZ", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9689", "collection": "thesis", "collection_id": "9689", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282016-162609209", "primary_object_url": { "basename": "Clark_Andrew J_Thesis_04292016_Final.pdf", "content": "final", "filesize": 7462710, "license": "other", "mime_type": "application/pdf", "url": "/9689/1/Clark_Andrew J_Thesis_04292016_Final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Delivery of Targeted Nanoparticles Across the Blood-Brain Barrier Using a Detachable Targeting Ligand", "author": [ { "family_name": "Clark", "given_name": "Andrew James", "orcid": "0000-0003-4240-7119", "clpid": "Clark-Andrew-James" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chronic diseases of the central nervous system are poorly treated due to the inability of most therapeutics to cross the blood-brain barrier. The blood-brain barrier is an anatomical and physiological barrier that severely restricts solute influx, including most drugs, from the blood to the brain. One promising method to overcome this obstacle is to use endogenous solute influx systems at the blood-brain barrier to transport drugs. Therapeutics designed to enter the brain through transcytosis by binding the transferrin receptor, however, are restricted within endothelial cells. The focus of this work was to develop a method to increase uptake of transferrin-containing nanoparticles into the brain by overcoming these restrictive processes.
\r\n\r\nTo accomplish this goal, nanoparticles were prepared with surface transferrin molecules bound through various liable chemical bonds. These nanoparticles were designed to shed the targeting molecule during transcytosis to allow increased accumulation of nanoparticles within the brain.
\r\n\r\nTransferrin was added to the surface of nanoparticles through either redox or pH sensitive chemistry. First, nanoparticles with transferrin bound through disulfide bonds were prepared. These nanoparticles showed decreased avidity for the transferrin receptor after exposure to reducing agents and increased ability to enter the brain in vivo compared to those lacking the disulfide link.
\r\n \r\nNext, transferrin was attached through a chemical bond that cleaves at mildly acidic pH. Nanoparticles containing a cleavable link between transferrin and gold nanoparticle cores were found to both cross an in vitro model of the blood-brain barrier and accumulate within the brain in significantly higher numbers than similar nanoparticles lacking the cleavable bond. Also, this increased accumulation was not seen when using this same strategy with an antibody to transferrin receptor, indicating that behavior of nanoparticles at the blood-brain barrier varies depending on what type of targeting ligand is used.
\r\n\r\nFinally, polymeric nanoparticles loaded with dopamine and utilizing a superior acid-cleavable targeting chemistry were investigated as a potential treatment for Parkinson\u2019s disease. These nanoparticles were capable of increasing dopamine quantities in the brains of healthy mice, highlighting the therapeutic potential of this design. Overall, this work describes a novel method to increase targeted nanoparticle accumulation in the brain.
\r\n", "doi": "10.7907/Z9WH2MZ6", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9758", "collection": "thesis", "collection_id": "9758", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252016-151033826", "primary_object_url": { "basename": "Thesis (Full).pdf", "content": "final", "filesize": 16638040, "license": "other", "mime_type": "application/pdf", "url": "/9758/104/Thesis (Full).pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms", "author": [ { "family_name": "Yuet", "given_name": "Kai P.", "orcid": "0000-0002-1381-8923", "clpid": "Yuet-Kai-P" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to \"click\" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.", "doi": "10.7907/Z9VD6WDH", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8893", "collection": "thesis", "collection_id": "8893", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272015-172038281", "primary_object_url": { "basename": "AmyFu2015_Thesis.pdf", "content": "final", "filesize": 22043453, "license": "other", "mime_type": "application/pdf", "url": "/8893/1/AmyFu2015_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mitigating Scarring and Inflammation during Corneal Wound Healing using Nanofiber-Hydrogel Scaffolds", "author": [ { "family_name": "Fu", "given_name": "Amy Hau Yu", "clpid": "Fu-Amy-Hau-Yu" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Due to the universal lack of donor tissue, there has been emerging interest in engineering materials to stimulate living cells to restore the features and functions of injured organs. We are particularly interested in developing materials for corneal use, where the necessity to maintain the tissue\u2019s transparency presents an additional challenge. Every year, there are 1.5 \u2013 2 million new cases of monocular blindness due to irregular healing of corneal injuries, dwarfing the approximately 150,000 corneal transplants performed. The large gap between the need and availability of cornea transplantation motivates us to develop a wound-healing scaffold that can prevent corneal blindness.
\r\n\r\nTo develop such a scaffold, it is necessary to regulate the cells responsible for repairing the damaged cornea, namely myofibroblasts, which are responsible for the disordered and non-refractive index matched scar that leads to corneal blindness. Using in vitro assays, we identified that protein nanofibers of certain orientation can promote cell migration and modulate the myofibroblast phenotype. The nanofibers are also transparent, easy to handle and non-cytotoxic. To adhere the nanofibers to a wound bed, we examined the use of two different in situ forming hydrogels: an artificial extracellular matrix protein (aECM)-based gel and a photo-crosslinkable heparin-based gel. Both hydrogels can be formed within minutes, are transparent upon gelation and are easily tunable.
\r\n\r\nUsing an in vivo mouse model for epithelial defects, we show that our corneal scaffolds (nanofibers together with hydrogel) are well-tolerated (no inflammatory response or turbidity) and support epithelium regrowth. We developed an ex vivo corneal tissue culture model where corneas that are wounded and treated with our scaffold can be cultured while retaining their ability to repair wounds for up to 21 days. Using this technique, we found that the aECM-based treatment induced a more favorable wound response than the heparin-based treatment, prompting us to further examine the efficacy of the aECM-based treatment in vivo using a rabbit model for stromal wounds. Results show that treated corneas have fewer myofibroblasts and immune cells than untreated ones, indicating that our corneal scaffold shows promise in promoting a calmer wound response and preventing corneal haze formation.
\r\n", "doi": "10.7907/Z9Q81B00", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8055", "collection": "thesis", "collection_id": "8055", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01202014-144143175", "primary_object_url": { "basename": "trudeau_dl_2014_140123.thesis.final.pdf", "content": "final", "filesize": 2728687, "license": "other", "mime_type": "application/pdf", "url": "/8055/1/trudeau_dl_2014_140123.thesis.final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Engineering Enzyme Systems by Recombination", "author": [ { "family_name": "Trudeau", "given_name": "Devin Lee", "clpid": "Trudeau-Devin-Lee" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Miller", "given_name": "Thomas F.", "orcid": "0000-0002-1882-5380", "clpid": "Miller-T-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.
\r\n\r\nChapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 \u00b0C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.
\r\n\r\nIn addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.
", "doi": "10.7907/F8T8-3S80", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8450", "collection": "thesis", "collection_id": "8450", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302014-173923900", "type": "thesis", "title": "Development of Semicrystalline Morphology of Poly(L-lactic Acid) during Processing of a Vascular Scaffold", "author": [ { "family_name": "Ailianou", "given_name": "Artemis", "clpid": "Ailianou-Artemis" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "New and promising treatments for coronary heart disease are enabled by vascular scaffolds made of poly(L-lactic acid) (PLLA), as demonstrated by Abbott Vascular\u2019s bioresorbable vascular scaffold. PLLA is a semicrystalline polymer whose degree of crystallinity and crystalline microstructure depend on the thermal and deformation history during processing. In turn, the semicrystalline morphology determines scaffold strength and biodegradation time. However, spatially-resolved information about the resulting material structure (crystallinity and crystal orientation) is needed to interpret in vivo observations.
\r\n \r\nThe first manufacturing step of the scaffold is tube expansion in a process similar to injection blow molding. Spatial uniformity of the tube microstructure is essential for the consistent production and performance of the final scaffold. For implantation into the artery, solid-state deformation below the glass transition temperature is imposed on a laser-cut subassembly to crimp it into a small diameter. Regions of localized strain during crimping are implicated in deployment behavior.
\r\n\r\nTo examine the semicrystalline microstructure development of the scaffold, we employed complementary techniques of scanning electron and polarized light microscopy, wide-angle X-ray scattering, and X-ray microdiffraction. These techniques enabled us to assess the microstructure at the micro and nano length scale. The results show that the expanded tube is very uniform in the azimuthal and axial directions and that radial variations are more pronounced. The crimping step dramatically changes the microstructure of the subassembly by imposing extreme elongation and compression. Spatial information on the degree and direction of chain orientation from X-ray microdiffraction data gives insight into the mechanism by which the PLLA dissipates the stresses during crimping, without fracture. Finally, analysis of the microstructure after deployment shows that it is inherited from the crimping step and contributes to the scaffold\u2019s successful implantation in vivo.
\r\n", "doi": "10.7907/Z9VT1Q26", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" } ]