Browsing by Author "Roess, Deborah A., committee member"
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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 Contributions of specific TRPS to myometrial calcium entry(Colorado State University. Libraries, 2010) Ulloa, Aida Erendira, author; Sanborn, Barbara M., advisor; Clay, Colin, committee member; Curthoys, Norman P., committee member; Kinnamon, Sue C., committee member; Roess, Deborah A., committee memberUnderstanding the mechanisms that regulate contractions in the myometrium during pregnancy may help avoid scenarios such as premature births. During labor, increases in intracellular calcium ([Ca 2+],) have been closely correlated with human myometrium contractions. Extracellular calcium enters the cell through voltage-operated and signal-regulated Ca2+-entry (SRCE) mechanisms and is involved in actions such as stimulating the contractile apparatus and replenishing intracellular Ca^^ stores. In SRCE, activation of some receptors and/or depletion of agonist-sensitive [Ca^^]j-stores stimulates Ca -uptake from the extracellular solution. This and other SRCE mechanisms are all important in providing regulation of calcium homeostasis. Ion channels potentially responsible for SRCE are the canonical transient receptor potential (TRPC) channels. The seven members (TRPC 1-7) are postulated to form hetero- or homotetramers, thus allowing for the formation of a variety of channels possessing different physiological properties. Additional TRPC channel regulation is also provided by STIMl, an endoplasmic reticulum Ca2+ sensor. My studies show that human myometrium expresses higher concentrations of TRPC4 and TRPCl mRNAs relative to other TRPCs as well as expressing STIMl proteins. Furthermore, I have found that specific TRPC proteins are involved in the SRCE pathways when studied in immortalized myometrial PHMl cells. The main objective of this work was to understand the functional role of TRPC 1 and TRPC4 channels in relation to Ca^^ signaling in order to elucidate their relative significance in myometrium. The individual roles for TRPCl and TRPC4 as well as the potential additive effects that a TRPCl plus TRPC4 double knockdown exerted on SRCE were studied in PHMl and primary human uterine smooth muscle (UtSMC) cells. This was achieved through RNAi mechanisms by expression of shRNAs targeting TRPCl, TRPC4, or TRPCl plus TRPC4. The role of STIMl in myometrial Ca2+ dynamics was also investigated by use of STIM1AERM, a dominant negative form of STIM1. The data presented here suggest that both TRPCl and TRPC4 are activated by similar G protein coupled receptor-stimulated Ca^^ entry mechanisms; however, no additive effects were observed by their combined knockdown. Additionally, thapsigargin- and OAGstimulated Ca entry were not affected by either the individual or combined knockdown of TRPCl and TRPC4. In contrast, STIMAERM appeared to induce an inhibitory effect on all three types of SRCE stimulation. These contributions, in addition to the important role in Ca2+ homeostasis played by voltage-operated channels and the influences of Ca^^ pumps, exchangers and potassium channels, provide the myometrium with the ability to respond in specific and precise ways to influences in its environment, both during pregnancy and at the time of parturition.Item Open Access Development of an insturmal method to evaluate insulin receptors and insulin-like growth factor-1 receptors homodimers and hybrid receptors in breast cancer(Colorado State University. Libraries, 2016) Shebani, Karima Ali, author; Roess, Deborah A., advisor; Roess, Deborah A., committee member; Barisas, George B., committee member; Miller, Charles W., committee memberBreast cancer is a major public health problem in the United States and many other parts of the world. It is the second most common cancer and second leading cause of cancer death among women in the US. Insulin receptors (IR) and insulin-like growth factor-1 receptors (IGF1R) are found in normal mammary gland cells where they are involved in normal development and differentiation of these cells. There is substantial clinical, epidemiological, and experimental evidence indicating that these receptors are also involved in initiation and progression of neoplasia of mammary gland cells. IR and IGF1R numbers are increased in breast cancers and raise the possibility that IR and IGF1R may have roles in the biology of these tumors. In this project, we have developed new methods to evaluate the presence of IR homodimers and hybrid receptors formed from IR and IGF1R monomers. In initial experiments we have used three cell lines of CHO that stably express IR-GFP. Using flow cytometry, we found that low expressing cells had ~ 62,000receptors/cell, moderately expressing cells had ~ 130,000receptors/cell, and high expressing cells had ~ 205,000 receptors/cell. We then used homo-transfer fluorescence resonance energy transfer (homo-FRET) methods to evaluate possible interactions between IR monomers in the IR homodimer or IR and IGF1R subunits in hybrid receptors. Our results suggest that IR exists as an apparent monomer in cells expressing low numbers of IR-GFP. In cell lines highly expressing IR-GFP, there are minimally dimers and higher order receptor oligomers present on the plasma membrane. These results suggest that when IR is highly expressed, as in the case in many breast cancers, the receptor is likely to be found as a IR homodimer rather than as a hybrid IR-IGF1R receptor. Further studies beyond the scope of this project will evaluate the effects of IGF1R expression on the formation of IR homodimers.Item Open Access Fluorine-containing fullerenes and endometallofullerenes: synthesis, structure, and spectroscopic characterization(Colorado State University. Libraries, 2010) Shustova, Natalia Borisovna, author; Strauss, Steven H., advisor; Anderson, Oren P., committee member; Szamel, Grzegorz, committee member; Elliott, Cecil Michael, committee member; Roess, Deborah A., committee memberMany new members of a relatively new class of exohedral fullerene derivatives with fluorine-containing electron-withdrawing groups have been prepared and studied by spectroscopic methods and X-ray crystallography. The fluorination and/or perfluoroalkylation reactions were performed with C60, C70, the higher hollow fullerenes C60+m (m = 14, 16, 18, 20, and 22), the endohedral metallofullerene Sc3N@C80-Ih(7), and the azafullerene dimer (C59N)2. Several efficient synthetic methods have been developed for perfluoroalkylation, which involved high-temperature reactions with AgCF3CO2 and with thermally or photochemically activated reactions with RFI reagents (RF = CF3, C2F5, n-C3F7, i-C3F7, n-C4F9, and n-C6F13). Structural studies of the C60(RF)n and C70(RF)n products demonstrated that variation of the size and structure of the RF radical led to the formation of derivatives with unprecedented addition patterns and hence unprecedented properties. Many of these derivatives were shown to have superior electron-accepting properties. Trifluoromethylation of a sample of insoluble hollow higher fullerenes resulted in the structural characterization of several new dodecakis(trifluoromethyl) fullerene compounds, and this led to the first experimental observation of fullerenes C74-D3h and C78-D3h(5). In the case of trifluoromethylation of (C59N)2, a strong effect of the heteroatom on the addition patterns of the products was discovered. The first X-ray crystal structure of a single regioisomer of C59N(CF3)5, as well as spectroscopic studies of C59N(CF3)7,9,11, revealed unexpected addition patterns which resemble that of Cs-C60X6 derivatives. The isolation and characterization of seventeen Sc3N@(C80-Ih)(CF3)n (even n = 2-16) compounds, including the X-ray structures of Sc3N@(C80-Ih(7))(CF3)10, Sc3N@(C80-Ih(7))(CF3)12, Sc3N@(C80-Ih(7))(CF3)14, and Sc3N@(C80-Ih(7))(CF3)16, have demonstrated for the first time a strong mutual effect of (i) the presence of the Sc3N cluster on the addition pattern and (ii) the addition pattern on the position of and structure of the Sc3N cluster.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 Rotation of cell surface and dissolved biomolecules examined by fluorescence imaging, time-tagged single-photon counting, and fluorescence depletion anisotropy(Colorado State University. Libraries, 2022) Pace, Jason M., author; Barisas, B. George, advisor; Crans, Debbie C., committee member; Roess, Deborah A., committee member; Van Orden, Alan K., committee memberIn this dissertation, I discuss our studies examining protein rotation both in solution and on single cells. Chapter I gives background on physics of rotational diffusion, the application of these measurements to cellular systems, and a general overview of the field, including a survey of techniques that have been used to measure rotation of membrane proteins. In the next two chapters, I discuss our research on the effect of various cell treatments known to perturb the dynamics of membrane proteins on the rotation of the high-affinity Type I IgE receptor (FcεRI) expressed on RBL-2H3 cells. I investigated effects on receptor rotation resulting from treatment with IgE antibody as well as from four treatments with IgE and an additional agent including DNP-BSA, paraformaldehyde, MβCD, and cytochalasin D. These agents have varied effects that I expect to cause a significant perturbation of the rotational dynamics of the receptor. These effects range from receptor crosslinking by DNP-BSA and paraformaldehyde which would be expected to hinder receptor rotation to effects on membrane cholesterol content and the underlying cytoskeleton in the cases of MβCD and cytochalasin D, the effects of which are more uncertain and thus of particular interest. I have investigated these phenomena using a single-particle fluorescence imaging approach and, alternatively, a time-tagged single photon counting approach. These topics are the subject of Chapters II and III respectively. These two approaches, while both designed with the intent to investigate the rotational dynamics of membrane proteins using fluorescence microscopy, share little in common with regards to their methods of data collection and analysis. The concepts behind them are completely different and they use an entirely different set of analysis programs. Chapter IV consists of a published manuscript entitled "Continuous fluorescence depletion anisotropy measurement of protein rotation" which describes our work using a newly-developed pump-probe technique to examine protein rotation in solution and extends this to single-cell measurements. In the continuous variant of fluorescence depletion anisotropy used here, the intensity and polarization of a laser beam are modulated continuously by a programmed acousto-optic modulator and Pockels cell respectively to produce the desired excitation waveform. We have used this method to examine rotation of eosin conjugates of carbonic anhydrase, BSA, and immunoglobulin G in 90% glycerol at varying temperatures. We have also explored the potential application of this method to single-cell measurements and recorded preliminary results on eosin-IgE-bound FcεRI. Generally, we found good agreement with time-resolved phosphorescence anisotropy measurements of rotation of solution-phase molecules and of cell surface FcεRI. Chapter V discusses future avenues worth exploring which would improve upon the methods presented in Chapters II and III. These include faster cameras to access shorter timescales, gold nanorods to improve the signal-to-noise ratio, and a method to obtain a true anisotropy in a microscope.Item Open Access Rotational motion and organization studies of cell membrane proteins(Colorado State University. Libraries, 2016) Zhang, Dongmei, author; Barisas, B. George, advisor; Van Orden, Alan, committee member; Henry, Chuck, committee member; Roess, Deborah A., committee member; Crans, Debbie, committee memberCell membranes are dynamic structures with complex organization. The complexity of the cell membrane arises from intrinsic membrane structure, membrane microdomains within the plasma membrane and the membrane cytoskeleton. Plasma membrane receptors are integral membrane proteins with diverse structures and functions which bind specific ligands to trigger cellular responses. Due to compartmentalization of the plasma membrane and the formation of membrane microdomains, receptors are distributed non-homogeneously in the cell membrane bilayer. Both lateral and rotational diffusion of membrane receptors reflects different kinds of intermolecular interactions within the plasma membrane environment. Understanding protein diffusion within the membrane is very important to further understanding biomolecular interactions in vivo during complex biological processes including receptor-mediated signaling. Rotational diffusion depends linearly on the in-membrane volume of the rotating proteins. Relative to lateral diffusion, rotational diffusion is a more sensitive probe of an individual molecule’s size and local environment. We have used asymmetric quantum dots (QD) to conduct imaging measurements of individual 2H3 cell Type I Fcε receptor rotation on timescales down to 10 msec per frame. We have also used time-tagged single photon counting measurements of individual QD to examine μsec timescales, although rapid timescales are limited by QD emission rates. In both approaches, decays of time-autocorrelation functions (TACF) for fluorescence polarization fluctuations extend into the millisecond timescale, as implied by time-resolved phosphorescence anisotropy results. Depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Imaging results demonstrate a range of rotational behavior among individual molecules. Such slow motions, not observable previously, may occur with large signaling complexes, which are important targets of study in cell biology. These slow motions appear to be a property of the membrane itself, not of the receptor state. Our results may indicate that individual mesoscale membrane regions rotate or librate with respect to the overall cell surface. The luteinizing hormone receptor (LHR) is a seven transmembrane domain receptor and a member of the GPCR family. It is located on luteal cells, granulosa and theca cells in females. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of mammalian reproduction. LHR becomes aggregated upon binding hCG when receptors are expressed at physiological numbers. Binding of hormone to LHR leads to activation of adenylate cyclase (AC) and an increase in intracellular cyclic AMP (cAMP). ICUE3 is an Epac-based cAMP sensor with two fluorophores, cyan fluorescent protein (CFP) and the YFP variant, cpVenus, and a membrane-targeting motif which can be palmitoylated. Upon binding cAMP, ICUE3 undergoes a conformational change that separates CFP and YFP, significantly reducing FRET and thus increasing the ratio of CFP to YFP fluorescence upon excitation with an arc lamp or 405nm laser source. Hence we have investigated hLHR signal transduction using the cyclic AMP reporter probe, ICUE3. A dual wavelength emission ratio (CFP/YFP) imaging method was used to detect a conformational change in ICUE3 upon binding cAMP. This technique is useful in understanding the sequence of intercellular events following hormone binding to receptor and in particular, the time course involved in signal transduction in a single cell. Our data suggested that CHO cells expressing ICUE3 and directly treated with different concentrations of cAMP with saponin can provide a dose-dependent relationship for changes in intracellular cAMP levels. Forskolin (50μM) causes maximal activation of the intracellular cAMP and an increase in the CFP/YFP emission ratio. In CHO cells expressing both ICUE3 and hLHR-mCherry, the CFP/YFP ratio increased in cells treated with forskolin and in hCG- treated cells. In flow cytometry studies, similar results were obtained when CHO cells expressed < 60k LHR-mCherry per cell. Our results indicate that ICUE3 can provide real time information on intracellular cAMP levels, and the ICUE3 is a reliable cAMP reporter can be used to examine various aspects of LH receptor-mediated signaling.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.