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Graduate Student Profile

Abstract: The consumption of tobacco products is a known contributor to a variety of health conditions and disease pathologies including cardiovascular and lung diseases. In addition to these well-known pathological effects of tobacco use, it has been established recently that tobacco consumption as well as nicotine consumption alone results in distinct, dysbiotic perturbations to the enteric microflora of the gastrointestinal tract. Previous research into the intestinal microbiome has demonstrated that these microbes are active contributors to the maintenance of a homeostatic and tolerogenic environment within the host. In fact, the presence of our enteric microflora is necessary for the proper development of a variety of critical internal systems, namely the immune and nervous systems. It is well known in literature that nicotine/tobacco consumption alters the intestinal microbiome in a dysbiotic fashion resulting in the development of inflammatory conditions, diseases of the digestive tract, and the development of stress-related disorders and depression. However, what has yet to be elucidated are the mechanisms by which nicotine/tobacco use alters the composition of the microflora to these dysbiotic states. In this dissertation, our research efforts focused on elucidating the mechanisms by which nicotine use alters the microbial composition of the gastrointestinal tract and what effect these compositional changes have on immune function. In order to accomplish our experimental goals we studied 1) the inhibitory properties of nicotine alongside other similar phytochemicals. This work revealed that nicotine does possess antimicrobial properties however, the concentrations at which nicotine was inhibitory were not physiologically relevant. Thus, we were able to dismiss inhibition by nicotine exposure as an explanation for the compositional changes seen in the literature. We next studied 2) bacterial products produced under nicotine stress and evaluated the impact these products had on innate immune cells to gauge the immune response under nicotine-exposed conditions. Here we found that exposure to nicotine and its primary metabolite, cotinine, resulted in differential protein packaging into extracellular vesicles in addition to hypervesiculation and an altered metabolic profile by the microbes studied. We also found that the plasticity of anti-inflammatory macrophages to a pro-inflammatory phenotype was impaired by nicotine in the presence of vesicles as a bacterial stimulus. Lastly, we studied 3) whether use of electronic nicotine delivery systems (ENDS) resulted in compositional changes similar to those seen in tobacco and nicotine use. We demonstrated that ENDS use appears to instigate compositional changes to the intestinal microflora similar to tobacco use and that these compositional changes are accompanied by inflammation of the gastrointestinal tract in murine models. In this manner, we were able to determine that nicotine/tobacco use likely alters microbial composition through a multifaceted approach, namely, through shifts in microbial metabolism thereby altering the pantry of available nutrients for consumption by other microbes and through shifts in immune response to a dominantly tolerogenic environment allowing for the growth and spread of a multitude of microbes without immune intervention.

Graduate Student Profile

Abstract: Plant-derived compounds have the potential to produce value-added compounds with a variety of applications. For example, the lignin part of the lignocellulosic biomass, produced in large quantities as waste from the paper and pulp industries, is a rich source of phenolics with potential applications in the renewable energy sector, pharmaceutical, and chemical industries. On the other hand, plant alkaloids are the primary source for developing plant-derived therapeutics. Unfortunately, the recalcitrant nature of plant cell walls, low extraction yields of small secondary metabolites, and the lack of effective analytical methods for a rapid and accurate identification of plant-based compounds and plant’s degradation products are the major limitations in plant-based valorization efforts.

In order to address some of these challenges, this dissertation focuses on utilizing different mass spectrometry-based techniques such as UHPLC-MS, GC-MS, and direct infusion high-resolution accurate orbitrap and ion trap mass spectrometry for the detection and structure elucidation of plant-based phenolics and alkaloids in order to contribute to ongoing efforts toward valorization of plant-based compounds. Mass spectrometry-based techniques are widely used in pharmaceutical and chemical industries, and have been emerged as one of the most promising analytical techniques for the analysis of plant-based compounds.

In the second chapter of this dissertation, a mass spectrometric method based on lithium cationization was developed to sequence lignin model oligomers with mixed bonding motifs, with a potential application in facilitating the structure elucidation of lignin degradation end products with β-β and β-O-4 linkages. In the third chapter, an important lignan, syringaresinol, was characterized in bourbon whiskey. The origin of syringaresinol was investigated using a model aging experiment to further our understanding of bourbon’s chemical composition. In chapter four, the development of a mild ethanosolv treatment combined with a GC-MS method enabled the detection of several different phenolic compounds in lignocellulosic biomass, which can be potentially used to rapidly compare different biomass samples for the valorization applications. Lastly, in chapter five, synthetic methods in combination with extensive mass spectrometry-based analysis were used to semi-synthesize new plant-based alkaloids with potential applications in drug discovery and development.

Overall, these studies confirm that mass spectrometry-based techniques provide a sensitive and robust analytical platform for the analysis of plant-based products.

Graduate Student Profile

Abstract: The bacterial flagellum is a whip-like structure that protrudes from the cell membrane and is one of the most complex and dynamic biological molecular machines that propels bacteria to swim toward beneficial environments and the sites of infection. It is composed of a basal body, a hook, and a long filament. The flagellar filament contains thousands of copies of the protein flagellin (FliC) monomer arranged helically and ending with a filament cap composed of oligomer protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction, and pathogenicity, such as adhesion to surfaces, secretion of effector molecules, penetration through tissue structures, and biofilms formation.

Bacterial flagellin is an important pathogen-associated molecular pattern (PAMP), which can activate both innate and adaptive immunity. Previous in vitro studies indicate that TLR5 is a major extracellular receptor for flagellin that mediates flagellin-induced production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-12, and tumor necrosis factor α (TNFα). Flagellin can also induce inflammasome activation through its intracellular receptor, the NLR family apoptosis inhibitory protein (NAIP) 5 and 6, leading to the generation of cytokines IL-1β and IL-18, as well as pyroptosis. Here, we found that inflammasome activation and subsequent pyroptosis, but not TLR5-mediated signal transduction, is responsible for flagellin-induced IL-6 and TNFα generation in vivo. Flagellin was fused to the cytosolic translocation domain of anthrax lethal factor (LFn) to enable efficient cytosolic delivery. LFn binds to anthrax protein protective agent (PA), which delivers the LFn-flagellin fusion protein into the cytoplasm through receptor-mediated endocytosis. Injection of LFn-flagellin with PA, but not LFn-flagellin alone by i. v., increased plasma concentrations of IL-1β, IL-6, and TNFα. LFn-flagellin/PA induced IL-1β, IL-6, and TNFα release was abolished in mice deficient in NAIPs, caspase-1, or GSDMD, but not TLR5. Depletion of monocytes and macrophages using clodronate inhibited LFn-flagellin/PA induced cytokine release. In addition, injection of the LFn fusion of another virulent factor, the T3SS rod protein EprJ from E. coli, together with PA also induced generation of IL-1β, IL-6, and TNFα in a caspase-1 and GSDMD dependent manner. Our data indicate that inflammasome activation leads to the generation of a broad range of inflammatory cytokines in vivo through pyroptosis, suggesting an important role of pyroptosis in cytokine storm.

Flagellin is a widespread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. Bacterial flagellin is composed of highly conserved D0 and D1 domain, as well as hypervariable D2 and D3 domain. It has been reported that deletion of the D0 domain of flagellin completely abrogates the activation of TLR5. D0 domain of flagellin alone can bind NAIP5/6, leading to activation of the NLRC4 inflammasome, while whether the D1 domain of flagellin plays any functional role in NAIP5/NLRC4 inflammasome activation remains elusive. Besides, flagellins from S.typhimurium, Yersiniosis enterocolitica, and Pseudomonas aeruginosa can bind to NAIP5/6 and activate inflammasome NLRC4, those from enteropathogenic E. coli, enterohaemorrhagic E. coli, Shigella flexneri, and Burkholderia thailandensis cannot interact with NAIP5/6 and are unable to activate the inflammasome NLRC4. Replacement of the C-terminal D1 domain of flagellin from P. aeruginosa with the C-terminal D1 domain of E. coli flagellin diminished inflammasome activation. These data reveal that the D1 domain also plays an important role in flagellin-induced NAIP/NLRC4 inflammasome activation.

KEYWORDS: bacteria flagellin, virulence factors, PAMP, TLR5, NAIP/NLRC4 inflammasome activation, pyroptosis, cytokines

Abstract: Despite the clinical success and proven efficacies of the conventional platinum-based drugs cisplatin, carboplatin, and oxaliplatin, these drugs suffer from a number of challenges that limit their more widespread therapeutic potential. These limitations, including toxic side effects and susceptibility to cancer drug resistance mechanisms, have prompted researchers to explore alternative metal complexes as anticancer agents. In this presentation, an overview of our work on the development and understanding of rhenium-containing organometallic complexes as potential drug candidates is discussed. We will disclose our discovery that a wide range of rhenium(I) tricarbonyl complexes exhibit potent in vitro anticancer activity via diverse biological mechanisms of action. Furthermore, several classes of rhenium(I) tricarbonyl complexes that we have investigated undergo photochemical processes that can be harnessed to trigger cancer cell death selectively upon irradiation or can be used for imaging applications. For this class of compounds, we have carried out detailed biological studies to determine their mechanisms of action. Our results indicate that subtle structural modifications of these compounds can lead to significant changes in their biological properties. Lastly, in vivo studies will be presented, demonstrating that the potential of these compounds as anticancer drug candidates exists beyond in vitro cellular experiments.

University of Alabama

Abstract: We aim to apply bioinorganic and organometallic chemistry to problems that relate to green chemistry and sustainability. We are exploring how protic and electron donor groups impact catalysis. We have pursued reactivity inspired by the need for energy storage, specifically carbon dioxide reduction. Recently, we designed new pincer ligands using N-heterocyclic carbene (NHC) and pyridinol rings that can change their properties by protonation and deprotonation, rather than lengthy synthesis. The most active transition metal catalysts with these pincers use methoxy groups which balance electron donor ability with stability. This has allowed for formation of ruthenium, cobalt, and nickel complexes that perform catalytic and light driven carbon dioxide reduction. We have also demonstrated that the OH derivatives can be switched on or off for catalysis with acid concentration. One of our ruthenium complexes is record setting in terms of reaction rates and selectivity. CO₂ reduction is of fundamental importance to the impending global energy crisis, and carbon dioxide reduction (when coupled with water oxidation) can allow for a sustainable method of energy storage in solar fuels. Furthermore, we have studied our hydroxyl substituted bipyridine ligands as a part of ruthenium based anticancer metallo-prodrugs. The ruthenium complexes are light activated and show selective toxicity towards cancer cells.  

Bio: Elizabeth T. Papish was born and raised on Long Island, NY. She studied chemistry at Cornell Univ. (BA, 1997) and Columbia Univ. (PhD, 2002). She has taught at Franklin & Marshall College (2002-3), Salisbury Univ. (Asst. Prof. 2003-2007), Drexel Univ. (Asst. Prof. 2007-2012, Assoc. Prof. 2012-2013), and at the Univ. of Alabama (Assoc. Prof. 2013-2019, Full Prof. 2019-present). Her research group studies bioinorganic and organometallic chemistry with an emphasis on designing new organic ligands for the use of transition metal complexes in energy related catalysis applications and for metal-based therapies for health applications. She is the recipient of an NSF CAREER award (2009) and has been honored with the "Outstanding Research Mentor of the Year Award" at Salisbury Univ. in 2007 and with the "College of Arts and Sciences Teaching Award" for excellence in teaching and mentorship from Drexel Univ. in 2012.  In 2013, Papish and her student received the "Division of Inorganic ÌÇÐÄvlog¹Ù·½Èë¿Ú Award for Undergraduate Research" from the American Chemical Society. Her research is currently supported by NSF and NIH.

Faculty Host: Dr. Aron Huckaba