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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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.
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.
A NOVEL IXODES SCAPULARIS PROTEIN DICTATES TICK HEMATOPHAGY AND CUTICLE INTEGRITY, IMPACTING TICK DEVELOPMENT
(2023) DUTTA, SHRABONI; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Ticks are prevalent throughout the world and are capable of transmitting a variety of pathogens (e.g., bacteria, protozoa, and viruses) to humans. Incidence rates for tick-borne diseases (TBD) are also increasing globally, and effective vaccinations to combat tick infestations and TBD transmission remain a critical unmet need. Of the six major tick genera that spread human illnesses worldwide, Ixodes ticks are the most prevalent. Specifically, Ixodes scapularis (also known as the blacklegged or deer tick) is an obligate blood-feeding arthropod that transmits several human and animal pathogens that include Borrelia burgdorferi sensu lato complex – the causative agent for Lyme disease. Unlike many hematophagous insects and soft ticks, I. scapularis (hard ticks) remain attached to their hosts for several days and are capable of uptaking bloodmeals that are 100 times greater than their initial body weight. A large and nutrient-dense bloodmeal is essential for their sub-adult and adult development processes and fecundity. However, the molecular and cellular processes that regulate tick blood feeding (hematophagy) and development have not been extensively elucidated. Therefore, our major objective is to characterize tick molecular components that are critical in the tick parasitism and life cycle in order to develop new strategies to combat tick infestations and spread of tick-borne diseases. Herein, we describe the structural and functional properties of a newly identified I. scapularis protein isolated from the partially fed nymphal ticks. Although the protein displays minor homology to proteins of known functions, structurally, it resembles some features of arthropod Odorant Binding Proteins (OBP). Therefore, we refer to this protein as, Ixodes Gut OBP (IGOBP). We show that the knockdown of IGOBP via RNA interference in ticks results in impaired blood feeding (hematophagy) and significantly decreases their post-fed weights. In addition, systemic IGOBP knockdown gives rise to aberrant phenotypes, significantly reduces tick molting rate, and compromises the structural integrity of the cuticle, specifically the flexible alloscutum components. Notably, IGOBP knockdown has profound effects on the molting efficacy and fitness of females than males. This is likely due to the fact that female adults consume a greater volume of bloodmeal than male adults, necessitating a more pronounced expansion of the alloscutum. Subsequently, our RNA sequencing data identifies multiple genes whose expressions are regulated by IGOBP. The underlying mechanism of possible IGOBP or associated gene functions may aid in identifying future targets for anti-tick vaccines. In summary, our studies characterized a novel I. scapularis protein revealing that the protein is essential for tick hematophagy and development. To the best of our knowledge, this is the first characterization of a tick odorant-binding protein (OBP), using structural and functional genomic tools that unearthed the unique and possibly multifunctional role of IGOBP in vector biology and parasitism. We anticipate that the presented data will enhance our fundamental understanding of tick biology and contribute to the development of potential anti-tick measures.
Role of Transient Receptor Potential Vanilloid 4 (TRPV4) Calcium-permeable Channels in Fibro-inflammatory Diseases
(2021) Goswami, Rishov; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Tissue fibrosis and foreign body response (FBR) have emerged as two major public health problems globally over the last few decades. While fibrosis is an outcome of a dysregulated wound healing process, FBR, a chronic inflammatory disease, develops when the body responds and reacts to the implantation of biological materials. Interestingly, recent studies have associated these non-specific inflammatory diseases with altering stiffness although the exact underlying mechanisms by which mechanical cues can regulate the diseases remain poorly understood. The objective of this thesis work is to determine how the changing of tissue stiffness and implant rigidity mediates disease progression of fibrosis and FBR respectively. Here we identify a novel role of a polymodal mechanosensitive calcium channel, Transient Receptor Potential Vanilloid 4 (TRPV4), as a potential cell membrane receptor/channel in the pathophysiology of FBR and skin fibrosis associated with Scleroderma, a multisystem idiopathic fibro-inflammatory connective tissue disorder. Our results showed that TRPV4 is over expressed in fibrotic skin tissue and colocalize with alpha-smooth muscle actin (a-SMA), a common myofibroblast marker. Using mouse model, we demonstrated that TRPV4 knockout mice are protected from bleomycin-induced skin fibrosis development. Additionally, in a separate mouse model, we showed that genetic ablation of the TRPV4 channel protects mice from implantation-induced macrophage foreign body giant cell (FBGC) formation, macrophage accumulation, and FBR development to biomaterials. The results of our studies also determined an essential role of TRPV4 for macrophage fusion and the mechanism by which TRPV4 and matrix stiffness leads to cytoskeletal remodeling through a feed-forward functional interaction generating cellular force to modulate FBGC formation. We also identified a mechanosensing domain of TRPV4 which is crucial for FBGC generation. Altogether, the results presented in this thesis suggest TRPV4 as a potential regulator of stiffness-dependent fibrosis and inflammation development, and multinucleated FBGC formation. The results of this thesis work proposes that interaction between TRPV4 and substrate stiffness leads to cytoskeletal remodeling and cellular force generation to modulate FBGC formation under FBR. Overall, the work presented in this thesis provides a better understanding about the role of mechanosensitive calcium channel TRPV4 in the regulation of fibro-inflammatory diseases and highlights the possibilities of therapeutically targeting of this channel for disease management.
Identification and functional analysis of a biflavone as a novel inhibitor of TRPV4-dependent atherogenic process in macrophages
(2021) Alharbi, Mazen Obaid; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cardiovascular disease is the major cause of death throughout the world. Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity throughout the world. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden macrophage foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Transient receptor potential vanilloid 4 (TRPV4), a non-selective Ca2+-permeant ion channel and a mechanosensor, is widely expressed in diverse cell types including macrophages. Accumulating reports from our laboratory and others on TRPV4 recognized this plasma membrane receptor/channel as an essential modulator of various physiological functions in cardiac, pulmonary, urinary, skeletal, digestive system, and central and peripheral nervous systems. Thus, it is expected that aberrant regulation of TRPV4 activity may lead to multiple pathological conditions such as cardiovascular disease, pulmonary disease, inflammation, neurological disorders, inflammatory bowel disease and wound healing. Previous studies by our group and others have reported that TRPV4 can be activated by numerous mechanical and biochemical stimuli including shear stress, osmolarity, temperature, and growth factors, as well as by alterations in matrix stiffness in vitro and in vivo. Recently, we reported that oxidized low-density lipoprotein-mediated and matrix stiffness-induced macrophage foam cell formation, a critical pathological process in atherosclerosis, is regulated in a TRPV4-dependent manner. Given that TRPV4 is a mechanosensitive channels and mechanical factors like hypertension, disrupted laminar flow of blood, and matrix stiffening are recognized pro-atherogenic factors, makes TRPV4 an important target for therapeutic intervention of atherosclerosis. The objectives of this proposal is to: i) identify natural inhibitor (s) of TRPV4 utilizing a fluorometric imaging plate reader-supported Ca2+ influx assay, ii) functionally characterize the identified compound, and iii) determine the mechanisms by which the identified compound blocks pro-atherogenic/inflammatory TRPV4 activity in macrophages. We expect that the results of this study may strengthen the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.
MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION
(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.
THE ROLE OF ESE-1 IN NON-SMALL CELL LUNG CANCER (NSCLC) CELLS
(2020) Lou, Zhiyuan; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lung cancer is the most life-threatening cancer in the world. The identification of the effective molecular target is essential for lung cancer prevention and therapy. Epithelial Specific ETS-1 (ESE-1) is a transcription factor associated with several types of cancer. However, the significance of ESE-1 in human non-small cell lung cancer (NSCLC) remains unclear. The objective of this dissertation was to investigate if ESE-1 expression influences the tumorigenic and metastatic activity of human non-small cell lung cancer (NSCLC) and to explore the mechanisms associated with tumorigenesis and epithelial mesenchymal transition (EMT). Overexpression of ESE-1 repressed the anchorage-independent growth of human NSCLC cells (H1299 and H1703) and led to an increase of G1 arrest and apoptosis, additionally, to repress invasion and migration. Xenograft study indicated that ESE-1 expression inhibited the formation and development of the tumor. In terms of mechanistic studies, overexpression of ESE-1 downregulates NF-κB transcriptional activity in both H1299 and H1703 cells. The downregulation might be associated with inhibition of NF-κB-p65 phosphorylation. ESE-1 is a downstream target of TGF-β-stimulated EMT. Downregulation of ESE-1 by TGF-β is dependent on Smad2/3, but not on Smad4 and other alternative pathways, including ERK, p38 MAPK, JNK, RAS, GSK3, PI3K, NF-ĸB, CDC42, PKC, and Rock signaling. We identified two putative Smad responsive elements (SRE) in the ESE-1 promoter. After cloning internal deletion and point mutated clones lacking distal and proximal SRE, which were localized at the distal and proximal regions of the ESE-1 promoter between -1500 to -713, the double mutation responsible for ESE-1 transcriptional downregulation with TGF-β induction. Moreover, EMT downstream target Snail reciprocally interacts with ESE-1. Our findings indicate that ESE-1 serves as a tumor repressor in ESE-1-null NSCLC cells, and we propose a potential use of ESE-1 as a target of lung cancer chemoprevention.
TRPV4, A CALCIUM-PERMEABLE CHANNEL, PLAYS A ROLE IN MATRIX STIFFNESS INDUCED MACROPHAGE POLARIZATION
(2020) Dutta, Bidisha; Rahaman, Shaik Ohidar; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Phenotypic polarization of macrophages is deemed essential in innate immunity and various pathophysiological processes, but little is known about how mechanical forces like matrix stiffness regulate the polarization and the associated signaling mechanisms. Here we report that calcium ion channel Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive receptor/channel, mediates the substrate stiffness-induced macrophage polarization. Using atomic force microscopy, we show that fibrosis-induced tissue stiffness is dependent on TRPV4. M1 macrophages were found to be the predominant macrophage subtype in stiffer tissues and loss of TRPV4 significantly decreased the level of M1 macrophages. These findings were further validated by our in vitro assays indicating that increase in substrate stiffness leads to an increased secretion of M1 proinflammatory mediators, which is further enhanced by the addition of soluble factors. Taken together, these findings provide new insights about the role of TRPV4 in matrix stiffness-induced macrophage polarization that can be explored in tissue engineering and in the development of targeted therapeutics.
Identification of ESE-1 as a novel molecular target of chemopreventive agents for colon cancer prevention
(2019) Lee, Jihye; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Epithelial specific ETS-1 (ESE-1) is one of the E26 transformation-specific transcription factor superfamily and could be considerable interest as a possible target for regulating diverse types of human cancer. Despite its clinical importance, the reported biological role of ESE-1 on cancer development and progression are still controversial and its underlying mechanisms of tumorigenesis remains unclear. The objectives of this dissertation are to elucidate the role of ESE-1 in tumorigenesis. With the evidence in cancer phenotypes, the underlying mechanisms of ESE-1 in colon cancer was also investigated. ESE-1 knockout mice increased azoxymethane (AOM) -induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF) compared to wild type mice. Overexpression of ESE-1 suppressed anchorage-independent growth and migration/invasion in human colon cancer cells and while knockdown of ESE-1 reversed anti-cancer activity. Full length ESE-1 was abundantly found in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) decreased the amount of nuclear ESE-1. Three lysine residues (318KKK320) in the NLS2 were critical for nuclear localization of ESE-1 and mediates tumor suppressive activity of ESE-1 through reduced beta-catenin transcriptional activity. We identified two anti-cancer natural compounds, epigallocatechin-3-gallate (EGCG) and patchouli alcohol as ESE-1 inducers. Both EGCG and patchouli alcohol increased expression of ESE-1 protein and mRNA in human colon cancer cells. Patchouli alcohol showed reduced the number of tumors and tumor load in Apcmin/+ colon cancer animal model although protein expression level of ESE-1 did not show significant difference. These findings suggest a potential use of ESE-1 as a novel and potential molecular target of natural anti-cancer phytochemicals for colon cancer prevention.
Investigating Copper Acquisition And Delivery via Transporters and a Pharmacological Chaperone in Copper-Deficient Worms and Mice
(2019) Yuan, Sai; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Copper (Cu) is a key micronutrient required for a variety of essential biochemical pathways. Systemic or tissue-specific Cu-deficiencies, caused by insufficient dietary Cu uptake or mutations in Cu transporting genes, result in impaired growth, neuropathy, ataxia, hypopigmentation, osteoporosis and anemia-like symptoms in mammals. How organisms regulate Cu homeostasis at the systemic levels in response to Cu deficiencies remain elusive. In this study, we use Caenorhabditis elegans (C. elegans), a genetically tractable, multi-tissue metazoan to explore Cu homeostasis and investigate these unknowns. The high-affinity Cu transporters encoded by CTR family genes are required for dietary Cu uptake and maintaining systemic Cu balance from yeast to mammals. However, little is known about Cu acquisition mechanisms in C. elegans. We identified ten CTR ortholog genes in C. elegans; of these, chca-1 was functionally characterized. Cu availability regulates transcription of chca-1 in both the intestine and hypodermis, and chca-1 is essential for normal growth, and reproduction in the worm. Additionally, altered Cu balance caused by the loss of CHCA-1 results in defects in Cu-responsive avoidance behavior. Identification of this CTR-like gene in C. elegans, which appears to be essential for normal Cu homeostasis in the worm, illustrates the importance of Cu delivery via CHCA-1 for normal metazoan development and behavioral phenotypes. In addition, we show that a Cu-binding pharmacological chaperone, elesclomol (ES), fully restores the developmental defects and Cu deficiencies in chca-1-depleted worms, as well as the lethality in worms lacking cua-1 expression (Cu exporter ATP7A ortholog), suggesting ES is able to efficiently deliver Cu from dietary sources to peripheral tissues through the intestine in C. elegans. Our study was further expanded to mammalian models such as cardiac-specific Ctr1-depleted (Ctr1hrt/hrt) mice. We found that ES administration fully restores the postnatal lethality, developmental defects and cardiac hypertrophy found in Ctr1hrt/hrt mice, as well as rescuing the secondary systemic Cu homeostasis responses, including aberrant ATP7A protein levels in the liver and intestine. Moreover, ES shows the potential ability to transport Cu across the blood-brain-barrier in in vitro studies. These results illustrate the capability of ES to rescue systemic Cu deficiency in worms and mice, independent of the presence of functional Cu transporters, and shed light on the therapeutic usage of ES in Cu-deficient human diseases.

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