The LIN-12/Notch signaling pathway is highly conserved in all animals, and is crucial for proper development. It is a key pathway in specifying cell fate in many cellular contexts, and dysregulation of the pathway can have deleterious consequences. Therefore, understanding how LIN-12/Notch signaling is regulated in different contexts has been a main area of interest in the field. Previous studies in different model organisms have identified many modes of regulation of the signaling pathway, one of which is endocytosis of the ligand and receptor. Here, I further investigated the role of endocytosis in LIN-12/Notch signaling in multiple developmental contexts in Caenorhabditis elegans. Work in Drosophila and vertebrates had previously established that ligand-mediated activation of Notch requires ubiquitination of the intracellular domain of the transmembrane ligand and the activity of the endocytic adaptor Epsin in the signaling cell. The consensus in the field is that Epsin-mediated endocytosis of mono-ubiquitinated ligand generates a pulling force that exposes a cleavage site in Notch for an ADAM protease, a critical step in signal transduction. In contrast, in this thesis, I examined two different transmembrane ligands in several different cell contexts and found that activation of LIN-12/Notch and the paralogous GLP-1/Notch in C. elegans does not require either Epsin-mediated endocytosis or ubiquitination of the intracellular domain of the ligand. Results obtained by a collaborator indicate that C. elegans ligand and receptor interactions are tuned to a lower force threshold than are Drosophila ligand and receptor interactions, potentially accounting for these differences. I also looked at the role of endocytosis in regulating LIN-12 signaling in the context of vulval development. The cell fate pattern of six vulval precursor cells (VPCs) is mediated by EGFR and LIN-12/Notch signaling. Previous work using multicopy transgenes in fixed specimens indicated that LIN-12 is post-translationally downregulated via endocytosis in response to EGFR activation in the VPC named P6.p, an event that appeared essential for ligands to activate LIN-12/Notch in neighboring VPCs. In this thesis, I manipulate the endogenous lin-12 gene and examine live specimens to show that LIN-12 appears to be regulated transcriptionally in P6.p and evidence that there may be additional potential endocytic motifs that may regulate LIN-12 in this context.

Copper (Cu) is required by marine microbes for essential biological processes, including photosynthesis and nitrogen fixation, but can be toxic above a certain threshold. Copper bioavailability in seawater is regulated by complexation with dissolved organic ligands of unknown source and structure. Culturing experiments have demonstrated the production of high-affinity Cu-binding ligands by marine algae in response to metal stress or limitation, suggesting they function either as metal ‘sponges’ to reduce copper toxicity or ‘carriers’ that promote uptake. The goal of my thesis research was to develop methods for the recovery and characterization of Cu ligands from seawater that could then be applied to natural samples to investigate sources and structures of recovered ligands. Using natural seawater spiked with model Cu ligands, I developed an immobilized Cu(II)-ion affinity chromatography (Cu(II)-IMAC) protocol which was shown to be effective in quantifying an operationally defined subset of natural Cu ligands. I then applied Cu(II)-IMAC to seawater collected along transects in the Canadian Arctic and NE Pacific Ocean to assess the abundance of this ligand pool across a diverse set of samples. Ligand distribution profiles and their covariance with other components of seawater (e.g. chlorophyll) were consistent with in situ biological production of some Cu-binding ligands. Model ligands spiked into seawater and recovered by Cu(II)-IMAC were also used to develop protocols for structural characterization of Cu ligands by solid-phase extraction (SPE) and tandem mass spectrometry (MS/MS). This research provides new tools for the isolation and characterization of copper ligands in natural samples, and new insights into the biogeochemical cycling and ecological significance of Cu in the ocean. / Graduate

The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.

Indiana University-Purdue University Indianapolis (IUPUI) / Molecular recognition plays an important role in biological systems. The purpose of this study was to get a better understanding of the process by incorporating computational tools.Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method and Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) method, the end-point free energy calculations provide the binding free energy the can be used to rank-order protein–ligand structures in virtual screening for compound or target identification. Free energy calculations were performed on a diverse set of 11 proteins bound to 14 small molecules was carried out for. A direct comparison was taken between the calculated free energy and the experimental isothermal titration calorimetry (ITC) data. Four and three systems in MM-GBSA and MM-PBSA calculations, respectively, reproduced the ITC free energy within 1 kcal•mol–1. MM-GBSA exhibited better rank-ordering with a Spearman ρ of 0.68 compared to 0.40 for MM-PBSA with dielectric constant (ε = 1). The rank-ordering performance of MM-PBSA improved with increasing ε (ρ = 0.91 for ε = 10), but the contributions of electrostatics became significantly lower at larger ε level, suggesting that the only nonpolar and entropy components contribute to the improved results. Our previously developed scoring function, Support Vector Regression Knowledge-Based (SVRKB), resulted in excellent rank-ordering (ρ = 0.81) when applied into MD simulations. Filtering MD snapshots by prescoring protein–ligand complexes with a machine learning-based approach (SVMSP) resulted in a significant improvement in the MM-PBSA results (ε = 1) from ρ = 0.40 to ρ = 0.81. Finally, the nonpolar components in the free energy calculations showed strong correlation to the ITC free energy while the electrostatic components did not; the computed entropies did not correlate with the ITC entropy. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein-ligand system in molecular recognition. SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We evaluate the performance of SVMSP in its ability to enrich chemical libraries docked to MD structures. Seven proteins from the Directory of Useful Decoys (DUD) were involved in the study. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models remarkably improved enrichment in two cases. We also explored approaches for a prior identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. SVMSP rescoring of protein–compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50,000 compounds docked to MD optimized structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5 μM. These compounds serve as leads for the design and synthesis of more potent and selective ALDH2 inhibitors.

This dissertation describes my efforts in the Nuckolls lab to expand synthetic methods of wet-chemistry superatom synthesis, superatom surface ligand and core modification, and assembly of superatoms into materials with useful, cumulative properties. This work builds off of previous work from the Nuckolls lab describing photolabile ligand substitution and use of this technique to covalently bind superatoms to form various materials such as polymers and weaved sheets. This work will focus on the Chevrel-type M₆E₈L₆ metal-chalcogenide cluster Co₆Se₈, modification of its outer stabilizing ligands, and fusion of its core with other Co₆Se₈ superatoms to form fused dimers. Chapter 1 consists of a review of background material that forms a foundational basis for this work. The field of superatoms and superatomic materials will first be covered to contextualize this work in the field at large. Then, the prior work on wet-chemistry synthesis of Co₆Se₈ superatoms with replaceable, photolabile carbonyl (CO) ligands will be discussed. Finally, previous dimensionally-controlled assembly of materials using these carbonylated superatoms will be covered. Chapter 2 consists of the discovery of a masking carbene ligand generated from trimethylsilyl diazomethane (TMSD) and its use to create a new, electronically-coupled superatom dimer species (Co₁₂Se1₆(PEt₃)₁₀) that shows evidence of quantum confinement akin to nanoparticles and nanoparticle assemblies. Chapter 3 consists of new ligand substitution and methods to synthetically functionalize the fused dimer introduced in Chapter 2. The reactive carbene-ligated cluster is used to add new functional groups that were previously inaccessible to these cobalt-selenide clusters. New multi-carbene clusters are demonstrated as well as the use of site-differentiated clusters to form functionalized fused dimers from bis-carbonyl clusters. Chapter 4 consists of an investigation of the carbene cluster and insights that may be used in the future to finally expand cluster fusion into a chain. A reversible bridging of the carbene ligand based on temperature and oxidation state is analyzed experimentally and computationally. This information is used to synthesize a series of new carbene clusters which are used to try and assemble electronically-coupled, fused Co₆Se₈ superatomic materials.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The bis(2-pyridylthio)methyl ligand, [Bptm], offers a synthetically convenient alternative to a variety of multidentate ligands, including most notably [Tptm] (tris(2-pyridylthio)methyl) and [BptmSTol] (bis(2-pyridylthio)(p-tolylthio)methyl), and, in contrast with [Tptm], necessarily coordinates to metal centers in a κ³ fashion. As such, numerous [Bptm] complexes of zinc have been synthesized and structurally characterized. In Chapter 1, we describe the reaction of the protonated ligand [Bptm]H with the homoleptic zinc compounds Me₂Zn and Zn[N(SiMe₃)₂]₂ to afford, respectively, [Bptm]ZnMe and [Bptm]ZnN(SiMe₃)₂; the latter has been used as a starting point for a wide range of reactivity.Most notably, the terminal zinc hydride, [Bptm]ZnH, can be accessed via either (i) metathesis of the zinc siloxide, [Bptm]ZnOSiPh₃, with either PhSiH₃ or HBpin, or (ii) direct metathesis of the zinc amide [Bptm]ZnN(SiMe₃)₂ with HBpin; the latter reactivity is not precedented and offers a novel approach for the synthesis of molecular zinc hydrides. Both [Bptm]ZnN(SiMe₃)2 and [Bptm]ZnH provide access to a variety of monomeric derivatives, including the zinc halides [Bptm]ZnX (X = Cl, Br, I) and the zinc isocyanate [Bptm]ZnNCO; the latter can be accessed directly via (i) metathesis of [Bptm]ZnH with Me₃SiNCO or (ii) a multistep reaction of [Bptm]ZnN(SiMe₃)₂ with CO₂. [Bptm]ZnH also undergoes insertion of CO₂ into its Zn—H bond to afford the zinc formate, [Bptm]ZnO₂CH, in which the formate moiety exhibits a monodentate binding mode in the solid state. This reactivity enables it to serve as a catalyst for the hydrofunctionalization of CO₂; specifically, [Bptm]ZnH catalyzes the hydrosilylation of CO₂ by (RO)₃SiH (R = Me, Et) at elevated temperatures to afford the respective silyl formates (RO)3SiO₂CH, as well as the hydroboration of CO₂ by HBpin at room temperature to afford the boryl formate HCO₂Bpin. In the absence of CO₂, [Bptm]ZnH also catalyzes the reduction of HCO₂Bpin to the methanol level, MeOBpin. Similarly, [Bptm]ZnH serves as an effective catalyst for the hydrosilylation and hydroboration of a variety of ketones and aldehydes. In all cases, hydroboration is more facile than the corresponding hydrosilylation. The [Bptm]Zn system has been investigated computationally, and the kinetics of insertion of CO₂ into the Zn—H bond of [Bptm]ZnH as well as the thermodynamics of the catalytic cycle have been examined. Further mechanistic studies examine two noteworthy spectroscopic features of the system, namely rapid exchange (i) between the zinc and boryl formates [Bptm]ZnO₂CH and HCO₂Bpin, as well as (ii) between [Bptm]ZnH and [Bptm]ZnO₂CH. Both of these exchange processes have been investigated with variable-temperature NMR spectroscopy; in particular, the former exchange resolves at low temperatures and can be confirmed by exchange spectroscopy. In addition to the aforementioned monomeric zinc halides [Bptm]ZnX (X = Cl, Br, I), the dimeric bridging zinc fluoride {[Bptm]Zn(μ-F)}₂ has been synthesized via reaction of Me3SnF with either [Bptm]ZnN(SiMe₃)₂ or [Bptm]ZnH, as outlined in Chapter 2. The dimeric nature of the fluoride in contrast with the other monomeric halides can be attributed to the significant polarity of the Zn—F bond. {[Bptm]Zn(μ-F)}2 also reacts with Me₃SiCF₃ to afford an unusual instance of a structurally characterized zinc trifluoromethyl complex, [Bptm]ZnCF₃. Chapter 3 discusses cadmium analogues to the [Bptm]Zn system, which provide a comparison and a contrast both with their zinc counterparts as well as with previously reported [Tptm]Cd complexes. While the cadmium amide [Bptm]CdN(SiMe₃)2 may be synthesized in a manner corresponding to that for its zinc analogue, the siloxides {[Bptm]Zn(μ-OSiR₃)}₂ (R = Me, Ph) form dimers that are distinct from the monomeric [Bptm]ZnOSiPh₃ and [Tptm]CdOSiPh₃, although similar to {[Tptm]Cd(μ-OSiMe₃)}₂. The distinctions between the [Bptm]Zn and [Bptm]Cd siloxides have been investigated computationally, indicating that the cadmium species show a thermodynamic preference for dimer formation, which can be attributed to the larger atomic radius of cadmium relative to zinc. Attempts to synthesize a cadmium hydride are interrupted by a Schlenk-type equilibrium giving way to the bis(ligand) complex [Bptm]2Cd and CdH₂, which in turn decomposes to Cd and H2. However, spectroscopic studies indicate that under CO₂, [Bptm]CdN(SiMe₃)₂ and HBpin react to trap a cadmium hydride species as the bridging formate derivative, [Bptm]Cd(μ-O₂CH)₂Bpin. The interaction of nitrogen-rich ligands with main group metals is further probed in Chapter 4, which describes the investigation of the coordination of 2,2’:6,2”-terpyridine (terpy) to magnesium compounds. Most prominently, unsubsituted terpy forms an adduct, terpyMg[N(SiMe₃)₂]₂, with the monomeric form of the magnesium amide {Mg[N(SiMe₃)₂]₂}₂. The adduct reacts with halide donors to form a series of mixed amide-halide complexes, terpyMg[N(SiMe₃)]X (X = Cl, Br, I), as well as a mixed amide-azide complex, terpyMg[N(SiMe₃)₂]N₃. These complexes represent the first instances of neutral monomeric terpyMg compounds that feature unsubstituted terpyridine. Structural comparisons of these complexes with one another as well as with comparable compounds are undertaken. Complexes of terpy with cadmium and zinc analogues, terpyCd[N(SiMe₃)₂]₂ and terpyZn [N(SiMe₃)₂]₂, are explored further, and DFT calculations are used to explore the strength of the interactions between the ligand and the metals in each case. Finally, in Chapter 5, attention is given to the recently reported zinc bromide complex featuring a zwitterionic carboxylate ligand, (Cbp)2ZnBr₂. The structure reported for this complex features several anomalous features, including abnormally long Zn—Br and Zn—O bonds, unusually small atomic displacement parameters for Zn, and a high R-value. This information led us to synthesize and investigate the cadmium counterpart, (Cbp)₂CdBr₂; we find that the cadmium complex possesses nearly identical structural parameters to the reported zinc complex, and when the cadmium is refined as zinc, the displacement parameter problems are reproduced. Therefore, we conclude that the reported structure is in fact that of (Cbp)₂CdBr₂, and report a revised structure for (Cbp)₂ZnBr₂.

Toll-like receptor (TLR) ligands and phytochemical feed additives (PFAs) were evaluated in this study to determine the effects of immune stimulation on gut immunity in the zebrafish, Danio rerio, and the channel catfish, Ictalurus punctatus. Rag1-/- (MT) zebrafish were used to study how the TLR ligands β-glucan and resiquimod (R848) affect the innate immune system in the gut of MT zebrafish. Enhanced expression of marker genes (NITR9, NCCRP-1 and MPEG-1) indicated stimulation of Natural Killer (NK) cells, non-specific cytotoxic cells (NCCs) and macrophages. After challenge with Edwardsiella ictaluri, MT zebrafish stimulated with β-glucan demonstrated higher survival and the presence of more macrophages/monocytes in the gut than control MT zebrafish. A PFA test diet containing a blend of prebiotic fiber, oregano, thyme, cinnamon essential oils, and Yucca schidigera (ONE Current™, OC) was fed to channel catfish for 3 months in ponds to determine the effect on channel catfish fingerling growth. Fish were fed in ponds and a tank bacterial challenge followed to test the efficacy of the product. Catfish fed OC demonstrated greater weight gain and feed conversion ratios, higher survival after challenge with E. ictaluri, greater phagocytosis or binding by macrophages and cytotoxic cells. Catfish fed OC also demonstrated greater gut surface area after 2 months feeding OC. To elucidate the effect(s) of each of the compounds in the OC diet on gut immune responses and to determine if PFAs can decrease bacterial colonization and replication within gut tissues, WT and MT zebrafish were fed diets containing different compounds included in OC. Quantification of live bacteria from gut and kidney tissue was determined after challenge with E. ictaluri. Expression levels of immune response genes were evaluated after ingestion of PFAs. Actifibe, Essential oil 25 ppm (EO 25) and Actifibe + EO demonstrated the lowest infection and colonization rate, upregulation of immune response genes, and significantly higher survival when challenged with E. ictaluri. This study demonstrates the potential for application of TLR ligand and feed administered PFAs to improve fish health. Our findings provide a more comprehensive understanding of host gut/pathogen interactions as well as suggestions for novel disease control measures.

<p> Voltage-gated potassium, sodium, and calcium channels play fundamental roles in cell physiology. They are targets for numerous drugs that are used to treat pain, cardiovascular, autoimmune, and other disorders. Atomic-resolution structures of ion channels and their complexes with ligands are necessary to understand the mechanisms of drug action of ligands. Electrophysiological and crystallographic studies have advanced our understanding of ion channels, but the binding sites, access pathways, and the mechanism of state-dependent action of medically important drugs remain unclear. During my graduate studies, I investigated the structure-function relationships of voltage-gated ion channels and their complexes with drugs by using energy calculations with experimental constraints. My work has helped resolve controversial interpretations of experiments addressing structural similarity between prokaryotic and eukaryotic K+ channels. Our model of the open Shaker K+ channel was confirmed by the later published X-ray structure of Kv1.2. Our Cav2.1 model reinterprets substituted-cysteine accessibility experiments, validates the proposed alignment between K+ and Ca2+ channels, and suggests a similar folding of voltage-gated K+ and Ca2+ channels. These results allowed me to model eukaryotic K+ and Na+ channels in the resting and open/slow-inactivated states, and to predict the binding sites of local anaesthetics, correolide, and chromanol 293B. In these studies, we proposed the involvement of metal ions in the binding of nucleophilic drugs and suggested that the deficiency of permeating ion(s) in the outer pore of the slow-inactivated channels stabilizes the ligands. Simultaneous studies of K+, Na+, and Ca2+ channels were advantageous because the information acquired from one family of ion channels was relevant to other families. My studies contributed to the growing knowledge about ion channels by offering structural information and suggesting mechanisms for the action of drugs. </p> / Thesis / Doctor of Philosophy (PhD)

Page 39 was included twice in the thesis. / <p> This thesis describes reactions involving low valent technetium nitrosyl complexes. O-Substituted hydroxylamines were reacted with [TcOCl4]- in methanol producing [Tc(NO)Cl4]-. NMR studies have shown that two species are present besides the starting material during this reaction. One of these species was confirmed by NMR to be the corresponding alcohol of the O-substituted hydroxylamine. The other species is believed to be a hydroxylamine intermediate that is in equilibrium with the final product, [Tc(NO)Cl4]-. A plausible mechanism for this reaction was proposed that included an oxo group attack of the α-carbon of the O-substituted hydroxylamine which would lead to the formation of the corresponding alcohol. In an attempt to confirm the mechanism, O-18 labeled [TcOCl4]- was synthesized, however, there is no conclusive evidence that the label is transferred to the corresponding alcohol at the present time. Substitution reactions of [Tc(NO)Cl4]- with phenanthroline and bipyridyl ligands were also investigated. Reactions with these ligands produced [Tc^(II)(NO)Cl3phen] (4a) and [Tc^(II)(NO)Cl3bipy] (5), respectively. The crystal structures of these complexes showed that the meridonial isomer is produced with one nitrogen atom of the bidentate ligand trans to the nitrosyl moiety. EPR spectra of these compounds confirm the Tc(II) oxidation state of the metal. All of the chloride ligands of 4a and 5 can be displaced using AgBF4 in acetonitrile solvent, which, in the case of bipyridyl, produces [Tc^(I)(NO)(bipy)2(MeCN)]2+ (6). Other technetium nitrosyl containing complexes are formed in these reactions and are currently awaiting x-ray structure determination.</p> / Thesis / Master of Science (MSc)