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: 

Site-selective modifications of target proteins using specifically designed small molecules is a powerful tool that has been extensively utilized for drug discovery. Small molecules can modify proteins either covalently or non-covalently depending on their structures and intrinsic chemical reactivity. Covalent chemical modification presents a more stable and often irreversible interaction with target proteins; unlike non-covalent binders, which form weak, reversible interactions with protein. Therefore, covalent modifiers represent an effective class of therapeutics due to their stability and irreversibility once bound to target proteins of interest. I hypothesized that tuning biocompatible, high-valent gold(III) complexes toward nucleophile-induced reductive elimination will lead to covalent protein modification by arylation. While most proteins are expressed amongst all cell types; protein overexpression is a common phenomenon in several cancer types due to their rapid proliferative phenotype and mutations compared to healthy non-cancerous cells. The nucleophilic amino acid side chains in proteins can be used as reactive handles for covalent modifications. Amongst the naturally occurring amino acids; cysteine, the most intrinsically nucleophilic, contains a highly reactive thiol functional group. This innate nucleophilicity provides a framework for covalent modification with electrophiles, which includes but is not limited to electron-deficient metal centers (e.g., Au and Pd).

Although there are previous reports successfully identifying transition metals as suitable chemical modifiers, specifically, tuning gold(III) complexes for selective binding offers a unique strategy for chemotherapeutics. Gold(III) metal centers are innately acidic and react with softer bases such as phosphorous and sulfur unlike the traditionally used late transition metals. Secondly, gold(III) complexes are known to target proteins over DNA, unlike other common transition metal complexes such as platinum and ruthenium. Combining the innate ability of gold(III) complexes to interact with proteins and the high affinity for cysteine thiols, rationale design of highly selective protein modifiers and efficient chemotherapeutics is possible.

My work focused on tuning the reactivity of cyclometalated gold(III) complexes for cysteine arylation and ligand-directed bioconjugation using Metal-mediated Ligand Affinity Chemistry (MLAC) have been elucidated to modify biomolecules including antibodies and undruggable protein targets such as KRAS. While developing cyclometalated gold(III) complexes discussed herein, a unique class chiral gold(III) complexes bearing diamine or phosphine ligands led to other applications including improved anticancer activity in comparison to first generation of gold(III) complexes. A key highlight is the development of stable organometallic gold(III) macrocycles with potent in vitro and in vivo anticancer action in aggressive cancer types including triple negative breast cancer (TNBC).  

KEYWORDS: Site-selective protein modification, gold complexes, covalent binders, cysteine arylation, anticancer

Graduate Student Profile

Abstract: A thiol molecule, 2,6-pyridinediamidoethanethiol (PB9), was synthesized based on the pyridine-2,6-dicarboxamide scaffold with appended cysteamine group. PB9 acts as an effective chelator for Pb(II) due to multiple binding sites (N3S2) through irreversible binding precipitating Pb(II). Removal of aqueous Pb(II) from solution was demonstrated by exploring the effects of time, initial PB9:Pb(II) ratios, pH, exposure time, and solution temperature. After 15 min the Pb(II) concentrations were reduced from 50 ppm to 0.3 ppm (99.4%) and 0.25 ppm (99.5%) for PB9:Pb ratios of 1:1 and 2:1, respectively.  Removal of >93% Pb(II) was observed over multiple pH values with negligible susceptibility for leaching over time. The thermodynamic studies reveal the removal of Pb(II) from solution is an entropically driven spontaneous process. Solution-state studies (UV-Vis, 1H-NMR, 13C NMR) along with solid-state (IR, Raman, and thermal studies) for L/Pb(II) compounds were performed. UV-vis displays a global maximum at 274 nm and a local maximum at 327 nm for ligand-to-metal charge transfer S- 3p -> Pb2+ 6p, and intraatomic Pb2+ 6s2 -> Pb2+ 6p transitions.  A Probable molecular structure designed is PB9 behaving like a bis-deprotonated ligand with an N3S2 donor set to give Pb(II) a pentagonal environment with non-stereochemically active s electrons is proposed.  However, the existence of a cyclic oligomeric (PB9)4(Pb)4 or polymer (PB9)?(Pb)? structure is evident by broad melting point, insolubility in most common solvents, and amorphous powder XRD. PB9 also exhibits high sensitivity and selectivity towards Fe3+ over other metal ions, acts as a naked-eye detector showing colorless to yellow, and by fluorescent quenching. The quenching efficiency found by Stern-Volmer is 7.42 ± 0.03 × 103 M-1 with a higher apparent association constant of 9.537 X 103 M-1. A linear range of Fe3+ (0- 80 µM) with a detection limit of 0.59 µM (0.003 ppm) was found. The obtained detection limit was much lower than the maximum allowance limit of Fe3+ (0.3 ppm) regulated by EPA in drinking water.  Since Pb(II) removal using PB9 was higher than 15 ppb (EPA limit), a separate study was conducted to explore the use of thiol molecule (AB9) which was already developed in our lab previously. Thus, 2,2'-(isophthalolybis(azanaediyl))bis-3-mercaptopropanoic acid (AB9) was coupled to amine-functionalized silica and silica-coated magnetic nanoparticles (with Fe3O4 core). Results revealed successful fabrication of AB9 on mesoporous silica and MNPs surfaces without introducing crystalline impurities. Indeed, an added advantage for AB9-MNP over AB9-silica is its superparamagnetic nature where a magnet was used to isolate the Pb(II)-containing (solid) composite from the treated water. The >99.9% removal of Pb(II) was obtained by AB9-MNPs with detectable Pb(II) dropping to below 15 ppb EPA level. The obtained equilibrium results were in good agreement with the Langmuir model suggesting a dominant chemical adsorption mechanism on AB9- composites with monolayer coverage with maximum adsorption capacities of  24.80 and 56.40 mg/g respectively for AB9-silica and AB9-MNP implying the thiol group improved the adsorption capacity of Pb(II). This eco-friendly modification with rapid magnetic separation makes these AB9-MNPs a good candidate for aqueous Pb(II) removal

Abstract: To develop an effective battery cathode material that can be useful for future batteries, the thermal stability and ion migration dynamics need to be well understood. In situ transmission electron microscopy (TEM) is a popular and proven technique to study the evolution of local structures during the dynamic processes in the cathode materials. This dissertation will demonstrate the application of high-resolution imaging and in situ heating and biasing in the TEM to study the structure and composition, morphology change, and ion migration in the cathode materials.   The three chapters in this dissertation will be focused on the two cathode materials: zeta (?) vanadium pentoxide, and chromium intercalated sodium manganese oxide. The first project demonstrates the effect of in situ heating method, nanowire size, sodium content, and vacuum condition on the thermal stability of zeta (?) vanadium pentoxide in real-time in the TEM. The second project concentrates on in situ biasing in the TEM to study the sodium ion migration, silver exsolution, and negative differential resistance phenomenon in the zeta (?) vanadium pentoxide. The third project concentrates on the synthesis and characterization of chromium incorporated sodium manganese oxide. The works presented here show the capability of in situ TEM imaging techniques to study the dynamic changes in the structure and composition of the nanomaterials during the heating and biasing processes.

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.

Jonathan Rivnay

Department of Biomedical Engineering and Simpson Querrey Institute

Northwestern University, Evanston, IL

Rivnay Group

Abstract: Direct measurement and stimulation of ionic, biomolecular, cellular, and tissue-scale activity is a staple of bioelectronic diagnosis and/or therapy. Such bi-directional interfacing can be enhanced by a unique set of properties imparted by organic electronic materials. These materials, based on conjugated polymers, can be adapted for use in biological settings and show significant molecular-level interaction with their local environment, readily swell, and provide soft, seamless mechanical matching with tissue. At the same time, their swelling and mixed conduction allows for enhanced ionic-electronic coupling for transduction of biosignals. Structure-transport properties allow us to better understand and design these active materials, providing further insight into the role of molecular design and processing on ionic and electronic transport, charging phenomena, and stability for the development of high-performance devices. Such properties stress the importance of bulk transport processes and serve to enable new capabilities in bioelectronics. In this talk I will discuss the design of new organic mixed conductors and future design rules for performance and stability. I will demonstrate how such materials properties relax design constraints and enable new device concepts and unique form factors, allowing for flexible amplification systems for electrophysiological recordings, and electroactive scaffolds to modulate tissue state and/or cell fate. New materials design continues to fill critical need gaps for challenging problems in bio-electronic interfacing.

Faculty Host: Dr. John Anthony

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.

**CANCELLED**

Marco Bonizzoni

Department of Chemistry and Biochemistry, The University of Alabama, Tuscaloosa, AL, USA.

Alabama Water Institute, The University of Alabama, Tuscaloosa, AL, USA.

E-mail: marco.bonizzoni@ua.edu

Abstract: Artificial supramolecular receptors often rely on weak intermolecular interactions for their chemical recognition properties, so they may struggle to work in competitive media, chief among 

which are water solutions. However, aqueous media are very important in analytical, environmental, and biomedical applications, so it is valuable to adapt our supramolecular tools to them. With the right tools, even the weakest noncovalent interactions can be pressed into service in aqueous media. We have been using water-soluble polymers (e.g. dendrimers, hydrogels, conjugated polymers) as scaffolds to build multivalent supramolecular sensors that take advantage of the large number of interactions and of the preorganization of receptor sites afforded by such scaffolds, resulting in improved affinity in buffered aqueous solutions near neutral pH. We have successfully built systems for the detection of interesting guest families, including carboxylate anions, simple saccharides, heavy metal cations, and polycyclic aromatic hydrocarbons. These are examples of a general approach with two key advantages. On the one hand, installing known receptor chemistry on a polymer scaffold affords a modular approach to multivalency with minimal design and synthesis effort. This improves the apparent strength of weaker interactions and allows them to overcome desolvation costs in water. On the other hand, water-soluble macromolecular scaffolds impart solubility to water-incompatible receptor families.

This simple approach is particularly valuable when designing chemical fingerprinting systems (sometimes referred to as an “electronic nose” or “tongue”) that typically require many different receptors, each one poorly selective, and recovers selectivity from judicious interpretation of the ensemble response.

**CANCELLED**

Abstract:

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism, in which endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in Electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens. Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: ET-FAD is bound in highly mobile domain II, which sits in a stable base created by domains I and III. Bf-FAD is buried in between domain I and III. The two flavins execute contrasting, complementary electron transfer reactions. Whereas one mediates single electron transfer (ET-FAD), the other accepts electrons pairwise (Bf-FAD), yet both flavins’ sites include a conserved Arg sidechain. R273 favors the ASQ of ET-FAD, whereas R165 near the Bf-FAD appears not to, possibly due to neutralization of its positive charge by nearby C174. R273 forms a pi- pi stacking interaction with ET-FAD whereas R165 appears to form hydrogen bond interactions with Bf-FAD. To learn whether the active site arginine residues each have different effects on their respective neighboring flavins, we replaced each of the Args in turn with chemically conservative, and divergent substitutions. Our data shows, R273 plays a vital role in BfETF by stabilizing the ASQ of the ET-FAD, whereas R165 favors binding of the Bf-FAD that is essential for electron bifurcation in RpalETF. Along with the electron bifurcation studies, we report an irreversible, pH dependent, site selective, enzyme mediated, anaerobic chemical modification of ET-FAD to a pink amino FAD, which opens a new perspective with which to understand the 726 nm band formed in bifurcating ETF.

Join the seminar here: https://uky.zoom.us/j/87175023115?pwd=dHlVZUtRRGVaY295eHVPWFJFa2l1dz09; Password: 764392