College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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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 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 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.