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1
Abia, Atogho Jude. "Polyaniline and Its Derivatives for Environmental Analysis." Digital Commons @ East Tennessee State University, 2006. https://dc.etsu.edu/etd/2240.
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Electrooxidation has been used to deposit thin film polyaniline as well as its derivative - thin film poly (ortho-phenylenediamine) (POPD) and poly (meta-phenylenediamine) (PMPD) on carbon electrodes, which are subsequently used to monitor the environmental heavy metal ions (Hg2+, Pb2+ ,Cd2+) through a rather unusual "blocking" of anodic stripping for these metals. Using Hg2+ as a model, its cyclic voltammogram for a modified glassy carbon electrode with the resultant polymer shows an enhanced cathodic peak that increases linearly with the analyte ion concentration. POPD also exhibits unique selective detection for organic species. Acetaminophen and uric acid can be preferentially detected over ascorbic acid from a mixture of these three compounds. In addition, the effect of carbon nanotubes incorporated in polyaniline (PAN) film is observed to have enhanced electrochemical catalytic activities on the remedy of environmental dichromate.
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D'Urbino, Davide. "Exploring the Effects of Different Classroom Environments on the Learning Process. Synthesis of Thiazole-Linked Porous Organic Polymers for CO2 Separation and Nitro-Aromatics Sensing." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4918.
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When attempting to study the learning process of undergraduate chemistry student, the classroom and any interaction that take place within it constitute the social context of interest. By studying how different approaches can foster different classroom environments, it is possible to approach course design from an informed and scientifically sound perspective. Thus, it becomes necessary to identify and quantify the factors that have a positive or negative effect on the classroom environment. Social comparison concerns, comfort levels and self-efficacy have been shown to be social factors that affect each other as well as the learning process and have therefore been deemed suitable for use in this study. POGIL, a pedagogic approach to teaching chemistry based on small-group work and active learning, has been shown to lead to positive academic outcomes and is currently employed by several faculties at Virginia Commonwealth University. This study seeks to investigate differences in the learning environment observed in lecture and POGIL based chemistry courses, by adapting Micari’s survey for measuring social comparison, comfort levels and self-efficacy in small-group science learning.
Reliance on the combustion of fossil-fuels, such as coal, oil and natural gas, as sources of energy has, since the industrial revolution, caused atmospheric CO2 to increase to the current level of 400ppm by volume; an increase of 25% from the 1960s when monitoring started. Climatologists predict that an increase to 450 ppm would have irreversible effects on the Earth’s environment and recommend that, in order to preserve the conditions in which civilization developed, levels be reduced to below 350 ppm. The use of porous organic polymers for capture and separation of CO2 from industrial sources has been at the forefront of research attempting to curb CO2 emission into the atmosphere. Benzimidazole based polymers have shown a high selectivity for CO2.7 To attempt to improve on the capture abilities of these polymers, we sought to synthesize sulfur containing analogs presenting thiazole moieties. Two such polymers were synthesized using a pyrene-based linker. Furthermore, the pyrene-derived fluorescence of these polymers enabled their use as chemosensors targeting nitroaromatic compounds and mercury
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Addy, Mary Akuyea. "Modified Organoclay Containing Chelating Ligand for Adsorption of Heavy Metals in Solution." Digital Commons @ East Tennessee State University, 2011. https://dc.etsu.edu/etd/1372.
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Presence of a chelating ligand in the clay structure significantly improves its ability to immobilize heavy metals from contaminated sludge or wastewater. Two-step modification procedure comprising sequential pillaring and grafting of chelating agent to the modified clay is involved.
Montmorillonite and kaolin were chosen as typical examples of expandable and non-expandable clays, correspondingly. Modifications with silica and ferric oxide were targeted on development of mesoporous structure. Laboratory tests of the organoclay efficiency for purification of wastewater were conducted with the most promising sample, i.e. organoclay with the highest specific loading of chelating agent. Experiments were conducted with model wastewater containing either individual or mixed cations of heavy metals.
The modified organoclay displayed a high adsorption capacity on heavy metal cations even in acidic media. The method of modification presented in this work can be used for synthesis of efficient adsorbents for applications in contaminated areas.
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Regmi, Suresh Chandra. "Monitoring Metal Containing Particulates Distribution on a College Campus Using Dandelion (Taraxacum officinale) Leaves as Receptors." Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etd/1976.
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This study aims to determine the distribution of particulates carrying heavy metals at selected sites on a college campus using dandelion leaves as collectors. As a comparison, sites far away from the campus surrounding Bristol Motor Speedway Car Racing Stadium were also monitored. To reduce the probability of memory effects from the long-term atmospheric deposition or absorption of metals from soil a seasonal plant, dandelion, was used to monitor the metal contents. The metals monitored are cadmium, calcium, copper, chromium, iron, lead, and zinc. Fourteen sites were monitored and samples were collected once a week initially (growing time of the plant), and later at 4-week interval from 28th March to 31st August of 2007. The metal contents of the nitric acid digested and appropriately diluted samples were determined by flame atomic absorption spectrometry using the regular standard calibration curve and also the standard addition method. From the results obtained, and the careful log of the weather and human activities at the different sites, it is concluded that human activities played a major role in the distribution of metal-laden particulates. Also dandelion leaves were proven to be viable collectors of these particulates without memory effects and as indicators of current particulates generated rather than a long-term cumulative one.
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Item, Ann Ejimole. "Determination of Selected Heavy Metals in Some Creeks in a Tennessee City." Digital Commons @ East Tennessee State University, 2011. https://dc.etsu.edu/etd/1249.
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Concentrations of Ni, Cu, Zn, Fe, Cd, and Pb were determined in six different creeks within a city in Tennessee using Atomic Absorption Spectrophotometer. Mean concentrations of Ni, Cu, Zn, Cd, Pb, and Fe in the sites examined reveal that they exceed the USEPA recommended limits. High concentrations of Cu (0.130 mg/L), Zn (13.7 mg/L), Ni (0.267 mg/L), and Cd (0.010 mg/L) were observed in site B and Fe (3.01 mg/L) in site E relative to other sites. The concentration of Pb (0.795 mg/l) was higher in site A. Higher concentrations of Cu, Zn, and Fe were detected in samples collected in the month of January and Cd in samples collected in the month of June. Pb and Ni concentrations did not show any significant difference with respect to dates of sample collection. Their presence in the environment on a particular day depends on the type and volume of human activities.
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Zhou, Guannan. "Polycondensation of Bridged Amino-Functionalized Trialkoxysilanes." Digital Commons @ East Tennessee State University, 2011. https://dc.etsu.edu/etd/1292.
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The reduction of CO2 emission has been a worldwide mission to resolve global warming predicament. Mesoporous silsesquioxanes, which stabilized by organobridges and has high content aminon absorption site, can serve as a potential CO2 adsorbent. Synthesis of such material is done by hydrolysis and polycondensation of trialkoxysilane. The fastest gelation was observed at reaction in the absence of acids or bases. However, addition of surfactant to the reaction mixture catalyzed formation of silsesquioxanes in acidic media. Obtained materials are strongly hydrophilic and possess a high thermostability. Study of particle size distributions showed that in all cases it was bimodal. The largest particles formed in basic media. Mesoporous silsesquioxanes were obtained from bridged alkyltrimethoxysilanes in the presence of surfactants.
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Edwards, Paula Kay. "The Correlation of the Concentration of Selected Metals Determined in Water and Fish Samples from a Public Pond." Digital Commons @ East Tennessee State University, 2010. https://dc.etsu.edu/etd/1774.
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The concentrations of heavy metals (Cd, Cu, Fe, Ni, Pb, Zn) were measured in water, and fish samples caught from the pond at Fishery Park in Unicoi County, TN. The water samples were collected once a week for 8 weeks. The amounts of metals in the muscle tissues, gill, and liver of the two fish species were measured. This was to determine if any correlation exists between the metal contents in water and those in the fish samples. The concentration ranges for the heavy metals found in the water samples are as follows: Zn 0.04-0.13; Cu, 0.00-0.00; Pb, 0.00-0.59; Cd, 0.0067-0.0071; Fe, 0.208-0.512; and Ni, 0.044-0.270 ppm. The concentration range for the heavy metals found in the fish tissues are as follows: Zn 0.0-0.48; Cu, 0.00-0.00; Pb, 0.00-0.43; Cd, 0.00-99.7; Fe, 25.7-1245.5; and Ni, 0.00-268.5 ppm. There was a strong correlation found between the water and fish tissue samples.
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Dotse, Charles Kafui. "Assessing Commercial Organic and Conventionally Grown Vegetables by Monitoring Selected Heavy Metals Found in Them." Digital Commons @ East Tennessee State University, 2010. https://dc.etsu.edu/etd/1715.
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Commercially available organic and conventionally grown vegetables were studied by quantitative determination of selected metals in them and to determine if any differences found are statistically significant. These findings can help the consumers to determine if the vegetable products are within the recommended maximum limits as proposed by the joint FAO/WHO Expert committee on organic foods designation. Eight edible vegetables were purchased from local stores in both the organic and conventionally grown categories. Samples were digested with concentrated nitric acid and the metals monitored were zinc, copper, lead, iron, cadmium, and nickel using flame atomic absorption. The concentration range for the heavy metals found are as follows: Zn, 2.04-69.4; Cu, 0.35-15.1; Pb, 0.00-3.99; Cd, 0.00-0.74; Fe, 2.52-319; and Ni, 39.9-53.8 μg/g. It was found that in general, conventional vegetables contain higher amounts of most of the heavy metals studied as compared to their organic counterparts. The study also showed that all vegetables products contain below the permissible limits for Zn, Cu, Ni, and Fe. For Pb all vegetables exceeded the safe limit except organic cucumber and conventional cabbage. For Cd, organic lettuce and green pepper, and conventional leafy green, green pepper, and spinach all exceeded the limit recommended by FAO/WHO.
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Burns, Molly Elise. "A Comparison of Solvent and Water-Borne Alkyd Coatings and the History of VOC Regulations in the United States." DigitalCommons@CalPoly, 2016. https://digitalcommons.calpoly.edu/theses/1741.
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A Comparison of Solvent and Water-Borne Alkyd Coatings Abstract
Conventional solvent based alkyd coatings have gone out of favor due to concerns over volatile organic compound (VOC) content. However, due to recent focus on renewable raw materials, alkyds are making a comeback in waterborne form. Water based alkyd coatings are known to have poor shelf stability and corrosion resistance, as well as other problems during the formulation process. This project focused on comparing solvent borne to two types of water-borne alkyds, water reducible alkyds and alkyds emulsions. The purpose was to understand the differences between the three types of alkyds in terms of their production and final properties. It was ultimately hoped that the formulations used for this project would prove to solve the problems normally experienced by waterborne alkyds.
After testing several chemical and physical properties, it was determined that the solvent borne alkyd coatings performed better than both water based systems in corrosion resistance, accelerated weathering, and shelf stability but the water reducible and emulsion alkyd coatings performed similarly to the solvent borne alkyd in gloss, contrast ratio, and durability. The VOC emissions for all three alkyd types were as expected; the solvent borne had the highest emission at 253 g/L, followed by water reducible with 166 g/L, and emulsion with 34 g/L.
The History of VOC Regulations in the United States Abstract
In another solvent based alkyd coating focused project within my research group, the question of the how volatile organic compound (VOC) regulation in the United States (U.S.) evolved came up. It quickly became apparent that no comprehensive answer to this question existed. Part two of this project is an attempt to answer this question in a comprehensive manner.
VOC regulations started in California in the late 1970s, and paints and coatings became a nationally regulated emission source by the 1990s. The U.S. government limited harmful emissions, such as smog and compounds contributing to ozone depletion, through Clean Air Acts. The first Clean Air Act was enacted in 1965, but it wasn’t until the Clean Air Act of 1990 that VOC emissions became a focus. VOCs are not inherently hazardous but are a source of concern because they serve as a precursor to the formation of damaging ground level ozone.
The Environmental Protection Agency (EPA) has established the minimum VOC emission limits in the Architectural and Industrial Maintenance (AIM) federal rule, but each state or state subdivision can enforce stricter limits within their borders. The strictest limits are set by the South Coast Air Quality Management District (SCAQMD) in Southern California, but other entities exist. This report thoroughly documents the history of VOC regulation in the United States by collecting, combining, organizing, and summarizing information gathered from various industries and government publications, agency members, and industrial and academic professionals.
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Yuan, Jing. "Quantitation of polyamines and metabolites in mouse erythroleukemia cells by mass spectrometry." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/2657.
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Polyamines are naturally occurring cellular polycations essential for cell growth and differentiation. This investigation focused on the quantitative analysis of polyamines and metabolites in mouse erythroleukemia (MEL) cells by mass spectrometry. Hexamethylene bisacetamide (HMBA) is a synthetic polyamine derivative known to induce differentiation of a variety of transformed cells such as MEL cells. A fast and sensitive quantitative method for HMBA and metabolites NADAH, DAH and AcHA was developed using atmosphere pressure chemical ionization mass spectrometry (APCI/MS) by flow injection analysis. Selected ion monitoring (SIM) mode was employed for the mass spectrometric detection and d 4 -DAH was used as the internal standard for quantitation. The intracellular concentrations of HMBA and metabolites were obtained in MEL cells treated with 5mM HMBA in the presence or absence of 500μM APAH, a potent N 8 -acetylspermidine deacetylase inhibitor. A significant increase in intracellular NADAH and decrease in DAH levels in MEL cells were observed in HMBA treatment in the presence of APAH. This result indicates that APAH inhibits the second deacetylation step in HMBA metabolism, the conversion of NADAH to DAH, but not the first deacetylation of HMBA to NADAH. Two histone deacetylase (HDAC) inhibitors including sodium butyrate (NaB) and m -carboxycinnamic acid bis-hydroxamide (CBHA) were also used as inducing agents for MEL cell differentiation. Both agents caused accumulation of hyperacetylated histone H4 and H2B in MEL cells at concentrations optimal for inducing differentiation, while neither HMBA nor APAH had detectable effect on the acetylation level of histones. A fast and sensitive method for five important polyamines including putrescine (PU), spermidine (SPD), spermine (SPM), N 1 -acetylspermidine (N 1 -AcSPD) and N 8 -acetylspermidine (N 8 -AcSPD) was also developed using APCI/MS by flow injection analysis. Selected reaction monitoring (SRM) mode was employed for the mass spectrometric detection and 1,7-diaminoheptane was used as the internal standard for quantitation. The intracellular polyamine concentrations was obtained in MEL cells treated with 5mM HMBA, 2mM NaB, 3μM CBHA and 500μM APAH respectively. A significant increase in N 8 -acetylspermidine levels was observed during 3hr to 4 days treatment with APAH. There was no change in N 8 -acetylspermidine levels in MEL cells treated either with NaB or CBHA. The results from the present study suggest APAH has a selective inhibitory effect on N 8 -acetylspermidine but not histone deacetylation. While HDAC inhibitors inhibit histone deacetylase but have no effect on N 8 -acetylspermidine deacetylation. In conclusion, despite the known similarities they share, the enzymes involved in the deacetylation process of N 8 -acetylspermidine and histones in MEL cells are different.
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Tejomurthula, Sravanthi. "Overexpression of Human Aryl Hydrocarbon Receptor in E.coli Using Two Different Solubility Enhancing Tags." Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/261.
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Dioxins such as TCDD are environment pollutants whose toxic effects are mediated via aryl hydrocarbon receptor (AhR) signaling pathway. AhR is a ligand sensitive transcription factor. The unbound AhR resides in cytoplasm as a complex containing p23, Hsp90 and XAP2. Upon ligand binding, AhR undergoes conformational change and translocates into the nucleus. Once the AhR dimerizes with AhR nuclear translocator (Arnt), the chaperone proteins in the complex get dissociated followed by the activation of transcription of various genes such as CYP1A1 and CYP1A2 by AhR-ARNT heterodimer. Various cancers have altered levels of AhR in the absence of ligand. Our current knowledge is only limited in the regulation of AhR protein levels in its ligand bound state. However, the mechanism involved in the regulation of AhR protein levels in the absence of ligand is still unknown. To make the study of AhR signaling pathway possible, our lab has been working on the expression of various AhR constructs in E.coli using recombinant DNA technology. As AhR forms inclusion bodies due to its poor solubility in the cytoplasm of the host bacteria, it is tagged as a “difficult to express” protein. Therefore, it is challenging to generate functional recombinant AhR protein. My thesis documents the expression of human AhR construct amino acid 108-400 using two different solubility enhancing tags (thioredoxin and maltose binding protein). Western blot data revealed that the soluble expression of the human AhR construct by thioredoxin solubility enhancing tag has outperformed the other.
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Patlolla, Karthik Reddy. "Predicting aqueous solubility of pharmaceutical agents by solid dispersion prepared by solvent evaporation method." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/268.
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Solubility of active pharmaceutical agents is a crucial process that determines drug absorption and ultimately its bioavailability. Many of the new therapeutically beneficial compounds discovered are lipophilic requiring various solubility enhancement strategies to improve their solubility. Among these strategies, solubility enhancement using solid dispersions is a leading method. To obtain a desirable increase in the solubility of a poorly-soluble compound, a good understanding of the molecular descriptors influencing the enhancement of solubility is essential. Therefore, the major research objective was to determine the descriptors which significantly influence the solubility enhancement by solid dispersions. After enhancing the solubility of selected poorly-soluble model compounds, a regression analysis was performed to determine the correlation of molecular descriptors of the active agent, polymer, and solvent with solubility enhancement. The partition co-efficient, hydrogen bonding and solubility parameters of polymer and water were found to influence the aqueous solubility of the poorly-soluble compounds. Aqueous solubility of a compound had an inverse relation with difference in solubility parameters of polymer and water. Similarly partition coefficient was found to be inversely related to aqueous solubility. However for an increase in hydrogen bond acceptors present in pharmaceutical agents increased their solubility, while the higher number hydrogen bond donors resulted in lower solubility. This complexity can be attributed to the contribution of hydrogen bonding in a crystal lattice and in aqueous environment. In conclusion, the contribution of partition co-efficient, solubility parameter and hydrogen bonding were found to be significant for a given set of poorly-soluble model compounds selected with a wide range of descriptors. Several models estimating aqueous solubility of compounds have been employed as screening tool in drug development process. However, all such models were developed to estimate aqueous solubility of pure active agents. Hence, the second research objective was to develop a model that could estimate aqueous solubility of Active Pharmaceutical Ingredient (API) in solubility-enhanced solid dispersions. Using multiple linear regression, a computational model was developed using the molecular descriptors of poorly-soluble compound, polymer and water. S=(2.02*HBA)-(3.37*??)-(11.56*log?P )-(0.9*HBD)+119.66 The model showed a regression (R2) value of 0.858. Upon validation, the model estimated the aqueous solubility of 79% of the compositions evaluated with within 20% variability.
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Yang, Dazhou. "Synthesis and biophysical evaluation of thiazole orange derivatives as DNA binding ligands." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/141.
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Guanine-rich telomeric DNA at the end of chromosomes can form a unique DNA tertiary structure - G-quadruplex, which is known to inhibit the binding of telomerase to telomeric regions in cancer cells and thus regulate unrestricted cancer cell growth. Hence, G-quadruplex DNA has recently become a promising target in oncology. The formation of G-quadruplex structures is greatly facilitated by G-quadruplex binding ligands such as Thiazole orange (TO). Compared with other G-quadruplex binding ligands, TO has an intriguing tunable fluorescence property. Upon binding to DNA, the fluorescence of TO can increase up to 1000-fold, making it an attractive probe for studying ligand-DNA interactions. However, the poor binding affinity and minimal binding selectivity towards different DNA conformations greatly limit its applications. My research focuses on developing G-quadruplex binding ligands using TO as a scaffold. In the first part of this work, we investigated the feasibility of increasing the TO binding affinity and selectivity toward G-quadruplex DNA by introducing side chains to the molecule. TO derivatives containing various side chains were successfully synthesized and characterized. Biophysical and biochemical studies with duplex and G-quadruplex DNA showed that tethering side chains to TO is an effective approach to tune its ability of binding to duplex or G-quadruplex DNA. Possible binding modes of the effective derivatives were studied using AutoDock. Their inhibition of telomerase activities was studied using the TRAP assay. The cytotoxicity of these derivatives toward three cancer cell lines was also investigated using the MTS assay. The second part of this work focuses on development of TO-based G-quadruplex DNA binding ligands that can bind to DNA via the dual recognition mode. TO was tethered with pyrene, naphthalene diimide, and anthraquinone respectively to yield three novel conjugates. Further investigation suggested that the conjugate of TO with naphthalene diimide (TO-NF) gave the best G-quadruplex binding affinity. It binds to G-quadruplex DNA via the end stack mode and strongly inhibits the telomerase activity. The cytotoxicity results will also be discussed in this presentation.
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Naidu, Prathyusha. "Catalase-loaded liposomal magnesium phosphate nanoparticles for intracellular protein delivery." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/266.
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Proteins are large biomolecules that have great therapeutic potential in treating many human diseases. Proteins exert higher specificity and more complicated functions; they are well endured and less inclined to evoke immune responses when compared to small molecule drugs. However, exogenous proteins when administered intravenously are prone to immune reactions. Chemical and enzymatic denaturation, and poor penetration into cells are some other challenges for clinical use of intracellular proteins. Proteins that enter cells through endocytosis will be eventually degraded in lysosomes if they do not escape the endosomal pathway before reaching lysosomes. Therefore, the development of protein delivery systems, including liposomal and/or polymeric nanoparticles would substantially facilitate the clinical use of proteins. This approach can protect the proteins from denaturation and immune reactions. Previously, our group has developed cationic lipid-coated magnesium phosphate nanoparticle (CAT-LP MgP NP) formulations to enhance the intracellular delivery of the protein, catalase. The objective of the current research is to improve the physicochemical properties of CAT-LP MgP NP. The magnesium phosphate (MgP) nanoparticles were prepared by water-in-oil micro emulsion precipitation. The cargo protein catalase was complexed with cationic liposome prepared by lipid hydration and extrusion. Then magnesium phosphate (MgP) nanoparticles were mixed with the catalase-complexed cationic liposome to form the final complexed CAT-LP MgP NP formulation. By sonication, extrusion and modification of the lipid composition, we have successfully prepared complexed CAT-LP MgP NP formulations of reduced size. The pH-sensitivity of the improved delivery system was observed at pH 6.0. Furthermore, the improved delivery system reduced the Reactive Oxygen Species (ROS) level inside EA.hy.926 cells (human umbilical vein endothelial cells) to 35% of the control, whereas the previously reported catalase formulation of our group reduced the ROS levels to 50%, indicating that the complexed formulation delivers functional catalase more efficiently into the EA.hy.926 cells. Complexed CAT-LP MgP NP with reduced size has delivered cargo protein more efficiently than encapsulated CAT-LP MgP NP.
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Kondepudi, Karthik Chalam. "Computational prediction of enhanced solubility of poorly aqueous soluble drugs prepared by hot melt method." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/267.
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Solubility is the concentration of a solute in a saturated solution at a given temperature and pressure. Solubility of a drug in aqueous media is a pre-requisite to achieve desired concentration of a drug in the systemic circulation. Low aqueous solubility is a major problem encountered with formulation development of recently designed new chemical entities. Solubility of poorly soluble drugs is enhanced by physical and chemical modifications of drug. Shake flask method is the most commonly used experimental method to determine solubility. However, this method has several limitations. A single solubility experiment can go on for several days and even weeks. Besides this, a large amount of drug is required to carry out the experiment. In order to overcome this and make initial screening easier, computational method can be used to predict solubility. In this study, the solubility of 12 small molecules of BCS class II having a wide range of physicochemical properties were studied to enhance their solubility by hot melt method. Three different grades of PEG (1450, 4000, 8000), PVP K17 and Urea as the hydrophilic carriers was employed for the solubility enhancement. The overall objective of this investigation is to develop a model that could estimate enhanced solubility using physicochemical descriptors. Multiple linear regression (MLR), a statistical tool, was used to generate a equation for the solubility by correlating physicochemical properties of the drug like- molecular size, logP, pKa, HBA, HBD, melting point, polar surface area, and number of rotatable bonds. Solubility enhancement is also influenced by the carrier used, we included the physicochemical properties of the carriers like molecular weight and solubility parameter in the development of the model. MLR analysis model, resulted in an equation, where, Log solubility = 5.982-0.010 MW (drug)-0.452 LogP-0.320 HBA-0.095 ?solubility parameter+0.015 MV. A regression analysis yielded a good fit with a regression value (adjusted R2) of 0.74. The model has been validated by leave one out method. This model has the potential to estimate the solubility of a physically modified drug in screening stages of drug development.
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Vutukuru, Naresh Kumar Reddy. "Apparent dissolution rate enhancement of poorly-water soluble drugs by adsorption technique." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/269.
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Nearly 70% of the new chemical entities (NCE’s) discovered are poorly-water soluble drugs and the number of poorly-water soluble drugs are increasing rapidly in the drug discovery. Most of the NCE’s are lipophilic and have dissolution rate issues. Low dissolution rate of the drugs result in poor bioavailability. To overcome poor bioavailability, an adsorption technique is developed to enhance the apparent dissolution rate of poorly-water soluble drugs. In this study, two poor-water soluble model drugs, ibuprofen and carvedilol were used. Methanol, DMF, DMSO and PEG400 were used as solvents and microcrystalline cellulose was used as an adsorbent. Pure model drugs, physical mixtures and prepared composites were characterized by using FTIR, DSC, XRD and dissolution testing. Results showed that the composites prepared with solvents DMF, DMSO and PEG400 showed enhancement in dissolution rates of two model drugs. Characterization of the composites prepared by using non-volatile solvents showed successful conversion of crystalline model drugs into solution state. Whereas, composites prepared by using volatile solvent showed similar results like physical mixtures and pure drug. Ibuprofen composites containing DMF, DMSO and PEG400 showed 9.4, 7.4 and 1.8 folds of increase in apparent dissolution rate, respectively. Whereas carvedilol composites containing DMF and DMSO showed 11.52 and 3.4 folds of increase in apparent dissolution rate. Four months of stability study were conducted on prepared composites at both 40°C and room temperature. It was observed that prepared composites were stable after 4 months and exhibited similar dissolution rate. In conclusion, the use of non-volatile solvents disrupted the crystal structure but also retained the drug in solution state which in turn enhanced the apparent dissolution rate of model drugs used. From the observed results we conclude that this method has a potential to replace existing techniques to enhance the apparent dissolution rate of the drug and stability of the composites.
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Vangala, Swathi. "Human Cytochrome P450 3A4 Over-Expressing IEC-18 and MDCK Cell Lines as an In-Vitro Model to Assess Gut Permeability and the Enzyme Metabolism." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/273.
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Purpose. The fate of an orally administered drug is dependent on many parameters before it can reach the systemic circulation, including drug absorption and first-pass metabolism in the gut and the liver. Mammalian cells lines such as MDCK and Caco-2 are commonly employed to assess drug permeability but they lack or have low expression level of drug metabolism enzyme expression such as CYP3A4, which contributes to significant first-pass gut and liver metabolism for many drugs. Consequently, these cell lines are not sufficient to integrate metabolism when assessing drug absorption. Here, we tested MDCK and IEC-18 cells transiently over-expressing CYP3A4 as models that can simultaneously assay a compound's permeability and metabolism potential in a single experiment. Method. A recombinant adenovirus carrying the hCYP3A4 cDNA was constructed according to Stratagene's AdEasy XL Adenoviral system. This adenovirus was used to transiently transfect hCYP3A4 into MDCK and IEC-18 cells. Western blot was performed to assess the level of hCYP3A4 expression in the wild type and CYP3A4 over-expressing IEC-18 and MDCK cells. In situ metabolism and transport studies were performed with wild-types and IEC-18-3A4 or MDCK-3A4 cells. Results. The amount of CYP3A4 present in MDCK-3A4 cells was 250 times to that of wild type cells which 1/4th the amount present in human liver microsomes. The amount of CYP3A4 present in IEC-18-3A4 cells was 150 times to that of wild type cells which 1/6th the amount present in human liver microsomes. In metabolism studies, there was higher formation of metabolites in cells transfected with hCYP3A4 compared to controls. In addition, apical to basal transport studies of several drugs in IEC-18-3A4 and MDCK-3A4 showed increased appearance of metabolites compared to the wild-type cells. Conclusions. This model may be a useful to assess the extent of drug absorption into systemic circulation after oral administration.
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Nannapaneni, Vijaysri. "Preparation of amorphous forms to increase the solubility of poorly soluble drugs using spray drying." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/274.
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Spray drying is widely used in enhancing the aqueous solubility of poorly soluble compounds. In this study, the mechanism of solubility enhancement was characterized using three model drugs-naproxen, ketoprofen and furosemide. Physical mixtures of the model drug with polyvinylpyrrolidine and spray dried composites were subjected to Fourier Transform Infrared Sprectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Powder X-ray Diffraction (XRPD). The data showed that the crystalline model drugs were converted to amorphous form upon spray drying, whereas the physical mixtures did not change their crystallinity. The effect of the amorphous forms produced by Spray drying on apparent solubility and intrinsic dissolution rate was determined. All the spray dried composites exhibited higher apparent solubility and intrinsic dissolution rate when compared to the pure drugs and their physical mixtures. The stability of the spray dried composites upon storage was also determined. The amorphous nature of the compounds in the spray dried composites were retained during 3 months storage as shown by FTIR, DSC and XRPD characterization and their apparent solubility and intrinsic dissolution rates also did not change.
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Ponnakanti, Himaja. "Soluble and Functional Overexpression of the Ligand Binding Domain of Mouse Aryl Hydrocarbon Receptor in E.Coli." Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/260.
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The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor whose toxicity and carcinogenesis is mediated through various polyaromatic hydrocarbons and other environmental pollutants. The role of AhR in carcinogenesis is an area of concern due its altered levels in various tumors. AhR binds structurally diverse ligands and may elicit different responses upon ligand binding. The crystal structure of mouse AhR PAS-A domain was already obtained due to the robustness of mouse AhR in comparison to human. There is a possibility of overexpressing mouse AhR ligand binding domain in its soluble and functional form, which could be used to perform ligand binding studies. This forms the aim of this thesis. Mouse AhR ligand binding domain was constructed as mAhR aa211-384, which was purified under native conditions with the use of 6 histidine tag but soluble overexpression was not possible. Thus a solubility enhancing tag called maltose binding protein (MBP) was used for purification of mAhR aa211-384 under native conditions, which still did not yield soluble overexpression. The strategy was modified to solubilize the protein by denaturation with the use of 8M Urea, which solubilized the protein but included an issue of protein binding to column. Subsequent use of an even stronger denaturant, 6M guanidine hydrochloride, solubilized most of the protein and purified mAhR aa211-384 in huge amount. Successful refolding of mAhR aa211-384 with the help of MBP was made possible by gradual reduction of denaturant in the presence of arginine, but 6 histidine tag failed to refold the protein. The refolded protein was tested for its secondary structure by circular dichroism. Thus, mAhR aa211-384 was solubilized and purified under denaturing conditions with the help of both 6 histidine and MBP, however efficient refolding of mAhR aa211-384 was only possible with the help of MBP but not 6 histidine. The MBP-refolded mAhR aa211-384 stayed in solution even after the removal of 0.1 M arginine, thus confirming the effectiveness of MBP in protein refolding in comparison to 6 histidine tag.
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Fang, Yunzhou. "A novel intracellular protein delivery system - Magnesium phosphate nanoparticles with cationic lipid coating for catalase intracellular delivery." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/270.
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Protein therapeutics have great potential in treating human disease, especially for those caused by alternations in the functions of intracellular proteins. However, clinical use of protein by intracellular delivery has been hampered by the instability due to proteins' physicochemical properties, and some barriers in the delivery pathway. This study was to prepare and test a novel intracellular protein delivery system - magnesium phosphate nanoparticles with cationic lipid coating for catalase intracellular delivery (LP MgP NP-CAT), and investigate whether it can release the encapsulated catalase to cytosol. LP MgP NP-CAT was designed, prepared and characterized, showing that it had an average diameter around 300 nm and zeta potential around +40mV. The pH - triggered catalase release from LP MgP NP-CAT was determined by a hydrogen peroxide degradation assay, where the concentration of remaining hydrogen peroxide was measured by UV-Vis spectroscopy, indicating catalase was released in response to the drop of pH, which was confirmed by the morphology change of LP MgP NP-CAT observed by transmission electron microscopy. The in vitro catalase release behavior was conducted on MCF-7 cells and EA.hy926 cells. LP MgP NP-CAT was delivered into MCF-7 cells and the release behavior was determined by the resultant resistance of the cells against hydrogen peroxide using MTS cell viability assay. The delivery of LP MgP NP-CAT into EA.hy926 cells was determined by the decrease of the reactive oxygen species level. Both of the studies showed that catalase was successfully delivered and released which is supported by the reduction of hydrogen peroxide.
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Sachdeva, Sameer. "Design and applications of antibody mimics against epidermal growth factor receptor." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/132.
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Antibodies have been widely used as reagents, homing devices, diagnostics and as therapeutic agents against different targets in clinic and research. Recently a number of monoclonal antibodies and their drug conjugates have been approved as therapeutic agents. While these molecules have great potential in various applications and therapeutics, extensive use of full length antibodies has been hampered by the high cost of production, large molecular weight and limited ability to penetrate tumor tissues. These limitations have led to the research for antibody alternatives with lower molecular weight, similar binding and affinity properties but without the lengthy and complicate process of generating antibodies. Some examples of these efforts include minibodies, fragment antigen binding (FAB), ScFv, and synthetic antibody mimics. Although these antibody alternatives have low molecular weight, as compared to the antibody, they are either derived from full size antibodies or by a long and tedious in vitro screening process. Therefore, a rational design of molecules that mimic antibody binding is a logical first step for the development of antibody alternatives. In this study, a novel approach to design antibody mimics without involving massive experimental screening was developed. The design was developed by mapping and identifying EGFR epitope region where Cetuximab CDR binds and modifying sequences using knob-socket computational model. The binding of antibody mimics were first analyzed by using MOE to obtain the binding energy, total and preserved interactions as compared to the interactions between EGFR and Cetuximab. Further, the designed antibody mimics were used to form a peptide drug conjugate (PDC). Antibody mimics were found to specifically bind and internalized by EGFR overexpressing cell lines with three to four folds higher than control cells. Antibody mimics showed binding in nanomolar range with Pep11 with binding affinity (K D ) of 252nM as shown by SPR studies. EGFR phosphorylation studies also showed that antibody mimics were able to inhibit the binding of EGF to the EGFR in a similar fashion as Cetuximab. Specific binding, affinity and functional activity of the antibody mimics demonstrated that these peptides were able to mimic all the three important characteristics of antibodies. Peptide drug conjugate (PDC) was found to be around 10 fold more potent as compared to the drug itself towards EGFR overexpressed cancer cells. PDC also showed more than 100 fold low potency against control cells. These studies demonstrated that a rational design of molecules to mimic the antibody characteristics is feasible. The antibody mimics were also successfully applied and used as targeting moiety to design peptide drug conjugates for efficient targeted drug delivery system than antibody drug conjugates.
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Sun, Jingjing. "Exploring the effect of alpha2 receptor on brain 5-HT via a mechanism-based pharmacodynamic model." Scholarly Commons, 2012. https://scholarlycommons.pacific.edu/uop_etds/154.
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Purpose: 5-hydroxytryptamine (5-HT) is an important neurotransmitter in depression. It is believed that α 1 and α 2 adrenoceptors mediate the 5-HT level in the brain. The mechanism is complex and not well explored. Especially in different combination treatments, the receptor systems may show varied modulation capability. Additionally, some research has suggested that α 2 heteroceptors may contribute to the time delay problem in dual depression treatment which is thought as the time needed for certain inhibition receptor to get desensitized. We hypothesized that the α 2 adrenoceptors had inhibition effect on 5-HT level in dorsal raphé nucleus (DRN), Prefrontal cortex (PFC) and Hippocampus (HP) with the dual reuptake inhibition. The present study was undertaken to explore the effect of BRL44408 (α 2 receptor antagonist) on 5-HT level in rat PFC, DRN and HP under dual antidepressant with blocking the α 1 receptor. Method: Serotonin reuptake inhibitor and norepinephrine reuptake inhibitor were used to mimic the dual reuptake inhibition antidepressant. To differentiate the α 2 adrenoceptors effect from al adrenoceptors effect, prazosin, an antagonist of α 1 adrenoceptors, was added to block α 1 adrenoceptors. Using the microdialysis method, the drug combination was examined in HP area and then DRN area to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results from DRN and PFC, a mechanism-based pharmacodynamic model was developed. Result: BRL44408 increased the serotonin (5-HT) level in rat PFC, DRN and HP to different degrees with the dual reuptake inhibition (p < 0.05). The overall model reasonably captured the time course of 5-HT in both DRN and PFC with different dose schemes of BRL44408. The model predicted EC50 of BRL44408 (0.0075 µM) for the α 2 heteroreceptor which control PFC 5-HT is close to the reported value of BRL44408 for α 2 adrenorceptor (0.008 µM). However, the model predicted EC50 of BRL44408 on the α 2 heteroreceptor which control DRN 5-HT need to be explained. Simulation result from this model suggested varied modulation capability of α 2 adrenoceptors on the 5-HT in DRN and the 5-HT in PFC. Conclusion: α 2 heteroceptor play a role in regulation 5-HT level under dual reuptake inhibition. Further exploration may bring a potential target for depression treatment. The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.
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Zhao, Liang. "Post-translational modifications of SEL24K from salmon eggs and ZPA from Xenopus laevis eggs." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/160.
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Post-translational modifications (PTMs) of proteins play significant roles in regulation of biological activities and signal transduction. Examining their diversity is critical for understanding the mechanisms of cellular regulations. Among the various techniques employed for identification of PTMs, mass spectrometry has become a more and more important tool for detecting and mapping these covalent modifications and quantifying their changes. The two projects described in this dissertation focus mainly on the method development for characterization of two major PTMs, disulfide bonds and glycosylation. In the first project, the disulfide bond pattern of a rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha was assigned unambiguously based on a multi-enzyme digestion strategy in combination with MALDI-TOF mass spectrometry analysis. The disulfide bond pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. More importantly, an interesting phenomenon of gas-phase scrambling of disulfide bonds was observed during MALDI mass spectrometry analysis and a possible mechanism for this surprising scrambling was proposed. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. In the second project, the glycosylation of a glycoprotein ZPA from the vitelline envelope of Xenopus laevis was determined by applying a strategy of general proteolysis coupled with mass spectrometry. The vitelline envelope glycoproteins were first separated through SDS-PAGE. A nonspecific in-gel pronase digestion was performed on the excised band of ZPA to produce informative small glycopeptides. Lectin affinity chromatography was used for the enrichment of these glycopeptides. An in-gel PNGase F digestion was also carried out to release the N-linked glycans from ZPA. The enriched glycopeptides and glycans were finally analyzed by MS and MS/MS techniques on MALDI-TOF and MALDI-TOF/TOF instruments.
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Liu, Xin. "Fliposomes: pH-sensitive liposomes comprising novel trans-2-aminocyclohexanol-based amphiphiles as conformational switches for the liposome mebrane." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/149.
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As a promising pH-triggerable molecular switch, trans -2-aminocyclohexanol (TACH) has a variety of applications. By introducing two hydrocarbon tails, multiple TACH-based lipids (flipids) have been designed and studied that are able to perform a drastic conformational flip upon protonation, loosening the stacking of hydrocarbon tails in lipid bilayers. Liposomes constructed from such flipids (fliposomes) can be disrupted by this acid-triggered conformational flip to cause a rapid release of a cargo specifically in areas of increased acidity (such as inflammation or ischemic tissues, solid tumor, and endosome pathway). A library of flipids has been built based on structural modifications of both amino headgroups and hydrophobic tails. A series of fliposomes have been constructed and their colloidal stability, capacity and pH-dependent leakage were investigated. A good correlation between the conformational switch of flipids studied by 1 H-NMR and the fliposomes' leakage indicated that the former is a cause for the latter. The obtained results showed that all the properties of fliposomes can be manipulated by selection of the amino headgroups structure and basicity, and the length and shape of hydrophobic tails, by using mixtures of different flipids or fliposomes, and by changing the content of flipids while constructing fliposomes. As a result, we prepared the pH-triggerable fliposomes with extraordinary characteristics: high stability in storage combined with instant release of their cargo in response to a weakly acidic medium. Fliposomes encapsulating the anticancer drug methotrexate (MTX) were applied to HeLa cells and demonstrated much higher cytotoxicity than the free drug and negative controls, indicating that they could conduct more efficient cellular delivery of MTX. The MTX-loaded fliposomes inhibited tumor growth in B16F1-melanoma-bearing nude mice compared to the control group, suggesting the anticancer activity of MTX delivered by pH-triggerable fliposomes in vivo. The results of research demonstrated the potential of fliposomes to serve as a viable drug delivery system.
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Liu, Wanbo. "Molecule recognition of nucleic acids, nucleosides, nucleotides, and their derivatives." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/150.
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It has long been known that the efficiency of anticancer drugs is limited by the emergence of resistance due to the evolving repair of such DNA lesions in malignant cells. Therefore, development of pharmaceutical agents, which can interfere with the DNA repair pathways, may represent a novel approach to enhance the cytotoxic effects of chemotherapy by reducing drug resistance. Abasic sites (AP sites) are the key intermediates in the BER pathway and promising targets for BER inhibition. In chapter 2, we report the synthesis of two small molecules specifically targeting at AP sites and the evaluation of their activity in terms of interstrand crosslinking formation. Our results show no covalent adduct is induced, which is due to the weak DNA binding affinity. In chapter 3, we try to use TFOs to deliver the interstrand crosslinking moiety to the AP site in a sequence specific manner. Two modified phosphoramidites were synthesized and incorporated into the 5' end of TFOs. The activity was evaluated by using various biophysical and biochemical experiments. The work reported in chapter 4 is focused on the G-quadruplex structure formed in the guanine rich telomeric sequence. Many studies have shown G4 ligands can induce and stabilize G-quadruplex within telomere region and inhibit the activity of telomerase that is overexpressed in 80-90% of cancer cells. Our results indicate that phenanthroline based metal complexes, Ni(Phen) 2 , have strong binding affinity and selectivity towards G-quadruplex over duplex DNA. The effect of Ni(Phen) 2 on telomerase activity and cytotoxicity towards cancer cells was also investigated. Calixarenes containing DNA building units such as nucleotides, nucleosides, and nucleobases have recently aroused much interest because of their versatile applications. In chapter 5, we report the synthesis of calix[4]arenes ( 5.11-5.14 ) functionalized with a single nucleobase (thymine, adenine, guanine, or cytosine) at the upper rim via click chemistry. Their complexation with alkali metal ions was examined using MALDI-TOF mass spectrometry and their molecular interactions were determined using 1 H NMR. All calix[4]arene derivatives show good complexation with alkali metal ions with apparent selectivity. The results also reveal that nucleobase-calix[4]arenes are capable of self-association in CDC1 3 and calix[4]arenes bearing complementary nucleobases can bind to each other via base pairing.
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Zheng, Yu. "Synthesis and conformational study of trans-2-aminocyclohexanol-based pH-triggered molecular switches and their application in gene delivery." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/153.
Full textAbstract:
Trans-2-Aminocyclohexanol (TACH) is a promising model for pH-triggerable molecular switches with a variety of potential applications. In particular, such a switch, when incorporated into cationic liposomes, provides a novel design of the pH-sensitive helper lipids for gene delivery. Protonation of TACH molecules results in a strong intramolecular hydrogen bond between the amino and its neighboring hydroxyl groups, which triggers a conformational flip, and forces changes of the relative position of other substituents on the ring. In this work, a library of TACH-lipids has been designed and built based on structural modifications of both hydrophilic headgroups and hydrophobic tails, and their conformational behavior has been studied by 1 H NMR. NMR-titration has been done to quantitatively monitor the conformational switch for TACH derivatives. It was discovered that conformational behavior of TACH-lipids is independent from the length or shape of their hydrophobic tails. Therefore, a simplified model was suggested based on TACH with diethyl groups instead of hydrocarbon tails. Conformational study of these models has demonstrated that the position of equilibrium shift A [special characters omitted] BH + can be effectively changed by altering structure of NR 2 R 3 group. Furthermore, the pH-induced conformational flip occurs in a certain pH range that mostly depends on the basicity of group NR 2 R 3 , allowing a broad tuning of the pH-sensitivity of TACH-based conformational switches in a wide range of acidity. The hydrophilic OH group was also modified to influence the conformational equilibrium. External stimuli including addition of acid, change of solvent and of the solution ionic strength also showed impact on conformation equilibrium to different extents. To explore the potential to serve as pH-sensitive helper lipids in gene delivery, a variety of TACH-lipids were incorporated into lipoplexes together with the cationic lipid DOTAP to mediate DNA transfection in Bl6F1 and HeLa cancer cell lines. The lipoplex comprising TACH-lipid 3o (R 1 = C 19 H 37 ; R 2 R 3 = CF 3 CH 2 NH) exhibited one to two orders of magnitude better transfection efficiency than the one with the conventional helper lipid DOPE while only inducing slight higher cytotoxicity. Thus, the lipid can be suggested as a novel helper lipid for efficient gene transfection with low cytotoxicity.
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Stowell, Yoshiko Katori. "Mechanistic study of nanoemulsion absorption and its application for permeation enhancement." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/139.
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Oil in water (o/w) nanoemulsion is a two-phase dispersed system in which the oil can incorporate poorly water soluble drugs to form a liquid dosage form. The enhancement of bioavailability with a use of nanoemulsion has often been reported empirically and speculated to be a result of the enhanced dissolution due to a larger surface area, however, the mechanism of nanoemulsion permeation was yet to be explored. The goal of this dissertation was to understand the mechanism of nanoemulsion permeation and to control permeation and bioavailability. The first objective was to delineate the effect of thermodynamic activities of the drug in nanoemulsion on the permeation through a barrier. The flux from nanoemulsion depended on the thermodynamic activities of both ionized and unionized species in the aqueous phase of nanoemulsion. A simple nanoemulsion was not favorable to enhance the permeation over the saturated solution due to the reduced thermodynamic activity. Thus, the second objective was to elucidate the role of transient supersaturation on permeation enhancement using nanoemulsion. In vitro permeation using self-nanoemulsifying drug delivery system (SNEDDS) was enhanced over the saturated solution due to the transient supersaturation; however the enhancement of bioavailability in rats was not due to the enhanced passive permeation. Therefore there was a need to increase or prolong the supersaturation. The third objective was to control the supersaturation to enhance in vitro permeation by formulation approaches. The optimum drug loading was determined based on the precipitation kinetics; however the ability to modulate the thermodynamic activity to enhance permeation by changing the drug loading was limited. The precipitation inhibitor, hydroxypropylmethyl cellulose was able to retard the precipitation and enhanced in vitro permeation due to the increased thermodynamic activity. The significance of this work was the systematic approach to understand the mechanism of nanoemulsion absorption and to utilize nanoemulsion for permeation enhancement. The knowledge gained in this work will help rationally design the formulation in the future.
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Estari, Rohit Kumar. "Effect of rutaecarpine on caffeine pharmacokinetics in rats." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/276.
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Many people like to drink caffeinated beverages, such as coffee or tea, but are sensitive to effects of caffeine. Therefore, they either avoid drinking caffeinated beverages altogether, or they avoid drinking them close to bedtime to prevent caffeine from interfering with their sleep. Ruta Cleanse and Ruta Sleep are natural supplements containing rutaecarpine that are designed to speed up the removal of caffeine from the blood. The recommendation is to take two capsules (equivalent to 100 mg rutaecarpine), as needed, to reduce caffeine level. Customers have reported positive effects, when taken 30 minutes to 2 hours prior. However, there is no scientific data to show how soon Ruta Cleanse and Ruta Sleep need to be taken in order for it to work. Therefore, we tested in rats the effect of single dose after 3, 6, 12, 24 hour and 7 doses (once a day, for seven days) of oral 100 mg/kg rutaecarpine (in suspension) induction on caffeine pharmacokinetics upon 15 mg/kg intravenous bolus and 20 mg/kg oral caffeine doses. The MROD data showed that as early as 3 hours after oral rutaecarpine administration, CYP1A2 activity in the liver tissue is increased by almost 3-fold compared to control rats and highest activity (9-fold compared to control) is found in the liver of rats administered with daily oral dose of rutaecarpine, for seven days. A suspension form of 100 mg/kg orally administered rutaecarpine significantly decreases the oral systemic exposure and mean residence time of caffeine and its metabolites (paraxanthine, theophylline and theobromine), as early as 3 hours before oral caffeine administration. The oral caffeine bioavailability (F) decreases by about 50% for the 3, 6 and 12-hour, 70% for the 24 hour and 80% for the one week daily rutaecarpine treatment groups. Currently we do not know the mechanism by which rutaecarpine significantly decreases the F values of caffeine upon oral administration. The systemic exposure of caffeine and its metabolites are also decreased when caffeine is given intravenously, though the effect is less pronounced compared to when caffeine is given orally. Interestingly, rutaecarpine achieves this effect without achieving detectable plasma level (less than 10 ng/mL). However, since the target organ for rutaecarpine is the liver, rutaecarpine can still induce CYP1A2 enzyme in the liver (as indicated by MROD data), without having to get absorbed into blood circulation.
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Shallal, Hassan M. "The discovery and anticancer preclinical investigation of novel piperazinylpyrimidine derivatives designed to target the human kinome." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/158.
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The current dissertation describes a multidisciplinary research project centered on the discovery and investigation of the anticancer activities exhibited by novel piperazinylpyrimidine derivatives designed to target kinases protein family. Primary screening of the antiproliferative effects implemented by these successfully synthesized new agents has resulted in the candidacy of 4 , 15 , and 16 as not only prototype representatives of the class, but surprisingly also as optimized agents for either globally cytotoxic, 16 , or selectively cytostatic, 4 and 15 , agents. Subjecting 4 , 15 , and 16 to screening tests aiming at measuring their binding to or their functional inhibition of selected sets of kinases has revealed the tendency of 4 to target PDGFR subfamily and the ability of 4 , 15 , and 16 to recognize CSNK1D. Docking as well as binding profiles comparative studies hypothesize 4 , 15 , and 16 as type-I kinase inhibitors. Further preclinical investigation of 15 against MDA-MB-468 triple negative breast cancer cell line revealed that 15 exhibits a time as well as dose-dependent antiproliferative activity mediated by the induction of both time and dose independent G2/M arrest and dose dependent apoptosis. Globally studying the molecular events accompanied with the 15 /MDA-MB-468 incidence has revealed the phosphorylation of TP53 and the consequent activation of its transcriptional activity as a hallmark molecular event relevant to the above observed effects on the cellular, cell cycle, and programmed cell death levels. Apart from the above experimentally oriented investigation, another theoretically driven inquiry was pursued aiming at studying the inherent ability of certain kinases to be more promiscuous towards binding to small molecules than others. Throughout the analysis of a reported dataset, dephosphorylated members of PDGFR subfamily were found to more potently bind to structurally diverse kinase inhibitors compared to INSR subfamily. A molecular dynamics study was performed to compare between the topological, energetic, and dynamic properties of the binding area usually targeted by kinase inhibitors in both KIT, as a representative of the more promiscuous PDGFR subfamily, and INSR, as a representative of the less promiscuous INSR subfamily. Interestingly enough, the binding area in both kinases showed significantly different properties which, to a large extent, can explain their different overall attitudes towards binding small molecules. As a representative example, the binding area of INSR tends to be more energetically self-stabilizing than that in KIT. Additionally, the topological analysis revealed that the binding are in KIT tends to be more rigid and to have bigger size than that of INSR. The current work has successfully cross-implemented experimental, theoretical, and computational studies aiming at the development of novel kinase inhibitors and/or promising anticancer preclinical candidates.
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Zhou, Zhu. "Exploring the effects of 5-HT2A and AMPA receptors on brain 5-HT via a mechanism-based pharmacodynamic model." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/143.
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Depression is a common mood disorder. Although major ethical challenges make it nearly impossible to invasively and directly measure serotonin (5-hydroxytryptamine, 5-HT) levels in human brains, neuroimaging technologies have shown macroscopic structural and functional abnormalities in the prefrontal cortex (PFC) of depressed patients. The monoamine hypothesis of depression is based on the neurotransmitter imbalance, such as deceased serotonin brain levels are implicated in the cause of depression. Research has focused on the control mechanisms involved in the dorsal raphé nucleus (DRN) which is the serotonergic control center located in the midbrain. We hypothesized that activation 5-HT 2A receptor in PFC would increase serotonin levels by an AMPA-dependent mechanism in both DRN and PFC. Enhancement of the 5-HT in DRN may inhibit 5-HT level in PFC by 5-HT 1A receptor. This becomes the full feedback loop system. While 5-HT levels in the PFC have been well studied, pathway that modulate this DRN pool through upstream cascade interactions leading to a downstream feedback loop have been difficult to elucidate. Developing a mechanism-based pharmacokinetics (PK) and pharmacodynamics (PD) model to quantitatively describe the effect of 5-HT 2A receptors regulation to serotonin in the DRN and PFC would help us to better understand the complex brain. 5-HT 2A receptor agonist and AMPA receptor agonist and antagonist were used to activate or block the related receptor. Male Wistar rats underwent neurosurgery for implantation of microdialysis (MD) probes. Three to five rats were randomly assigned to experimental arms. Using the MD method, the drug combination was examined to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results, a mechanism-based PD model was developed. Phoenix WinNonlin ® and Berkeley Madonna™ were used for model estimation, external validation with secondary data set, and simulation. The result supports the possibility of a 5-HT 2A /AMPA feedback control circuit that originates in the PFC and modulates DRN and PFC 5-HT levels through feedback coupling of 5-HT. The time-course profiles of 5-HT in both DRN and PFC was well modeled and model parameters were estimated with good precision (CV% ranged from 1.37% to 35.03%). The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.
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Malla, Ritu. "Role of PRAS40 in mammalian target of rapamycin (mTOR) modulation in cancer and insulin resistance." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/129.
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Dysregulation of PI3K-AKT-mTOR pathway has been reported in various pathologies, such as cancer and insulin resistance. The proline-rich AKT substrate of 40-kDa (PRAS40), also known as AKT substrate 1 (AKT1S1), lies at the crossroads of these cascades and inhibits the activity of the mTOR complex 1 (mTORC1) kinase. Firstly, our findings showed that disruption of PRAS40, a substrate of AKT and component of mTORC1, alters glucose homeostasis and prevent hyperglycemia in the streptozotocin (STZ)-induced diabetes mouse model. PRAS40 ablation resulted in a mild lowering of blood glucose levels and glycated hemoglobin (HbA1C), a lowered insulin requirement, and improved glucose tolerance in untreated PRAS40 gene knockout (PRAS40 (-/-)) as compared to wild-type (PRAS40 (+/+)) mice. PRAS40 deletion significantly attenuates hyperglycemia in STZ-induced PRAS40 (-/-) mice through increased hepatic AKT and mTORC1 signaling, a lowered serum insulin requirement, and altered hepatic GLUT4 levels. Furthermore, we investigated the role of PRAS40 in possible feedback mechanisms, and altered AKT/PRAS40/mTOR signaling in the pathogenesis of tumor progression. For this we probed new datasets extracted from Oncomine, a cancer microarray database containing datasets derived from patient samples, to further understand the role of PRAS40 (AKT1S1). These data strongly supports the previous findings that PRAS40 may serve as a potential therapeutic target for various cancers. Elevated levels of HER2 and PRAS40 are found in some human breast cancers. To directly test the importance of these genetic events in mammary tumorigenesis, we assessed whether disruption of PRAS40 could alter mammary tumor occurrence in HER2 overexpressing mice. HER2 overexpressing mice expressing the activated rat Erbb2 (c-neu) oncogene under the direction of the MMTV promoter was bred with Cre-recombined homozygous (PRAS40-/-) mice. We examined mammary tumor development in the presence (PRAS40+/+) or absence (PRAS40-/-) of PRAS40 using this double transgenic mouse mammary tumor model. Loss of PRAS40 resulted in a delayed mammary tumor onset, improved tumor-free survival, and reduced mammary pre-cancerous lesions in PRAS40-/- versus PRAS40+/+ HER2 overexpressing mice. These results suggest that PRAS40 knockdown could be an attractive target and adjuvant therapy in HER2-positive breast cancers.
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Wiser, Lauren Sample. "Mechanisms of polymer adsorption in nanoparticle stabilization for poorly water soluble compounds." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/159.
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In this dissertation, the mechanisms of nanosuspension stabilization via polymer adsorption on nanoparticle surface were investigated. As the electrokinetic behavior and colloidal stability depend on the surface characteristics, altering the surface adsorbed polymers affords the different surface properties of nanoparticles and leads to the insight on the mechanism of nanoparticle stabilization. Drug nanosuspensions were prepared by wet milling of drug with water as medium and polymers as stabilizers. Block copolymers were evaluated based on varying the hydrophobic and hydrophilic amounts, polymer concentration, and polymer affinity differences onto the nanoparticle surface. Specifically, block copolymers of ethylene oxide (EO) and propylene oxide (PO) with different EO chain lengths were used to modify the nanoparticle surface and investigate the mechanisms of stabilization by varying the ratio of hydrophobic (PO) and hydrophilic (EO) units. It was hypothesized that the PO chain of block copolymers adsorb at the solid-solution interface and the EO chain provides steric hindrance preventing aggregation. Block copolymer adsorption layer thicknesses were experimentally determined with adsorption layer thicknesses increasing from 4.7 to 9.5 nm as the number of EO increase from 26 to 133 monomer units. Nanoparticle aggregation occurred with insufficient polymer monolayer coverage and electrokinetic zeta potential greater than -20 mV. The amount of block copolymers on the surface of nanoparticles was quantified and the affinity of polymer adsorption increased as the copolymer hydrophobic units increased. The amount adsorbed and affinity provides a qualitative ranking of the affinities between a specific polymer and nanoparticle substrate to provide a method in determining the mechanism of stabilization, where specific functional groups for adsorption could be selected for maximum nanoparticle stability. A molecular modeling was conducted to visualize and support the mathematical model and the proposed mechanism of block copolymer adsorption onto a nanoparticle surface. The time lapse molecular modeling of a block copolymer in an aqueous media showed the hydrophobic units adsorbing onto the nanoparticle surface with the hydrophilic units projecting into the aqueous media. For the first time in pharmaceutical research, a systematic series of studies were conducted to elucidate the mechanisms of adsorption with both surface charge and polymer affinity analyses. A series of studies evaluating the adsorption properties polymer stabilizers provided useful information on how a block copolymer comprised of both hydrophilic and hydrophobic domains adsorbs onto an active pharmaceutical ingredient. A systematic set of experimental techniques were presented with novel analysis tools and predictors to construct stable nanoparticle formulations.
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Kaur, Navdeep. "Sublingual drug delivery: In vitro-in vivo correlation." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/148.
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Administration of drugs sublingually allows direct absorption into the systemic circulation which results in quick onset of action and a higher bioavailability as a consequence of by-passing first pass metabolism. Absorption of drugs across sublingual mucosa is typically determined by means of in vitro permeation studies using excised sublingual tissue during early phases of drug development. Although in vitro set up has been designed to mimic in vivo system yet the results of in vitro studies often deviate from in vivo results. Therefore, it is not known if the in vitro studies can be used as surrogate for in vivo studies in a predictable manner. To understand the relationship between in vitro and in vivo system for sublingual drug delivery, the first objective of this dissertation research was to investigate difference/similarities between in vitro and in vivo system by performing parallel in vitro and in vivo studies and establish a correlation. Five model drugs possessing diverse physicochemical properties and New Zealand White rabbits were used for these studies. Comparison of time course of absorption revealed a significant difference in time lag between in vivo (less than 5 min) and in vitro (30-120 min) systems. However, the derived absorption parameter permeability coefficient was similar in in vitro and in vivo system for caffeine: (2.10±0.22)×10 –5 , (2.06±0.47)×10 –5 ; Naproxen: (1.91±0.44)×10 –5 , (2.34±0.26)×10 –5 ; Propranolol: (2.93±0.52)×10 –5 , (3.51±0.75)×10 –5 ; Verapamil: (3.95±0.29)×10 –5 , (4.75±0.81)×10 –5 and Atenolol: (2.01±0.68)×10 –6 , (2.95±0.32)×10 –6 cm/s, respectively (p>0.05). The discrepancy between in vitro and in vivo system was hypothesized in this study to be due to the difference in thickness and role of extensive microcirculation in the two systems. Histological evaluation revealed the presence of rich vasculature 10-20 μm below the epithelium which is responsible for quick removal of drug permeating the epithelium (100-150 μm) of sublingual mucosa and reaching systemic circulation in an in vivo system. In contrast, in in vitro system the permeated drug can only be detected after crossing the excised sublingual tissue of 250±50 μm thickness. A mathematical model based on the monolayer (epithelium) and bilayer (epithelium+connective tissue) nature of the membrane representing in vivo and in vitro system, respectively demonstrated the nature of membrane to be responsible for difference in time lag but similar permeability coefficient. To be able to predict in vivo result using in vitro data, the second objective of this dissertation research was to develop a predictive pharmacokinetic model based on the established in vitro in vivo correlation (IVIVC) of sublingual absorption parameters across two systems. Predicted plasma concentration-time profiles of propranolol, verapamil, naproxen, atenolol and caffeine were found to be in good agreement with the experimental profile with the coefficient of determination of 0.85, 0.80, 0.97, 0.98 and 0.88, respectively. The applicability of the model was further evaluated by predicting in vivo performance of Zolpidem and Propranolol following sublingual administration in human beings and comparing area under the plasma concentration-time curve. Percent prediction error was 12.02% and less than 10% (4.69, 6.69, 5.02 for 1, 1.75 and 3 mg dose, respectively) for Propranolol and Zolpidem, respectively. The final objective of this dissertation was to extend the established IVIVC to other suitable animal models such as pig for assessing sublingual absorption. Histological evaluation revealed the similarity in the structure of sublingual mucosa of pig and New Zealand White rabbit. Similar transport characteristics (p>0.05) of model drugs across sublingual mucosae of two species were observed indicating the possibility of using them interchangeably. In conclusion, a rational attempt was made in this dissertation research to identify the root cause of the discrepancy between in vitro and in vivo system and establish a correlation correcting the discrepancies. The established IVIVC and predictive pharmacokinetic model will help in rationale design and development of new sublingual formulations and will be a valuable tool in the preclinical phase of early drug development stage.
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Shah, Khyati Niral. "Mechanism of tamoxifen resistance in breast cancer." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/138.
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Acquired tamoxifen resistance develops in the majority of hormone responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells, with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biological outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNAi-mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated pro-apoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM2 inhibitor Didox significantly reduced cell growth in vitro and in vivo. To further establish a functional association between RRM2 expression and tamoxifen resistance in breast cancer cells, gain of function studies were performed by overexpressing RRM2 in MCF-7 cells. Overexpression of RRM2 profoundly reduced tamoxifen sensitivity and down-regulated ER-&agr; in otherwise tamoxifen sensitive breast cancer cells. Furthermore, breast cancer cells with high RRM2 had elevated Her-2 and EGFR expression, modulated ER-&agr; signaling and NFκB expression. These findings also indicate that it may be possible to use RRM2 as a prognostic factor in breast cancer patients under tamoxifen therapy, and can be considered a potential therapeutic target in tumors that have acquired resistance to tamoxifen. Finally, inhibition of RRM2 by drug Didox effectively eradicates the tamoxifen resistant population, revealing a potential beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance. In conclusion, the findings of this work delineate the important role of RRM2 in Akt induced and acquired tamoxifen resistance in breast cancer. It also provides a preclinical rationale for evaluating tamoxifen in combination with Didox for breast cancer treatment.
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Gyanani, Vijay. "Turning stealth liposomes into cationic liposomes for anticancer drug delivery." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/147.
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Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0.
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Han, Xiaoyuan. "Sex differences in aortic endothelial function of diabetic rats: Possible involvement of superoxide and nitric oxide production." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/136.
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Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate whether there were sex differences in rat aortic endothelial function in diabetes, and to examine the potential roles of superoxide and nitric oxide (NO) in this sex-specific effect. Two diabetic animal models were used: streptozotocin (STZ)-induced type 1 diabetic rats (at early and intermediate stages of disease) and Zucker type 2 diabetic fatty (ZDF) rats. Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) was measured in aortic rings pre-contracted with phenylephrine (PE) before and after pretreatment with MnTmPYP (10 mM), a superoxide scavenger, or apocynin (100 μM), a NADPH oxidase (Nox) inhibitor. Constrictor response curves (CRC) to PE (10 -8 to 10 -5 M) were also generated before and after pretreatment with L-NAME (200 μM), an endothelial nitric oxide synthase (eNOS) inhibitor, in the presence of indomethacin. In addition, the level of Nox (a potent source of superoxide) and eNOS mRNA expression were determined using real-time RT-PCR. STZ-induced diabetes impaired EDV to ACh to a greater extent in female than male aortae both at early and intermediate stage of disease (1- and 8- week, respectively). Incubation of aortic rings with L-NAME potentiated PE responses in all groups, but aortae from control females showed a greater potentiation of the PE response after NOS inhibition compared with others. STZ-diabetes reduced the extent of PE potentiation after L-NAME and the aortic eNOS mRNA expression in females to the same levels as seen in males. In addition, pre-incubation with MnTMPyP enhanced sensitivity to ACh only in diabetic females one week after STZ induction. Similarly, the levels of Nox1 mRNA expression were enhanced in STZ-induced diabetic females. Type 2 diabetes significantly impaired EDV in aortic rings from females; however, it potentiated the relaxation in male rats. Moreover, type 2 diabetes enhanced the extent of PE potentiation after blocking NOS with L-NAME in females. Pre-incubation of aortic rings with apocynin increased EDV only in diabetic female group. Accordingly, the levels of Nox1, Nox4 and eNOS mRNA expression were substantially enhanced in aorta of female ZDF rats compared to those in lean animals. In a conclusion, our data suggest that an elevation of superoxide and alteration of NO production may in part contribute to the predisposition of the female aorta to injury in diabetes.
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Mai, Yvonne M. "Use of various health care providers and the associated clinical and humanistic outcomes in an ambulatory Medicare population." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/265.
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Background: The use of complementary and alternative medicine (CAM) and other non-physician health care providers (dentists, optometrists, etc.) has steadily increased in the United States; however, the associated outcomes reported in the Medicare beneficiary population are limited. Objective: To evaluate the utilization of different healthcare providers by Medicare beneficiaries and assess resultant beneficiary outcomes. Methods: Fourteen outreach events targeting Medicare beneficiaries were conducted throughout Northern/Central California during the 2014 open enrollment period. Trained student pharmacists (working under licensed pharmacist supervision) provided beneficiaries with comprehensive medication therapy management (MTM) services. During each intervention, demographic, quality-of-life, health behavior and health provider/service utilization data were collected. Results: Of 620 respondents, 525 (84%) and 84 (14%) reported using at least one non-physician healthcare professional or CAM provider, respectively. Beneficiaries who reported using non-physician healthcare providers were significantly (p < 0.05) more likely to indicate being ‘very confident’ in managing their chronic health conditions. The number of providers seen with prescriptive authority was positively correlated with the number of prescription medications taken (r s =0.342, p < 0.001). The total number of providers seen was positively correlated with the number of drug-related issues identified (r s = 0.179, p < 0.001). Conclusion: Many beneficiaries have multiple chronic conditions and increasingly utilize a variety of healthcare professionals. As such, bridging the communication chasm between these professionals can improve humanistic outcomes and minimize medication related issues of Medicare beneficiaries. Coordinated care, a key strategy for improving healthcare delivery under the Affordable Care Act, is a step in the right direction.
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Saraf, Poonam S. "RGD based peptide amphiphiles as drug carriers for cancer targeting." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/137.
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Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to α v β 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment in comparison to free drug in Detroit 551 cells. In A2058 melanoma xenograft mice model, the Paclitaxel-RGD micelles exhibited a significant inhibition of tumor growth in comparison to control treatment for both alternate day and twice weekly treatments. The studies showed the feasibility of using the non covalent peptide based self-assemblies as vehicles for targeted delivery in cancer.
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Penchala, Sravan C. "Characterization of AG10, a potent stabilizer of transthyretin, and its application in enhancing in vivo half-life of therapeutic peptides." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/130.
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The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African Americans, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 years causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the characterization of AG10 , a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10 , combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life of therapeutic peptides. The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of a model peptide Gonadotropin Releasing Hormone (GnRH) and its analog GnRH-A without compromising their potency. Apart from GnRH, we have used other peptides to study their proteolytic stability in vitro . Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo . We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.
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Cao, William Sam. "Characterization and application of human pluripotent stem cell-derived neurons to evaluate the risk of developmental neurotoxicity with antiepileptic drugs in vitro." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/131.
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The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.
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Arikatla, Swetha. "Effect of Tumor Microenvironmental Conditions on Non Small Cell Lung Cancer." Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/126.
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Tumor microenvironmental conditions play a vital role in promoting metastasis and tumor recurrence. Due to inefficient vasculature, cancer cells experience hypoxia, glucose deprivation and low pH even during the early stages of tumor growth. Tumor cells are proposed to adapt to these microenvironmental conditions by acquiring increased migratory and invasion potential and tumor initiating ability. Our research addresses the effect of these biochemical factors of the tumor microenvironment (TME) on motility, epithelial to mesenchymal transition (EMT) and stemness of non-small cell lung cancer (NSCLC). NCI-H292 and NCI-H1650 NSCLC cell lines were used to measure the effect of the above mentioned TME conditions. Apart from acidic pH, low glucose and hypoxia, the effect of high glucose conditions was also measured on H292 and H1650 cell lines. Acidic pH, high and low glucose conditions were observed to have no effect on the motility, EMT and stemness of H1650 cell line. Hence, use of this cell line was discontinued and no further treatment conditions were tested on this cell line. In H292 cell line, acidic pH, low glucose and tumor like conditions combined together (acidic pH + low glucose + hypoxia) [AP+LG+HYP] significantly decreased motility whereas hypoxia significantly increased the motility of H292 cells. High glucose did not affect the motility of H292 cells. Although N-cadherin, a mesenchymal marker, expression was significantly upregulated by acidic pH, high and low glucose conditions, no direct correlation was observed between N-cadherin expression and motility. E-cadherin expression was not affected by acidic pH, high and low glucose conditions. An increase in N-cadherin expression and no change in E-cadherin expression under these conditions might be an indication of partial EMT. Hypoxia and AP+LG+HYP did not alter the expression of E-cadherin and N-cadherin. Although expression of vimentin, another mesenchymal marker, and Sox2, a cancer stem cell marker (CSC), was observed at the mRNA level, no expression of vimentin and Sox2 proteins was observed in H292 cells under any of these treatment conditions. The expression of OCT4, another CSC marker, was also not observed at the protein level in H292 cells. HIF-1α expression was observed in H292 cells under normoxic conditions and was unaffected by hypoxia and AP+LG+HYP. Therefore our research indicates that the effect of these TME conditions might be different on different cancer cell lines or cancer types. Not all cancers may depend on EMT for metastasis. An increase in metastasis under hypoxia may be independent of HIF-1α.
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Xu, Lu. "New quinazoline analogues as NF-κB activation inhibitors." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/152.
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NF-κB is a transcription factor protein complex that plays an important role in some cancers and inflammatory responses. It can enhance the proliferation rate, reduce apoptosis, as well as create more blood flow to ensure the survival of cancer. Thus blocking the NF-κB pathway has potential therapeutic benefit. We designed a series of compounds based on quinazoline scaffold pharmacophore model which may have high binding affinity with p50 subunit of NF-κB. The compound series with phenyl substitution at position 2 of quinazoline proved to be more effective at inhibiting NF-κB function both theoretically and experimentally. These compounds also reduce the proliferation of numerous tumor cell lines and the mean GI50 for representative compound 2a is 2.88μM on NCI 60 cell lines. Compound 2a can induce significant apoptosis at the concentration of 1μM. The exploration of the mechanism of action of these compounds found that 2a does not inhibit kinases upstream of NF-κB and does not inhibit p65 translocation from the cytosol to the nucleus as 2b does. However 2a inhibits NF-κB dependent Luciferase expression as well as NF-κB target genes better than 2b. This may suggest that 2a inhibits the NF-κB pathway by directly blocking gene transcription, while 2b acts at cytoplasmic stage.
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Gokgoz, Kilic Sinem. "Dynamic fugacity modeling in environmental systems." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/22557.
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Thesis (M. S.)--Civil and Environmental Engineering, Georgia Institute of Technology, 2008.Committee Chair: Aral, Mustafa; Committee Member: Guan, Jiabao; Committee Member: Pavlostathis, Spyros; Committee Member: Uzer, Turgay; Committee Member: Yiacoumi, Sotira.
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Dong, Jin. "First-principle based pharmacokinetic modeling." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/128.
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Predicting drug concentrations in the blood and at the site of action is the hottest topic in pharmacokinetics (PK). In vitro-in vivo extrapolation (IVIVE) and physiological based pharmacokinetics (PBPK) models are two major PK prediction strategies. However, both IVIVE and PBPK models are considered as immature methodologies due to their poor predictability. The goal of the research is to investigate the discrepancies within IVIVE and PBPK predictions according to first-principles: convection, diffusion, metabolism, and carrier-mediated transport. In Chapter 2, non-permeability limited hepatic elimination under perfusion steady state is examined. The well-stirred model is re-derived from the convection-dispersion-elimination equation when both dispersion and concentration gradient are ignored and re-named as the zero-gradient model. Pang and Rowland’s lidocaine data are re-analyzed. Their data analysis was based on an unfair comparison of the zero-gradient and parallel- tube models at two different efficiency number ranges. The interference of sensitivity greatly biased the comparison. I also show that both theoretical discussions and experimental results indicate that apparent intrinsic clearance and intrinsic clearance could be affected by blood flow and protein binding. In Chapter 3, I discuss permeability limited hepatic elimination under perfusion steady state. Permeability limited elimination is classified to diffusion dominated, carrier-mediated transport mediated, and mixed effects based on drug passage mechanisms. Each of these three drug passage classes is sub-divided to sink condition and finite volume condition based on the boundary conditions of drug passage. In Chapter 4, the discrepancies within IVIVE for both non-permeability limited and permeability limited drugs are explored. The deficiencies in assay design and data analysis of common in vitro metabolism assays are investigated. The scaling/converting equations for both non-permeability limited and permeability limited drugs are derived. In Chapter 5, I focus on transient PK models. Numerical analysis using finite difference and finite volume methods are introduced into the derivation and discussion of transient PBPK models. In addition, the use of partition coefficient in the non-eliminating tissue/organ models is discussed.
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Zhang, Changfeng. "Investigation of the endoplsmic reticulum calcium stores for their potential roles in neuroprotection using the NG115-401L neuronal cell line model." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/142.
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There is significant interest in the field of neuroscience to gain a better understanding of how neurons die in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have used the neuronal cell line NG115-401L with unique calcium signaling characteristics to test the hypothesis that improving calcium loading into the endoplasmic reticulum (ER) to increase ER calcium levels acts as a possible neuroprotective response. We approached this problem using both pharmacological and genetic approaches targeting the central mediator of calcium uptake in the ER localized sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) enzyme. The pharmacological studies involved use of the ginger root compound 6-gingerol, which to date is the best documented agent for activating SERCA enzymes in heart and skeletal muscle. However, in our experiments, gingerol did not appear to activate NG115-401L SERCA pumps; indeed, the compound produced a response more like that of a SERCA inhibitor inducing a rapid ER calcium depletion. In addition, gingerol stimulated robust calcium influx responses, an unexpected result given the NG115-401L neural cell line is uniquely deficient in calcium influx pathways. Our genetic approach involved expressing the stromal interaction molecule 1 (STIM1) protein in the NG115-401L cell, which is also an ER localized protein that serves as a pivotal calcium influx channel regulator. NG115-40lL neurons present a native deficiency of STIM1 expression in a background phenotype with well characterized perturbations in ER calcium regulation and control of calcium influx pathways. Thus, STIM1 may be predicted to increase ER calcium levels, conferring protection against neuron cell death due to ER calcium store defects. STIM1 expression reconstituted the corrupted calcium influx pathway in NG115-401L neurons, which conferred neuroprotective responses to ER calcium perturbation, mitochondrial oxidative stress and subsequent cell death. Our results argue for unique and undiscovered regulatory effects of gingerol on the ER calcium circulation system, and suggest that the expression of STIM1 in these neurons protects against ER stress and oxidative stress via reconstruction of cellular calcium homeostasis.
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Morgan, Judith. "Part I: Oxidation of Heavy Metal Sulfides in Relation to the Environment; Part II: Fundamental Theory & Experiments Concerning Gas Chromatography & Mass Spectrometry." TopSCHOLAR®, 1990. https://digitalcommons.wku.edu/theses/2672.
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Oxidation of heavy metal sulfides is a thermodynamically spontaneous process. Because of this, metal sulfides in the presence of oxygen are not stable. Currently there are over 100 streams and rivers, within the U.S., contaminated with heavy metal mine drainage; therefore an approved method of restoration is necessary. Precipitation of heavy metals as sulfides using H2S as a reductant has been favorably reviewed as a restorative technique to clean up mining areas. However, using this technique on the laboratory scale does not prove to be a viable answer and shows strong pH dependence.
In the past three years the ILI (Instrumentation and Laboratory Improvement) has awarded funding to over 50 institutions, in the U.S., for the purchase of GC/MS. Therefore, there is a great need for laboratory experiments to properly train students in this field in American universities. A strong theoretical treatment for sophomore-level students is presented within this thesis and from this, three experiments have been developed in order to educate young professionals seeking a career in the field of chemistry.
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Su, Dan. "Rational design, characterization and in vivo studies of antibody mimics against HER2." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/133.
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Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.
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Wang, Yu. "Mechanistic study of aryl hydrocarbon receptor nuclear translocator (ARNT)-mediated signaling." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/151.
Full textAbstract:
A novel aryl hydrocarbon receptor nuclear translocator (ARNT)-interacting peptide (Ainpl) was characterized from human liver cDNA library using phage display. Ainpl suppresses hypoxia inducible factor-1a (HIF-1α) signaling pathway through an ARNTdependent manner. HIF-1α is known to be overexpressed in more than 90% of solid tumors, and the inhibition of HIF-1α is proved as an effective approach to suppress tumor growth. ARNT, as the obligatory heterodimeric partner of HIF-1α for downstream gene activation, was used as a bait to screen for Ainpl. Ainpl specifically interacts with the helix-loop-helix (HLH) subdomain of ARNT, but not with HIF-1α. GFP-Ainpl is localized in both cytoplasm and nucleus, and suppresses HIF-1α signaling by two mechanisms: (1) cytoplasmic GFP-Ainp 1 retains ARNT in the cell cytoplasm and (2) nuclear GFP-Ainpl inhibits HIF-1α/ARNT heterodimerization. The suppression of Ainpl on HIF-1α signaling was reversed by introducing ARNT into the cells using transient transfection. We further utilized HIV TAT protein transduction domain to deliver 6His-TAT-Ainpl into three different cancer cell lines (Hep3B, HeLa, MCF-7), and found that 6His-TAT-Ainpl co-localizes with ARNT in the cell nucleus. 6His-TATAinpl can be detected inside the cells after 30 min of transduction, and can reach the maximum level at 2 h. 6His-TAT-Ainp 1 remained detectable in the cells up to 96 h and had a half life of 24 h after transduction. In addition, 6His-TAT-Ainp 1 suppresses HIF-1α downstream genes at both message and protein levels in a dose-dependent manner. Taken together, molecules that target the HIF-1α and ARNT interface can be developed as viable drugs to suppress HIF-1α signaling.
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Xie, Jinghang. "Study of the aryl hydrocarbon receptor as a target for rational drug design." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/140.
Full textAbstract:
The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12-24 KDa in size—namely D1 (aa 84-295), D2 (aa 84-192) and D3 (aa 191-295)—to suppress the Arnt function. We observed that all three constructs interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. In chapter 2, a Pichia Pastoris expression system was constructed expressing codon optimized human full length AhR. This codon optimization is necessary for overexpression of huAhR. RT-PCR data showed that the codon optimized mRNA was more stably expressed than wild types. Overexpressed huAhR protein was degraded by proteinase when using a regular P. Pastoris strain yJC100 whereas the proteinase deficient ySMD1163 maintained a much higher level of huAhR. P. Pastoris expressed huAhR was natively purified and analyzed. Coimmunopricipitation assay shows its interaction with endogenous Arnt. A ligand-dependent gel shift was also observed. In addition, we performed an in vitro coprecipitation assay to study its binding to endogenous cyp1b1 DREs. The result shows that the DRE3, known as a critical DRE for cyp1b1 transcriptional activity, has the highest binding affinity to AhR/Arnt complex. Taking together, we constructed a novel P. Pastoris expression system to overexpress human full length AhR. Purified huAhR is a good reagent for studing its ligand and DNA binding. In chapter 3, an adeno-associated virus (AAV) expression system was constructed to express an AhR deletion contruct CΔ553 (aa1-295) for tumor injection. Western blot shows the expression of CΔ553 (aa1-295) in hela cells infected by AAV-553, but the low yield of AAV-553 limited its application on tumor treatment. Possible solutions were discussed for future work.
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Wohl, Christopher John Jr. "Photochemical Applications to the Study of Complexity Phospholipid Bilayer Environments." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/1269.
Full textAbstract:
The physical and biophysical properties of a biological membrane model, phosphatidylcholine bilayers, were investigated using novel spiropyran/merocyanine molecular probes. The femtosecond to second dynamics of this system's photochemistry enabled bilayer viscosity and free volume to be studied over a broad time scale. Spiropyrans/merocyanines with different polarity were synthesized by changing the substitution of the indole moiety enabling determination of the trans-membrane properties of the bilayer. In addition, transient grating spectroscopy was used to study thermal energy transfer in phospholipid bilayers on a picosecond time scale.Femtosecond transient absorption spectroscopy was used to study the photo-induced spiropyran ring-opening and isomerization reactions that produce the highly polar merocyanine species. The hindered rotation of the merocyanine bridge results in several metastable merocyanine isomers. The merocyanine ground state was determined to be populated predominantly by two isomers (TTC and TTT). Selective photoexcitation of these isomers results in excited state isomerization producing a third isomer (τ = 60 ps). Merocyanine thermal ring-closing was observed on a seconds time scale. Reaction kinetics, and solvatochromic and photochromic properties of merocyanines and spiropyrans were used to determine the bilayer physical properties. Bilayer viscosity was determined from merocyanine isomerization kinetics. Phospholipid bilayer free volume (the unoccupied volume enclosed in the bilayer) was determined from a modified Kramers' analysis. The greatest free volume was found in the extreme interior of the bilayer, while the head-group region exhibited the least free volume in qualitative agreement with molecular dynamics simulations of these bilayer systems. Free volumes determined via ps experiments were lower than those determined on a seconds time scale due to reduced acyl chain dynamics on the ps time scale.Femtosecond transient grating spectroscopy was used to study the rate of thermal energy transfer from photo-excited porphyrin molecules to the surrounding solvent. Thermal energy transfer was observed as photo-acoustic waves propelled through the system upon relaxation of photo-excited porphyrin molecules in aqueous solution and embedded in bilayers. For liposome solutions, a bimodal energy transfer model was developed. The determined rate constants suggest that energy transfer occurs predominantly via thermal diffusion and vibrational energy transfer, while lipid dynamics (isomerizations) are not involved.