Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item INVESTIGATION OF CYCLIC DINUCLEOTIDE HOMEOSTASIS AND THE HYDROLYSIS OF THEIR LINEAR INTERMEDIATES IN BACTERIA(2019) Weiss, Cordelia Anne; Winkler, Wade C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis of cyclic dinucleotides as signals is one strategy bacteria use to sense and adjust to environmental changes. Cyclases synthesize the cyclic dinucleotide, while phosphodiesterases cleave it to yield a linear diribonucleotide, which is recycled into monoribonucleotides by other enzymes. For many bacteria, cyclic di-GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile community. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. Bacillus subtilis is a model for the study of bacterial differentiation, yet how c-di-GMP functions in this organism is not fully understood. This work began with construction of a fluorescent reporter to measure c-di-GMP abundance in B. subtilis, which showed that c-di-GMP levels are strikingly different among differentiated subpopulations. These data highlight how single-cell approaches can be used to analyze metabolic trends within bacterial populations and demonstrate that for some bacteria, c-di-GMP levels are adjusted heterogeneously across bulk populations. The enzymes Orn, NrnA, NrnB, and NrnC have been proposed to act as general 3’-5’ exoribonucleases that preferentially process ‘short’ oligoribonucleotides. Intriguingly, Orn also performs a crucial role in c-di-GMP homeostasis by processing the pGpG generated from c-di-GMP production. To discover the molecular basis for Orn’s ability to ‘select’ short RNAs, and to elucidate the relationship between Orn and the diribonucleotide pGpG, we combined structural, biochemical, and in vivo analyses of RNA cleavage. These data reveal that Orn is not a general exoribonuclease of short RNA oligoribonucleotides, as previously believed, but instead acts as a dedicated ‘diribonucleotidase’. Our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of diribonucleotides, which affect cellular physiology and viability. Examination of the roles of NrnA and NrnB is underway. We conducted an initial study to determine if NrnA and NrnB are redundant proteins, as has been proposed, and if they might also act as ‘diribonucleotidases’. These data show that they exhibit different substrate preferences and that they may have unique cellular functions. Therefore this work changes the perception of the role(s) Orn plays and that a re-evaluation of ‘short’ RNases is needed.Item THE MECHANISMS AND ROLES OF POST-TRANSLATIONAL PROCESSING OF THE DROSOPHILA FIBROBLAST GROWTH FACTOR BRANCHLESS DURING DEVELOPMENT(2019) Sohr, Alexander Ronayne; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During embryonic development, cells communicate with each other to cooperate to form organized tissues. Cells spatiotemporally coordinate with each other by communicating with signaling proteins such as Fibroblast Growth Factor (FGF) that travel from source to target cells to activate various functions. To better understand cell communication during tissue morphogenesis, this study aimed to address a fundamental question: how different cellular and molecular mechanisms in signal-producing cells prepare and release signals at the correct time and location and at an appropriate level. This research focuses on the intercellular communication of the Drosophila FGF Branchless (Bnl) to elucidate this question. Bnl is dynamically produced in restricted groups of cells to induce morphogenesis of tracheal airway epithelial tubes. Tracheal cells receive the signal over distance by extending long receptor-containing filopodia, or cytonemes, to dynamically contact the Bnl-source. This work discovered two post-translational modifications of Bnl that regulate its polarized intracellular trafficking and cytoneme-mediated intercellular dispersal. During intracellular trafficking through the source cell Golgi network, Bnl is endo-proteolytically cleaved at a single site by the protease Furin-1. This cleavage activates polarized intracellular trafficking of the truncated signal exclusively to the surface of the source cells that faces the recipient tracheal cells. Thus, the intracellular cleavage acts as a switch to catalyze the efficient trafficking of the signal to the correct location from where cytonemes can subsequently receive it. Secondly, in the endoplasmic reticulum of source cells, Bnl is modified with a glycosylphosphatidylinositol (GPI) moiety at its C-terminus. This lipid moiety tethers Bnl molecules to the outer leaflet of the cell membrane, inhibiting its free release and ensuring signal exchange solely by direct physical contacts established by cytonemes. Therefore, this study discovered how Bnl is prepared by the source cells to ensure its subsequent target-specific intercellular dispersion through cytonemes. Conserved FGF family proteins are essential for regulating a broad spectrum of biological functions and defects in spatiotemporal levels of FGF signaling leads to severe diseases. Given the conservation of developmental signaling mechanisms in all organisms, the discovery of new regulatory mechanisms of FGF signaling has fundamental implications for understanding development and disease in humans.Item Novel Models for Studying Trophoblast Development and Placental Pathologies(2019) Pence, Laramie; Telugu, Bhanu; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Placental development begins in the mammalian blastocyst, when the first lineage specification event commits one cell population to making extraembryonic tissues, including the placenta, and commits another cell population to making the embryo proper. The mouse is an excellent animal model to study these early events and how the resulting placenta organ supports normal fetal development and a healthy pregnancy in the mother. The studies included in this Dissertation use the mouse to understand the role of long non-coding RNAs during early placental development, and to create a lineage biasing model that takes advantage of what is known about the first lineage specification event in mammals. Using expression analysis and the CRISPR/Cas9 system to create a knockout mouse strain, a placental-specific lncRNA was discovered and shown to be expressed in derivatives of the ectoplacental cone. Additionally, using the line age bias model to cause biased ablation of Hif1α in the placenta has revealed a role for fetal vs. placental contribution of resulting phenotypes.Item SYSTEMIC AND TRANSGENERATIONAL REGULATION OF GENE EXPRESSION BY SMALL RNAS IN C. ELEGANS(2019) Raman, Pravrutha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Development of an organism requires information contained minimally within a single cell. This information is inherited in two forms- the genome sequence and regulatory molecules. Little is understood about the types of the regulatory molecules inherited or the impact of parental experiences on them. However, environmental stimuli can alter gene expression without changing DNA sequence and these changes can be inherited suggesting heritable regulatory molecules are influenced by parental experience. Such changes could require communication of regulatory information between cells within an animal (systemic regulation) and across generations via germ cells (transgenerational regulation). Double- stranded RNA (dsRNA) introduced to an animal can silence a gene of matching sequence within that animal and this silencing can persist in progeny suggesting that RNA has the potential to transfer gene-specific regulatory information. Using RNA silencing in C. elegans, we identify conditions that facilitate systemic and transgenerational regulation of gene expression. Previous work suggested that two forms of dsRNA, short and long, could move between somatic cells to cause systemic silencing. However, we show that the movement of short dsRNA is not an obligatory feature of systemic silencing and that long dsRNA introduced by feeding likely enters every cell to cause silencing. Silencing by dsRNA can also be communicated to the germ cells, however this does not guarantee persistence of silencing in descendants. Even the same target sequence expressed from different genetic contexts shows varying susceptibility to transgenerational silencing. Most tested genes recover from silencing in a few generations suggestive of mechanisms that repair changes induced in ancestors. We characterize a unique gene that is exceptionally susceptible to transgenerational silencing that lasts for >200 generations and find that non-genomic signals mediate its expression pattern in every generation. A forward genetic screen to isolate mutants exhibiting re-activation of gene expression (Rage) after many generations of silencing revealed additional defects indicative of endogenous processes that utilize transgenerational silencing mechanisms. We speculate that homeostatic mechanisms that prevent or preserve induced changes maintain form and function across generations in living systems.Item NEISSERIA GONORRHOEAE MODULATES INFECTIVITY BASED ON PROPERTIES OF HUMAN CERVICAL EPITHELIA AND PHASE VARIABLE BACTERIAL SURFACE STRUCTURES(2019) Yu, Qian; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) infection in the human female reproductive tract causes various clinical outcomes, from no symptom to severe complications. The major barrier to a better understanding of GC infection in women is the lack of experimental system closely mimicking in vivo infection. Here, I developed a human cervical tissue explant model, which maintains the heterogeneity of the cervical epithelium. Using this model, my thesis research examined the impact of the heterogeneity of the cervical epithelium and the phase variation of GC surface structures on GC infectivity. My research revealed that GC preferentially colonize the ectocervix and the transformation zone (TZ), but exclusively penetrate into the subepithelial tissues of the TZ and endocervix. Pili are essential for GC colonization in all regions of the cervix. Expression of Opa isoforms that bind to the host receptors CEACAM (OpaCEA) enhances GC colonization in the ecto/endocervix but inhibits GC penetration into the endocervix. However, GC infectivity in the TZ does not respond to Opa phase variation, due to the low expression level and intracellular location of CEACAMs in the TZ epithelial cells. OpaCEA enhances GC colonization in the ecto/endocervix by inhibiting epithelial exfoliation and suppresses GC penetration into the endocervical subepithelium by inhibiting GC-induced disassembly of the apical junction. Opa-mediated modulation of GC infectivity depends on the immune receptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and its downstream phosphatase SHP. The effect of epithelial cell polarity on GC invasion was studied using a cell line model. My results show that GC invade more efficiently into non-polarized than polarized epithelial cells without changing the adhesion efficiency. Opa (phase variable) expression enhances both adhesion and invasion in both non-polarized and polarized cells. In non-polarized cells, Opa expression induces F-actin accumulation and microvilli elongation underneath GC microcolonies, suggesting an actin-mediated uptake of GC. In contrast, GC expressing no Opa reduce F-actin and demolish microvilli underneath microcolonies in both polarized epithelial cell line and endocervical epithelial cells potentially by increasing calcium flux, NMII activation and the redistribution of actin nucleation factor Arp2/3 from the apical surface. Taking together, my research demonstrates that both the heterogeneity of the cervical epithelium and the phase variation of bacterial surface structures regulate GC infectivity in the human cervix, either dominated by colonization or penetration, consequently influencing the clinical outcomes of the infection.Item Investigating the role(s) of mammalian heme transport by HRG1(2019) Pek, Rini; Hamza, Iqbal; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recycling of hemoglobin from damaged or senescent red blood cells (RBCs) contributes almost 90% of daily body iron requirements in humans for bone marrow erythropoiesis. Previously, our cell biological studies have shown that HRG1, a four transmembrane protein first discovered in C. elegans, facilitates the transport of heme within reticuloendothelial system (RES) macrophages during the turnover of RBCs, a process termed erythrophagocytosis. HRG1 transports heme from the phagolysosomes into the cytosol where heme is degraded to liberate iron for erythropoiesis. Here we show that mice deficient for HRG1 are defective in heme- iron recycling by RES macrophages, resulting in over ten-fold excess heme accumulation as visible dark pigments within lysosomal compartments that are 10- 100 times larger than normal. The sequestered heme is tolerated by macrophages through polymerization into crystallized hemozoin, a phenomenon typically observed in blood-feeding parasites as a detoxification method to protect against heme toxicity. HRG1-/- mice display ineffective bone marrow erythropoiesis which results in a reduction in hematocrit and extramedullary erythropoiesis in the spleen. Under iron- deficient conditions HRG1-/- mice are unable to utilize hemozoin as an iron source to sustain erythropoiesis, causing severe anemia and lethality. Our studies establish that polymerizing cytotoxic heme into hemozoin is a previously-unanticipated heme tolerance pathway in mammals.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.Item Orthologous Gene Swapping and Experimental Evolution Provide Novel Way to Study Essential Poxvirus Genes(2018) Stuart, Carey A; DeStefano, Jeffrey J; Moss, Bernard; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The transcriptional program of poxviruses is divided into early, intermediate and late phases enabled by a multisubunit DNA-dependent RNA polymerase and stage-specific transcription factors that recognize cognate promoters. Although promoter sequences are highly conserved among the different chordopoxvirus genera, the transcription factors exhibit considerable amino acid divergence that parallels the evolutionary distance of the host species. Thus, the large/small subunits of the intermediate transcription factors (ITFs) of salmon gill poxvirus, crocodilepox, canarypox, and myxoma have 23/29, 40/31, 51/38 and 58/65 % amino acid identity, respectively, to the vaccinia virus (VACV) orthologs. The purpose of the present study was to determine the functional interchangeability of the ITF subunits and their putative interactions with other elements of the transcriptional machinery. A quantitative readout of ITF function using firefly luciferase (Fluc) was obtained. The activity of the large subunit orthologs was greater than that of the small subunit orthologs, with both sets following the degree of sequence similarity in relation to VACV. The same pattern was obtained with both heterospecific (e.g., myxoma large and VACV small subunits) and homospecific (e.g., myxoma large and small subunits) pairings, suggesting inefficient interactions with other elements of the transcription system. When recombinant hybrid VACV expressing the Myxoma virus (MYXV) ortholog of the small subunit (A8) were blind passaged multiple times, their replicative abilities were enhanced. Complete genome sequencing of the virus populations revealed five mutations present in the two largest subunits of the viral RNA polymerase (RNAP) and two predicted expression-enhancing mutations around the translation initiation site of the MYXV A8 ortholog. Amplicon sequencing was used to quantify the frequency of each mutation in its respective population, which revealed that they increased as passaging occurred. This indicated a correlation with increased fitness, which then needed to be confirmed, so these mutations were all experimentally introduced into the original hybrid virus and demonstrated to enhance virus replication independently. These mutations were then characterized to determine their specific effects on the viral RNAP (vRNAP) and viral replication and transcription. This approach could have broader applications for studying essential genes in poxviruses and other viruses as well.Item PROGRAMMED TRANSLATIONAL RECODING SIGNALS AS A THERAPEUTIC TARGET AGAINST ALPHAVIRUSES(2018) Kendra, Joseph Aaron; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While infection from communicable diseases has posed a longstanding threat to human health throughout history, the modern realities of population expansion, global travel, and climate change have facilitated the rapid emergence and worldwide distribution of RNA viruses at an unprecedented scale. Of particular concern are the alphaviruses, mosquito borne viruses from the Togaviridae family. These viruses were previously relegated to rare outbreaks in isolated forested regions but have dramatically spread across the globe in the past decade. One of these viruses, Venezuelan equine encephalitis virus (VEEV), is a noted bioterror threat due to its ability for aerosol transmission and successful weaponization during the Cold War. While no FDA approved drugs exist against alphaviruses, their reliance on programmed translational recoding mechanisms to regulate gene expression presents a potential vulnerability for therapeutic exploitation. Two instances of translational recoding have been identified but poorly characterized in the alphavirus genome. The first is a termination codon readthrough (TCR) event required for expression of the alphavirus replicase. The second is a programmed -1 ribosomal frameshift (-1 PRF) that produces a C-terminally extended variant of viroporin 6K. In this work, the cis-acting RNA elements that mitigate alphavirus recoding were functionally and structurally characterized. The predicted TCR and -1 PRF sequences were cloned into dual luciferase reporter vectors and their ability to promote efficient recoding was verified in several mammalian cell lines. Chemical probing assays elucidated the presence of highly structured stemloop elements downstream of the alphavirus recoding sites, which function as a kinetic trap for elongating ribosomes. Notably, mutations that abrogate efficient -1 PRF not only attenuated pathogenesis of VEEV in mice, but also provided protective immunity to subsequent wild-type challenge. These findings suggest a novel approach to the development of a safe and effective live attenuated vaccine strategy against VEEV, closely related alphaviruses, and potentially all viruses that rely on translational recoding mechanisms for optimal gene expression.Item INVESTIGATION OF DEFECTIVE CELL SIGNALING CASCADE INVOLVED IN THE OSTEOGENESIS IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2018) Choi, Ji Young; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human bone homeostasis is maintained through constant bone remodeling, which balances bone formation by osteoblasts and bone resorption by osteoclasts. Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass that manifests in a high risk of fractures and an atypical skeletal geometry, suggesting impaired bone remodeling. HGPS is a premature aging disease caused by truncated lamin A that is permanently farnesylated. The mutant lamin A is referred as progerin. Several previous clinical reports discussed abnormal skeletal development of the children with HGPS, but the molecular mechanistic study on defective osteogenesis of HGPS stem cells need to be further elucidated. The major aim of my dissertation research is to investigate dysfunction in stem cell differentiation due to aberrant cell signaling in osteoprogenitor cells that express progerin. To achieve this aim, the study demonstrates both in vitro and in vivo models of HGPS to support defective mechanism of the canonical WNT/β-catenin pathway, seemingly at the level of efficiency of nuclear import of β-catenin and impaired osteoblast differentiation. Restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. In particular, HGPS patient-derived induced pluripotent stem cells (iPSCs)-osteoprogenitors and primary mesenchymal stem cells (MSCs) expressing the HGPS mutant progerin display defects in osteoblast differentiation, characterized by deficits in alkaline phosphatase activity and mineralizing capacity. Mechanistic investigation reveals that canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in nuclear active β-catenin protein levels and reciprocal aberrant cytoplasmic accumulation which causes reduced transcriptional activity for osteogenesis. Non-farnesylation of progerin in MSCs attains higher level of active β-catenin protein expression and consequently increasing the signaling, enhancing mineralization capacity and ameliorating the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. This study implicates β-catenin signaling cascade as a therapeutic target for restoring defective skeletal microarchitecture in HGPS. Given the fundamental nature of WNT/β-catenin signaling to stem cell renewal and lineage allocation, the findings from this dissertation may provide broader inferences for the treatment options in HGPS.