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 MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.Item 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.Item 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.Item SUBAQUEOUS SOILS OF CHESAPEAKE BAY: DISTRIBUTION, GENESIS, AND THE PEDOLOGICAL IMPACTS OF SEA-LEVEL ALTERNATIONS(2020) Wessel, Barret Morgan; Rabenhorst, Martin C; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Soils and sediments make up a substantial portion of the resource base that supports human societies and other life on Earth, yet in the subaqueous environment our understanding of these materials pales in comparison to our understanding and management of upland soils. We must develop an understanding of how subaqueous soils/sediments are distributed, how they form and change over time, and how they will be impacted by rising sea-levels as a result of climate change if we are to wisely manage these resources. The goal of this study is to improve this understanding in Chesapeake Bay subestuaries. The Rhode River subestuary was first surveyed to identify rates of bathymetric change in these settings and to characterize the common material types found in these settings. Bathymetric change was evaluated using hydrographic surveys dating back to 1846, and though the river bottom does change slowly, it has been more or less stable during the years evaluated. Several types of morphologically distinct materials make up the soil profiles in Rhode River. Materials highest in organic matter are easy to identify in the field, and commonly become ultra-acidic if disturbed. Also present were submerged upland soils, colored and structured like soils in the surrounding landscape. To better understand the impacts of submergence on these materials, a sampling campaign was conducted on shallow marine sediments, reclaimed land, and restored aquatic environments under both seawater and freshwater. This demonstrated that shallow marine sediments develop upland soil features and biogeochemical characteristics within 150 years of drainage, and that these characteristics do indeed persist in the subsoil two years after submergence. Topsoil changes more radically, releasing anomalous amounts of Fe while accumulating anomalous amounts of reduced S minerals, a process exacerbated by seawater flooding. Using these results, a soil-landscape conceptual model was developed and used to predict subaqueous soil distribution in the West River subestuary. These predictions were evaluated with a sampling campaign, and found to be significant. This model can now be used in other subestuaries to quickly and efficiently survey subaqueous soils, supporting the development of future land-use interpretations in these environments.Item 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.Item Quantifying the Ecosystem Metabolism of a Tidal Estuary as a Consequence of Aeration(2019) Gotthardt, Zachary; Harris, Lora A; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)As anthropogenic activity affects shallow estuaries it is imperative to quantify how these systems respond to changing conditions. Ecosystem metabolism is an integrative, metric to measure how ecosystems change, and can act as the focus of comparative experiments. We leveraged an aeration system, to examine the ecosystem metabolism of the estuary through comparative experiments. The aeration system allows us to study a normoxic, eutrophic ecosystem. Chapter 1 explains the causes and effects of eutrophication, with an emphasis on the connection between hypoxia and eutrophication. In chapter 2, we describe an experiment focused on quantifying the ecosystem metabolism in a tidal, eutrophic estuary where engineered aeration has been operational since the 1980s. The aeration system provides an ideal site for addressing some of the difficulties inherent to studying eutrophication. In our experiments, we observed evidence of chemoautotrophy when the aerators were operational. Bottle methods and open water methods provided conflicting results.Item TRPV4, a calcium-permeable channel, regulates oxidized LDL-induced macrophage foam cell formation(2017) Goswami, Rishov; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Accumulation of lipid-laden “macrophage foam cell” in the arterial wall is the hallmark of atherosclerosis that leads to the highest number of cardiovascular disease-related deaths in United States. Membrane scavenger receptors such as SR-A, and CD36 play important role in controlling oxidized low-density lipoprotein binding and uptake, and, thereby, in macrophage foam cell formation. Recent studies also put emphasis on the role of mechanical factors, such as matrix stiffness, in the regulation of macrophage function and atherogenesis. However, the identity of a plasma membrane mechanosensor and the underlying mechanisms that may promote atherogenesis is yet to be identified. We have found that a calcium-permeable plasma membrane protein TRPV4, a mechanosensor, may play an essential role in regulating macrophage foam cell formation, a critical process in atherosclerosis. We have also found that TRPV4 is essential for oxLDL uptake, but not for its binding. Altogether, herein, we demonstrate that TRPV4 plays a critical role in macrophage-foam-cell formation by regulating oxLDL uptake in cells.Item The Role of Adipocyte Lipid Droplet Lipolysis in Thermogenesis and Metabolic Health(2017) Shin, Hyunsu; Yu, Liqing; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)My thesis was focused on the role of Comparative Gene Identification-58 (CGI-58)-mediated adipocyte lipid droplet (LD) lipolysis in thermogenesis and metabolic health. LD lipolysis in energy-dissipating brown adipose tissue (BAT) was believed to play a central role in cold-induced non-shivering thermogenesis, but this concept has not been tested in whole animal in vivo. We created a mouse line that lacks BAT CGI-58, a coactivator of Adipose Triglyceride Lipase (ATGL) that initiates the first step of cytosolic LD lipolysis by cleaving a fatty acyl chain from a triglyceride (TG) molecule. We found that BAT-specific CGI-58 knockout (BAT-KO) mice defend against the cold normally when food is absent, despite a defect in BAT LD lipolysis. Interestingly, BAT-KO versus control mice display higher body temperature when food is present during cold exposure. This cold adaptation in BAT-KO mice is associated with increases in BAT glucose uptake, insulin sensitivity, white adipose tissue (WAT) browning, energy expenditure, and sympathetic innervation. To identify the sources of fuels for thermogenesis of BAT-KO mice in the fasted state, we hypothesized that WAT lipolysis is a major source of thermogenic fuels during fasting. To test this hypothesis, we genetically inactivated CGI-58 expression in the whole fat tissues (both BAT and WAT) of mice (FAT-KO mice). We observed that FAT-KO mice are cold sensitive when food is absent, but tolerate cold normally when food is present, demonstrating that WAT lipolysis is essential for cold-induced thermogenesis during fasting and that dietary nutrients can substitute WAT lipolysis for fueling whole-body thermogenesis. Intriguingly, FAT-KO mice display a dramatic increase in cardiac glucose uptake under both basal and insulin-stimulated conditions, which is associated with significant increases in glucose tolerance, insulin sensitivity, and cardiac expression levels of natriuretic peptides. In conclusion, our studies demonstrate that 1) BAT LD lipolysis is not essential for cold-induced whole-body thermogenesis due to increased BAT uptake of circulating fuels and WAT browning; 2) WAT lipolysis is required for fueling thermogenesis during fasting; and 3) Adipose lipolysis is critically implicated in whole-body energy metabolism and cardiac function.Item Denovo synthesized fatty acids as regulators of milk fat synthesis(2011) Vyas, Diwakar; Erdman, Richard A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objectives of the dissertation research were to determine the role of denovo synthesized fatty acids (DNFA) in the regulation of milk fat synthesis. Milk fat responses to increasing amounts of short- and medium-chain fatty acids (SMCFA), added in the proportion as synthesized denovo, were studied in lactating dairy cows. The results showed a significant linear increase in milk fat concentration with SMCFA supplementation. However, milk fat yield was similar for all treatments. A subsequent study was aimed at increasing the availability of SMCFA during trans-10, cis-12 CLA-induced milk fat depression (MFD) in lactating dairy cows to determine whether SMCFA can rescue part of CLA-induced MFD. Post-ruminal infusion of butterfat (BF) was used as a source of SMCFA. The BF treatment was compared to a mixture of fats containing only the long-chain FA (LCFA) with or without trans-10, cis-12 CLA infusion. Milk fat content and yield were significantly reduced with trans-10, cis-12 CLA. However, increased availability of SMCFA with BF infusion had no effects on milk fat yield and concentration. Trans-10, cis-12 CLA significantly reduced the mRNA expression of transcription factor SREBP-1c along with its downstream targets including ACC,FASN, LPL, SCD and AGPAT. The increased availability of SMCFA had no effect on either lipogenic gene or protein expression suggesting that nutritional manipulation was not sufficient to rescue trans-10, cis-12 CLA-induced MFD. Finally, the effects of combination of a Rosiglitazone (ROSI), a PPAR-γ agonist, and trans-10, cis-12 CLA were examined on mammary and hepatic lipogenesis in lactating mice. Mammary lipogenesis was significantly reduced with trans-10, cis-12 CLA, reducing the milk fat content and mRNA expression of lipogenic transcription factors SREBP1-c and PPAR- γ. Trans-10, cis-12 CLA significantly increased hepatic lipid accumulation, while the mRNA expression of SREBP1-c and PPAR- γ were not altered. On the contrary, ROSI had no effects on mammary lipogenesis. However, ROSI significantly rescued trans-10, cis-12 CLA-induced hepatic steatosis.