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UC San Diego Electronic Theses and Dissertations

Interrogating in vivo Mechanisms of Kupffer Cell Transcriptional Regulation Using Natural Genetic Variation

(2022)

Tissue macrophages are essential for the maintenance of organ homeostasis, but the cellular mechanisms specifying their phenotype are poorly understood. Here, we leverage natural genetic variation between inbred mouse strains as a tool to perturb Kupffer cell transcriptional regulation. We show that natural genetic variation disrupts Kupffer cell gene expression through cis-mediated disruption of transcription factor binding motifs and via trans-acting differences in the activity of gene regulatory machinery. We further divide trans-acting genetic variation into non-cell autonomous variation driven by changes in the Kupffer cell environment, and cell autonomous variation driven by changes in intracellular pathway activity. We show that careful evaluation of each mode of genetic variation can reveal signaling pathways specifying Kupffer cell identity in vivo. Collectively, this work demonstrates a novel approach for understanding how genetic diversity impacts tissue macrophage behavior.

Modular Mycobacterium: Decomposition of Mycobacterium Tuberculosis RNA-Seq Data through Independent Component Analysis

(2021)

Mycobacterium Tuberculosis is an infectious disease and a serious public health concern due to the organism’s adaptive transcriptional response to environmental stresses via the transcriptional regulatory network (TRN). (Galagan et al., 2013) While many studies seek to better characterize specific portions of the M. tuberculosis TRN, a systems level characterization and analysis of interactions between the controlling transcription factors has yet to be done. Here, we utilize unsupervised machine learning to compartmentalize and describe the transcription factors and regulatory interactions of M. tuberculosis’s TRN, allowing us to create a model for how the bacterium responds to environmental stresses.(Boot et al., 2018; Serafini et al., 2019) By applying Independent Component Analysis (ICA) to over 650 transcriptomic samples, we obtained 80 independently regulated gene sets known as “I-modulons” that help to explain the variance in the organisms transcriptional response. This ICA structure helps to elucidate the function of previously undescribed regulons, as well as the transcriptional shifts that occur during environmental changes such as shifting carbon sources, oxidative stress, and virulence events. Additionally, this analysis has also uncovered an inherent cluster of transcriptional regulons that connects several important metabolic systems, including lipid catalysis, cholesterol catalysis, and sulfur metabolism. This system-wide analysis of the organism’s TRN can help inform future research on effective ways to study and manipulate the transcriptional regulation of M. Tuberculosis.

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The More The Brighter: Coupling and Emission Tunability in High-beta Telecom-band Semiconductor Nanolasers

(2021)

The emergence of nanolasers over the past two decades has helped enable a plethora ofnovel applications such as optical communications, on-chip interconnects, sensing and superresolution imaging. Particularly for the field of computing/communication, nanolasers have become an intriguing area of research as photonic integrated circuits (PICs) of the future would require nanoscale light sources. To that end, nanolasers based on a variety of architectures and underlying physics have been demonstrated in the literature. Metallo-dielectric nanolasers (MDNLs) are particularly attractive since they combine the advantages of ultrasmall footprints and low thresholds offered by other nanolaser types while still offering electromagnetic isolation, telecom-band operation and current injection. In this dissertation, we mainly focus on exploring additional attributes of MDNLs that further lend credence to their suitability for dense integration on-chip. One of these desirable traits we study includes reversible wavelength tuning (upto 8.35 nm) and intensity modulation of an MDNL based on an external electric field (Chapter 2). More importantly, we report that this electric-field based intensity modulation can be performed at high-speeds of upto 400 MHz (limited only by the detector bandwidth). A second characteristic appropriate for dense integration involves investigating the presence of coupling when two MDNLs are designed in proximity on-chip (Chapter 3). Our results indicate that not only does coupling occur, but it can also be inhibited if independent operation of the emitters is required. We further explore the concept of coupling but with regards to phase-locking two high-b, laterally coupled lasers (Chapter 4). We found that high b values, that are usually only exhibited in nanolasers such as MDNLs, help significantly increase the stable phase-locking regions for two coupled lasers. Additionally, high b can also lead to a wider range of phase differences attainable for a stable nanolaser system (p) compared to what has been demonstrated for commercially available semiconductor lasers (p/10). Finally, we review some unique applications that have already been made possible by the inevitable next step in the nanolaser technology of integrating into dense arrays (Chapter 5) and we briefly discuss a couple of future directions that are worth pursuing in the nanolaser-arrays research field (Chapter 6).

Cover page of Identifying and characterizing de novo tandem repeat mutations and their contribution to autism spectrum disorders

Identifying and characterizing de novo tandem repeat mutations and their contribution to autism spectrum disorders

(2021)

Genetic factors are known to make a large contribution to the risk of Autism Spectrum Disorders (ASD). The heritability of ASD is estimated to be over 50%, and it is estimated that de novo rare variants contribute in about 30% of simplex autism-affected cases. To date, population sequencing studies have been limited to analyzing single nucleotide variants (SNVs), small insertions and deletions (indels), or copy number variants (CNVs). This dissertation expands genetic research to further identify potential genomic regions and pathogenic mutations associated with ASD. Tandem repeats (TRs) are a class of repetitive structural variants composed of 1-20 base pair repeating units. TRs exhibit mutation rates that are orders of magnitude higher than SNPs, indels, or CNVs (6), and thus represent one of the largest sources of human genomic variability (4,5). TRs are often associated with diseases characterized by neurological and developmental symptoms (7–9). for example, Fragile X Syndrome, the most prevalent genetic cause of ASD. To date, direct studies of de novo TR mutations have been limited in population genetic studies. In this dissertation, I present a framework for population-scale characterization of genome-wide de novo TR mutations and their contribution to the genetic etiology of ASD. In my first chapter, I present my bioinformatics pipeline using MonSTR to analyze whole genome sequencing data to identify high- confidence, germline de novo TRs within parent-offspring trios. MonSTR, a novel statistical method, takes genotype likelihood values reported by a TR variant caller as input and estimates the posterior probability of a mutation resulting in a repeat copy number change at each TR loci in each child. In the following chapters, I present the results from identifying de novo TR mutations in autism- affected and unaffected children. I characterize patterns of TR mutational mechanism in the general population, in which I found an average of 54 de novo TRs per individual. I show that ASD affected individuals have a higher number of de novo TR mutations, specifically in regulatory regions and brain-related genes, as well as larger sized mutations, compared to matched unaffected siblings. Lastly, I applied a novel natural selection-based method (SISTR) to identify deleterious de novo TR mutations, and show that autism probands are enriched for rare and pathogenic TR mutations. Overall, this dissertation presents and applies a novel framework for identifying and prioritizing de novo TR mutations in order to better understand TR mutational mechanisms and the genetic etiology of ASD.

The effects of pregravid body mass index and gestational weight gain on indicators of placental health

(2021)

Background: Pregravid obesity and abnormal gestational weight gain added to the physiological stresses of pregnancy have been shown to be associated with increased risks of morbidity and mortality for both the infant and mother. However, their effects on placental health are still uncertain. The placenta and the umbilical cord facilitate the interchange of nutrients between the maternal-fetal dyad. Recent research suggests that the placenta influences the metabolic environment in utero, which can in turn affect birth outcomes. Therefore, understanding how obesity affects the health of the placenta is vital. Methods: This dissertation consists of three studies using data from the University of California San Diego Perinatal Biospecimen Repository Cohort. Study 1 evaluated the effect maternal BMI has on indicators of placental health and assessed GDM as a mediator within the casual pathway. Study 2 assessed the effect gestational weight gain has on indicators of placental health. Study 3 built upon results from the first study by stratifying analyses to determine if the effect of maternal BMI on indicators of placental health were modified by fetal sex. The attenuation of the effect of obese maternal BMI on placental outcomes in the presence of chronic villitis was also evaluated in Study 3. Results: We found that maternal obesity was significantly associated with increased risk of larger placentas, longer umbilical cords, and chronic villitis (Study 1). Women with excessive gestational weight gain were shown to have higher risks of longer umbilical cords, especially in pregnancies with male fetuses (Study 2). We found that in pregnancies with female fetuses, the risk of chronic villitis is increased in women with obesity while other measurements of abnormal placental health were not at an increased risk. This varied in pregnancies with male fetuses, where the risk of larger placentas and longer umbilical cords were increased in women with maternal obesity, but no increased risk of chronic villitis was reported (Study 3). Discussion: Maternal BMI and gestational weight gain are associated with indicators of placental health. Each can be used to help identify women at high risk for abnormal placentas and provide the opportunity for increased surveillance and early intervention.

Cover page of Bioinspired liquid crystal elastomer (LCE) based soft actuators with multimodal actuation

Bioinspired liquid crystal elastomer (LCE) based soft actuators with multimodal actuation

(2021)

Inspired by the biology, soft robots have drawn tremendous attention due to its large and continuous deformation, friendly human-machine interaction, large number of degrees of freedom (DOFs), capability of absorbing energy. They have been explored in broad applications ranging from dexterous soft gripper to the novel assistive devices. In the recent decade, numerous soft actuating materials and deformable structures have been developed to construct soft robots, including hydrogels, shape memory polymers (SMPs), dielectric elastomer actuators (DEAs), fluid elastomer actuators (FEAs) and magnetic actuators. However, those materials and structures have well-known limitations such as slow actuation speed, irreversibility, high voltage input and bulky controlling systems. Liquid crystal elastomers (LCEs), as newly emerging soft actuating materials, exhibit large and reversible deformation and versatile actuation modes. Based on the molecular structure, LCE can be viewed as a combination of liquid crystal molecules and polymer networks. When the LCE is heated above the critical temperature, it can generate large deformation because of the nematic-isotropic phase transition. However, in terms of the practical use of LCE, a few challenges exist such as lack of programmable operation and slow responsive speed for LCEs, which need to be addressed.

In this dissertation, we first integrate flexible heating wire into LCE tube, forming electrically controlled soft tubular actuator. By selectively applying low electrical voltage, this soft tubular actuator can exhibit multiple actuation modes, such as different directional bending and homogeneous contraction. The LCE soft tubular actuator can also be integrated to construct untethered robot that can execute multiple functionalities. To address the slow responsive speed of LCE based soft actuator, we embed microfluidic channel into LCE, forming vascular LCE soft actuator. Through alternatively injecting hot and cold fluid into its internal fluidic channel, the vascular LCE soft actuator can generate fast actuation as well as recovery. In addition, by introducing the disulfide bonds into the LCE materials, the newly obtained vascular LCE based soft actuator has shown repairability and recyclability. Finally, we use electrospinning technique to fabricate LCE microfiber that can be actuated by NIR light. We demonstrate that the electrospun LCE fiber can be easily integrated to micro-robotic system and machine as artificial muscle fiber.

The Role of Rap1 Binding to Talin1 in Promoting Integrin Activation in T Lymphocytes

(2021)

Integrins are essential transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix adhesions and induce bidirectional signaling across the cell membrane to regulate cell functions in lymphocytes. Ras-related protein 1 (Rap1) is a small GTPase known to regulate the recruitment and tethering of Talin1 to the plasma membrane to initiate integrin activation. Previous studies have shown that Rap1 can bind to Talin1 F0 and F1 domains directly to regulate integrin activation in platelets. However, the function of such interaction remains unclear in integrin activation in T lymphocytes. In our study, we examined mice bearing point mutations in Talin1 F0 and F1 domains, which block Rap1 direct binding to Talin1 without disturbing Talin1 expression, and found that the direct interaction between Rap1 and Talin1 is pivotal in both CD4+ T cells and regulatory T (Treg) cells integrin activations. Furthermore, by cross-breeding mice bearing F0F1 double mutations with other transgenic mice strains, we also tested whether the binding of Rap1 and Talin1 could compensate for known integrin activation pathways such as the Rap1-RIAM-Talin1 axis in T lymphocytes. We found that the direct interaction between Rap1 and Talin1 is redundant with the Rap1-RIAM/Lamellipodin-Talin1 pathways on T lymphocytes and that the overexpression of Rap1-GTP-interacting adaptor molecule (RIAM) could compensate for the loss of Rap1 and Talin1 direct binding in CD4+ T cells.

Chemical Studies Toward the Total Synthesis of Lagunamide A Incorporating the Application of Diastereoselective Vinylogous Mukaiyama Aldol Reaction via Kinetic Resolution Methodology

(2021)

Lagunamide A is a cyclic depsipeptide isolated from marine cyanobacterium Langbya majuscula from deep oceans of Pulau Hantu Besar, Singapore. Upon isolation it was revealed to be a potent antimalarial, cytotoxic against leukemia and colon cancer. Latest Structure Activity Relationship research shows Lagunamide A to be active against various cancer cells including A549, HeLa, U2OS, HepG2, BEL-7404, BGC-823, HCT116, MCF-7, HL-60, and A375; with IC50 values ranging from 4.7 nM to 19.8 nM. The diversity exemplifies the natural products potential for future therapeutics as other cyclic depsipeptides have in the past. The investigation of various Vinylogous Mukaiyama Aldol Reactions (VMAR) of β-oxyaldehydes led to the efficient construction of the polyketide carbon framework of Lagunamide A. Solution state synthesis of multiple peptides fragments of Lagunamide A were achieved to avoid epimerization of crucial stereocenters. The peptide portion and the polyketide framework were converged leading to precious intermediates. HR-MS data shows the detection of Lagunamide A although a modified synthetic route would be needed for larger quantities of the natural product. An investigation of Vinylogous Mukaiyama Aldol Reaction was done to test if kinetic resolution could be achieved using Kobayashi’s protocol. An unexplored territory of VMAR is reacting vinylketene N,O-acetal with a racemic mixture of aldehydes to produce stereotriades. Differences in selectivity were observed depending on the size of α-substitutions. Single diastereomers of stereotriades can be produced using this platform of VMAR.

Exploring Cancer Persister Cell Vulnerabilities

(2021)

One major problem plaguing the medical community is patient relapse of cancer following targeted drug therapy. In a majority of patients with non-small-cell lung carcinoma, this process was shown to occur in as little as one year following treatment. Here we investigate the role that molecules, such as antioxidants, and cellular processes, such as DNA death, damage, and response, have in the underlying mechanistic basis for acquiring drug resistance. Our findings suggest that antioxidants are not capable of adequately preventing the acquisition of drug resistance, pointing toward R.O.S.-independent mechanisms of acquisition of drug resistance mutations. Additionally, we show that while the inhibition of DNA damage response and repair pathways significantly prevent the outgrowth of cancerous cells in the presence of drug, there is no difference in response from drug naïve versus drug-tolerant cancer cells. From these findings, we conclude that despite their reported disabled DNA repair machinery, drug-tolerant persister cells are not sensitized to death via inhibition of DNA damage response genes. Additionally, we elucidate the mechanism through which Disulfiram, a drug clinically approved for alcoholism which was recently reported to kill persister cells, induces persister cell-specific lethality. We find that Disulfiram does not kill persister cells through ALDH inhibition, as previously reported, but rather through an oxidative-apoptotic mechanism. By furthering the understanding of factors involved in the tumor’s acquisition of drug resistance, we provide insight into potential mechanisms to target through the development of new treatments aimed at preventing the occurrence of cancer relapses.

Biophysical investigation of acyl-carrier and SARS-CoV-2 protein communication mechanisms

(2021)

Proteins are one of the primary macromolecules that carry out the many essential functions of life. They catalyze chemical reactions, provide structural integrity, and perform biological motor functions. Proteins dynamically interact with surrounding molecules by diffusing, binding, and changing conformations, signaling their stage in biochemical processes. Interpreting these signals is key to harnessing control over protein function for beneficial purposes, such as inhibiting disease related proteins and synthesizing desired molecules including biofuels and therapeutics. This dissertation investigates the communication mechanisms of two protein classes: acyl carrier proteins (ACPs) and SARS-CoV-2 viral proteins. The methods and fundamental properties uncovered herein are expected to be applicable to a broad range of protein systems.