Cell Biology & Molecular Genetics Theses and Dissertations

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    (2023) Chey , Mary Somontha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The form and function of an organism rely on the recreation of similar gene expression patterns in every generation. The information for these expression patterns is stored in a single cell (e.g., zygote) in two forms – the genome sequence and the spatial arrangements of gene regulators. The interactions of regulators and the genome form intricate networks with different regulatory architectures. However, a change in the environment can impact gene expression by disrupting the physical and/or chemical properties of regulators or interactions without mutations in DNA sequence. Such epigenetic information can be transmitted across generations, but how long these effects can last is unclear. Here, we investigate the regulatory elements that promote transient or permanent epigenetic effects by analyzing the properties of a recombinant two-gene operon that expresses the fluorescent proteins mCherry and GFP and is susceptible to long-term RNA silencing in the nematode C. elegans. We reveal that 1) multiple mechanisms regulate transgenerational gene silencing and 2) the presence of the mCherry sequence can perturb RNA regulation within the germline to facilitate heritable epigenetic changes. Previous studies showed that the Argonaute protein HRDE-1 is required for the maintenance of silencing in the germline initiated by double-stranded (ds)RNAs and that poly-UG (pUG)-RNAs are key intermediates generated from the target mRNA. We found that loss of HRDE-1 can selectively rescue the expression of one cistron in a two-gene operon, suggesting that the two cistrons are not regulated by the same silencing pathway, but rather by a chromatin-independent mechanism that requires an unknown regulator. Surprisingly, we detected distinct populations of pUG-RNAs associated with expressed and silenced genes, suggesting that pUG-RNAs could potentially prime expressed genes for long-term silencing. Consistently, total RNA sequencing revealed trace amounts of anti-sense RNAs against mCherry and gfp that could trigger the production of pUG-RNAs. Examining the endogenous genes perturbed by the presence of mCherry suggests that long-term RNA silencing relies on the synergy between the sensing and processing of dsRNAs. Together, our results provide insights into the regulatory architectures and mechanisms of heritable gene silencing that occur without genetic mutations.
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    Characterizing the full complement of antimicrobial resistance genes and linking the resistance genes and plasmid to source bacteria
    (2023) sarria, saul; Song, Jiuzhou; Tadesse, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Antimicrobial resistance (AMR) is a global public health threat. Selective pressure exerted by antimicrobial use has been the major driving force, more than 2.8 million AMR infections occur yearly in the United States. The intestinal microbiome is an important reservoir of antimicrobial resistance genes. Yet, little is known about the true diversity of the antimicrobial resistance genes in food animal intestinal microbiomes. We employed deep shotgun metagenomic sequencing and proximity ligation (Hi-C) library sequencing to characterize the resistome, assemble genomes from metagenomic samples, and accurately attribute antibiotic resistance genes and plasmids to host bacteria cells. We randomly selected 21 cecal samples from food animal sources (cattle n= 6, swine n= 6, chicken n= 3, and turkey n= 6). We generated more than 75 million reads/sample for Hi-C and more than 100 million reads/sample for shotgun metagenomic sequence reads. Bioinformatics analysis revealed over 200 bins containing metagenome assembled genomes (MAGs) with different levels of completeness, novelty scores, and contamination based on CheckM. A total of 245 previously uncharacterized genomes were reconstructed with high level of confidence (>90% Completeness, >90% Novelty, < 5% contamination). Of the 245 newly reconstructed MAGs, 24 were at bacteria taxonomic rank level, 5 at phyla (Actinobacteria; 11 genomes. Firmicutes; 3 genomes. Bacteroidetes; 17 genomes. Euryarchaeota; 2 genome), 4 at class (Bacilli; 1 genome. Clostridia; 9 genomes. Deltaproteobacteria; 3 genomes. Gammaproteobacteria; 1 genome), 5 at order level (Actinomycetales; 2 genomes. Bacteroidales; 25 genomes. Clostridiales; 114 genomes. Lactobacillales; 2 genomes. Selenomonadales; 2 genomes), and 3 at family level (Lachnospiraceae, 24 genomes. Spirochaetaceae; 1 genome. Spirochaetaceae; 2 genomes). We identified over 400 antimicrobial resistance genes representing 22 antimicrobial classes including: aminoglycoside (40 gene variants), beta-lactams (37 gene variants), bleomycin (2 gene variants), colistin (3 gene variants), fosfomycin (4 gene variants), glycopeptide (6 gene variants), lincosamide (9 gene variants), lincosamide/streptogramin (2 gene variants), macrolide (16 gene variants), macrolide/lincosamide/streptogramin (4 gene variants), nitroimidazole (1 gene variant), phenicol (9 gene variants), phenicol/oxazolidinone (1 gene variant), phenicol/quinolone (2 gene variants), pleuromutilin ( 1 gene variant), quinolone (5 gene variants), streptogramin (1 gene variant), streptothricin (3 gene variants), sulfonamide (3 gene variants), tetracycline (25 gene variants), and trimethoprim (8 gene variants). Plasmid characterization using Hi-C proximity ligation and shotgun metagenomics allowed the identification of 146 plasmids (>= 85% completeness, >= 90% reference sequence similarity), and over 13000 plasmid-contigs (<85% completeness, < 90% reference sequence similarity). Shotgun metagenomics provide valuable insights into the diversity and identity of the resistome present in a microbiome, while Hi-C generates millions of paired-end reads linking DNA fragments in close proximity. When shotgun metagenomics is coupled with the Hi-C proximity ligation approach it shows a great capability in genome binning and simultaneous retrieval of high-quality MAGs from a single sample, thusly enabling the link of resistance genes and plasmids to host bacterial cells and facilitating the public health management decisions aimed at reducing the source and exposure routes of AMR to humans.
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    Engineering physiologically-relevant model systems to understand the requirements of rhinovirus C infection
    (2023) Goldstein, Monty Eli; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Rhinovirus (RV) is the most prevalent etiologic agent of the common cold, and infections by RV species C (RV-C) are often associated with more severe illness, and have been strongly correlated with childhood development of asthma. Due to lack of in vitro and in vivo model systems capable of supporting the RV-C life cycle, few details of RV-C biology are understood about this recently discovered, clinically-relevant respiratory pathogen. To reveal the nature of virus-host interactions and study viral pathogenesis, the application of physiologically-relevant model systems that capture relevant cell types, differentiation states, and microenvironmental cues is essential. Applying these principles to our investigations of RV-C, I engineered in vitro and in vivo model systems to better understand the requirement of specific host factors for RV-C replication in human and mouse cells. Specifically, I utilized a pseudostratified in vitro model of human airway epithelium (HAE) to study RV-C replication, and applied CRISPR/Cas9 technology in these cultures to assess the specific role for stimulator of interferon genes (STING) in promoting viral replication. Since RV-C species tropism is highly restricted, I then applied our knowledge of RV-C replication in HAE cultures towards building an improved RV-C mouse model. Here, I first characterized RV-C replication in mouse lung cells in vitro, and demonstrated that human STING expression enhanced viral replication; second, I applied these findings in vivo, where I generated a transgenic mouse expressing the human ortholog of the RV-C receptor, cadherin-related family member 3 (CDHR3), along with human STING. While these mice lack overt symptoms typically associated with viral infection, they exhibited significantly increased viral replication 24 hours-post infection. Finally, to support ongoing efforts to further develop these mice as a robust small animal model of RV-C, I developed several novel cell lines which represent important tools to interrogate the impacts of other host factors on RV-C replication in mouse cells, which upon validation, can be re-engineered into these transgenic mice.
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    (2023) Crawford, David Robert; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In this dissertation I describe four projects investigating different aspects of the variability of gene expression in human cancers. In the first chapter, we analyze epidemiological incidence rates for autoimmune diseases and cancers across numerous populations and find that sex biases in incidence rates are positively correlated between autoimmune diseases and cancers arising from the same tissue. We find that across these tissues the expression of protein-codingmitochondrial genes is positively correlated with both autoimmune disease and cancer incidence rate sex biases, suggesting a possible direction for further investigation. In the second chapter, I construct a computational pipeline to conduct unbiased searches in large databases for possible events accounting for cancer neopeptides predicted by mass spectrometry. I identify several ribosomal frameshift-derived neopeptides from HLA-peptidomics data and discuss future approaches for further improving the accuracy and flexibility of our approach. In the third chapter, I compare the power of different multivariate Cox proportional hazards survival models based on gene- and below-gene-level expression measures to predict genes whose expression in tumor samples at diagnosis affects subsequent survival of cancer patients. I find that models based on both gene-level expression and isoform-level expression (whether transcript abundance or relative transcript abundance) identify the greatest number of statistically significant genes of interest. Finally, in the fourth chapter I briefly explore how heteroformity and entropy measures can be used to examine differences in mRNA splicing diversity at numerous levels of comparison. I propose some simple visualizations that harness these measures to display patterns in splicing diversity.
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    (2023) Li, Yujia; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    During tissue morphogenesis, cells must precisely coordinate their functions over distance by communicating with secreted paracrine signaling proteins, such as the fibroblast growth factors (FGFs). The spatiotemporal regulations of FGF signaling are critical for development and homeostasis in all organisms. However, the mechanisms that control the location, timing, and level of FGF secretion and ensure its target-specific delivery over distance are poorly understood. This thesis has addressed this fundamental question by using a Drosophila FGF Branchless (Bnl) during tracheal development. Bnl is produced in a small group of wing-disc cells. Bnl regulates the development of a disc-associated tracheal branch, the air-sac-primordium (ASP), a vertebrate lung bud analog. Although FGFs are secreted diffusible signals, previous work has shown that the inter-organ dispersion of Bnl from the source to the ASP is highly polarized, target-specific, and is mediated over distance via long polarized actin-based filopodia, named cytonemes. The contact-dependent cytoneme-mediated Bnl delivery is programmed by the glycosylphosphatidylinositol (GPI) anchoring of Bnl. A GPI anchor tethers Bnl to the outer leaflet of the source cell membrane and inhibits free/random Bnl secretion. Surprisingly, GPI anchoring also facilitates Bnl’s release, but only through the cytoneme contact sites, thereby activating MAPK signaling exclusively in those recipient cells that establish contact with its source. However, the molecular mechanisms that might spatiotemporally activate the release of the membrane-anchored Bnl at the cytoneme contact sites remained unknown. This work addresses this knowledge gap by using a combination of cell biological, biochemical, and genetic analyses. Visualization of the distribution of a fluorescently-tagged Bnl revealed that a proteolytic cleavage within the Bnl backbone is essential for its target-specific release. Phenotypic analyses of an uncleavable Bnl mutant generated by genome editing suggested that the proteolytic event that releases Bnl is essential for Bnl signaling and morphogenesis. An in vitro cell-culture-based screening for candidate sheddases identified five membrane-localized enzymes that specifically shed Bnl from the cell surface. These enzymes include two matrix metalloproteinases, MMP1 and MMP2, a pro-protein convertase, Furin1, and two ADAM family metalloproteases, Meltrin and Tace. An inherently disordered region within the Bnl backbone was identified as the substrate for the proteolytic cleavage. Serial mutagenesis in this region revealed four minimal zones required for enzymes to act on Bnl. Interestingly, one of the minimal regions at the juxta-membrane position harbors the Bnl cleavage site, and the other three zones apparently play regulatory roles. Further work on MMPs using mosaic gain-of-function analyses confirmed MMP-mediated Bnl shedding in vivo. However, MMPs are not expressed in the Bnl source. Consequently, Bnl is not freely released. In contrast, MMPs are expressed in the ASP and are localized on the recipient ASP cytonemes. in vitro cell culture experiments that reconstituted contact-dependent Btl-Bnl interactions between Bnl-source and recipient cells confirmed that MMPs are selectively recruited by the recipient cells to the signaling contact sites and activate Bnl shedding in trans. These results explained why and how Bnl is released only via the cytoneme contact sites. Notably, MMP1 and MMP2 are GPI-anchored proteins but are also catalytically active in secreted soluble forms. Therefore, how is Bnl prevented from non-specific shedding by the extracellular soluble MMPs? in vitro and in vivo experiments using GPI-modified and non-GPI-modified MMPs revealed that only the membrane-tethered MMPs that are retained on the cell surface could efficiently shed cell surface Bnl. These findings supported a working model suggesting that recipient ASP cells extend receptor-containing cytonemes and establish contact with the Bnl-source via the receptor-ligand interaction. Due to the GPI-anchoring, MMPs are presented on the surface of ASP cytonemes and are delivered via these cytonemes to their contact sites, leading to the contact-dependent target-specific Bnl trans-shedding. These results provide new insights into how contact-mediated signal dispersion via cytonemes can be modulated by cell surface sheddases. Additional work on Furin1 and ADAM proteases showed that Tace and Meltrin shed Bnl in a cell-non-autonomous manner, like MMPs. In contrast, Furin1 is required only in the source cells for Bnl shedding. How and under what contexts Furin1 and ADAM proteases are activated to shed Bnl needs to be explored in the future. Collectively, this work characterized a novel enzymatic Bnl shedding mechanism and provided insights into how Bnl sheddases might be controlled in space and time to ensure cytoneme-mediated Bnl exchange.
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    Investigation of progerin expression in non-Hutchinson-Gilford Progeria Syndrome individuals
    (2023) Yu, Reynold; Cao, Kan; Mount, Steve; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Hutchinson-Gilford Progerin Syndrome (HGPS) is a premature aging disease caused by a point mutation in the LMNA gene, which encodes A-type lamins. This mutation activates a cryptic splice donor in exon 11 and leads to the production of a toxic lamin variant called progerin. Interestingly, small amounts of progerin have also been found in cells and tissues of normal individuals. Here we examine the expression of progerin in publicly available RNA-seq data from normal individuals of the GTEx project. Among the 30 available tissues, progerin expression in normal individuals is highest in sun-exposed skin samples, and its expression in different tissues of the same donor is correlated. In addition, telomere shortening is significantly correlated with progerin expression. Transcriptome-wide correlation analyses suggest that the level of progerin expression is highly correlated with switches in gene isoform expression patterns, perhaps reflecting widespread isoform shifts in these samples. Differential expression analyses show that progerin expression is correlated with significant changes in the level of transcripts from genes involved in splicing regulation and a significant reduction of mitochondrial transcripts. Interestingly, 5’ splice sites whose use is correlated (either positively or negatively) with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5’ splice site. Furthermore, introns whose alternative splicing is correlated with progerin have reduced GC content. Together, our study suggests that progerin expression in normal individuals is part of a global shift in splicing patterns and provides insight into the mechanism behind these changes.
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    (2023) Alvarado-Martinez, Zabdiel; Biswas, Debabrata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Salmonella enterica serovar Typhimurium (ST) remain one of the main bacterial pathogens responsible for illnesses, hospitalizations, and deaths in the USA. Its ubiquitous prevalence in nature, invasive pattern and increasing antibiotic resistance make it a public health threat, warranting the discovery of novel antimicrobials that can be implemented as either treatments or as forms of control. Plant-derived compounds have been proposed as potential antimicrobials that can be used against gram-negative pathogens, with phenolic acids being of interest for their prevalence in nature and bioactivity. This research studied the effects of gallic acid (GA), protocatechuic acid (PA) and vanillic acid (VA) against ST. Findings showed these compounds to be able to inhibit bacterial growth in vitro, while also showing a reduction in the expression of key virulence genes, without inducing resistance over multiple passages. Further studies using a human epithelial cell line for studying host-pathogen interactions, showed their capability to reduce the number of ST that were able to invade the host cells. Further studies were performed in cecal fluid to test their potency in more complex environments and assess their effects on the microbiome. When in cecal fluid, compounds showed a reduced inhibitory potency compared to in vitro, but still exerted antimicrobial pressure against ST. When analyzing relative abundance of other bacteria through 16S-rRNA gene sequencing, there was an overall decrease in the Protobacteria phylum, while no significant negative effect was seen for other phyla like that of the Firmicutes and Actinobacteria. Experiments to determine the mechanism of action against ST showed these phenolic acids to permeabilize the cell plasma membrane, in addition to reducing cell wall synthesis. Scanning electron microscopy showed treated bacteria to have dents at the polar ends of the cell, while others were found in a duplet formation, suggesting further disruption of specific bacterial functions associated to cell division and structure. These findings suggest that despite their similarities, these compounds are capable of exerting different types of antimicrobial pressure against ST that could better inform their future use as control measures against ST, and their potential use case based on the desired outcome.
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    Molecular and Biophysical Bases of Intracellular Electric Fields in Pollen Tubes
    (2022) Oliveira Nunes, Custódio; Feijό, José A.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Pollen tubes are the male gametophyte of flowering plants. They are arguably one of the fastest-growing cells in nature and inherently an excellent model for studying cellular processes like apical growth, polarity, and chemotropism. Pollen tube development is associated with a unique choreography of ion fluxes and cytosolic ion gradients of Cl-, Ca2+, H+, and K+, creating a unique electrochemical environment, where alternating depolarizing ionic currents at their growing apex are spatially separated from hyperpolarizing currents in their shank. We hypothesize that these electrical differences generated by the opposite ionic patterns could sustain a standing membrane potential gradient at the growing apex. In agreement with evidence from other cellular electrotaxis phenomena, we further hypothesize that a standing electric field gradient could be mechanistic in terms of cell polarity and chemotropism of pollen tubes.Here we show, for the first time, the existence of a standing membrane potential gradient in pollen tubes, confirmed in three different species, thus suggesting a conserved role in apical growth. This conclusion was achieved using three complementary methods, two membrane potential dyes with opposite fluorescence kinetics, and a genetic probe for cytosolic potassium (K+). The K+ gradient is focused at the pollen tube tip, and is compatible with previous information on the individual ion features. Of relevance, K+ shows a negative gradient from the tip, the first ever described in a living cell, suggestive of K+ apical efflux that contributes to the depolarized state. Quantifications of the fluorescent dyes estimate an apical depolarization of approximately 30mV compared to the shank. Screening of ion-channel mutants inducing male-fertility phenotypes supports the hypothesis that this bioelectric oddity is mechanistic for pollen tube’s critical functions, fast invasive growth and chemotropism. Furthermore, we determined that anionic lipids determine the emergence of the pollen tube and correlate with the apical depolarization area, suggesting that they may act as physical determinants of the growing apex. These results open important questions in our understanding of the bioelectrical processes determining cell growth, polarity, morphogenesis, and chemotropic reactions.
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    (2023) Xue, Huijing; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Alzheimer’s Disease (AD) is one of the most common causes of dementia. Twopathological features of AD include amyloid plaques and neurofibrillary tangles. The mechanism underlying the disease's onset and progression remains unclear. Lamin A is an essential component of the nuclear lamina, and nuclear lamina plays a vital role in essential cell functions. Specific mutations in lamin A yield a truncated protein called progerin that causes Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disease. Despite the low expression of lamin A in the brain, several studies reported abnormal lamin A accumulation in patients' hippocampus through the different stages of AD. Besides, there are a lot of common phenotypes between AD and HGPS. Meanwhile, one of the challenges of studying AD is the model issue. It is difficult to recapitulate all AD pathology in a single model, and most models are time-consuming. This dissertation focuses on goals: (1) exploring the potential role of lamin A in AD and (2) facilitating the AD model development. To investigate the potential role of lamin A in AD, I overexpressed either lamin A or progerin in neural cells and checked the phenotypes in Chapter II. Early cell death is closely associated with neuronal loss in AD. After ectopically expressing lamin A in neural cells, early cell death was slightly increased. Progerin could worsen these phenotypes. Oxidative stress and cell cycle re-entry are early events in neurodegeneration and are associated with increased cell death. With the ectopic expression of lamin A, neural cells exhibited slightly elevated oxidative stress and significantly increased cell cycle reactivation. Both two events were significantly increased with exogenous progerin. These results provide insights into how lamin A is involved in neurodegeneration. Besides, progerin addition could further disrupt cellular homeostasis and therefore provide a potential environment for modeling late-onset disease. Most of the current cellular models for AD require several months to display AD phenotypic features, mainly because of the lack of an aging environment in the in vitro cell culture, which is an essential player in age-related neurodegeneration. To provide the aging environment for modeling AD, I examined the impacts of exogenous progerin expression on the neural progenitor cells carrying familial AD mutations (FAD) in Chapter III. Exogenous progerin could accelerate hallmark AD phenotype exhibition from 8-16 weeks to 3-4 weeks, including increased tau phosphorylation and Aβ42/Aβ40 ratio in 2D cell culture, and accumulation of amyloid plaques in 3D cell culture. Additional AD cellular phenotypes, including elevated cell death and cell cycle re-entry, were significantly increased after progerin intervention as well. Together, these results indicated that the approach with progerin expression could create an accelerated model for modeling AD development and future drug screening.
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    Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode
    (2022) Bhanja, Anshuman; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The B cell-mediated humoral immune response is critical in fighting off invading pathogens and potentially harmful foreign substances. B cells detect antigens through the B cell receptor (BCR). The binding of cognate antigen to the BCR induces a signaling response, a critical initiation and regulatory step for B cell activation and differentiation. The actin cytoskeleton has been shown to play essential roles in BCR signaling. When encountering membrane-associated antigens, actin amplifies signaling by driving B cell spreading and BCR clustering, while promoting signal attenuation by causing B cell contraction. This signal attenuation is essential for curtailing the activation of autoreactive B cells. However, the mechanism by which the actin cytoskeleton switches BCR signaling from amplification to attenuation was unknown. My thesis research examined the mechanisms by which actin reorganization transitions B cells from spreading to contracting and B cell contraction switches BCR signaling from amplification to attenuation, using mouse splenic B cells, a functionalized planar lipid bilayer system, and total internal reflection fluorescence microscopy. Our results show that branched actin polymerized by Arp2/3 is required for B cell transition from spreading to contraction after driving B cell spreading. Ubiquitously expressed Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP), but not the haematopoietically specific WASP, activates the branched actin polymerization and generates inner actin foci from lamellipodial actin networks, by sustaining their lifetime and centripetal movement. N-WASP-dependent inner actin foci are necessary for recruiting non-muscle myosin II, creating an actomyosin ring-like structure at the periphery of the membrane contact region to drive B cell contraction. B cell contraction primarily increases the BCR molecular density in individual BCR-antigen clusters, measured by the peak fluorescence intensity. Inhibition of B cell contraction by Arp2/3 inhibitor and B cell-specific N-WASP knockout (cNKO) reduced the increasing rates of BCR molecular density. Increased molecular density caused by B cell contraction leads to decreases in the levels of phosphorylated BCR, the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. However, the levels of total Syk and SHIP-1 have a different relationship with BCR density in individual clusters: Syk does not decrease until a high threshold of BCR density, which can be achieved only by contracting B cells, but SHIP-1 consistently reduces with the increase in BCR molecular density. Inhibiting B cell contraction by cNKO reduces the molecular density of BCR clusters but does not affect the relationship of the Syk and SHIP-1 levels with BCR molecular density in clusters. Taken together, our results suggest that the actin cytoskeleton reorganizes from the lamellipodial branched actin networks to centripetally moving actin foci, enabling actomyosin ring-like structure formation, through N-WASP-activated Arp2/3. Actomyosin-mediated B cell contraction attenuates BCR signaling by increasing receptor molecular density in individual BCR clusters, which causes the dissociation of both stimulatory and inhibitory signaling molecules. My thesis research results reveal a novel negative regulatory mechanism for BCR signaling, an essential checkpoint for generating pathogen-specific and suppressing self-reactive antibody responses.
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    Genome-wide identification and analysis of imprinted genes in strawberry seed development
    (2022) Joldersma, Dirk; Liu, Zhongchi Anne; Taneyhill, Lisa Anne; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The activation of zygotic gene expression is of fundamental importance to reproductive biology, but its regulation remains poorly understood. Within Angiosperm plants, fertilization occurs simultaneously in two locations, the embryo and its genetic twin, the endosperm, a nutritive tissue that is a defining feature of Angiosperm reproduction. Auxin hormone synthesized in the endosperm is essential to seed and fruit development. In the diploid strawberry Fragaria vesca, that auxin synthesis is regulated by FveAGL62, which is expressed specifically after fertilization in endosperm. How fertilization activates FveAGL62 expression in the endosperm, however, is presently unknown. I investigated the hypothesis that epigenetically regulated maternally- and paternally expressed genes (MEGs and PEGs, and together, “imprinted genes”) regulate the expression of FveAGL62. I hybridized two F. vesca accessions, isolated the endosperm from the F1 seeds, and sequenced the transcriptome of the F1 endosperm—a result facilitated by strawberry’s uniquely accessible seed. To identify imprinted genes within the endosperm, I assembled and annotated the genome of the maternal parent, F. vesca accession “Yellow Wonder” (FvYW5AF7), a model for the commercial strawberry. The paternal parent genome was obtained from a collaborator. 809 PEGs and 825 MEGs were identified from RNA sequencing reads that align uniquely to the maternal or paternal genome. MEGs are enriched in genes catabolizing auxin and hence limit seed growth, while PEGs are enriched in genes involved in histone modification, thereby promoting cell differentiation and seed growth. The distinct roles of MEGs and PEGs supports and can be explained by parental conflict and kinship theories, which predict a maternal genome tends to restrict progeny consumption of maternal resources, while a paternal genome will encourage such consumption. In contrast to findings in other species, I find that the endosperm-specific auxin biosynthetic gene FveYUC10 is maternally expressed, but while its imprinting status has changed, it may still function as a fertilization sensor. The maternally expressed gene FveMYB98 contains a binding domain that targets motifs present in FveAGL62’s promoter and its homolog binds AtAGL62 promoter in Arabidopsis. With collaborators, I showed that overexpression and CRISPR knockout of FveMYB98 changes seed size. Transient expression, yeast one hybrid and quantitative PCR analyses suggest that FveMYB98 represses FveYUC10 expression directly and FveAGL62 expression indirectly. These results suggest that FveMYB98 expression is a vehicle for maternal regulation of the level of auxin in the endosperm and thereby endosperm proliferation and seed size. My dissertation research has produced a new genome assembly of a model strawberry, a transcriptome of strawberry endosperm, and identified imprinted genes at genomic scale. I find FveMYB98 regulates seed size—a function echoed broadly within MEG and PEG classes—providing supporting evidence for the parental conflict theory within the developing progeny. These results improve our understanding of zygotic expression in developing seeds, addressing a fundamental scientific gap and, more tangibly, may enable future production of fertilization-independent seeds and seedless fruits. 
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    (2022) Hsu, Amy Pepper; Mosser, David M; Holland, Steven M; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. While the majority of those infected have minor symptoms or remain asymptomatic, illness requiring medical attention occurs in approximately 30%, with <1% developing extrapulmonary dissemination. To address why some individuals allow dissemination, we performed whole-exome sequencing on an exploratory cohort of 67 DCM patients. Using standard genetic analysis for identification of novel or rare Mendelian mutations only two patients were identified, both with STAT3 premature termination codons causing haploinsufficiency. Since Coccidioides are geographically isolated, I explored the possibility that dissemination could be a combination of more common genetic variants plus exposure. Defects in sensing and response to -glucan, the major component of Coccidioides cell wall, were seen in 34/67 (50.7%) cases. Damaging variants in CLEC7A, encoding DECTIN-1, (n=14) and PLCG2 (n=11) were associated with impaired production of -glucan-stimulated TNF from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed over-representation of the specific variants, PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Lastly, I identified a novel pathway of pulmonary-epithelial fungal recognition by DECTIN-1 leading to activation of the NADPH oxidase complex, DUOX1/DUOXA1. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Together these studies highlight the importance of fungal recognition and response for control of infections. Patients with DCM have impaired -glucan sensing or response affecting TNF and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.
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    (2022) Braccia, Domenick James; Hall, Brantley; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The human gut is home to trillions of microorganisms that routinely interact with their human host in both beneficial and detrimental ways. The advent of next-generation sequencing and high-throughput “omics” technologies has created new opportunities to examine the role that the human gut microbiome plays on human health, especially in regard to gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In my dissertation, I utilize genomic, transcriptomic, metabolomic, and protein sequence datasets to characterize three health-relevant functions of the human gut microbiome. First, I performed a multi-omic, bioinformatic analysis to identify the bacterial enzyme, bilirubin reductase. While bilirubin reduction to urobilinogen and stercobilinogen is a well-known function of the human gut microbiome, the enzyme(s) responsible for the conversion of bilirubin to non-toxic reduced products have yet to be fully characterized. In this chapter, I review how I leveraged publicly available metabolomic, metagenomic, and metatranscriptomic data to explore over 2 million putative reductase genes and identify a candidate operon encoding bilirubin reductase. Second, I examined sources of microbial hydrogen sulfide (H2S) production by bacteria of the human gut microbiome. H2S is a sulfuric gas produced by various bacterial phyla of the human gut microbiome and is implicated in the etiology of gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In this chapter, I show via bioinformatic analysis that the capacity to produce H2S via cysteine degradation is ubiquitous in the human gut. Third, I explored bacterial prodrug activation required for the activation of immune system modulators such as sulfasalazine. After curating amino acid sequences of known azoreducing genes and performing a protein sequence search across the Unified Human Gastrointestinal Genomes (UHGG) collection containing 4,644 genomes, I identified putative azoreducing and non-azoreducing bacterial strains to be experimentally validated. Together, these results highlight a successful mult-omic approach to characterizing three diverse but health-relevant functions of the human gut microbiome.
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    (2022) Li, Muzi; Liu, Zhongchi; Mount, Stephen; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Rosaceae is a plant family with over 3,000 species including a number of economically important fruit-bearing species. Although plants in Rosaceae family have similar basic flower structure, their fruit flesh comes from distinct floral tissues. In drupe fruit, such as peach and plum, the ovary wall becomes enlarged and fleshy. In pome fruit, such as apple and pear, the fruit fleshy is mainly derived from the hypanthium that encases the ovary. In drupetum fruit, such as raspberry, numerous unfused ovaries each grow into a fleshy drupelet. In achenetum fruit, such as strawberry, the numerous unfused ovaries eventually dry up, but the receptacle, the stem tip that supports these ovaries, instead develops into the fruit flesh. By investigating and comparing the transcriptomes from these four Rosaceae fruits, peach (Prunus persica), apple (Malus x domestica), strawberry (Fragaria vesca), and raspberry (Rubus idaeus), at the earliest stages of fruit development, we gain important insights into the genetic mechanisms underlying fleshy fruit diversity. The expression of B class MADS-box genes, PISTILLATA, APETALA3 and TM6, shows negative correlation with the ability to form fleshy fruit tissues. Based on RNA transcript and phylogenetic analysis, FBP9, a MADS-box gene related to the E class, appears to be necessary but insufficient for flesh formation. In addition to the regulatory roles MADS-box genes play in fruit identity specification, extensive lignification of the strawberry ovary wall may contribute to the inability of strawberry ovary to become fleshy. Finally, a database (ROsaceae Fruit Transcriptome database, ROFT) is established for researchers to query for orthologous genes and their expression patterns during fruit development in the four species as well as to query for the tissue-specific and tissue- and stage-specific genes. Together, these findings provide the framework for functional investigations of fruit type specification and insights into the evolution of diverse fruit types in the Rosaceae family. The knowledge gained will advance our understanding in the evolution of fleshy fruits, a defining feature of angiosperm, and enable the creation of new fruit types for consumers.
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    (2022) Turner, Randi; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Translational reprogramming is a key component of the bacterial stress response and is a function of mRNA stability, protein turnover and proteolysis. Total proteome measurements give a view of the stable proteome but can fail to capture dynamic changes under stress, including incomplete polypeptides that result from cleaved mRNAs or stalled translation events. Bacteria employ a nearly ubiquitous native ribosome rescue system, transfer-messenger RNA (tmRNA), that rapidly resolves stalled translational complexes and tags the incomplete polypeptides for degradation. Characterization of these tmRNA-tagged polypeptides could reveal previously unknown aspects of the bacterial stress response. To address this information gap, we have developed a synthetic tmRNA platform that reprograms the native system to allow for co-translational labeling of the incomplete polypeptides in live bacteria. A short tag reading frame (TRF) encoded on native tmRNA facilitates the addition of a natural peptidyl degradation tag to the polypeptides, and therefore offers an attractive modular domain to introduce synthetic peptide tag sequences and study the “degradome”. To study translational remodeling under stress, we modified the native tmRNA with an 6x-HIS isolation tag with the specific purpose of stabilizing, isolating, and characterizing the degradome in Escherichia coli. Using our inducible system, we have successfully isolated 6xHis-tagged proteins, verified dynamic controlled tagging, assessed broad-spectrum tag introduction with mass spectrometry. Our results capture known tmRNA substrates and excitingly show that tagged protein profiles are markedly different under stress. We investigated the shifting degradome in cells experiencing translational stress associated with serine starvation induced by serine hydroxamate. In cells lacking RelE, the mRNA interferease toxin that cleaves mRNA in the ribosome A site, we find a dramatic shift away from catalytic protein degradation and distinct, disparate enrichment of ribosomal proteins in the degradome under stress. These latter results suggest a new specific role for RelE in regulating ribosome protein abundance under translational stress conditions
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    (2022) Wang, Fuxi; Liu, Zhongchi ZL; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Plants are important for human survival and the environment. They provide oxygen, food, medicine and fuel. Understanding the development of plants has been a fundamental research question. Among all the plant tissues, the most important ones are the meristems. Sitting at the tip of the shoot and the root are the shoot apical meristem (SAM) and the root apical meristem (RAM). The shoot apical meristem gives rise to the above-ground organs like leaves and flowers while the root apical meristem produces all the root tissues that help to anchor the plants and transport water and nutrients. As the meristem is capable of producing new organs throughout the lifespan of a plant, the study of meristem maintenance and development provides the key to the understanding of plant development.Arabidopsis transcription factor TSO1 plays an essential role for the proper development of shoot apical meristem and root apical meristem. TSO1 encodes a protein with a cysteine-rich repeats domain and TSO1 is a potential component of a cell cycle regulating complex, the DREAM complex. The tso1-1 mutant has fasciated SAM due to shoot meristem cell over-proliferation and complete sterility due to lack of differentiated female and male floral organs. Interestingly, the tso1-1 mutant also produces shorter root than the wild type, presumably caused by early differentiation of the cells in the RAM. A prior mutagenesis screen identified two major suppressors of tso1-1. Characterization of these tso1-1 suppressor mutations provides important insights to the understanding of TSO1-regulatory pathways. My dissertation project focuses on analyzing one of these suppressors that was shown to be a mutated type-A cyclin gene named CYCA3;4. Mutations in CYCA3;4 suppress the shoot phenotype but not the root phenotype of tso1-1. The suppressed plants can produce normal floral organs and become partially fertile. Using transgenic method, I showed that the expression of CYCA3;4 was increased in the tso1-1 SAM, and overexpression of CYCA3;4 in the tso1-3 mutant enhanced the fertility defect, suggesting that overexpression of CYCA3;4 partially mediates the tso1-1 shoot phenotype. In addition, I provided evidence supporting that TSO1 likely represses CYCA3;4 gene expression indirectly through MYB3R1, whose mutations also suppress tso1-1 mutants. My dissertation provides an important link between TSO1, a potential cell cycle regulatory complex component and meristem regulator, and cyclin A, a protein directly involved in cell cycle regulation. This link provides an important mechanistic insight into how plant meristems maintain their identity by limiting their cell division activity. To further investigate the mechanism of TSO1 action in the root, I collaborated with two other scientists to profile the gene expression in the tso1-1 root at single cell level. I compared the single cell RNA sequencing data of tso1-1 and wild type roots and identified molecular defects in the tso1-1 root vasculature. Correspondingly, the known regulators of vasculature development, the HD-ZIP III genes, are ectopically expressed in some of the vascular cells in the tso1-1 root. It suggests that the defects of root vasculature may be attributed to mis-expressed HD-ZIP III genes in the tso1-1 mutant. The HD-ZIPIII function was previously linked to their regulation of cytokinin biosynthesis genes, which were ectopically expressed in tso1-1 roots as revealed by our scRNA-seq data. Together, our data suggest that the over-production of cytokinin might be the cause of tso1-1 short root phenotype. In summary, my dissertation research revealed previously unknown links between TSO1 and cell cycle regulation in the shoot and root meristems as well as the molecular mechanisms of TSO1 function in the root vascular development at single cell level. These findings have furthered our understanding of how cell cycle regulation is integrated with plant development.
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    (2022) Guest, Johnathan Daniel; Pierce, Brian G.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Antibody recognition of antigens is a unique class of protein-protein interactions, and increased knowledge regarding the determinants of these interactions has advanced fields such as computational vaccine design and protein docking. However, the diversity and flexibility of antibodies and antigens can hinder generation of potent vaccine immunogens or prediction of correct antibody-antigen interfaces, slowing progress in the design of vaccines and antibody therapeutics. In this thesis, we present strategies to design vaccine candidates for a difficult viral target and describe expanded resources for benchmarking and training antibody-antigen docking and affinity prediction algorithms.We utilized rational design to develop candidate immunogens for a vaccine against hepatitis C virus (HCV), which represents a global disease burden despite recent advances in antiviral treatments. This design strategy produced a soluble and secreted E1E2 glycoprotein heterodimer with native-like antigenicity and immunogenicity by fusing ectodomains with a leucine zipper scaffold and a furin cleavage site. We developed additional constructs that incorporated synthetic or non-eukaryotic scaffolds or alternative ectodomains that included consensus sequences designed using a large reference database. Finally, we utilized previously published data on HCV antibody neutralization and E1E2 mutagenesis to predict residues that impact antibody neutralization and E1E2 heterodimerization, offering potential insights that can aid vaccine design. To improve our knowledge of and accuracy in modeling antibody-antigen recognition, we assembled a set of antibody-antigen complex structures from the Protein Data Bank (PDB) that expanded Docking Benchmark 5, a widely used benchmark for protein docking. These complexes more than doubled the number of antibody-antigen structures in the benchmark and, based on tests of current algorithms, highlight significant challenges for docking and affinity prediction. Building on this resource, we assembled and curated a dataset of ~400 antibody-antigen affinities and corresponding structures, forming an expanded and updated benchmark to guide ΔG prediction of antibody-antigen interactions. Using this dataset, we retrained combinations of terms from existing scoring functions and potentials, demonstrating that this resource can be used to improve antibody-antigen ΔG prediction. Overall, these findings can advance HCV vaccine design and antibody-antigen docking and affinity prediction, helping to better elucidate the determinants of antibody-antigen interactions and to better display vaccine immunogens for induction of neutralizing antibodies.
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    (2022) Iverson, Ethan; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The human airway epithelium represents the primary site of infection for many respiratory viruses, including influenza A virus (IAV). To safeguard this tissue and maintain the functionality of the lung, humans possess a two-layer, extracellular, mucus barrier composed predominantly of individual proteins termed mucins. Additionally, underlying epithelial cells produce interferons upon virus detection that promote the establishment of a local antiviral state through autocrine and paracrine signaling. However, despite these protective measures, IAV continues to cause significant annual morbidity and mortality across the globe. Therefore, we sought to further investigate how specific mucin molecules interact with IAV, and how interferon drives intrinsic antiviral defense in the context of a human airway epithelial (HAE) culture system. By utilizing fluorescently-labeled influenza virus particles we further elucidate the adhesive interactions between mucus and influenza virus while also detailing, for the first time, real-time IAV diffusivity within patient-derived mucus samples. These results reveal that the polymeric structure of mucus greatly influences the mobility of IAV within human secreted mucus. Additionally, we investigate the interaction between influenza virus and tethered mucin 1 (MUC1), finding that MUC1 expression is enhanced by virus-driven inflammation and interferon signaling. Moreover, by establishing a genetically-tractable airway epithelial model, we detail the protective role MUC1 plays in preventing the initial establishment and spread of influenza virus in HAE. Specifically, we find that the loss of MUC1 significantly enhances IAV uptake and spread. Finally, we observe that the directionality of IFN exposure at airway epithelial surfaces impacts the magnitude of protection against IAV and SARS-CoV-2. We then detail the cellular composition of our HAE culture system and define a shared IFN response profile across all HAE component cell types as well as cell type-specific interferon stimulated genes. Together our work provides novel insight into the innate and intrinsic anti-viral properties of the human airway epithelium.
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    (2022) Kelly, Jamie; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Programmed -1 ribosomal frameshifting (-1 PRF) is a molecular mechanism that redirects translating ribosomes into a new reading frame. It is widely used by RNA viruses to conserve genome space while expanding the viral proteome and it can help regulate gene expression in eukaryotic cells. Strict regulation of both programmed and non-programmed frameshift events are essential to translational fidelity. This dissertation explores the -1 PRF element of SARS coronavirus 2 (SARS-CoV-2) and the -1 PRF inhibitor, Shiftless. We comparatively analyzed the structural and functional conservation of -1 PRF elements in SARS-CoV and SARS-CoV-2. Both -1 PRF structure and frameshift efficiency were highly conserved between the two viruses and a small molecule effective against SARS-CoV -1 PRF significantly decreased frameshift efficiency in SARS-CoV-2. This suggests -1 PRF is an attractive antiviral target and could be a useful tool to combat the SARS-CoV-2 pandemic or future outbreaks of similar coronaviruses. The innate immune system targets viral frameshifting using an interferon-stimulated -1 PRF inhibitor called Shiftless (SFL) that binds, arrests, and terminates translation of -1 frameshifted ribosomes. We found that SFL is not only expressed in response to interferon but that it may have a role in general translational fidelity. SFL is constitutively expressed at low levels in human-derived cell lines and its effects are not limited to -1 PRF signals. Disruption of SFL homeostasis results in reciprocal 2-fold changes to recoding efficiencies in a panel of human and viral-derived translational recoding signals, decreases reporter gene expression, and decreases mRNA steady state abundances. Additionally, SFL over or under expression combined with knockdown of prominent ribosome-associated protein quality control (RQC) proteins reveals that SFL is epistatic to RQC. These results suggest that SFL has a role in general translational fidelity monitoring for spontaneously frameshifted ribosomes in addition to its role as a member of the innate immune response.
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    (2022) Mao, Xiaojing; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. In this dissertation, I study the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, I investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. Chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to early-stage epithelial development markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS. One important feature in both HGPS and normal aging is the elevated levels of Reactive Oxygen Species (ROS), which are generated from metabolic pathways to cause oxidative damage to macromolecules within the cells. Although peroxisomal bioreactions can generate free radicals as their byproducts, many metabolic enzymes within the peroxisomes play critical roles as ROS scavengers, particularly catalase. In this dissertation, I observed impaired peroxisomes-targeting protein trafficking, which suggested that the poorly assembled peroxisomes might cause high oxidative stress, contributing to the premature senescent phenotype in HGPS. I also investigated the ROS clearance efficiency by peroxisomal enzymes and found a significantly decreased catalase expression in HGPS. Furthermore, I evaluated the effects of two promising HGPS-treatment drugs Methylene Blue and RAD001 (Everolimus, a rapamycin analog), on catalase in HGPS fibroblasts. I found that both drugs effectively reduced cellular ROS levels. As a well-known antioxidant, MB did not affect catalase expression or activity. Interestingly, the RAD001 treatment significantly upregulated catalase activity in HGPS cells. This is the first characterization of peroxisomal function in HGPS and provides new insights into the cellular aspects of HGPS and the ongoing clinical trial.