Cell Biology & Molecular Genetics Theses and Dissertations

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Mixture Models for Nucleic Acid Sequence Feature Analysis
(2023) Wang, Bixuan; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Signals in nucleotide sequences play a crucial role in interactions among macromolecules and the regulation of biological functional processes such as transcription, the splicing of messenger RNA precursors and translation. Recognition of signals in nucleotide sequences is the first step in functional annotation, which is critical for the identification of deleterious mutations and the identification of targets for disease treatment. One of the essential steps in gene expression, RNA splicing removes introns from newly transcribed RNA, ligating exons to generate mature RNA. Splicing involves the formation and recycling of the spliceosome, a large macromolecular complex whose assembly requires complex coordination by splicing factors through the recognition of RNA-protein binding sites. One potential method to reveal unknown subtypes of samples and identify distinctively distributed features is by applying a mixture model called the admixture model or Latent Dirichlet Allocation (LDA), which allows samples to have partial memberships of different clusters that can be interpreted for functional motif identification. By applying mixture models to RNA sequences, I found splicing signals such as the polypyrimidine tract and the branch point in intron sequences. Mixture models also showed motifs associated with reading frames from coding sequences, which further revealed potential coding regions from 5’ untranslated regions and long non-coding RNAs. Dynamic single-molecule imaging of nascent RNAs coupled with multiple genome-wide assays reveals that splicing happens far more often than expected, and partial intron removal can be captured prior to completion of the entire transcript. I hypothesize that the spliceosome progressively removes large introns in small pieces through 'recursive splicing' instead of removing the whole intron at once. However, the sequence features that distinguish sites of recursive splicing from canonical splice sites remain to be discovered. Here, I applied mixture models to sequences from human introns to identify sequence features associated with recursive splicing. This method helped me to recognize and visualize splicing signals from annotated intron sequences and identify potential coding sequences from human 5' untranslated regions and long non-coding RNA. After applying mixture models to the sequences surrounding recursive and canonical splicing sites, I found that transcripts where large introns can be recursively spliced can be distinguished from those without recursive splicing by the presence of CG-rich motifs flanking 5' splice sites upstream of first introns, and the absence of DNA methylation at these sites.In addition to applications of mixture models, I also explored RNA Bind-N-Seq data reflecting the binding activities of the splicing factor U2AF and found that the recursive 3' splice sites have higher U2AF binding affinities than the downstream canonical 3'SS. The observations suggest that, first, mixture models have the potential to identify functional motifs, including subtle signals in sequences such as the branch sites that only occur in a subgroup of introns. Second, the usage of recursive splicing sites is associated with sequence features in the first exons of the transcripts, suggesting a testable model for the regulation of recursive splicing in human introns.
HIGH RESOLUTION MODELING OF ANTIBODY AND T CELL RECEPTOR RECOGNITION USING DEEP LEARNING
(2024) Yin, Rui; Pierce, Brian G; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Antibodies and T cell receptors (TCRs) are crucial for the immune system's ability to recognize and combat pathogens and cancer cells. High resolution structures of antibody-antigen complexes and TCR-peptide-MHC (TCR-pMHC) complexes provide key insights into their targeting. This knowledge has enabled the structure-based design of vaccines against viruses and pathogens, and therapeutics against cancer, immunological disorders, and viral infection. However, the vast diversity of the immune repertoire, along with limited resources and time constraints, makes experimentally determining the structures of most antibody-antigen and TCR-pMHC interactions challenging. To support these experimental efforts, computational approaches have been developed to model the structures of these protein-protein interactions. Despite decades of development, an accurate predictive understanding of the structural basis of antibody and TCR targeting remains a challenge. Recently, deep learning algorithms have shown major promise in the field of molecular modeling, due to their ability to analyze and learn complex non-linear features underlying molecular systems. For my research, I harnessed the power of deep learning tools toward predictive modeling of antibody and TCR recognition. First, I examined the structural and physiochemical features underlying antibody-antigen recognition for antibodies that interact with the SARS-CoV-2 receptor-binding domain (RBD). Then, as a critical step toward the development of highly accurate modeling tools, I conducted a thorough benchmarking of the state-of-the-art deep learning algorithm, AlphaFold, in modeling protein-protein complexes. Focusing on antibody-antigen complexes, I identified critical areas where AlphaFold's modeling capabilities could be enhanced. Next, I developed improvements of AlphaFold to perform accurate modeling of TCR-pMHC complexes, leading to the TCRmodel2 algorithm, which is available to the community as a public web server. This was followed by an effort to explore the use of increased sampling to improve AlphaFold success, which generated near-native predictions for approximately half of antibody-antigen test cases and nearly all TCR-pMHC test cases. These advances in modeling accuracy constitute a leap forward in our predictive understanding of immune recognition and can serve as a step toward successful design of more effective vaccines and therapeutics.
TLR9 Activation as Immunotherapy in a Murine Model of Metastatic Lymphangioleiomyomatosis
(2024) Amosu, Oluwamayowa; Maisel, Katharina; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Pulmonary Lymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age, marked by abnormal growth of smooth muscle-like cells leading to cystic lung destruction. Rapamycin, the only approved treatment for LAM, slows disease progression but ~40% of patients have partial or no response to treatment. There is an urgent need for new treatments. Research shows that LAM has hallmarks of cancer, like expression of immune checkpoint receptors, and is responsive to immune checkpoint inhibition in mouse models. This suggests that other anti-cancer strategies could be effective in treating LAM. In this thesis, we investigated toll like receptor (TLR) activation using intranasal administration of CpG, a TLR9 agonist, as LAM immunotherapy. We used a mouse model of metastatic LAM to determine survival after biweekly intranasal CpG therapy (10µg/ 5µg) with and without systemic α-PD-1, rapamycin, or α-CD317 therapy. We used ELISA to measure the cytokine profile and flow cytometry to quantify cell populations and characterize differences in the immune response between CpG-treated and untreated LAM lungs. We found that CpG treatment enhanced median survival from 32 to 60 days in murine LAM. Survival benefit of CpG treatment was inversely dose-dependent and more effective during early stages of disease. CpG-treatment was synergistic with both α-PD-1 checkpoint inhibition and rapamycin, with survival increasing from 60 days (CpG) to 71 days (CpG + α-PD-1) and 100 days (CpG + Rapamycin). Histological analysis showed that CpG treatment decreased the LAM nodule burden but inevitably caused tissue inflammation. Efficacy of CpG treatment in LAM is facilitated in part by plasmacytoid dendritic cells through decreased regulatory T cell numbers, priming of Th17 cells, and increased secretion of inflammatory and cytotoxic cytokines by CD8 T cells. Our findings suggest that adjuvant immunotherapy, like CpG, may offer new treatment strategies for LAM that are compatible with the current standard of care, rapamycin.
AN INTERSECTING NETWORK OF REGULATORS IS REQUIRED FOR RNA SILENCING AND NUCLEAR INTEGRITY IN C. ELEGANS
(2024) Knudsen-Palmer, Daphne R; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Regulation of gene expression is required for an organism to develop, maintain homeostasis, and respond to environmental stimuli. While each cell in a multicellular organism contains the same genetic information, the epigenetic control of the expression of genes at different times is crucial for processes such as cell differentiation, division, and for allowing for cells to carry out different functions from one another. One type of such epigenetic regulation is mediated by small non-coding RNAs. Introduction of double-stranded RNA (dsRNA) and subsequent production of small interfering RNAs can result in sequence-specific mRNA silencing, creating the potential for highly specific therapeutics and pesticides. However, some targets are more easily silenced than others, and the mechanisms of silencing are not fully understood. Here we investigate regulators of small RNA-mediated silencing in the nematode C. elegans and find that they function in an intersecting network, allowing the potential for regulators to contribute to the silencing of any target. Quantitative modeling suggests that the production and turnover rates of a target at steady-state can affect the ease with which a target can be knocked down, and experimentally we found that changing the cis-regulatory sequences of a target can make it more susceptible to silencing. We found restricted production of RNA silencing intermediates, allowing for the recovery of a target in response to dsRNA, which we observed experimentally in non-dividing cells. In addition to roles in response to dsRNA, we report that disruption of small RNA-based regulation can result in germline nuclear defects. In the absence of the intrinsically disordered and perinuclear granule-forming protein MUT-16, some of the nuclei in the syncytial germline appear enlarged, suggesting that small RNA-based regulation may be playing an active role in maintaining nuclear size. Taken together, these findings suggest that (1) regulators of small RNA silencing can contribute to the silencing of all targets as part of an intersecting network, as opposed to operating in specialized pathways and (2) small RNA-based regulation is required for nuclear integrity, providing a paradigm for studying control of nuclear size, where enlarged nuclei can be compared with wildtype nuclei in a shared syncytium. We speculate that these findings will improve understanding of RNA silencing across species and provide insight into understanding how nuclear size is controlled, a fundamental ability of all eukaryotes.
Methods for Efficient Processing and Comprehensive Analysis of Single Cell Sequencing Data
(2024) He, Dongze; Patro, Rob R.P.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Over the past decade, the rapid development of single-cell RNA-sequencing (scRNA-seq) technology has revolutionized the understanding of cellular differentiation, heterogeneity, transcriptional dynamics, and, many other biological processes. Despite the explosive growth of data analysis methods that aid in biological discovery, there are still many unsolved questions in raw data processing (also known as preprocessing) of scRNA-seq data --- the procedure for analyzing the raw sequenced fragments to generate the quantitative measurements of gene expression. In this dissertation, we first describe a computational ecosystem we developed that provides an end-to-end pipeline for accurately and efficiently processing single-cell sequencing data. Then, we will discuss the computational and analytical challenges we found during the development of alevin-fry and the solutions we provided for tackling these challenges. Chapters 2 and 3 demonstrate the computational successes we achieved for single-cell data processing. In Chapter 2, we present a novel computational framework, alevin-fry, for rapid, accurate, and memory-frugal quantification of single-cell sequencing data. In Chapter 3, we discuss an augmented execution context, simpleaf, of alevin-fry that not only provides a simplified user interface to the alevin-fry framework, but also offers many high-level simplifications for single-cell data processing, and for assisting with data provenance propagation and reproducible analyses. Our results demonstrate that, with the help of alevin-fry and simpleaf, we are able to process single-cell data from both "standard'' chemistries, as well as from more advanced and complex data types, and achieve the same level of accuracy as existing best-in-class methods, while being substantially faster and more memory efficient. Chapter 4 introduces Forseti, a mechanistic model to probabilistically assign a splicing status to scRNA-seq reads. As the first probabilistic and mechanistic model for solving the ambiguity of splicing status in tagged-end, short-read scRNA-seq data, we show that Forseti can be used to accurately and efficiently infer the splicing status of scRNA-seq reads, and to help identify the correct gene origin for multigene-mapped reads. In Chapter 5, we describe the results of a comprehensive analysis of "off-target'' reads (reads whose mappings cannot be accounted for under the presumed and intended components of the underlying protocol) in scRNA-seq. Overall, our results suggest that off-target scRNA-seq reads contain underappreciated information about various transcriptional activities. These observations about yet-unexploited information in existing scRNA-seq data will help guide and motivate the community to improve current algorithms and analysis methods, and to develop novel approaches that utilize off-target reads to extend the reach and accuracy of single-cell data analysis pipelines.
INVESTIGATING GENE REGULATORY ARCHITECTURES THAT DICTATE TRANSGENERATIONAL EPIGENETIC EFFECTS IN C. ELEGANS
(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.
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.
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.
STUDIES ON VARIABILITY IN CANCER GENE EXPRESSION: FROM SINGLE PROTEINS TO POPULATIONS
(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.
CHARACTERIZATION OF THE SHEDDASE-MEDIATED RELEASE OF A DROSOPHILA MEMBRANE-ANCHORED FGF THROUGH CYTONEMES
(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.
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.
ANTIBACTERIAL MECHANISM OF PLANT-DERIVED PHENOLICS AGAINST SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
(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.
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.
DEVELOPMENT OF AN ACCELERATED ALZHEIMER’S DISEASE IN VITRO MODEL WITH THE ADDITION OF PROGERIN
(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.
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.
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.
IDENTIFICATION AND ANALYSIS OF GENETIC VARIANTS PERMITTING DISSEMINATED COCCIDIOIDOMYCOSIS
(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.
A MULTI-OMICS APPROACH TO CHARACTERIZING THREE HEALTH RELEVANT FUNCTIONS OF THE HUMAN GUT MICROBIOME
(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.
INVESTIGATING MOLECULAR MECHANISMS SPECIFYING DIVERSE ROSACEAE FRUIT TYPES THROUGH COMPARATIVE TRANSCRIPTOMIC ANALYSIS
(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.
A SYNTHETIC TMRNA PLATFORM FOR ELUCIDATION OF BACTERIAL PROTEOME REMODELING UNDER STRESS
(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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