Browsing by Author "Roess, Deborah, committee member"
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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 Characterization of interactions of lipoquinone derivatives within model membrane systems(Colorado State University. Libraries, 2021) Bublitz, Gaia Rachel, author; Crans, Debbie, advisor; Cohen, Robert, advisor; Santangelo, Thomas, committee member; Roess, Deborah, committee memberMenaquinones (MK) are electron carriers composed of a naphthoquinone moiety and an isoprene side chain of variable length and saturation. These molecules are the only quinone derivatives present in the electron transport systems of all Gram-positive bacteria and some Gram-negative anaerobes. Subsequently, MK plays a critical role in respiration for pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis. Although the physiological function and relevance of MK as a redox cofactor have been established, its chemical interactions within the plasma membrane and the effects of these properties on MK-mediated electron transport are still obscure. These unknowns are reflected in existing literature, as MK is commonly depicted in an extended conformation, although in vitro and in vivo studies suggest that biomolecules with alkyl moieties assume folded conformations in native environments (Ko et al., 2011; Trembleau et al., 2003). In this study, we implemented 1D 1H and 2D 1H-1H NMR spectroscopic techniques to characterize the location and 3D conformation of MK-2 within a L-α-phosphatidylcholine liposome model. MK-2, a truncated menaquinone analog, was selected due to its limited rotational variability and previous characterization in a simple monolayer lipid system (Koehn et al., 2018). Our data suggests that MK-2 is largely incorporated into the phospholipid bilayer, with an aqueous subspecies residing at the polar membrane interface in a concentration-dependent manner. 2D NOESY spectroscopic analysis supports the interpretation that both the aqueous form and the membrane-associated form of MK-2 assume a folded conformation. These findings provide a reference for the study of the properties of MK derivatives with longer isoprene chains, which are analogous to functional MK variants in native environments.Item Open Access Fundamental studies of reverse micellear aggreagates by multinuclear and multidimensional NMR spectroscopy(Colorado State University. Libraries, 2012) Sedgwick, Myles, author; Levinger, Nancy, advisor; Crans, Debbie, advisor; Henry, Chuck, committee member; Roess, Deborah, committee member; Van Orden, Alan, committee memberSelf-assembled reverse micellar aggregates using cationic, anionic and non-ionic surfactants have been investigated by multinuclear and multidimensional NMR. By utilizing 51V NMR chemical shifts and line widths of decavanadate, the local proton concentration and characteristics of the reverse micellar environment are measured. There is a distinct environmental change on the interior of the reverse micelle depending on the surfactant used. 51V NMR signals for decavanadate inside an Igepal CO-520, non-ionic surfactant, reverse micelle display sharp signals indicating the decavanadate experiences water like environment. Conversely, 51V NMR signals for decavanadate inside an Igepal CO-610/430 mixed reverse micelle show significant broadening of the decavanadate signal indicating that the environment inside the reverse micelle in which the decavanadate resides is more viscous. These data provide a description in that the water pool of non-ionic surfactants can be compared. Time resolved anisotropy decays, ultrafast time-resolved transient absorption, and 2D NMR spectroscopy have been used to study proton transfer reactions in the interiors of Igepal-CO 520, CTAB and AOT reverse micelles. For ѡ0 = 10 reverse micelles formed with anionic AOT surfactant, the HPTS proton transfer dynamics are similar to dynamics in bulk aqueous solution, and the corresponding 1H 2D NOESY NMR spectra display no cross peaks between HPTS and AOT consistent with the HPTS residing, well-hydrated by water, in the interior of the reverse micelle water pool. In contrast, ultrafast transient absorption experiments show no evidence for HPTS photoinduced proton transfer reaction in reverse micelles formed with the cationic CTAB surfactant. In CTAB reverse micelles, clear cross peaks between HPTS and CTAB in the 2D NMR spectra show that HPTS embeds in the interface. Similar behavior is observed for HPTS in Igepal reverse micelles as in CTAB reverse micelles and we interpret the slowed dynamics in the same manner. The 2D NMR spectra for HPTS in Igepal-CO 520 reverse micelles shows interaction that imply the HPTS molecule is rested near the interface inside the reverse micelle. Dynamic light scattering (DLS) and 1H NMR spectroscopic experiments suggest that the assembly of the reverse micellar aggregates depends on non-polar solvent and co-surfactant used. Two different self-assembled particles form in the AOT/cholesterol /water in cyclohexane, where in the similar system of AOT/cholesterol /water in 1-octanol there is only one particle present. In microemulsions employing 1-octanol as the continuous medium, AOT reverse micelles form in a dispersed solution of cholesterol in 1-octanol. Although the size distribution of self-assembled particles is well-known for many different systems, evidence for simultaneous formation of two distinctly sized particles in solution that are chemically different is unprecedented. By utilizing optical spectroscopic techniques, 2D NMR, and DLS, the structure of the non-ionic reverse micelles have been characterized. The impact of adding cholesterol, a biologically relevant molecule, has on the structure of the reverse micellar solutions has also been shown.Item Open Access Role of homotropic association of luteinizing hormone receptors in hormone mediated signaling(Colorado State University. Libraries, 2012) Crenshaw, Shirley Ann, author; Barisas, B. George, advisor; van Orden, Alan, committee member; Rickey, Dawn, committee member; Levinger, Nancy, committee member; Roess, Deborah, committee memberG protein-coupled receptors (GPCR) are plasma membrane receptors involved in signal transduction and are an important target for drug discovery. Luteinizing hormone receptors (LHR) are GPCRs found on the reproductive organs of both males and females and promote spermatogenesis and ovulation. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of the mammalian reproduction system and other GPCR systems. Studies in the past suggested that these receptors oligomerize after hormone binding, but recent studies performed with LHRs suggest that these receptors maybe constitutively oligomerized in the endoplasmic reticulum and on the plasma membrane. However, these experiments were performed on receptors expressed by transient transfection and using bioluminescence resonance energy transfer (BRET). These methods have potential weaknesses. Transient transfections typically yield a fraction of cells with very high receptor expression and BRET measurements are strongly weighted towards those cells. Hence, this overall approach may have yielded misleading results. Fluorescence energy transfer (FRET) is a similar technique to BRET but has advantages such as allowing imaging examination of single cells. Using FRET, LHR oligomerization was evaluated on cells treated with human chorionic gonadotropin (hCG) or deglycosylated-hCG, hormones which activate and inhibit the receptor function, respectively. FRET measurements demonstrated that, on the surfaces of transiently transfected cells, LHRs exhibit substantial intermolecular FRET which is very slightly increased by hCG treatment and very slightly reduced by exposure to DG-hCG. Closer examination of these data showed that all observed FRET depended linearly on receptor expression and approach zero at low expression levels. This suggests that FRET between LHR on these transiently-transfected cells may arise from inter-molecular proximity induced non-specifically by high receptor surface concentrations. To evaluate the receptor density on cells flow cytometry was used. Flow cytometry revealed that transiently-transfected LHRs are expressed over a broad range of surface densities, including very high expression levels. Using a mathematical model, the FRET efficiencies expected for various receptor surface densities were calculated. These calculations suggest that expression levels observed cytometrically could cause substantial amounts of FRET from molecular crowding and, particularly if the receptors are additionally concentrated in lipid rafts, most of the observed FRET signal could be attributed to non-specific concentration effects.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 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.Item Open Access The use of model membrane techniques for the analysis of interactions, conformation and redox properties of menaquinones and other small molecules(Colorado State University. Libraries, 2021) Doucette, Kaitlin A., author; Crans, Debbie, advisor; Crick, Dean, committee member; Roess, Deborah, committee member; Menoni, Carmen, committee memberThis thesis explores the use of model membranes to solve complex problems in determining the placement, conformation, and electrochemical properties of hydrophobic compounds as they interact with a model membrane. Menaquinone, an electron transporter commonly found in Gram-positive and Gram-negative obligate anaerobes, consists of a naphthoquinone head group and isoprene tail of variable length and saturation. Chapter two shows the use of liposomal model membranes to solubilize menaquinone analogues of variable length and saturation for aqueous electrochemical studies characterizing half-wave potentials, reversibility, and diffusion coefficients to examine its redox properties in connection to its role as an electron transporter. This work shows a distinct odd-even effect with respect to the isoprene chain length of the compound and its electrochemical properties. The exploration of this project is continued in chapter three, in which the conformation and placement of menaquinone-2 is determined in the context of a phosphatidylcholine liposome using 1D and 2D 1H NMR. Finally, chapter four explores the use of a reverse micellar model membrane for determining the placement of glycine and short glycine peptides to explore its placement near a membrane with regards to its role as a neurotransmitter and to explore its potential role in antimicrobial peptides. The experiments contained herein show that model membranes are a useful tool for the study of hydrophobic compounds and molecules commonly found within a cellular membrane.