Theses and Dissertations from UMD
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Item ZIKA VIRUS RECRUITS CELLULAR PROTEINS TO SUPPORT ITS REPLICATION(2024) Chang, Peixi; Zhang, Yanjin YJ; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Zika virus (ZIKV) is a mosquito-borne pathogen with a massive impact on global public health due to its association with severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. The ZIKV epidemic in the Americas in 2015-2016 and its continuing spread in tropical regions have highlighted the urgent need to understand the molecular mechanisms of viral replication to develop effective antiviral strategies. However, many aspects of how ZIKV interacts with host cells remain unclear. This study identifies and characterizes host factors contributing to ZIKV replication. First, karyopherin alpha 6 (KPNA6) contributes to ZIKV replication by interacting with the ZIKV non-structural protein NS2B. Characterization and mutational analyses identified two essential amino acid residues within NS2B that are critical for interacting with KPNA6. The substitution of these two residues of NS2B in an infectious ZIKV cDNA clone resulted in a significant reduction in viral replication, suggesting that the NS2B-KPNA6 interaction plays a vital role in the viral life cycle. Further studies found that KPNA6 contributes to ZIKV RNA synthesis. Mass spectrometry analysis of the KPNA6 interactome showed that KPNA6 interacts with proteins involved in RNA synthesis, suggesting that ZIKV recruits these factors by promoting KPNA6-binding. Second, this study developed an effective method to isolate the ZIKV replication complex, a membranous structure where viral RNA is synthesized. Proteomic analysis of the isolated complex led to identifying numerous host proteins associated with the viral replication machinery. Among these proteins, human replication factor C subunit 2 (RFC2), an accessory factor involved in DNA replication and repair, was discovered to facilitate ZIKV replication, making it a potential target for therapeutic interventions. In conclusion, this study reveals crucial host factors essential for ZIKV infection and replication and provides insights into the ZIKV-cell interactions. These findings offer new possibilities for developing novel antiviral strategies for controlling future viral outbreaks.Item ROLE OF TRPV4 MECHANOSENSING REGULATING MACROPHAGE FUNCTIONS IN INFLAMMATORY DISEASES(2024) Dutta, Bidisha; Rahaman, Shaik O; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are the most versatile cells of the hematopoietic system with roles in homeostasis, host-tissue development, innate immune response and tissue repair. Although the inflammatory activation and maintenance signals are tightly regulated, an imbalance in them results in unchecked inflammation resulting in cellular and tissue damage. Macrophages can affect most if not all phases of inflammation owing to their ability to adopt distinct functional states, secrete cytokines and phagocytose pathogens and debris. Recent evidence suggests that beyond biochemical cues, mechanical forces, like changing matrix stiffness in the tissue microenvironment, can shape immune cell functions involved in inflammation. These cells convert mechanical stimuli to biochemical signals in a process called mechanotransduction, regulating a multitude of cellular functions. However, knowledge about the molecular mediators of mechanotransduction and their functions in macrophage phenotypic and functional change is largely missing, highlighting the need for studying mechanosensory molecules such as ion channels. The present study focuses on the role of a specific mechanosensitive ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4), in the regulation of macrophage mediated inflammatory responses. Given its emerging role in inflammatory diseases like fibrosis, arthritis, foreign body response (FBR), TRPV4’s contribution to macrophage behavior in inflammation is of growing interest. Employing cellular imaging and molecular biology techniques such as Ca2+ influx assays, immunohistochemistry, immunoblotting, and single nuclei RNA sequencing we delineate mechanisms by which biomechanical stimuli-mediated activation of TRPV4 affects macrophage function. We elucidate TRPV4’s role in macrophage mechanotransduction, providing a mechanistic understanding of inflammatory disease pathophysiology which could lead to the development of potential therapeutics for disease intervention.Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.Item Impact of salinity on morphology, growth, and pigment profiles of Scenedesmus obliquus HTB1 under ambient air and elevated CO2 (10%) conditions(2024) Jiao, Fanglue; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Certain microalgal species tolerate high CO2 concentrations and proliferate faster with elevated CO2 than with ambient air. This feature makes them attractive for carbon sequestration, a tool for mitigating climate change due to increasing atmospheric CO2. Scenedesmus species are among these microalgae. Scenedesmus obliquus strain HTB1 is a microalgal strain isolated from the Baltimore Inner Harbor (brackish water) and has shown a faster growth with 10% CO2 compared to air. However, how HTB1 grows under different salinity and if the salt response is affected by elevated CO2 remains elusive. Two experiments were set up to address these questions. The first experiment tested the impact of salinity gradient (0, 17.5, 20, 22.5, 25, 27.5, and 30 ppt) on HTB1 under ambient air. With increasing salinity, HTB1 cells became smaller, and the cultures changed color from green to brown, yellowish brown, and then to pale white. The pigment analysis showed that HTB1 reduced several pigments (i.e. zeaxanthin, lutein, chlorophyll b) in response to salt stress. However, HTB1 produced higher concentrations of canthaxanthin under the salt stress. The growth of HTB1 decreased with increasing salinity and was inhibited when the salinity was greater than 22.5 ppt. In the second experiment, we compared the impact of salinity (0, 10, and 20 ppt) on HTB1 under air and 10% CO2, respectively. HTB1 cultures showed little color change with increasing salinity under 10% CO2. In contrast, the change of culture color from dark green to brown was observed with increasing salinity when HTB1 was grown with air. Interestingly, the growth of HTB1 was less inhibited with salt under 10% CO2 than with air, suggesting that elevated CO2 mitigates the salt stress of HTB1. Lutein and canthaxanthin increased with increasing salinity when HTB1 was grown with 10% CO2. Our results indicate that increased salinity affects the growth of Scenedesmus obliquus HTB1 more with air than with 10% CO2. This study provides insight into the impact of salt stress on algal morphology, growth, and pigment composition.Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item Three Clostridium species with Health Imparting Properties: In vitro Screening for Probiotic Potential(2024) Mochama, Victor Moronge; Obanda, Diana; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research aimed to unlock the probiotic potential of the genus Clostridium, which is often overshadowed by the predominant focus on pathogenic species. The study specifically targeted three promising Clostridium species: C. disporicum, C. celatum, and C. vincentii, which have shown potential in mitigating diet-induced obesity. Despite the challenges presented by the anaerobic growth requirements of Clostridium bacteria, the study capitalized on their capacity to sporulate. This characteristic provides an avenue to use them as probiotics, with resilient and dormant spores capable of surviving food processing and harsh stomach conditions. The resilience of these spores was examined by exposing them to oxygen, heat, gastrointestinal juices, and bile salts. The spores survived oxygen exposure, exhibited resilience to both bile salts and gastric acids, and demonstrated a survival temperature of 70°C. When exposed to suitable germination conditions in vitro, the spores successfully germinated. The study assessed the colonization potential of the bacteria by evaluating their adhesion ability, and all bacteria were found to have the adhesion ability. Furthermore, a safety assessment was conducted by examining hemolytic activity and antibiotic susceptibility to selected antibiotics. The bacteria were found to be susceptible to the antibiotics and did not exhibit hemolytic activity. Bile salt hydrolase (BSH) activity and antibacterial activities were also assessed, and none of the bacteria exhibited BSH activity or antibacterial activity. Antioxidant tests revealed that C. vincentii had the highest antioxidant properties. Assessment of anti-inflammatory properties showed that C. celatum downregulated the gene expression of cytokine inflammation markers IL-6, IL-1, and iNOS while upregulating TGF-β expression. In summary all 3 bacterial species showed good probiotic potential from the in vitro tests. Particularly the formation of resistant spores that later germinated to vegetative cells that produced molecular patterns with antioxidant and anti-inflammatory properties. This necessitates further studies on their probiotic properties.Item Characterization of chromatin assembly dynamics mediated by the histone H3.3 chaperone HIRA and implications of innate immunity during Human Papillomavirus infection(2024) Della Fera, Ashley Nichole; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The circular double-stranded DNA genome of Human papillomavirus (HPV) is chromatinized throughout its viral lifecycle and relies on numerous host chromatin assembly processes, epigenetic modifications, and immune evasion to ensure genomic stability and productive infection. Despite its chromatinization, the HPV genome remains susceptible to innate immune pathways that sense and respond to foreign DNA. In this work next generation sequencing (RNAseq) was utilized to profile changes in the host transcriptome following cellular differentiation and HPV infection in keratinocyte cell lines. Global alterations in keratinocyte differentiation were observed upon HPV infection, and unexpectedly, upregulation of innate immune signaling upon differentiation. Recent findings indicate that packaged HPV genomes are enriched in histone H3.3. Notably, the replication-independent histone H3.3 chaperone HIRA has been implicated in several pro- and anti-viral responses, but its function during HPV infection has yet to be elucidated. Using in-situ approaches, the role of HIRA during the late phase of the HPV lifecycle was evaluated, which showed that HIRA and other chromatin assembly factors localize to sites of HPV replication. Here the requirements for this localization were further characterized, and the impacts of HIRA on HPV genome amplification and viral transcription during the late stage of the HPV life cycle were assessed. Moreover, histone H3.3 phosphorylated at serine 31 was shown to be highly associated with HPV replication factories. HIRA, in part through association with the PML nuclear body associated protein Sp100, has also been reported to promote innate immune responses following infection with other DNA viruses. Here, HIRA localization to PML-NBs was identified to increase following stimulation with IFN in an Sp100-dependent manner. However, while Sp100 is required for localization of HIRA at PML-NBs, it was not required for HIRA localization at sites of HPV replication. In summary, this work highlights the broad changes in the host transcriptome following cellular differentiation and HPV infection, elucidates a previously undescribed role for histone H3.3 chaperone HIRA during the late phase of the HPV life cycle, and further characterizes the relationship between HIRA and Sp100 at PML-NBs.Item USING DNA LOOPING PROTEINS TO ENHANCE HOMOLOGY DIRECTED REPAIR IN VIVO FOLLOWING A CAS9 INDUCED DOUBLE STRAND BREAK(2024) Ferencz, Ian Theodore; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genome engineering methods that start with a CRISPR/Cas9 targeted genomic DNA double strand break proceed through cellular DNA repair mechanisms after the induction of the break. Imprecise nonhomologous end-joining (NHEJ) is useful for knockouts, but precise homology-directed repair (HDR) is necessary for gain of function changes. NHEJ tends to be more efficient, so directing the cell to knock in a precise sequence via HDR is an active area of research. The system we have designed uses a bivalent protein to recruit HDR donor DNA to the site of a specific DNA double strand break induced by a Cas9/sgRNA nuclease. Previously described leucine zipper dual-binding (LZD) proteins areused because they are small and stable. The system was designed to reduce the effort needed for screening, shorten the time required for the repair process, and/or decrease the amount of donor DNA needed, reducing potential off-target effects. We developed a model system in Saccharomyces cerevisiae to measure gene disruption and HDR frequencies in yeast that contain combinations of non-replicating donor DNA plasmid or linear DNA, expression plasmids of four LZD variants, and a plasmid expressing Cas9 and an sgRNA targeting either the AGC1 or ADE2 genes. The donor DNA includes a gene coding for G418 resistance in yeast. It also includes an INV-2 site recognized by the C-terminal DNA binding domain of LZDs adjacent to suboptimal regions of homology to the target gene. The N-terminal DNA binding domain of the LZDs recognizes an endogenous CREB site near the target gene. The desired recombinants are scored by their inability to grow on acetate as a sole carbon source (for AGC1) or their red color (ADE2), accompanied by resistance to G418. We believe that LZD enhancement can become a simple and valuable adjunct to any other method of improving the efficiency of HDR, in any system. We were able to show that the inclusion of LZD73 in recombination experiments increased the number of colonies presenting with the desired phenotype and genotype nearly eight-fold in the absence of a designed DNA break. We also provide evidence suggesting that the presence of LZD73 has a slight positive effect on the efficiency of Cas9 targeting. Desired recombinants were recovered after Cas9/sgRNA cleavage in an experiment where there was no apparent recombination in the absence of LZD73. Future work on this project includes optimization of the homologous sequences to improve background recombination so a more quantitative measure of the improvement observed in the presence of LZD proteins. This work can be transferred laterally to enhance other recombination-based methods in other organisms: the LZD proteins could be analogous to an adjuvant that increases overall efficiency.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.