Abstract: Oxidative Phosphorylation occurs within the inner mitochondrial membrane producing ATP for the cell’s energy needs. The Electron Transport Chain carries out the transfer of electrons from electron carriers through a series of proteins to form a proton gradient across the membrane. This gradient acts as the energy source needed to put a phosphate onto ADP. With the large amounts of free electrons and oxygen within the mitochondria, an inevitable by-product of free radicals in the form of superoxide are produced. Superoxide (O-?2) is an extremely reactive radical that can go on to perform further reactions leading to the formation of more radicals. When these radicals and reactive oxygen species are kept in balance they act as signaling molecules for the cell. A network of antioxidant proteins helps the cell to keep this balance. However, when there is an overload of radicals and ROS within the cell leading to oxidative damage and becoming detrimental to the cell’s ability to survive.  The brain is made up of different types of cells, neurons, and glia, that are rich in polyunsaturated fatty acids and have an abundance of O2. The combination of these things is what allows the brain to work with such high function, but it is also this combination that can lead to unfavorable reactions. The abundance of O2 allows for higher changes of free radicals and reactive oxygen species which can interact with the polyunsaturated fatty acids in a process called lipid peroxidation. Lipid peroxidation results in the formation of 4-hydroxynonenal (HNE) which has deleterious effects within the cell.  HNE adducts to proteins on a cysteine, lysine, or histidine. When these amino acids are located towards the inside of the protein it causes a conformational change of the protein and therefore a loss in function.  The adduction of HNE to proteins has been observed in different brain related diseases such as Glioblastoma and Alzheimer Disease.  Glioblastoma is one of the most difficult forms of cancer to treat due to location of the tumor and its resistance to radiation. Oxidative damage within the tumor cells does not seem to cause the same deadly effects as it would to the surrounding cells. When tumor cells have a large amount of oxidative damage there is a need to rid the cell of the affected proteins. This is in the form of extracellular vesicles (EVs). The findings of this dissertation elucidate the mechanism by which these EVs aid in the progression of the tumor cells. EVs bleb from the surface of the cell carrying the HNE adducted proteins into the extracellular space and encounter the surrounding glial cells and neurons. When the EVs are taken up by the glial cells, such as astrocytes, this induces the production of ROS in the form of hydrogen peroxide. This ROS is key in inducing proliferation of the tumor cells and furthering radiation resistance.  Alzheimer Disease has also been shown to have HNE adducted proteins and oxidative damage. One such pathway is affected in Alzheimer disease, the Pentose Phosphate Pathway, depending on the apolipoprotein E (ApoE) allele status of the cell. There are three variations of this gene, E2, E3, or E4, with the most common being E3. Those who have ApoE4 have a higher risk of developing Alzheimer Disease. ApoE4 allele is also seen in conjunction with higher oxidative damage and HNE production within the cell. The findings in this dissertation show the correlation between the ApoE allele status and the oxidative damage.

Abstract Title: The outer membrane of Gram-negative bacteria (GN) makes them distinct among superbugs that are associated with the development of antibiotic resistance. The outer membrane, and inner membrane, separated by the periplasm, form a double-membrane barrier to the entry of antibiotics into the cell.  Several studies have been conducted to examine the role of outer membrane modifications such as porins, lipopolysaccharides, and efflux pumps on antibiotic resistance. However, there is a paucity of knowledge on how antibiotics behave in the periplasm, in order to gain access into their target region. My thesis focuses on understanding the mechanism of antibiotic permeability through the cellular envelope of Gram-negative bacteria.   I studied the distribution of fluoroquinolones in the two aqueous compartments (periplasm and cytoplasm) of Escherichia coli using fluorescence intensity measurement and minimum inhibitory concentration (MIC) test.  We treated the bacteria cells with each antibiotic, allowed the antibiotic to accumulate in the cells, fractionated the cells and quantified the concentration of accumulated antibiotic through the measurement of its fluorescence intensity. The compound accumulation assay showed that the efflux-deficient strain (?tolC) accumulated more antibiotic than the wild type (WT) strain, for all nine fluoroquinolones we tested. An analysis of the subfractions showed a greater accumulation of the antibiotic in the periplasm than in the cytoplasm. A positive correlation was seen between the MIC ratio (WT/?tolC) and the cytoplasmic accumulation ratio (?tolC/WT). This is an indication that the efficacy of a drug is a combined function of its ability to accumulate in the target region and inhibit the target.  I also studied the impact of osmo-regulated periplasm glucans (OPGs) on antibiotic susceptibility in GN.  We created E. coli and Salmonella typhimurium strains deficient in OPG production. We also created an E. coli strain that produced neutral OPGs. The drug susceptibility test showed that the strains that are either deficient in OPG production or produce neutral OPGs were less susceptible to the positively charged aminoglycosides compared to the WT strain. A similar response was observed when the bacteria strains were treated with the fluoroquinolones and tetracyclines. We speculate that this behavior is due to the net positive charge carried by these antibiotics from complexes formed with Mg2+. In contrast, the strains grew slower in the presence of a negatively charged cerufoxime. The observed MIC changes were not due to a leaky membrane. The OPG deficient strains produced significantly reduced amount of OPGs compared to the parent strain. Our study demonstrated that charge plays a significant role in OPG-mediated susceptibility to antibiotic.   We also probed the role of OPGs on copper homeostasis in Gram-negative bacteria. We found that the disruption of the opgGH operon had an impact on the tolerance of GN bacteria to copper. Copper quenched the fluorescence of cytosolic GFP faster in the OPG- deficient strains compared to the WT strain. The GFP-quenching effect of copper ions was diminished with the increase of ionic strength, indicating that Cu2+ penetration into the cytoplasm slowed down under high salt condition. 

Graduate Student Profile

Abstract: Lignocellulosic biomass is pivotal in the development of renewable energy sources and materials essential to mitigate the exploitation of fossil fuels causing environmental pollution issues. The conversion of biomass into fuel requires the hydrolysis of cellulose and a biproduct of this process is the isolation of lignin as biorefinery waste. Lignin is a complex high molecular weight polymer whose structure remains undefined and critically limits potential industrial applications of lignocellulosic biomass. The advancement of analytical methods for structural elucidation of lignin and its ensemble of phenolic compounds is therefore essential to advance this field. While a variety of analytical methods play an integral role in developing our understanding of lignin, only mass spectrometry can provide exact information on the substructure of lignin, the sequence of monolignols, and linkage types. In this dissertation, the supramolecular interactions of a variety of model lignin monomers and dimers are characterized to improve mass spectrometric analysis and potential applications of lignin as a renewable source of valuable phenolics.  Mass spectrometry (MS) requires the conversion of analytes into detectable gas-phase ions, and the most widely used ionization technique for biological compounds is electrospray ionization (ESI). The primary challenge facing ESI-MS analysis of lignin is ionization because lignin compounds do not readily accept protons for positive mode analysis and negative mode analysis causes destabilization and in-source fragmentation. While protonation is unsuccessful, lithium adduction has recently been discovered as a promising method for ESI-MS sequencing of lignin compounds. Consequently, the gas-phase lithium cation basicity of synthetic monolignols and dimers were characterized by ESI-MS to improve sequencing techniques and future applications of lithium adduction.  Lignin also presents a challenge in biomass processing due to its inhibition of the enzymatic hydrolysis of cellulose for biofuel production. Supramolecular guest-host interactions have the potential to isolate lignin compounds from biomass fractions through the formation of inclusion complexes and the development of selective materials. In this work a cyclodextrin host was selected based on its remarkable ability to encapsulate guest molecules and availability on the industrial scale. The binding strength between guest and host was evaluated for lignin model dimers with cyclodextrin by ESI-MS for comparison with our collaborators ITC and computational results. The retention of electrostatically bound complexes during the ESI-MS process and lithium adduct impacts are also extensively evaluated.  Lignin compounds and metabolites additionally show biological activity, and therefore the separation of diastereomers is of interest for pharmaceutical applications. To advance biological studies, the success of chromatographic separations (HPLC) of lignin model dimers and their diastereomers is evaluated. The separative method is coupled to MS with post-column lithium adduction to identify lignin dimers. Novel determinations of lignin dimer partition coefficients are also presented, a measure of hydrophobicity important for biological studies and chromatographic method development. These fundamental characterizations of lignin model compounds are essential for the continued advancement of renewable energy and materials derived from lignocellulosic biomass.

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

Graduate Student Profile

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Abstract: Earth’s atmosphere is a multicomponent system comprising gases, aerosols, cloud, and their interaction with sunlight impacts radiative forcing. Biomass burning and anthropogenic emissions releases volatile phenolic compounds at the atmosphere as primary organic aerosol. These phenolic compounds can undergo atmospheric processing such as chemical oxidation, and photochemical aging providing precursors for the formation of secondary organic aerosol. This dissertation ensembles laboratory studies of oxidative processing of group of representative phenolic compounds by O3, HO•, and NO3 at the relevant environmental conditions. Aerosolized microdroplets of phenolic aldehydes undergoes electron transfer reaction generating HO• at the online electrospray mass spectrometry (OESI) reactor when exposed to 0.045 ppmv to 5 ppmv O3(g) at the microsecond contact time, generating hydroxy-substituents of the parent molecules. Ozonolysis of  phenolic compounds and ring-functionalized product phenols produces compounds containing carboxylic, and ester-like functionalities. These phenolic compounds were then deposited on ZnSe FTIR windows and exposed to 1 L min-1 of 0.20 ppmv to 800 ppmv O3(g) for a longer timescale in a flow through reactor and analyzed by FTIR spectroscopy, UV-visible spectroscopy, ultrahigh pressure liquid chromatography (UHPLC) with UV-visible and mass spectrometry (MS) detection, ion chromatography (IC) with conductivity and MS detection, and nuclear magnetic resonance (NMR) spectroscopy for 1H and 13C nuclei and tow-dimensional heteronuclear single quantum coherence (HSQC) experiments. The reaction products were dominated by functionalized and oligomeric compounds. Syringic acid was used as a common standard to compare the responses of UHPLC-MS, IC-MS and NMR analysis and quantify methoxy-aromatic product compounds and syringaldehyde was used to compare results from UHPLC-MS and NMR, and pseudo quantify aromatic aldehydic compounds. The uptake of O3(g) by the phenolic compounds increased with increasing relative humidity (RH). The decay kinetics showed non-linear dependence against increasing molar ratio of O3(g).  Phenolic compounds were also studied for oxidation with NO3. When aerosolized microdroplet of phenolic compounds such as catechols were exposed to NO3, produced from the mixing of NO2(g) and O3(g) at the OESI-MS reactor, nitroaromatic compounds (NAC) were produced. Under variable pH (4.05 to 8.07) conditions, all these compounds generated NAC. Catechol thin film was deposited over ZnSe windows and oxidized by 1 L min-1 of NO2(g) and O3(g) mix, in the flow through reactor for longer times scale. Formation of 4-nitrocatechol (4NC) was observed after exposure to oxidant mixture of 200 ppbv NO2(g) and 50 ppbv O3(g) at 0% RH. 4NC production was highest at 0% RH and at elevated RH the reaction was dominated by O3(g) as observed by the increased production of cis,cis-muconic acid. Exposure to high ppbv oxidant mix such as 10700 ppbv NO2(g) and 2500 ppbv O3(g) at 0% RH showed generation of NAC and oligomers. Decay of catechol against increasing oxidant molar ratio showed non-linear dependence at 0% RH. All these results show the crucial role of daytime oxidant O3(g), and HO•, and nighttime oxidant NO3 on the oxidative processing of phenolic compounds. Considering these reaction pathways and kinetics parameters in future climate modelling would reduce the gap between field observation and computer simulation predictions.

Wilson Group

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

Papish Group

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 Chemistry Award for Undergraduate Research" from the American Chemical Society. Her research is currently supported by NSF and NIH.

Faculty Host: Dr. Aron Huckaba

The Department of Chemistry Graduate Student Association (GSA) would like to invite you to a tailgating event on Saturday, November 20 from 10:00am-11:30am on the Tobacco Research, Lawn 1. This event is co-hosted by the Sri Lankan Student Association. A grill and some non-alcoholic beverages will be available. If you choose to bring your own alcohol, you must remain in compliance with University Alcohol Policies.

The Tobacco Research, Lawn 1 is located on the corner of University Drive and Cooper Drive, directly adjacent to the Kentucky Tobacco Research and Development Center. You may view it on the campus map here.

We hope to see you there this Saturday!

Department of Chemistry Graduate Student Association

Sri Lankan Student Association

Faculty host: Dr. Anne-Frances-Miller

Zoom links coming soon!