Browsing by Author "Crans, Debbie C., advisor"
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Item Open Access Analytical spectroscopy method development to study mechanisms of Alzheimer's and tuberculosis diseases(Colorado State University. Libraries, 2020) Beuning, Cheryle Nicole, author; Crans, Debbie C., advisor; Levinger, Nancy E., committee member; Barisas, George, committee member; Fisher, Ellen R., committee member; Zabel, Mark, committee memberThis dissertation covers the analytical method development created to study and enhance the knowledge of two specific disease mechanisms important to Alzheimer's disease and Mycobacterium tuberculosis. There are two parts in this dissertation where Part 1 is entitled Measurement of The Kinetic Rate Constants of Interpeptidic Divalent Transition Metal Ion Exchange in Neurodegenerative Disease. Part 2 is entitled The Electrochemistry of Truncated Menaquinone Electron Transporters with Saturated Isoprene Side Chains Important in Tuberculosis. These diseases appear on the World Health Organization's top 10 leading causes of death worldwide. The amyloid-beta (Aβ) peptides are associated with Alzheimer's disease, where neurodegeneration is caused by the aggregation of the peptide into senile plaques within neuronal synaptic cleft spaces. Alzheimer's disease currently has no cure due to its multi-causative pathology. One disease mechanism is the coordination of divalent metal ions to the peptide. Specifically, Aβ coordinates Cu(II) and Zn(II) ions that can enhance the aggregation of Aβ into plaques. These metal ions are highly regulated within the human body and are usually found bound to peptides and not as free ions. Therefore, the Aβ must sequester the metals from other proteins and peptides. The primary research in this dissertation advances fluorescence method development to measure interpeptidic Cu(II) exchange kinetics to be able to measure this type of disease mechanism. The small peptides GHK (Gly – His – Lys) and DAHK (Asp – Ala – His – Lys) both chelate Cu(II) with nM affinity, have biological relevance as they are motifs found in human blood like Aβ, and chelate Cu(II) with similar nitrogen-rich binding ligands as Aβ. By substituting non-coordinating lysine residues with fluorescent tryptophan, the interpeptidic exchange rates can be measured since tryptophan fluorescence is statically quenched when within 14 angstroms of a paramagnetic bound Cu(II). Thus Cu(II) transfer from Cu(H-1GHW) to either GHK or DAHK can be monitored by recovered GHW fluorescence as the Cu(II) is exchanged and second-order kinetic rate constants were determined. This methodology was then used to monitor the Cu(II) exchange from truncated Cu(Aβ1-16) and Cu(Aβ1-28) complexes to GHW and DAHW, where second-order reaction kinetic rate constants were determined. While in the exchanges between Cu(H-1GHW) with GHK/DAHK the second-order rate constants were on the magnitude of 102 or 101 M-1s-1, respectively, the exchanges from Cu(Aβ) complexes were 2-3 orders of magnitude larger, 104 M-1s-1 (to GHW and DAHW). These differences in rate constant magnitude arise from the fact that the affinity of GHW (KA = 1013 M-1) for Cu(II) is larger than Aβ (KA =1010 M-1). This method development is an important step to an accurate measurement of the interpeptidic exchange between Aβ peptides, including in their fibril and plaque formations. Since senile plaques are found in synaptic cleft spaces with nanometer distances between neurons, a model system was generated to study coordination reactions at the nanoscale. In order to do this, the metal ion would need to be released in a controlled manner. Studies of metal ion burst reactions through the use of photocages can simulate bursts of ions like those seen in the synaptic cleft. Zn(II) is often released in its ionic form within the synapse in its function as a neurotransmitter, so we simulated a burst of Zn(II) ions by using a photocage, [Zn(NTAdeCage)]- which releases Zn(II) when irradiated with light. The photocage was irradiated to release Zn(II) then we followed its reaction progress with an in situ chelator, Zincon, in reverse micelles and in bulk aqueous buffer. The coordination reaction was 1.4 times faster in an aqueous buffer than in reverse micelles, despite the Zn(II) and Zincon being closer in the nanoparticle. These observations suggested that there is an impact on coordination reactivity within a highly heterogeneous environment with a cell-like membrane, which is due to the partitioning of each ligand. We observe that the photocage stays in the water pool of the reverse micelle and the Zincon partitions into the membrane interface. Thus, the coordination reactivity is diminished, likely due to the need for Zn(II) to diffuse to the Zincon, crossing a highly organized Stern layer to encounter the Zincon. Whereas in aqueous buffer, these are free to encounter each other despite being hundreds of nanometers apart. These proof of concept studies are integral to studying initial binding dynamics of metal ions with peptides at the nanoscale present in cells and neuronal synapses. Tuberculosis is a pathogenic bacterium which despite having a curable medication, can be drug-resistant. Menaquinone (MK) analogs with regiospecific partial saturation in their isoprenyl side chain, such as MK-9(II-H2), are found in many types of bacteria, including pathogenic Mycobacterium tuberculosis and function as electron transport lipids cycling between quinone and quinol forms within the electron transport system. While the function of MK is well established, the role of regiospecific partial saturation in the isoprenyl side chain on MK remains unclear and may be related to the redox function. Recently, an enzyme in M. tuberculosis called MenJ was shown to selectively saturate the second isoprene unit of MK-9 to MK-9(II-H2). The knockout expression of this enzyme was shown to be essential to the survival of the bacterium. A series of synthesized truncated MK-n analogs were investigated using a systematic statistical approach to test the effects of regiospecific saturation on the redox potentials. Using principal component analysis on the experimental redox potentials, the effects of saturation of the isoprene tail on the redox potentials were identified. The partial saturation of the second isoprene unit resulted in more positive redox potentials, requiring less energy to reduce the quinone. While full saturation of the isoprene tail resulted in the most negative potentials measured, requiring more energy to reduce the quinone. These observations give insight into why these partially saturated menaquinones are conserved in nature.Item Open Access Antibacterial growth effects and speciation of several vanadium salts and complexes(Colorado State University. Libraries, 2023) Arhouma, Zeyad Kamal, author; Crans, Debbie C., advisor; Crick, Dean, committee member; Roess, Deborah, committee member; Jackson, Mary, committee memberVanadium (V) is a first-row transition metal ion that acts as a phosphatase inhibitor with a wide variety of biochemical and physiological functions. The ability of vanadium to form stable polyoxovanadates (POVs) and organometallic complexes has attracted attention for studying the properties and effects of these compounds in various biological systems. In my research, I used the bacterium species Mycobacterium smegmatis (M. smeg), which has undergone reclassification and is now classified as Mycolicibacterium smegmatis. Despite the taxonomic change, both the previous and current classifications use the same abbreviation, M. smeg. I also carried out some studies in Mycobacterium tuberculosis (M. tb). In my work, I explored the properties of several types of vanadium compounds including salts, oxometalates, and coordination complexes to investigate how they impact cellular growth. The first chapter of this dissertation focuses on determining the growth inhibitory effects of decavanadate (V10) and rapidly exchanging oxovanadates on the growth of two mycobacterial species: M. tb and M. smeg. Speciation analysis, utilizing 51V NMR spectroscopy, was employed to document that one specific oxometalate exhibits greater potency as a growth inhibitor for these mycobacterial species compared to other oxovanadates, indicating selectivity in its cellular interaction. Oxometalates have been involved in numerous applications in biological and medical studies, including their ability in addressing the phase-problem in X-ray crystallography of the ribosome. This study investigated the effect of different vanadate salts on the growth of M. smeg and M. tb, highlighting the critical role of speciation in the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10O286−) anion was found to be a stronger growth inhibitor for these bacteria compared to the colorless oxovanadate derived from sodium metavanadate. The 51V NMR spectroscopy and speciation calculations were employed to monitor the vanadium(V) speciation in the growth media and its conversion among species under growth conditions. Our results show that the decavanadate was 200-20 times more potent in inhibiting growth dependent the consideration of molecules or total vanadium content. The findings presented in this work are particularly important in the context of the numerous applications of polyoxometalates in biological and medical studies. The second chapter focuses on investigating the inhibitory effects of two monosubstituted decavanadates (V10): monoplatino(IV)nonavanadate(V) ([H2PtIVV9O28]5−, V9Pt), and by MoIV in monomolybdo(VI)nonavanadate(V) ([MoVIV9O28]5−,V9Mo) on the growth of M. smeg. The inhibitory effects of V9Pt and V9Mo were examined against the growth of M. smeg with EC50 values of 0.0048 mM and 0.015 mM, respectively. These values were compared to the reported inhibitory value of decavanadate ([V10O28]6−/[HV10O28]5−, V10) on M. smeg (EC50 = 0.0037 mM). Time-dependent 51V NMR spectroscopic studies were carried out for all three polyanions in aqueous solution, biological medium (7H9), and heated and non-heated supernatant. These studies aimed to evaluate their stability in their respective media, monitor their hydrolysis over time to form different oxovanadates, and calculate the corresponding EC50 values. The results presented in this study indicate that the two related derived decavanadate derivatives (V9Pt and V9Mo) and V10 exhibited greater potency as growth inhibitors of M. smeg, compared to monomeric vanadate (V1). The spectroscopic characterization conducted in the growth medium led to the conclusion that both the decavanadate structure and its properties play significant roles in their growth effects. In the third chapter in this dissertation, we investigated the growth effects of an anticarcinogenic non-toxic Schiff base oxidovanadium(V) complex (N-(salicylideneaminato)-N'-(2-hydroxyethyl) ethane-1,2-diamine) coordinated to the 3,5-di-tert-butylcatecholato ligand on a representative bacterium, M. smeg.. In addition, we synthesized a series of the Schiff base V-complexes based on previously reported methods and examined the effect of complexes as well as the free catecholates on the bacterial growth. To determine the inhibition activity of these complexes on M. smeg., the biological studies were complemented by spectroscopic studies using UV-Vis spectrophotometry and NMR spectroscopy. These spectroscopic studies determine which complexes remained intact under biologically relevant conditions. In this work, we examine (1) the growth effects of Schiff base oxidovanadium complexes coordinated to a catechol, (2) the growth effects of the respective free catecholates on M. smeg., and (3) the effects of the scaffold. These studies allowed us to demonstrate that some metal coordination complex exhibited greater potency than the ligand alone under biological conditions, whereas others showed greater effects of the free catecholate ligand and in one case the effects were similar of complex and catecholate ligand. The findings from these studies revealed that the observed effects of the Schiff base V-catecholate complex were influenced by the properties of the catechol, including toxicity, hydrophobicity, and steric factors. Finally, the fourth chapter presents preliminary research data on the antimicrobial effects of two pseudospherical mixed-valence polyoxovanadates (MV-POVs), namely K(NH4)4[H6PVIV2VV12O42]·11H2O (V14) and (Me4N)6[VIV8VV7O36(Cl)] (V15) on the growth of M. smeg. These MV-POVs showed complex effects on cell growth, as many of these systems are not stable under biological conditions. To investigate the vanadium(V) speciation in aqueous solutions and growth media, as well as to monitor any conversion among species under growth conditions, 51V NMR spectroscopy was employed. The 51V NMR spectra revealed some hydrolysis and more extensive oxidation of vanadium(IV) in V14 compared to V15 in both aqueous solutions and media. The studies show that both MV-POVs are effective growth inhibitors. The combined findings from the studies described in all the chapters of this dissertation indicate that the stability of the vanadium compound and its structure plays a significant role in the ability of the vanadium complexes to inhibit bacterial growth. These studies highlight the importance of speciation in the biological activity of vanadium complexes.Item Open Access Applications of inorganic nanoparticles in diabetes(Colorado State University. Libraries, 2016) Elhabush, Nada Atiya Omar, author; Crans, Debbie C., advisor; Barisas, George B., committee member; Roess, Deborah A., committee memberDiabetes Mellitus (DM) is an endocrine and metabolic disease that has become a global emergency because of the rapid rise in morbidity and mortality rates worldwide. Since the direct delivery of biomolecules, such as insulin, to treat DM is inefficient and subjected to enzymatic degradation, nanotechnology and nanomedicine research have been devoted to the development of more effective methods to treat DM. Nanoparticles (NP), organic, inorganic, or hybrid, have served as potential carrier for safe and efficient transport for insulin. Additionally, several NP have biological activities that help treat and/or prevent DM and diabetes complications, such as antioxidant, anti-apoptotic, or insulin-mimetic activities. Moreover, physicochemical properties of some NP allow them to be used in diagnostic tools for potential diagnosis or monitoring purposes. This work highlights the applications of inorganic NP such as, gold, selenium, silver, calcium phosphate, zinc oxide, cerium oxide, and iron oxide and in the treatment or diagnosis of DM.Item Open Access Changing dogma regarding the conformation of electron transferring menaquinone (MK)(Colorado State University. Libraries, 2017) Magallanes, Estela Serrano, author; Crans, Debbie C., advisor; Menoni, Carmen S., committee member; Tsunoda, Susan, committee memberMenaquinone-9 (MK-9) is the natural substrate containing a naphthoquinone and an isoprenyl side-chain with nine isoprene units that carry out the electron transfer for the Mycobacterium tuberculosis. We present studies aiming to understand the chemical and biochemical properties of hydrophobic MK molecules. Specifically, we are investigating the MK derivative with two isoprene units, MK-2, because it provides us with the base structure containing the naphthoquinone unit and the isoprene side-chain. Its synthesis is relatively simple because the precursors are commercially available, which allows for large scale preparation and detailed characterization of the molecular structure under different conditions. Using 1D and 2D 1H NMR studies we are establishing that MKs have different conformations depending on the specific environmental conditions. Similarly, we show using 1H-1H 2D NOESY NMR studies that the association of MK with the surfactant- water interface of reverse micelles, which is a model membrane system, modify the conformation of the menaquinone derivative. Finally, the redox potentials of MK-2 was measured in the three different solvents (DMSO, CH3CN and pyridine). We hypothesize that the redox potential is correlated to the conformational of the MK. We observed that the redox potentials varied with solvent. The observed folded structures of MK derivatives stand in contrast to the linear conformation shown in life science text books.Item Open Access Conversion of lipid biomass to liquid hydrocarbons via pericyclic decarboxylations of α,β- and β,γ-unsaturated fatty acids using polycyclic aromatic hydrocarbon (PAH) solvent systems(Colorado State University. Libraries, 2014) Romanishan, Michelle, author; Crans, Debbie C., advisor; Henry, Charles S., committee member; Barisas, George, committee member; Van Orden, Alan, committee member; Reardon, Kenneth, committee memberDevelopment of a new process for converting lipid biomass, containing α,β- and β,γ-unsaturated fatty acids, to liquid hydrocarbon fuels (LHF) of varying carbon number is described in this dissertation. The theme for LHF production at present revolves around utilizing a catalytic system that requires high temperatures and pressures as well as multiple processing steps. The cost attributed to these types of processes has been a hindrance in moving the economy towards a cost-effective renewable fuel. Investigating possible catalyst-free processing techniques has led to the discovery of a lower energy reaction that utilizes specific unsaturated fatty acids into a cheap, high boiling point solvent system that has the ability to produce pure alkenes as liquid hydrocarbon fuels when heated to reflux temperature of the fatty acid. This sustainable process has been proven to decarboxylate α,β- and β,γ-unsaturated fatty acids via a pericyclic rearrangement. Using a high boiling, polycyclic aromatic hydrocarbon (PAH) solvent, such as phenanthrene or pyrene, pure alkene products in high yields have been obtained from heating α,β- or β,γ-unsaturated fatty acids to a temperature no higher than reflux of the acid. The successful process development and subsequent conversion of lipid-like biomass will be discussed at length and confirmed by ¹H NMR and GC/MS.Item Embargo Hydrophobic vanadium complexes for use in anticancer activity(Colorado State University. Libraries, 2023) Murakami, Heide, author; Crans, Debbie C., advisor; Reynolds, Melissa M., committee member; Zadrozny, Joseph M., committee member; Crick, Dean C., committee memberThis dissertation contains the development, synthesis, and characterization of vanadium metal complexes in both biological environment and organic solutions for the purpose of novel medical treatments. As cancer is a disease that causes uncontrollable growth of mutated cells over time, treatments need to also improve to account for an ever-increasing cancer types. Although platinum-based drugs have found successful in treating multiple forms of cancer, inorganic metal based drugs are relatively uncommon in the medical field. It was found in 2020, that a vanadium compound was more potent than cisplatin and efforts were made to investigate and improve that compound. The compound is a ternary V(V) complex that consists of VOL1L2 where L1 is N-(salicylideneaminato)-N'-(2-hydroxyethyl)ethane-1,2-diamine and L2 is 3,5-di-tert-butylcatechol. It is believed that that hydrophobicity of the catechol ligand was significant in keeping the compound intact long enough to cause cytotoxicity in bone cancer cells. In Chapter 1, the biological effects of vanadium and the reasoning that vanadium may be used as a potent medical treatment for various illnesses such as bone cancers, brain cancers and/or tuberculosis are investigated. Chapter 2 describes the synthesis and characterization of halogenated version of the original vanadium Schiff base complexes to test how electronegativity affects the activity as well as testing how modification of the salicyaldehyde portion affects the metal complex. Chapter 3 reports the development of adamantanol catechol ligands to improve hydrophobicity in the vanadium Schiff base complex. Chapter 4 explains the speciation of working with a metal complex in a biological system by comparing two different vanadium systems and how they hydrolyze and form multiple different species in various environments. Chapter 5 summarizes the conclusions and proposes future works based on the research done that can move new projects forward.Item Open Access Interactions of biologically active molecules, cofactors, and drugs with model membranes(Colorado State University. Libraries, 2021) Van Cleave, Cameron, author; Crans, Debbie C., advisor; Van Orden, Alan K., committee member; Levinger, Nancy E., committee member; Roess, Deborah A., committee memberThe cell membrane is important for the structure, function, and overall homeostasis of the cell. It consists mainly of phospholipids which have different physicochemical and material properties. As such, molecular interactions between the membrane's components and its environment are of importance. This manuscript explores the interactions of different classes of molecules with model membrane systems to gain a fundamental understanding. Chapter 1 provides background on the cell membrane and current models as well as an introduction to lipoquinones and small molecule drugs. Chapter 2 discusses the interactions of menadione and menadiol with Langmuir monolayers and reverse micelles. Menadiol and menadione are representative of the headgroup of menaquinones, a class of electron transporter, hence they are redox active. We hypothesized that the respective locations of menadione and menadiol within the membrane would vary due to their different physicochemical properties. We used Langmuir monolayers and NMR of reverse micelles to explore the location and association of menadione and menadiol with model membrane interfaces. Chapter 3 investigates the location, association, and conformation of truncated menaquinones with Langmuir monolayers and simulated bilayers. Previous work found that truncated menaquinones fold at the interface of a reverse micelle, so we hypothesized that subtle differences in folding would cause variations in location and association with phospholipids. We used a combination of Langmuir monolayers and molecular dynamics simulations to probe location, association, and conformation of truncated menaquinone homologues, MK-1 through MK-4, in a phospholipid membrane. Chapter 4 explores the pH-dependent effects of two anti-tubercule molecules at the membrane interface. Recent studies have suggested that pyrazinoic acid behaves as a protonophore and we further explored this suggestion while simultaneously exploring physicochemical properties of pyrazinoic acid and pyrazinamide. This chapter utilized a combination of Langmuir monolayers, NMR, and fluorescence leakage studies to characterize the molecular interactions of pyrazinoic acid with model membranes so that POA could be compared to a previous study with benzoic acid, a known protonophore.Item Open Access Investigation of surface interactions of pyrazinamide and pyrazinoic acid with synthetic and natural lipid membrane model systems(Colorado State University. Libraries, 2022) Gasparovic, Nathaniel, author; Crans, Debbie C., advisor; Henry, Chuck, committee member; Ryan, Elizabeth, committee memberPyrazinamide (PZA) is a pro-drug used in the treatment of tuberculosis. Upon administration of the drug, it is converted to its active form of pyrazinoic acid (POA) by the tuberculosis bacterium; this is believed to be the biologically active form of the drug which exerts anti-tubercular activity. However, it is generally accepted that both compounds interact with and transverse the membrane, with POA potentially functioning as a protonophore and lowering the intracellular mycobacterial pH. To investigate the interactions of PZA and POA in model membranes, we employed Langmuir monolayers to investigate the potential membrane-disrupting effects of PZA and POA on a model membrane. At physiological pH, neither PZA nor POA disrupted the membrane, although a difference in compressibility was observed. At acidic pH, POA became more disruptive but only at high, non-physiological concentrations. 1H NMR spectroscopy of a microemulsion system was used to investigate the location of PZA and POA in the interface in different protonation states. The neutral POA species was found to preferentially reside in the interface while the charged species remained in the interfacial water. Finally, the effects of PZA and charged POA on the bilayer in liposomes were investigated. A leakage assay on fluorophore-filled liposomes showed that PZA and POA do not induce leakage in the membrane at physiological conditions.Item Open Access Physical and chemical characteristics behind membrane interactions of small molecules and electron transporters(Colorado State University. Libraries, 2018) Peters, Benjamin J., author; Crans, Debbie C., advisor; Crick, Dean C., committee member; Reynolds, Melissa M., committee member; Ross, Eric D., committee memberThere are many types of molecules that interact with and within membranes whereas many factors can dictate how they interact with membranes. Often, the interactions with the membrane interface can affect the mechanism of action of these molecules. Here, the interactions of small molecules and an electron transporter with model membranes under varying conditions are described. In the first chapter, the pH dependence of membrane association of a commonly used food preservative, benzoic acid was discussed and compared to the mechanism of action of general weak acid preservatives. Next the interactions of many structurally very similar compounds with model membranes were compared. These studies outline the importance of both the environment and that by just altering the molecules slightly, the interactions of the molecules can be changed. Chapter 4 outlines the importance of lipid density on the interactions of the electron transporter used within the electron transport system of Mycobacterium tuberculosis (menaquinone-9) to show that menaquinone is capable of membrane transport of protons and electrons. Together, these studies show how interactions and diffusion across membranes are not straight forward and more research is necessary to fully understand the interactions of molecules with cell membranes.Item Open Access Selenium speciation determined by ICPMS: effects on fish diversity, solubility, and bioavailability to the bryophyte Hygrohypnum ochraceum in Fountain Creek, Colorado(Colorado State University. Libraries, 2019) Carsella, James S., author; Crans, Debbie C., advisor; Bonetti, Sandra J., committee member; Laybourn, Paul J., committee member; Ryan, Elizabeth P., committee memberSelenium (Se) is a micronutrient that can be present in high levels in aquatic environments which may result in toxic effects observed in aquatic wildlife. The levels in Fountain Creek Colorado are of special concern as these levels are above the EPA limit of 5 µg/L. The high Se levels are a result of the exposure of the water to Pierre Shale deposits that underlie parts of the creek. The effects of this creek water on fish diversity were examined at different locations along the creek. The hypothesis tested was that high Se present in the water and bryophytes should be an indicator of fish species diversity. In addition, the possibility of low toxicity resulting from Se species was explored. The speciation analysis determined the levels of Se(IV) and SE(VI) at 12 sites and the statistical results show that sites with higher Se(IV)/Se total exhibit lower fish diversity and fish number than the other sites. There is a statistically significant difference in Ca, Mg, and Se levels in each of the 3 main tributaries in the Fountain Creek Watershed. The tributaries are Monument Creek and Upper Fountain Creek, which join at a confluence near Eighth Street in the city of Colorado Springs, Colorado, to form the Lower Fountain Creek which empties into the Arkansas River in eastern Pueblo, Colorado. The following factors were considered in determining the forms of Se that could exist: Ca2+, Mg2+, SeO42-, SeO32-, and carbonates in addition to the reported thermodynamic relationships. There is a correlation that exists between water hardness and Se level. This correlation can be described in terms of the formation of a soluble CaSeO4. The formation of CaSeO4 is assisted by the increase in Ca2+ by the presence of Mg2+ regardless of the equilibria with the Ca2+ level reducing carbonate. The bryophyte Hygrohypnum ochraceum has been shown to accumulate zinc, cadmium, and lead is often found growing near acid mine drainages. This natural ability to accumulate metals makes H. ochraceum an good organism to use in the study of heavy metals and metalloids in the environment. In a previous work the bryophytes were shown to differentially uptake Se based on season. In this study the seasonality of the uptake of Se was examined and it is suggested to be related to an Iron (Fe) cofactor. The H. ochraceum cultures were placed in the creek for 10 days, harvested, dried and digested according to EPA Method 3052. The resulting digestates were analyzed using EPA Method 6020a for ICPMS metal determination. The results show that Fe and Se uptake are correlated. The hypothesis that was developed from these results is that Fe is needed by the plant for Se uptake from the creek water. Indeed H. ochraceum demonstrated statistically significant log-linear uptake of Se in the presence of dissolved Fe (R2=0.8488, p=0.002). Se uptake was negatively sloped in the fall compared to the linear relationship in the spring. It was determined that the Fe in the water went from a soluble form to an insoluble form. We failed to reject the null hypothesis that Fe is not required in a soluble form for the bryophyte H. ochraceum to uptake Se. Further examination did show that a significantly different slope exists between the Se(IV) uptake and the Se(VI) uptake. There was not a significant difference between the total dissolved Se and Se(VI). In the future directions, this work could be extended to look at specific markers for Se induced stress in the fish populations that may aid in determining the cause for a lack of diversity in some areas as the habitat along the reaches is similar for that reach. Targeted research of water chemistry could investigate the interesting solubility phenomenon giving rise to the seasonal variation of the Fe in Fountain Creek. Finally, the use of plants as bioremediation in the high Se areas could be further investigated with the knowledge that plant nutritional needs must be accounted for when using plants such as H. ochraceum as biological indicators or biological remediators.Item Open Access Simplified membrane-like systems describing the physical behaviors of cholesterol and anti-diabetic drugs(Colorado State University. Libraries, 2013) Trujillo, Alejandro M., author; Crans, Debbie C., advisor; Roess, Deborah A., committee member; Frye, Melinda A., committee member; Van Orden, Alan K., committee memberThis work evaluates the properties contributing to natural membrane permeability by using simplified systems. Absorption mechanisms are a critical step in evaluating the action of orally active drugs. Reverse micelles (RMs) were used as a membrane-like model to analyze the permeation through spectroscopy. The properties exerted by the ligand and ligand substituents were evaluated in the context of membrane permeation. The polydentate ligand of anti diabetic dipicolinatooxvanadium(V) [VO2dipic])-, 2,6-pyridinedicarboxylate (dipic2-) was observed for permeability in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) RMs. Measurements using proton nuclear magnetic resonance (1H NMR) spectroscopy revealed the permeation and hydrophobic stability at physiological pH for dipic2-. Substituents, NH2, OH, H, Cl, NO2 were evaluated forinfluencing the stability and permeability of [VO2(dipic)]-; in AOT RMs. Using infrared spectroscopy (IR), substituent changes significantly influenced the permeation of the vanadium complex series. Properties contributing to the membrane permeation of drugs may also be altered by membrane composition. Cholesterol is present in intestinal membranes and is known to possess properties reducing permeability. A system composed of cetyltrimethylammonium bromide (CTAB), 1-pentanol, cholesterol, and an aqueous phase formed RMs characterized by NMR and dynamic light scattering (DLS). Cholesterol altered the RM structure and proton transfer rates between the water and 1-pentanol of the system. Combined, this work illustrates that ligands, substituents, and membrane components influence the uptake of orally administered drugs.Item Open Access Strategies for targeting cancer: small molecules, epigenetics and drug design(Colorado State University. Libraries, 2020) Hassell, Kelly N., author; Crans, Debbie C., advisor; Brown, Mark A., advisor; Roess, Deborah, committee member; Menoni, Carmen, committee memberCancer has plagued our human population since its early characterizations as abnormal cells and tissues in the mid-1900s. Initial treatment models included surgical removal of cancerous tissues. In the late 1960s, surgical removal and localized radiation were the only available options for treatment until the development of chemotherapeutics. These chemical cocktail treatments, designed to kill cancer cells, started in the late-1900s and even today remain a major line of defense in fighting this disease. The goal of the research described in this dissertation was to investigate current methodologies and techniques used to treat cancer; treatments utilizing chemotherapeutics, small molecule interactions, metallocage drug delivery, epigenetics and protein activity inhibition. The first part of my research focused on the significance of Cisplatin as a chemotherapeutic. My findings indicate the unexpected speciation of platinum in the human body as a revelation to be utilized in novel drug design. In my reverse micelle study, the hydrolysis of the Schiff-base compound was observed to be dependent on the size of reverse micelles; resulting in partial phase selectivity. The reverse micelle model provided ample support for engineering various types of liposomal delivery options for insoluble compounds like Cisplatin. My metallocage research explored the idea of utilizing a self-assembling Cisplatin protective capsule with fluorophores, equipped to monitor real-time cancer cell death as well as drug delivery. My findings support the efficacy of metallocages for delivery of cancer therapeutics and the necessity for continued methodology development for clinical applications. The second part of my research focused on the use of epigenetics for gene expression regulation and protein activity inhibition. My findings reported the most recent status of drugs developed using histone deacetylases (HDAC) and histone deacetylases inhibitors (HDACi) for targeting specific cancers. And in my final chapter of SET-domain proteins, my research focused on comparing the methyltransferase activity inhibition of SMYD3 in two different cancer cells lines. The data showed the A549 lung cell line is slightly more sensitive to the SMYD3 activity inhibitor. This dissertation describes work that has increased our collective understanding of cancer therapeutics. Furthermore, it vastly supports future cancer treatment investigations utilizing both small molecules and bioinformatics.Item Open Access Synthesis and exploration of biologically important, hydrophobic, redox-active molecules: investigation of partial saturation of mycobacterial electron transport lipids(Colorado State University. Libraries, 2019) Koehn, Jordan T., author; Crans, Debbie C., advisor; Reynolds, Melissa M., committee member; Shi, Yian, committee member; Crick, Dean C., committee memberThere are many types of molecules that are biologically important because they either carry out crucial functions or exhibit exploitable biological activity. Some of the most interesting and challenging molecules to work with are those that are redox-active and hydrophobic or water insoluble. Herein, the synthesis and investigation of two classes of hydrophobic redox-active molecules are explored. Chapter one provides background on menaquinone (MK) and vanadium chemistry and primes the reader for the subsequent chapters. Chapter two describes the synthesis and characterization of truncated MK derivatives with varying isoprenyl side chain length and degrees of saturation. Chapter three explores the conformational flexibility of the isoprenyl side chain of MK and shows that a truncated MK analog, MK-2, can adopt folded conformations in hydrophobic environments and within a model membrane system. Chapter four isolates the conformational and chemical effect of saturation of the isoprenyl side chain on MK and shows that saturation minimally affects folded conformations of truncated MK derivatives but remarkably, a 20 mV redox potential difference was observed between unsaturated MK-1 and the saturated analog MK-1(H2). Then in chapter five, hydrophobicity and steric bulk are explored as properties to enhance membrane affinity and anti-cancer properties of Schiff base vanadium(V) catecholate complexes, where the hydrophobic [VO(Hshed)(ditertbutylcatechol)] complex was found to have enhanced hydrolytic stability and potent activity against a bone cancer cell line. Together, the findings of the studies presented herein help to further understand how the conformation and the degree of saturation in the isoprenyl side chain of MK affects the recognition, reactivity, and function of MK within the electron transport system of pathogenic bacteria. These studies are important because they begin to explain and provide a working model behind the chemical rationale as to why partially saturated MK-9 is observed in pathogenic M. tuberculosis. Furthermore, the studies with the hydrophobic vanadium(V) catecholate metallo-complexes underpin a drug design concept exploiting hydrolytic stability imparted by hydrophobicity and steric bulk of a non-innocent ligand.Item Open Access Synthesis of biologically relevant molecules(Colorado State University. Libraries, 2023) Braasch-Turi, Margaret, author; Crans, Debbie C., advisor; McNally, Andy, committee member; Prieto, Amy, committee member; Prenni, Jessica, committee memberNatural products total synthesis and bioorganic chemistry rely on organic synthesis to produce the compounds for biological study. Natural products and some biomolecules are naturally found in extremely low concentrations. Organic synthesis made it possible to acquire the amounts needed for biological studies. The research described herein discusses both areas. In the body of this document, the bioorganic work regarding lipoquinones, particularly ubiquinones and menaquinones, is described. Appendix IV described the natural products work. Chapter 1 provides a more detailed explanation as to the circumstances that led to two different areas of research. Chapter 2 serves as the introduction to the bioorganic research. In this chapter, a review of the literature surrounding ubiquinones, plastoquinones, and menaquinones is complied with their properties at the forefront. Lipoquinones are incredibly hydrophobic molecules, and such properties are often ignored or misinterpreted in the literature. The review compares reported similarities and differences among the lipoquinones with respect to their headgroups, isoprene sidechain length, conformations, and location of lipoquinones in membrane environments. The review also highlights the need for and encourages more experimental studies to validate the computational work in the field. Chapter 3 discusses the conformation and location of ubiquinone-2 in AOT reverse micelles. Previous work with menaquinone-2 determined the truncated lipoquinone derivative adopted a folded conformation in organic solvents and in AOT reverse micelles and suggest menaquinone-2 is located near the lipid-water interface. We hypothesized ubiquinone-2 would also adopt a folded conformation in the membrane and be located near the lipid-water interface, but closer to the bulk water than menaquinone-2. We used 1D and 2D NMR spectroscopic methods to explore the solvent and membrane conformations and membrane location of ubiquinone 2. The conformations and locations of ubiquinone-2 were compared to menaquinone-2, and the location of ubiquinone-2 was found to be slightly closer to the interface than menaquinone-2. Chapter 4 provides a review of the literature regarding the synthesis of naphthoquinone derivatives. There are five main synthetic approaches that have been used to synthesize naphthoquinone derivatives. The categories are (1) nucleophilic ring methods, (2) sidechain homologations and extensions, (3) metal-mediated and radical reactions, (4) electrophilic ring, and (5) pericyclic reactions. The advantages and disadvantages of each approach are discussed regarding selectivity, number of steps, yield, and overall safety. Some approaches are simpler to carry out for the non-expert and successfully yield product, although stereospecificity and yields of the reactions are less, whereas other routes are higher yielding. Chapter 5 discusses the exploratory synthesis of menaquinone derivatives. The established Friedel-Crafts approach has poor regioselectivity, poor stereoretention of the first isoprene unit in the sidechain, and universally low yields. Using the knowledge gained in Chapter 4, a pericyclic approach using Diels-Alder adducts was used to exert regiocontrol of sidechain and maintain the stereochemistry of the sidechain. A convergent route was designed to provide access to a diverse library of sidechains to include E and Z isomers of the first isoprene unit and varying degrees of saturation along the sidechain. Appendix IV discusses the progress towards the total synthesis of versiquinazoline A and versiquinazoline B, alkaloids with anti-cancer properties and a unique pyrazinoquinazolinedione (6-6-6) and imidazoindolone (5-5-6) scaffold. Through this synthesis, the non-proteinogenic amino acid, 1-amino-1-cyclopropycarboxylic acid, was prepared to be used in the synthesis of versiquinazoline B. The synthesis of the 5-5-6 ring system explored the use of many peptide coupling conditions to afford a sterically hindered amide bond. After frequent unsuccessful trials, steps toward the total synthesis of versiquinazoline A were taken using alanine instead of 1-amino-1-cyclopropylcarboxylic acid. After successful amide coupling, the reoxidation of the aromatic sing system was explored using 2,3-dichloro-5,6-dicyanoquinone. This project ended prematurely due to the advent of COVID-19 and the passing of my advisor, Dr. Robert M. Williams.Item Open Access The PH-dependent activity and the protonophoric mechanism of pyrazinoic acid and its structural analogues(Colorado State University. Libraries, 2019) Piedade Neto Guerra Fontes, Fabio Levi, author; Crick, Dean C., advisor; Crans, Debbie C., advisor; Roess, Deborah, committee member; Ross, Eric, committee memberPyrazinamide is an anti tubercle drug used in the standard treatment regimen against Mycobacterium tuberculosis, the cause of tuberculosis infections. The mechanism of action of pyrazinamide requires its enzymatic conversion to pyrazinoate, but the final molecular target of pyrazinoate is controversial. Pyrazinamide also exhibits pH dependent activity in vitro, but the phenomenon was seldom explained by the mechanisms of action proposed in literature. Moreover, pyrazinamide is known to synergize with other anti mycobacterial drugs in vivo but reports of this synergistic activity in vitro are scarce. The work presented here aimed to gain insight on the mechanism of action of pyrazinamide that explains both its activity and the pH dependent behavior in vitro, while seeking to understand the synergism of pyrazinamide in vitro. The results presented here show the pH dependent activity is not caused by pH sensitivity of M. tuberculosis, as the data demonstrates the bacilli are able to maintain pH homeostasis in a pH range between 5.5 and 7.3. Additionally, M. tuberculosis actively replicates in physiologically extreme pH environments (pH 5.5 and pH 8.5), albeit at a slower rate than at neutral pH values. Mycobacterial cultures treated with pyrazinoic acid showed growth inhibition that correlates with the relative concentration of the acid (but not of its conjugated base, pyrazinoate). Treatment with pyrazinoic acid also leads to concentration dependent acidification of the cytoplasm of mycobacterial bacilli and concentration dependent dissipation of the electric potential across the cytosolic membrane. These results led to the conclusion that pyrazinoic acid, but not pyrazinoate, is the active form of pyrazinamide. The mechanism involves the enzymatic conversion of pyrazinamide into pyrazinoate. Pyrazinoate then crosses the cytosolic membrane and is exposed to the acidic environment, where an acid base equilibrium is established with pyrazinoic acid. Pyrazinoic acid crosses the membrane and reaches the cytosol, where the more neutral pH leads to the loss of the proton it carried from the extracellular environment. The acid base equilibrium outside the cell generates a higher relative concentration of pyrazinoic acid as the pH of the environment becomes more acidic, leading to the pH dependent activity of pyrazinamide. The work presented here demonstrates the pH dependence is not replicated by other anti tubercle drugs, such as rifampin, isoniazid or bedaquiline. However, structural analogues of pyrazinoic acid, such as salicylic acid, and a known protonophore, carbonyl cyanide m chlorophenyl hydrazone, mimic the pH dependent growth inhibition of pyrazinoic acid. A model for the pH dependent activity of these compounds was derived, based on chemical assumptions. The model demonstrates that pyrazinoic acid acts as a protonophore, causing the disruption of proton motive force, and that this mechanism is only possible if no other cellular target exists. The synergism of pyrazinoic acid with rifampin and isoniazid in vitro was determined, using the median effect principle, at different pH environments. Additionally, salicylic acid was tested in combination with rifampin or isoniazid to determined if the drug drug interactions of pyrazinoic acid with these drugs was mimicked by its structural analogues, like its mechanism was shown to be. The results indicate pyrazinoic acid behaves additively with both rifampin and isoniazid in vitro, which was seen in salicylic acid as well. The data indicates that the drug drug interactions of pyrazinoic acid are replicated by salicylic acid. Additionally, the results suggest the synergism of pyrazinamide in vivo may originate from some type of host effect that was not present in the in vitro studies conducted.