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1
Peterson, Amity. "Characterization and Pharmacogenetics of Hepatic Phase I Exemestane Metabolism." Thesis, Washington State University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10266284.
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Exemestane (EXE) is an endocrine therapy used to combat postmenopausal breast cancer. Several studies have reported substantial differences in clinical outcomes between EXE-treated patients, as well as inexplicable variability in serum concentrations of EXE and its major metabolite, 17β-dihydroexemestane (17β-DHE). For many pharmaceuticals, drug response is influenced by patient-specific genetic factors related to xenobiotic metabolism. Thus, it is possible that allelic variation in genes involved in EXE metabolism contributes to inter-individual differences in patient outcomes, possibly through differential EXE clearance or varied rates of metabolite formation. Historically, knowledge of phase I EXE metabolism has been extremely limited with significant ambiguity surrounding the identity of the specific hepatic enzymes involved. To address this gap in knowledge, in vitro studies were undertaken to better characterize hepatic phase I EXE metabolism and in particular, to assess the impact of genetic variation in drug-metabolizing enzymes on the production of EXE metabolites with inhibitory activity against aromatase.
The first part of this dissertation describes the identification of phase I EXE metabolites and details their capacity to suppress estrogen synthesis. Four metabolites, including 17β-DHE, were detected in incubations of EXE with pooled human liver microsomes. 17β-DHE and a novel metabolite, 17α-DHE, were formed in incubations of EXE with pooled human liver cytosol. The identities of phase I EXE metabolites were confirmed through comparison to reference compounds using UPLC/MS/MS. Anti-aromatase activity assays (AAA) revealed that 17β-DHE is the only phase I EXE metabolite formed by human liver fractions that appreciably impedes estrogen formation. AAA also suggest that the inhibitory potency of EXE is unaffected by common nonsynonymous polymorphisms in aromatase. The latter half of this dissertation identifies hepatic enzymes that are likely to participate in phase I EXE metabolism. In vitro assays show that CBR1, AKR1Cs, and multiple hepatic CYP450s predominantly reduce EXE to 17β-DHE with minor formation of additional inactive metabolites. Kinetic assays comparing 17β-DHE formation by each wildtype enzyme to its common variant allozymes show that specific genotypes are associated with altered EXE metabolism in vitro. However, additional investigations are needed to determine the prognostic value of these associations for predicting in vivo EXE response.
2
Fraser, Graeme L. "Characterization of [delta] opioid receptor function in rat brain by pharmacological and antisense techniques." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38454.
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The opioid family of G-protein coupled receptors comprises four known receptor subtype genes (delta, mu, kappa, ORL1) and further receptor heterogeneity within each opioid receptor subfamily has been proposed. All four genes are expressed throughout the central nervous system and are believed to modulate a variety of behavioural responses including analgesia. Opiate drugs such as morphine that are selective for the mu receptor subtype are effective analgesics, but their chronic use is limited by the appearance of side effects such as respiratory depression, constipation and dependence. Consequently, the analgesic potential of agonists selective for other opioid receptors is under investigation. In this regard, previous studies suggest that delta agonists mediate antinociception, yet produce fewer adverse effects than mu agonists. To further investigate the cloned delta opioid receptor (DOR) as a target for novel analgesics, the pharmacological role of DOR in brain was evaluated in rats.First, we characterized delta agonist binding sites and receptor activation in rat brain membranes. We also introduced a novel antagonist radioligand, [125I]AR-M100613, to label tissues with low delta opioid receptor expression in order to support follow-up studies where radioligand binding was performed on rat brain membranes following antisense treatment. Second, we examined the behavioural response to delta agonists in rats. Deltorphin II and SNC80 (i.c.v.) were shown to induce antinociception in acute pain assays, and to reverse hyperalgesia following tissue inflammation induced by Freund's adjuvant with even greater potency. These findings indicate that delta receptors play an enhanced role in the modulation of descending pain pathways following tissue injury. Deltorphin II and SNC80 (i.c.v. ) were also shown to induce hyperlocomotor activity. Third we used antisense studies to demonstrate that the antinociceptive and locomotor stimulant effects of delta agonists are modulated by the cloned delta opioid receptor (DOR). In contrast to other delta agonists, the antinociceptive effects of DPDPE were not modulated by DOR antisense treatment but rather were blocked by a selective mu antagonist (CTOP) suggesting that DPDPE may activate mu sites in the brain rather than an alternate delta receptor subtype. Finally, we demonstrated that peptide nucleic acids (PNA, i.c.v.) can act as target-specific and sequence-selective antisense agents. In total, these findings demonstrate that DOR is an appropriate target for the development of novel analgesics and that PNA can serve as effective antisense agents for the determination of gene function for CNS targets.
3
Li, Bing. "Drug solubility studies by using combined solubilization techniques." Diss., The University of Arizona, 2000. http://hdl.handle.net/10150/284037.
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This study focuses on the development of mathematical models that explain and predict the combined effects of pH control and complexants (cyclodextrin), pH control and surfactants, as well as pH control and cosolvents on the total solubility of the drug. The total solubility of the drug is expressed as the sum of the concentrations of individual species. In complexant-water and surfactant-water systems free unionized drug [Dᵤ] and free ionized drug [Dᵢ] are present along with either complexed unionized drug [DᵤL] and complexed ionized drug [DᵢL], or micellar unionized drug [DᵤM] and micellar ionized drug [DᵢM], respectively. On the other hand, in cosolvent-water system the only species present are free unionized drug [Dcᵤ] and free ionized drug [Dcᵢ]. The equations developed show that a pH change favoring ionization of the drug not only increases the concentration of the ionized species in water, but also increases the concentration of the ionized species in cyclodextrins, micelles, or cosolvents. In fact, the concentration of the ionized species in the complexant, micelle, or cosolvent can be greater than those of the unionized species. The solubility data of flavopiridol and several other drugs reported in the literature support these conclusions. A mathematical model is also developed to describe the combined effect of cosolvency and complexation on non-polar drug solubilization. The total drug solubility is determined by the summation of three drug species present in the solution: free drug [D], drug-ligand binary complex [DL], and drug-ligand-cosolvent ternary complex [DLC]. The proposed equation describes the dependencies of these three species upon the intrinsic drug solubility, [Dᵤ], the cosolvent solubilizing power, sigma, the binary and ternary intrinsic complexation constants, K(b)ⁱⁿᵗ and K(t)ⁱⁿᵗ, and the cosolvent destabilizing powers for the binary and the ternary complexes, ρ(b) and ρ(t). The equation explains the decline in the solubility of fluasterone (a non-polar drug) produced by low cosolvent concentrations as well as the increase in the solubility produced by high cosolvent concentrations that are observed at all cyclodextrin concentrations.
4
Gezginci, Mikail Hakan. "Synthesis and in vitro antimycobacterial activity of some pyridine and pyrazinecarboxylic acid isosteres." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/284057.
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Tuberculosis has been one of the most commonly encountered infectious diseases of humans throughout history. The discovery and introduction of effective treatments in the 1940s resulted in a decline of the incidence of the disease until the 1980s, which marked a sharp turn in this trend with an increase in the number of reported cases in the world. The prevalence of mycobacterial infections in poorer nations and centers of urban decay, and especially in immunologically compromised patients indicates the need for new and better treatments. In this study, to contribute to the worldwide effort to discover more effective medications against tuberculosis, 26 previously unreported compounds were synthesized and tested against Mycobacterium tuberculosis. The synthesized compounds were designed to act as bioisosteres or prodrugs of pyridine and pyrazinecarboxylic acid derivatives and contained acidic or neutral hetero- or carbocyclic ring systems attached to pyridine or pyrazine rings at different positions. The synthesized ring systems included 1,2,4-oxadiazole-5-ones, 1,2,4-thiadiazole-5-ones, 1,2,4-oxadiazole-5-thiones, 1,3,4-oxathiazoline-2-ones and cyclobutene-1,2-diones, which were documented in the literature to act as carboxylic acid isosteres or could act as prodrugs thereof. Pivaloyloxymethyl derivatives of the compounds were also prepared in order to increase their lipophilicity and therefore improve their bioavailability. The synthesized compounds were expected to be biotransformed by esterases or amidases to the active species after penetration of the mycobacterial cell wan. Biological and pharmacokinetic properties of the compounds were compared with the unmodified polar isosteres of pyrazinoic and nicotinic acids. The majority of the tested compounds exhibited activities ranging from 0.5 to 8 times the activity of pyrazinamide, one of the lead compounds that inspired their design.
5
Cai, Yuli 1961. "Disposition kinetics of Amphotericin-B in rats." Thesis, The University of Arizona, 1991. http://hdl.handle.net/10150/291689.
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The disposition kinetics of Amphotericin-B (Am-B) were investigated in rats in three different dose groups. Each group contained four rats. The serum and urine Am-B concentrations were analyzed by reversed-phase HPLC. There were no significant differences for total body clearance and half-lives as a function of dose. Those observations suggested systemic dose-independent behavior of Am-B. However, renal clearance of Am-B decreased significantly as Am-B dose increased; whereas, no renal damage was observed during the experiment. The present studies suggested that renal clearance was dose-dependent and that there may be a saturable tubular secretion process for Am-B renal excretion.
6
Custodio, Joseph M. "Predicting intestinal transporter effects in food-drug interactions and the role of food on drug absorption." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324590.
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Kim, Shinja Rhea. "Pharmacokinectic and pharmacodynamic aspects of cocaine and its interaction with ethanol." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/289511.
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The main purpose of the research described in this dissertation was to evaluate various aspects of cocaine in pharmacokinetics and pharmacodynamics including: physiologically based-pharmacokinetics modeling; the influence of ethanol on cocaine disposition. Further, cocaine and cocaethylene (CE) were compared using pharmacokinetic-pharmacodynamic (PK-PD) models. Lastly, PK-PD models after cocaine and a combination of cocaine and ethanol dose were developed. Cocaine was administered by iv with or without ethanol in rats. CE was formed only in the group of rats given cocaine in the presence of ethanol. The extent of benzoylecgonine formation from cocaine significantly suppressed in the presence of ethanol. There were no statistical differences in cocaine disposition kinetics following iv cocaine dose in the presence or absence of ethanol. The PB-PK model was developed to describe cocaine disposition in the rat, dog, monkey and ultimately for scaling to humans using information developed in animals. The model gave a good prediction of tissue concentration-time profiles in animals. The prediction of the plasma concentration-time data in humans was poor when using the same tissue-to-blood-partition coefficients (R) obtained in rats. However, an excellent prediction was obtained after R was adjusted for differences in the apparent volume of distribution at steady state (rat vs. humans). The PK-PD model for cocaine or CE was developed by analyzing literature data. CE appears to be less potent in producing euphorigenic effects and equipotent to cocaine in producing physiological effects (e.g., cardiovascular function). The sigmoid Emax model was selected to describe the relationship between the physiological and euphorigenic effects produced by cocaine, ethanol and CE and their respective concentrations in the effect compartment. This model gave a good prediction for those effects. It appears that increased heart rate and "cocaine high" after a combination dose of cocaine and ethanol compared to cocaine alone was due to both the increase in cocaine concentration and the CE formed following ethanol exposure. Similarly, increased effect of "any high" or "good effect" after a combined dose appears to be due to cocaine (in the presence of ethanol), ethanol and CE formed in the presence of cocaine and ethanol.
8
Keiser, Michael James. "Relating protein pharmacology by ligand chemistry." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378494.
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Riviere, Kareen. "Investigation of the enhancement of drug synergy by co-delivery in targeted liposomes." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390075.
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Furbacher, Todd Raymond. "Bioassay-guided isolation of potential antineoplastic natural products from Southwestern plants." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/279927.
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This dissertation details the investigation of numerous plants for potential antineoplastic compounds. 144 plants (391 extracts) were prescreened with an assortment of assays. The pre-screens included an Agrobacterium tumefaciens/potato disk gall tumor inhibition assay, a Saccharomyces cerevisiae mutant topoisomerase assay, and an Escherichia coli plasmid scission assay. Bioassay-guided fractionation was conducted on three plants, Phoradendron juniperinum, Psorothamnus thompsoniae , and Acourtia thurberi, using a different assay for each. Phoradendron juniperinum (Viscaceae) was screened with a plasmid scission assay and the novel compound, 5-caffeoyl-epi-quinic acid (I) was isolated, the first chlorogenic acid to be reported for the genus. Chemical structure was established using NMR and MS data and published structural information. The EC₅₀ for 5-caffeoyl- epi-quinic acid-mediated plasmid DNA cleavage was 76 μM. Fractionation of Psorothamnus thompsoniae (Fabaceae) was directed using the potato disk assay. The active component was dalrubone (II). P. emoryi was fractionated to obtain dalrubone and to search for related compounds. 5-Methoxydalrubone (III) was isolated and tested with dalrubone in cell line assays. 5-Methoxydalrubone was active against MCF-7 (IC₅₀ = 28.2 μM), while dalrubone (IC₅₀ = 1.3 mM) was not. Neither compound significantly inhibited the growth of NCI-H460 or SF-268. Acourtia thurberi (Asteraceae) was active in the yeast mutant assay. Fractionation yielded the sesquiterpene, 14,15-diacetoxy-,8-hydroxy-,3-(3-methylbutanoyl)-14, 15-epoxy-isocedrene (IV). This compound was weakly active against the topoisomerase II sensitive yeast strain, RS321N, with an IC12 of 342 μg/mL. The isocedrene was active in the yeast assay but inactive against human topoisomerase IIalpha. Ten celastroloids (unsaturated, oxygenated D:A-friedo-nor-oleanane triterpenoids from Sri Lankan Celastraceae) and their derivatives, some of which were also weakly active against RS321N, were tested for activity against human topoisomerase IIalpha. Demethylzeylasterone (ex. Kokoona zeylanica) strongly inhibited topo IIalpha with an IC50 of 17.6 μM. All others, including the structurally similar zeylasterone, possessed no activity at 100 μM. Demethylzeylasterone was determined to be a "catalytic inhibitor," preventing DNA from binding to the enzyme while not interacting with the DNA itself. Demethylzeylasterone selectively inhibits the MCF-7 breast cancer cell line with an IC50 of 12.5 μM.
11
Chance, Jeffrey John. "Liquid-phase sensing strategies for the thickness shear mode acoustic wave sensor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0009/NQ36964.pdf.
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Adams, James Corey. "Mapping drug chemistry from the viewpoint of small molecule metabolism." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3359572.
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Thesis (Ph. D.)--University of California, San Francisco, 2009.Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3519. Adviser: Patricia C. Babbitt. Includes supplementary digital materials.
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Akther, Farjana. "Sexual Dimorphism in Aortic Function of UC Davis Type 2 Diabetes Mellitus Rat Model: Estrogen Specific Responses." Scholarly Commons, 2019. https://scholarlycommons.pacific.edu/uop_etds/3600.
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Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate the effects of estrogen as well as type 2 diabetes (T2D) on aortic function in rats with respect to sex. To test the effects of T2D and sex, UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) rat model was used. To study the effects of estrogen, ovariectomized Sprague- Dawley female rats and UCD-T2DM rats at pre-diabetic stage were used and the rats were implanted subcutaneously either with placebo or 17 β-estradiol pellets (60 days release, 1.5mg/pellets). The plasma analytes for metabolic parameters and aortic responses to vasodilator and vasoconstrictor agents were determined. The expression of molecules associated with vascular response (e.g. endothelial nitric oxide (NO) synthase (eNOS), Nox1, Nox4, intermediate conductance calcium-dependent potassium channels (IKCa) and small conductance calcium-dependent potassium channels (SKCa)) were also evaluated in aortic tissue.
The main objectives of the study were whether 1) sex differences exist in the development of abnormal vascular responses of UCD-T2DM rats, 2) there were changes in the relative contributions of endothelium-derived relaxing factors (EDRFs) in modulating vascular reactivity of aorta, and 3) estrogen replacement improves the aortic function of ovariectomized UCD-T2DM rats at pre-diabetic stage.
In the study of examining the effect of sex and T2D, diabetes significantly impaired relaxation responses to ACh and SNP in aortic rings from female UCD-T2DM rats, however, potentiated the relaxation in males. The responsiveness to PE was significantly enhanced in both diabetic groups regardless of sex. Accordingly, the basal nitric oxide (NO), as indicated by the potentiation of the response to PE after L-NAME, was reduced in aorta of both diabetic groups. Blocking of COX, sGC and NOS completely abolished the relaxation response in female diabetic group whereas male diabetic animals showed a significant remaining relaxation response to ACh. Further incubation of aortic rings of male animals with TEA or TRAM 34 blunted the relaxation responses to ACh in both control and diabetic groups. However, the inhibitory effects of TEA or TRAM 34 on the ACh-induced relaxation in male UCD-T2DM group was greater than their respective controls. By contrast, ACh responses were not affected following incubation with Apamin in either group of male rats. Moreover, protein expression of IKca were significantly higher in male diabetic group compared with the respective controls.
In the estrogen replacement study, treatment with E2 markedly enhanced the ACh responses of aortic rings in both control and pre-diabetic groups compared to respective placebo treated group. Moreover, effect of E2 in improving the ACh induced relaxation response was significantly higher in control group compared with pre-diabetic animals. The responsiveness to PE were significantly reduced in both E2 treated groups. Basal NO level was significantly higher in both E2 replaced groups but in control group the level was significantly higher than the pre-diabetic rats. Also, protein expression level of Nox1 were decreased in E2 treated control and pre-diabetic group but eNOS were enhanced only in E2 treated control groups.
In conclusion, this study suggests that the effects of type 2 diabetes on aortic ring are sex specific and we showed a differential contribution of EDRFs in male UCD-T2DM rats. Furthermore, our data suggests that elevated eNOS and decreased Nox1protein level may contribute to the higher impact of estrogen in ovariectomized control groups compared to the pre-diabetic rats.
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Cao, Jieyun. "The role of sex hormones on monocarboxylate transporter expression in tissues related to drug disposition." Scholarly Commons, 2019. https://scholarlycommons.pacific.edu/uop_etds/3599.
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Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport monocarboxylates such as ketone bodies, lactate and pyruvate, as well as drugs such as gamma-hydroxybutyric acid. CD147 acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane trafficking. Previously, it has been shown that MCT expression changes under different sex hormone conditions in skeletal muscle and Sertoli cells. However, it is unknown if MCTs, SMCTs or CD147 demonstrate sex differences in tissues where they play an important role in drug disposition. Monocarboxylate transporter substrates GHB and valproic acid have demonstrated sex differences in pharmacokinetic profiles. We hypothesize that sex hormones regulate monocarboxylate transporters and CD147 expression in drug disposition tissues, including the liver, intestine and kidney.
The purpose of the current study is to evaluate sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 mRNA and protein expression in drug disposition tissues. Liver, kidney and intestinal segments (duodenum, jejunum and ileum) were harvested from estrus cycle staged female rats, ovariectomized (OVX) females, males and castrated (CST) male rats. Hormone replacement experiments were performed to investigate testosterone and 17β-estradiol dependent regulation of renal MCTs, SMCT1 and CD147 in OVX females and CST males. mRNA of MCT1, MCT4, SMCT1 and CD147 was evaluated by real time quantitative PCR. Whole cell protein and membrane protein was extracted, target protein expression was evaluated by western blot.
We have demonstrated sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 in the liver, intestine regions and kidney occurs in a tissue specific manner. mRNA, protein expression and membrane localization of monocarboxylate transporters and CD147 were regulated differently by sex hormones. Sex differences in MCTs and SMCTs expression are important determinants of drug disposition in the body and sex differences in their regulation may contribute to differences in drug pharmacokinetics.
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Rice, Peter J. "Understanding Drug Action: An Introduction to Pharmacology." Digital Commons @ East Tennessee State University, 2014. http://amzn.com/1582121125.
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This highly readable introduction to the science of pharmacology assumes only a modest understanding of biology, chemistry, and human physiology. Author Peter J. Rice provides readers with a survey of the scientific understanding of drug action. He discusses pharmacology at a basic scientific level to build a framework of how drugs work, and he supplements this discussion with information on some representative drugs that are used clinically. KEY FEATURES: Concise and systematic introduction to the science of pharmacology; Knowledge objectives in each chapter; Glossary of key terms in each chapter; Review questions in each chapter with answers provided in the back of the book; Tables listing brand and generic drug names and dosage forms by drug class; More than 170 illustrations that supplement the text.https://dc.etsu.edu/etsu_books/1033/thumbnail.jpg
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Pappas, Beverly. "Mechanistic Study of p23-Mediated Aryl Hydrocarbon Receptor Expression." Scholarly Commons, 2018. https://scholarlycommons.pacific.edu/uop_etds/3131.
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The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins and isothermal titration calorimetry showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1–216 and p23 amino acid 1–110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation. However, the increased ubiquitination is not through the small ubiquitin-like modifier (SUMO) signaling pathway.
Troubleshooting and optimization were paramount for understanding and evaluating the p23 and AHR interaction. Specifically, the p23 mutant purification, p23: Hsp90 interaction, transient transfection, p23: AHR assay, and ITC study were phases of this research that required extensive time and critical thinking. These topics were further detailed to outline the specific problems encountered and the various steps taken to alleviate or optimize these issues.
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Sheng, Wanhui. "AN EXTENSION OF PLANARIAN BEHAVIORAL MODEL: CANNABINOID PHYSICAL DEPENDENCE AND WITHDRAWAL." Master's thesis, Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/402376.
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Pharmaceutical SciencesM.S.
Background: Planarians have mammalian-like neurotransmitter systems and have been established as a novel in vivo model for neuropharmacology. In previous research, planarians exposed to the cannabinoid receptor (CB-R) agonist WIN 55,212-2 (10 μmol/L) for 1 h displayed a significant (p < 0.05) decrease in spontaneous locomotor velocity (pLMV) when subsequently tested in drug-free, but not in drug-containing, water. This demonstrated abstinence-induced withdrawal from a CB-R agonist as a manifestation of the development of physical dependence. Purpose: The purpose of the present study was to extend previous work and to further establish a cannabinoid behavioral model with planarians. Specifically, the goals included (i) confirm the work with WIN 55,212-2 and extend to a second agonist (ii) interfere with agonist-induced physical dependence using several CB-R antagonists, (ii) demonstrate antagonist-induced precipitated withdrawal behavior, and (iii) try to induce withdrawal behavior from CB-R agonists using UV light. Methods: Two CB agonists (WIN 55,212-2 and JWH251) and four CB antagonists (AM251, AM281, SLV319 and SR144528) were used. Planarians were placed individually in CB-R agonist or agonist + antagonist mixtures for 20 and 30 min of exposure (with or without UV radiation), and withdrawal was quantified by measuring pLMV in drug-free vs drug-containing water (with or without UV light irradiation). Results: (i) Four different CB1-R antagonists (AM251, AM281, SLV319 and SR144528) dose-relatedly blocked development of physical dependence induced by two different CB-R agonists (WIN 55,212-2 and JWH251). (ii) None of the same four antagonists (AM251, AM281, SLV319 and SR144528) precipitated withdrawal. (iii) Short wavelength (254 nm), but not long wavelength (366 nm), UV light attenuated abstinence-induced withdrawal from WIN 55,212-2, while short wavelength UV light induced moderate withdrawal behavior. Conclusions: The results confirm the use of a planarian model as a simple yet robust way to study development of physical dependence to cannabinoid agonists. The model is more rapid and sensitive than the usual rodent models. The effect of UV irradiation adds to the supposition that the results are receptor-related. The results also give rise to the surprising suggestion, within the limitations of the methodology, that development of cannabinoid physical dependence and antagonist-induced precipitated withdrawal might be separable phenomena in planarians.
Temple University--Theses
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Guillemard, Véronique. "Design and chemical synthesis of selective cancer therapeutics." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85073.
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The clinical use of chemotherapeutic agents against malignant tumors is successful in many cases but suffers from major drawbacks. One drawback is the lack of selectivity which leads to severe side effects and limited efficacy, and another is the emergence/selection of drug-resistance. To limit non-specific toxicity and to improve the efficiency of cancer therapy, "tumor markers", which are proteins generally overexpressed on the surface of tumor cells, can be selectively targeted.Growth factor receptors are one of the most extensively studied groups of tumor markers. The implication of growth factor receptors in the pathogenesis of cancer has clearly been established and therefore, provides a rationale for therapeutic intervention. The targeting of cytotoxic substances to defined cell populations with "magic bullets" is an old idea that raised high expectations but also disappointment. Over the past decade, newly gained understanding of mechanisms for targeted therapy have brought new hopes. Pharmacological agents that selectively target and block the action of growth factors and their receptors have been attempted, such as monoclonal antibodies (mAbs) (whole molecule or fragments), bispecific antibodies, mAbs conjugated to drugs, toxins or radioisotopes, small peptidic and peptidomimetic molecules in free form or conjugated to drugs, anti-sense oligonucleotides, immunoliposomes-encapsulated drugs, and small molecule inhibitors. We designed, synthesized and characterized new chemotherapeutic agents consisting of Paclitaxel or Doxorubicin as anti-cancer drugs chemically coupled to growth factor receptor-selective monoclonal antibodies or small peptides as targeting agents. We show that the conjugates were selective and specific towards the targeted receptors, and had significant increased efficiency compared to parent drugs. More importantly, the conjugates were able to bypass p-glycoprotein-mediated resistance both in vitro and in vivo. These findings have considerable importance since drug resistance is a major cause of cancer treatment failure.
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McReynolds, Kathrine Dawn. "1. Development of a novel ELISA for the testing of glycobioconjugates as anti-HIV agents 2. Synthesis of potential inhibitors of the HIV entry mechanism 3. Probing the secondary structural characteristics of oligosaccharides utilizing circular dichroism." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/283988.
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AIDS, or acquired immunodeficiency syndrome, is caused by the human immunodeficiency virus (HIV). HIV is a retrovirus that is capable of rapid genetic mutation, which makes the virus and the disease difficult to treat. Several drug therapies are currently available, in the form of viral enzyme inhibitors. Other inhibitors of the viral entry and replication process are being investigated to enhance the drug therapy arsenal. Our research has focused on the development of HIV entry inhibitors. We are working towards the development of novel carbohydrate-based agents that are capable of binding the gp120 protein on the viral surface, such that viral entry into an uninfected host cell is prevented. In order for our research to progress, a qualitative method by which our synthetic compounds could be evaluated for gp120 binding was sought. We have developed a unique ELISA (enzyme-linked immunosorbent assay) that indicates whether or not a compound has binding affinity for the viral protein. A TIRF (total internal reflection fluorescence) microscopy method, has been developed as part of a collaborative effort with the laboratories of Professors Saavedra and O'Brien, to assess active compounds for quantitative equilibrium binding constants to gp120. We have synthesized several carbohydrate-based molecules targeted to one or more of the binding sites on the surface of gp120; the galactosylceramide site, the V3 loop, and the CD4 binding site. Utilizing both the ELISA and TIRF methods, we have succeeded in probing the binding profile of gp120. Circular dichroism studies have also been employed to evaluate the secondary structural characteristics of oligomeric carbohydrate materials. Molecules with helical properties have potential as CD4 binding site inhibitors. The long term goals of this project involve the synthesis and gp120 binding evaluation of novel carbohydrate-based materials to serve as entry inhibitors of the HIV replication process. A possible application of this project lies in the development of compounds capable of binding to more than one site on the protein. A variation of this goal involves the tethering of various compounds with specificities to different sites on gp120, for the purpose of inhibiting multiple binding sites on the protein.
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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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Vyas, Piyush Manhur. "Sulfonamide-induced cutaneous drug reactions: role of bioactivation, oxidative stress and folate deficiency." Diss., University of Iowa, 2006. https://ir.uiowa.edu/etd/81.
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Sulfonamide- and sulfone-induced hypersensitivity reactions are thought to be mediated through bioactivation of parent drug molecule(s) to their respective reactive metabolite(s). In order to explain the cutaneous drug reactions caused by sulfonamides and sulfone, a mechanism can be proposed by which the bioactivation of these drugs in keratinocytes of the skin forms reactive hydroxylamine metabolites that can covalently bind to cellular proteins, which in turn act as antigens leading to the cascade of immune reactions resulting in a cutaneous drug reaction. In order to probe the proposed mechanism, we determined the enzymes responsible for the bioactivation of these parent drugs to their hydroxylamine metabolites in cultured human keratinocytes. It was found that flavin containing monooxygenases and peroxidases play an important role in the bioactivation of these drugs in keratinocytes. We also confirmed the presence of these enzymes in keratinocytes. Interestingly, though cytochrome P450s are important in the oxidation of parent arylamine xenobiotics to their hydroxylamine metabolites in the liver, they do not appear to play a significant role in the bioactivation of these drugs in keratinocytes. The hydroxylamine metabolites of sulfamethoxazole and dapsone can undergo autooxidation, generating reactive free radicals. Our studies showed that both of these metabolites elevate oxidative stress in keratinocytes by forming reactive oxygen species. Though the cytotoxicity induced by these metabolites is not correlated with the extent of oxidative stress, the generation of reactive oxygen species may be important finding as these species can act as danger signals that activate antigen presenting cells in the skin. As a possible explanation for the idiosyncratic nature of these reactions, folate deficiency was studied as a potential risk factor. However, the results of these studies suggested that deficiency of folic acid in keratinocytes does not predispose such cells to the toxicity associated with the parent drugs or their metabolites. Unexplored is the potential role of such deficiency on the immune response itself.
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Hui, Tina Hsiao-Tin. "Design and test of a pharmacologic screen for the adaptogenic properties of plant drugs : a master's thesis." Scholarly Commons, 1992. https://scholarlycommons.pacific.edu/uop_etds/2231.
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Panax ginseng C.A. Mey. (Araliaceae), which has been known in China for more than 4000 years, occupies a particular place among tonic remedies. Pharmacological investigations of the roots have shown that the basic effect of ginseng's action is its capacity to increase the nonspecific resistance of the organism to various untoward influences. In addition to ginseng, another plant of the Araliaceae family is used in a similar manner and is known as Siberian ginseng, Eleutherococcus senticosus (Rupr. et Maxim.) Maxim.). Its roots have been very extensively investigated by Russian scientists. Ginseng is reported to be shorter in duration and weaker in activity when compared to E. semicosus in respect to stimulant and tonic effects. 13 Chinese scientists have claimed that China Produced E. senticosus can exert a definite anti fatigue action. Less extensive studies have been reported on the berries of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae). for its adaptogenic properties.
The main objective of the work reported in the following pages was to design and test a simpler and easier animal test model whereby multiple physiologic parameters such as Tn EKG and respiratory rates could be continuously and concurrently monitored. It was desired that the test model could be used repeatedly and rapidly to serve as a screen for all potential adaptogenic plants and their derivatives and extracts.
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Akbar, Shahid. "Hypothermic activity of repin, a sesquiterpene lactone from Centaurea solstitialis." Scholarly Commons, 1993. https://scholarlycommons.pacific.edu/uop_etds/2757.
Full textAbstract:
Powdered aerial parts of the weed yellow starthistle (Centaurea solstitialis, Compositae) and the methanol extract, alkaloidal and fatty fractions of the methanol extract of the aerial parts, injected intraperitoneally, produced a highly significant hypothermia in conscious normal rats. Other significant symptomatology included sedation, decreases in spontaneous motor activity, depressed respiration, mydriasis, ear blanching, enophthalmos and anuria. Four sesquiterpene lactones of the guaianolide type (repin, solstitialin-A, janerin and cynaropicrin), constituents of yellow starthistle, showed similar activities in rats. The predominant effects were hypothermia, sedation and enophthalmos. Both repin and janerin, in equidoses of 3.1-31 mg/kg, produced similar profiles of generalized effects and hypothermia, whereas solstitialin-A and cynaropicrin did not show well-defined dose-response relationships. The lethal doses of the methanol extract, repin, solstitialin-A, janerin and cynaropicrin were 1000, 31, 310, 31 and 310 mg/kg, respectively. Studies were carried out to document the effects of various potential antagonists and agonists on the repin-induced hypothermic effect. Rats were pretreated intraperitoneally with atropine sulfate (10 and 20 mg/kg), atropine methylbromide (20 mg/kg), propranolol (10 and 20 mg/kg), metergoline (0.5 mg/kg), ketanserin tartrate (0.2 mg/kg), diphenhydramine HCl (10 mg/kg) and apomorphine HCl (0.5 mg/kg). No significant effects of pretreatment were evident up to 2 h but statistically significant partial reversals of repin-induced hypothermia by atropine sulfate (20 mg/kg), metergoline, ketanserin, diphenhydramine and apomorphine were observed beginning 3 h after repin injection. Propranolol pretreatment resulted in a significant potentiation of repin's hypothermic effect. The sleeping time in mice induced by intraperitoneal sodium pentobarbital was markedly potentiated by pretreatment with repin. Receptor binding studies showed that repin facilitated the binding of GABA$\sb{\rm A},$ bombesin and neuropeptide Y without any significant binding of repin to any of the 36 receptors and binding sites tested.
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Zhang, Tianmin. "Effects of Ainp2 and beta-tubulin on the Arnt-dependent signaling pathways." Scholarly Commons, 2010. https://scholarlycommons.pacific.edu/uop_etds/2645.
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Aryl hydrocarbon receptor nuclear translocator (Arnt) belongs to the basic helix-loop-helix/Per-Arnt-Sim protein family. Our lab is interested in identifying the protein factors that modulate the Arnt-dependent pathway. We identified a novel Arnt-interacting peptide Ainp2 with an estimated size of 9 kDa. The endogenous Ainp2 protein is not found in MCF-7 cells. Another protein that is of interest to us is β-tubulin. Beta-tubulin is the monomeric subunit of microtubules. I have presented my thesis in two parts: the first part is focused on Ainp2 and the second part is on β-tubulin. The purpose of the first part of my thesis was to investigate whether Ainp2 affects the Arnt-dependent estrogen receptor (ER) pathway via an Arnt-related mechanism. Transient transfection studies in MCF-7 cells revealed that transfected Arnt increases E2-induced, ERE-driven luciferase activity in a dose-dependent manner; however, the enhancement effect of Arnt is significantly suppressed in the presence of Ainp2. The Ainp2 protein was successfully expressed in Sf9 cells and was affinity purified by the TALON metal resin method. Data from co-immunoprecipitation experiments showed that Ainp2 suppresses the interaction between Arnt and ERα in the presence or absence of E2. The aim of the second part of my thesis was to explore the effect of β-tubulin on the Arnt-dependent aryl hydrocarbon receptor (AhR) pathway. My data indicates that human β4-tubulin inhibits 3MC-driven, AhR-dependent luciferase expression. This suppressive effect of β4-tubulin is likely caused by a reduction in the nuclear Arnt content resulting from Arnt retention in the cytoplasm. The findings are certainly intriguing that the Arnt-mediated pathway can be modulated by either Ainp2 or β-tubulin. Limiting the nuclear Arnt function can be an approach to block Arnt-dependent signaling events which are crucial for cancer growth. These findings provide a means for rational drug development.
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Matos, Francisca Fatima. "Opioid Receptor Effects Of Two Aminotetralin Derivatives In Guinea Pig Ileum Longitudinal Muscle And Mouse Vas Deferens Preparations." Scholarly Commons, 1987. https://scholarlycommons.pacific.edu/uop_etds/3394.
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Two substituted analogs of 3-amino-2,2-dimethyltetralin, namely 3-dimethylamino-7-hydroxy-2,2-dimethyl-1-tetralone HBr (J) and 3-dimethylamino-7-hydroxy-2,2-dimethyl-1-tetralol (MRSAL) were evaluated for opioid agonist and antagonist activity using the electrically-stimulated guinea pig ileum longitudinal muscle and mouse vas deferens preparations. The effects of these compounds in these tissues were compared to those induced by several opioid prototype agonists at mu, kappa and delta sites (normorphine, dihydromorphine, ethylketocyclazocine, U-50,488H, beta -endorphin, dynorphin 1-13, leu-enkephalin and DADLE) and one opioid antagonist (naloxone). The results of these experiments demonstrated that compound J inhibited contractions in a concentration-dependent manner as an opioid agonist and its effects were antagonized by naloxone in both preparations. The agonist effects of J were also irreversibly antagonized by beta -funaltrexamine pretreatment suggesting a preference for mu receptors. On the other hand, MRSAL was able to antagonize all the opioid agonist prototypes in a concentration-dependent manner, but with varying affinities. The differential opioid receptor selectivity for compound J was studied based on: (i) its agonist potency in guinea pig ileum longitudinal muscle versus the mouse vas deferens; (ii) its irreversible antagonism by beta -funaltrexamine in guinea pig ileum; and (iii) by the calculation of the apparent dissociation constant (Ke) of naloxone for this agonist in both preparations. The opioid receptor preference for MRSAL was based on its potency in antagonizing the opioid agonist prototypes by calculating its Ke value. Based on these criteria, compound J behaved as a mu receptor agonist while MRSAL had preference for mu rather than kappa or delta receptors.
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ARORA, DEEPIKA. "IN VITRO MODELS FOR INHALED CORTICOSTEROID (ICS) AEROSOLS: A STUDY OF THEIR BIOPHARMACEUTICS AND PHARMACOLOGY." VCU Scholars Compass, 2008. http://scholarscompass.vcu.edu/etd/1650.
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Lung cellular disposition and anti-inflammatory pharmacology of inhaled corticosteroids (ICSs) is complex, comprised of a cascade of aerosol deposition and dissolution, followed by cellular uptake for local pharmacological action. This project hypothesized that the kinetics of dissolution for certain ICS aerosols generated from inhaler products were kinetically rate-determined for their cellular uptake and local pharmacological action. A novel dissolution testing system was developed to determine the dissolution kinetics for the ICS aerosols. A total of 5 ICSs aerosols generated from 6 inhaler products were collected in 2.1-3.3 or 4.7-5.8 µm of aerodynamic diameters at 0.7-19.8 µg on filter membranes by impaction using the Andersen cascade impactor. The filter membrane was then placed on the donor side of the transwell insert, with its face down, and the ICS dissolution in the limited 40 µL of the donor fluid was monitored over time. The dissolution kinetics overall conformed to the rank order of the aqueous solubility, while also being affected by ICS aerosol’s mass, size, formulation and dosage forms. For the readily soluble triamcinolone acetonide (TA), the kinetics was first-order, reaching ≥89 % dissolution in 5 h. In contrast, for the least soluble fluticasone propionate (FP), the kinetics was zero-order, reaching only 3 % dissolution in 10 h. The project then developed an air-interface culture of human bronchial epithelial cell line, Calu-3. Well-differentiated monolayers were formed with sufficiently “tight” barrier for restrictive solute diffusion while their mucosal surface was maintained semi-dry with 39.7±12.1 µL of the mucosal lining fluid in the 4.5 cm2 transwells. These monolayers were transfected with reporter plasmid of pNFκB-Luc to assess in vitro anti-inflammation via repression of pro-inflammatory NFκB by direct FP or TA aerosol deposition. The FP aerosols at 0.9 µg successfully exhibited significant 35.7±6.3 % repression. Notably, however, an identical ~0.5 µg of FP and TA aerosols caused comparable 15.5±2.2 and 10.4±2.6 % repression, respectively, despite FP’s 10-fold greater “intrinsic” anti-inflammatory potency over TA, reported in the literature. This was attributed to FP’s slow dissolution resulting in only 4.7 % cellular uptake, compared to 32.6 % for the TA aerosols. Hence, the FP aerosols were shown to be rate-determined by dissolution on the lung cell surface, resulting in reduced anti-inflammatory actions, which was not the case for the readily soluble TA aerosols.
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Zheng, Yujuan. "The role of aryl hydrocarbon receptor (AHR) in drug-drug interaction and the expression of AHR in Pichia Pastoris." Scholarly Commons, 2019. https://scholarlycommons.pacific.edu/uop_etds/3580.
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The aryl hydrocarbon receptor is a ligand-activated transcription factor that is involved in many important functions in the body. To study the role and function of AHR, an abundant amount of in vitro expressed and purified protein is needed. A baculovirus insect expression system is commonly employed to express AHR, however, there are several drawbacks with this method, such as mutation potential and high cost. A better in overexpression system is needed and we hypothesize that Pichia pastoris, a yeast expression system, could stably express AHR and ARNT (aryl hydrocarbon receptor nuclear translocator) in sufficient amount with reasonable cost. Codon optimized human AHR and ARNT genes were separately transformed into the Pichia pastoris genome and expressed. Co-immunoprecipitation, gel-shift assay and western analysis indicate Pichia pastoris was able to stably overexpress functional AHR and ARNT proteins in comparable yield and lower cost compared to baculovirus insect expression system and the expressed proteins were used to develop a new in vitro method to study AHR and ARNT binding.
Pharmacokinetic studies were performed to investigate the role of AHR in rutacarpine-caffeine interactions. Oral RUT pretreatment was shown to reduce oral caffeine area under the curve (AUC) in rats, due to an increase in caffeine clearance (CL) and a decrease in oral bioavailability (F). RUT, an AHR ligand, increases caffeine CL by inducing Cyp1A2 enzyme, but the mechanism by which RUT reduces caffeine F is not understood. We hypothesize that it is also mediated via AHR pathway. To test the hypothesis, wild type (WT) and AHR knock out (KO) mice were administered caffeine IV and orally, with and without VEH or RUT pretreatment. As expected, PK data show higher caffeine CL and lower F values in WT, but similar CL and F values in AHR KO mice, upon RUT co-administration.
Rats study, in which with pretreatment of vehicle, AHR ligands: RUT, beta-naphthoflavone or indole-3-carbinol before caffeine was dosed orally is consistent with mice study, that all three AHR agonists tested were able to reduce oral caffeine AUCs in rats.
RUT reduces caffeine’s oral bioavailability is through AHR signaling pathway, however, However, the mechanism by which AHR mediates the reduced caffeine F is not known.
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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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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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Chen, Jinyun. "REGULATION OF INTRACELLULAR ARYL HYDROCARBON RECEPTOR PROTEIN LEVELS." Scholarly Commons, 2020. https://scholarlycommons.pacific.edu/uop_etds/3675.
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The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which controls tumor growth and metastasis, T cell differentiation, and liver development. Expression levels of this receptor protein are sensitive to the cellular p23 protein levels in immortalized cancer cell lines. As little as 30% reduction of the p23 cellular content can suppress the AHR function. Here we reported that down-regulation of the p23 protein content in normal, untransformed human bronchial/tracheal epithelial cells to 48% of its content also suppresses the AHR protein levels to 54% of its content. This p23-mediated suppression of AHR is responsible for the repression of (1) the ligand-dependent induction of the cyp1a1 gene transcription; (2) the benzo[a]pyrene- or cigarette smoke condensate-induced CYP1A1 enzyme activity, and (3) the benzo[a]pyrene and cigarette smoke condensate-mediated production of reactive oxygen species. Reduction of the p23 content does not alter expression of oxidative stress genes or production of PGE2. Down-regulation of p23 suppresses the AHR protein levels in two other untransformed cell types, namely human breast MCF-10A and mouse immune regulatory Tr1 cells. Collectively, down-regulation of p23 suppresses the AHR protein levels in normal and untransformed cells and can in principle protect our lung epithelial cells from AHR-dependent oxidative damage caused by exposure to agents from environment and cigarette smoking.
The AHR is expressed in triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with the estrogen receptor signaling. Normally the AHR is degraded shortly after ligand activation via the action of 26S proteasome. Here we report that the piperazinylpyrimidine compound Q18 triggers AHR protein degradation which is mediated through chaperone-mediated autophagy in triple-negative breast cancer cells (MDA-MB-468 and MDA-MB-231). This lysosomal degradation of AHR exhibits the following characteristics: (1) not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (2) inhibited by progesterone receptor B but not estrogen receptor alpha; (3) reversed by chloroquine but not MG132; (4) required LAMP2A; (5) triggered by 6 amino-nicotinamide and starvation and (6) involved AHR-LAMP2A interaction mediated by 6 amino-nicotinamide and starvation. The NEKFF sequence localized at amino acid 558 of human AHR is a KFERQ-like motif of chaperone-mediated autophagy, essential for the LAMP2A-mediated AHR protein degradation.
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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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Treki, Mahmud Sighayer. "Formulation and evaluation of propylene glycol monostearate microspheres for sustained release of nitrofurantoin." Scholarly Commons, 1988. https://scholarlycommons.pacific.edu/uop_etds/3366.
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Sustained release of nitrofurantoin (NFT) from microspheres of propylene glycol monostearate (PGM) and of PGM and ethoxylated stearyl alcohol (ESA) prepared by the meltable dispersion and cooling process was investigated. The microspheres (30-850 mu) were nonsticky, discrete and free flowing. Both the rate and extent of in vitro release of NFT (from NFT-PGM microspheres in distilled water at 37 degrees C under constant agitation at 50 rpm) declined with decreasing NFT/PGM ratio. The effect of incorporating ESA over the range of 0.01 to 0.05% w/w of PGM in formulations with NFT/PGM ratios of 1:1, 1:1.5 and 1:4, studied under similar experimental conditions revealed that NFT release was maximum in the range of 0.02 to 0.035% ESA. The effect of pH on NFT release from microspheres with 0.03% ESA and without ESA at NFT/PGM ratios of 1:1 and 1:4, was investigated in buffer solutions at pH values of 1.2, 5.8 and 7.2. The pH-dependent solubility and dissolution of NFT and PGM, in addition to NFT/PGM ratio, were found to control the rate and extent of NFT release. Both scanning electron photomicrography and contact angle measurements suggested that about 0.03% ESA was critical for the formulations studied. The in vitro evaluation of adhesion of various polymers to rabbit stomach tissue was investigated. The polymers, CLD, ACDISOL, polycarbophil and calcium polycarbophil were investigated for potential use as bioadhesives. The modified balance method developed and used to evaluate the polymers adhesion to rabbit stomach tissue in vitro at different pH levels was reproducible and could detect change in adhesive force as little as 10 mg weight. Maximum adhesion for polymers CLD and polycarbophil was observed at pH 5.8, while that of ACDISOL was at pH 7.2. The polymer, calcium polycarbophil, showed essentially no adhesion at all. The results revealed that CLD was superior and ACDISOL was inferior to polycarbophil under all three pH conditions investigated. In vitro release studies of the drug from microspheres mixed in various proportions with bioadhesive polymer CLD in phosphate buffer at pH 5.8 indicated that the presence of the polymer did not significantly hinder the drug release. (Abstract shortened with permission of author.)
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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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Huang, Wei Hua. "Chemical investigation on root barks of Oplopanax horridus." Thesis, University of Macau, 2012. http://umaclib3.umac.mo/record=b2590373.
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Berry, Elicia Bee Ean. "Intracellular signalling by arachidonic acid metabolites." Thesis, University of Auckland, 2006. http://hdl.handle.net/2292/3111.
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In intrauterine tissues, pro-inflammatory cytokines and prostaglandins (PGs) have been identified as key mediators in the maintenance of pregnancy and parturition. The rise in PGD2 detected in the amniotic fluid before labour prompted the research presented in this thesis which describes the effects of 15-deoxy Δ12,14 -prostaglandin J2 (15d-PGJ2), a non-enzymatic metabolite of PGD2, on amnion-derived WISH and JEG3 choriocarcinoma cells as models of the amnion and chorion trophoblasts, respectively.15d-PGJ2 induced apoptosis in both cell lines in a concentration-dependent fashion (2.5-10 µM). Apoptosis was characterised by condensation of chromatin (visualised after Hoechst 33342 staining), appearance of nucleosomal DNA fragmentation upon electrophoresis and flow cytometry analysis, and activation of caspase-3. Apoptotic cell death was inhibited in the presence of serum (0.5% w/v) and albumin, not serumderived growth factors (insulin growth factor (IGF)-1, IGF-2 or epidermal growth factor (EGF), was determined as the key survival factor. Since 15d-PGJ2 is reported to activate peroxisome proliferator activated receptor (PPAR)-γ, the activities of PPARs were assessed using JEG3 cells transfected with a PPAR-response element reporter construct (pTK-PPREx3-luc). The PPAR-γ-specific ligand, rosiglitazone, induced PPRE mediated activity in a concentration-dependent manner, while the PPAR-γ-specific irreversible inhibitor, GW9662, fully inhibited this induction. However, GW9662 only partially inhibited 15d-PGJ2-induced luciferase activity, suggesting that 15d-PGJ2 may also activate either of the other isoforms. The expressions of PPAR-α and -δ were identified in amnion, choriodecidua and placental membranes and PPAR-δ was significantly increased all tissues with labour. PPAR-α expression was reduced in chorio-decidua, but was significantly higher in placenta with labour. The changes observed with labour suggest that regulation of PPAR expression and function may have a role in the mechanisms that maintain pregnancy or initiate labour. The anti-inflammatory effects of 15d-PGJ2 were also investigated by measuring interleukin (IL)-1β-stimulated prostaglandin and cytokine productions by WISH cells after treatment with 15d-PGJ2 for 3 hours. 15d-PGJ2 exerted differential effects that were dependent upon its concentration. At low nanomolar physiologic concentrations (1-10 nM), 15d-PGJ2 inhibited IL-1β-stimulated PGE2, but not cytokine (IL-6/IL-8) production or cyclooxygenase (COX)-2 expression. This effect was attenuated by GW9662, by transfection with dominant negative PPAR constructs, and was reproduced by rosiglitazone. At micromolar (1-10 µM) concentrations, 15d-PGJ2 inhibited IL-1β-stimulated COX-2, PGE2 and cytokine productions and these effects were not blocked by GW9662 or mimicked by rosiglitazone. GW501516 (PPAR-δ agonist) also inhibited IL-1β-stimulated PGE2 production, but only at high concentrations (1 µM). IL-1β-induced NF-kB DNA binding activity was significantly inhibited by 15d-PGJ2 (10 µM) and GW501516 (1 µM), but increased by rosiglitazone (10 µM). In conclusion, this is the first report of an effect of 15d-PGJ2 at low nanomolar physiologic concentrations and 15d-PGJ2 mediates its actions through PPAR-γ (<0.1 µM) and PPAR-γ-independent(1-10 µM) pathways, the latter through inhibition of NF-kB and/or activation of PPAR-δ. Further studies on the effect of physiologic concentrations of 15d-PGJ2 on primary gestational tissues will provide understanding on the role(s) 15d-PGJ2 plays in fetal membrane remodelling and its involvement in the inflammatory processes associated with labour and parturition.Whole document restricted, but available by request, use the feedback form to request access.
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Collier, Abby Cherise. "Expression and activity of enzymes in the human placenta: pharmacological & toxicological consequences in AZT therapy." Thesis, University of Auckland, 2002. http://hdl.handle.net/2292/3138.
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The aim of this thesis was to study xenobiotic metabolising enzymes (XME) in the human placenta, in particular the uridine diphosphate glucuronosyltransferases' (UGTs) role in the metabolism, transfer and disposition of the drug AZT, and to develop the human placental perfusion model into the first trimester. UGT, β-glucuronidase, Cytochrome p4501A (CYPIA) and CYP reductase were present and active in the human placenta. CYP2E1 protein was expressed but not active. CYPIA, CYP2EI and the UGT2B subfamily were expressed across gestation, but the UGTIA subfamily was only expressed in first trimester placentas. The localisation of XME (the syncytiotrophoblast layer bordering the placental villi) did not change with gestation but enzyme activity and affinity did. Greater activify but lower affinity of UGT and CYPIA were observed in first trimester placentas than at term. In contrast, β-glucuronidase had a high affinity, low activity profile in early gestation but the opposite at term. UGT and CYPIA activities in the first trimester placenta were induced by maternal cigarette smoking and may be synergistically induced by combined alcohol consumption and smoking. A significant correlation with higher UGT activity and earlier gestational age was observed. CYPIA exhibited a significant, negative correlation with maternal age across gestation. Maternal variables had no effect on β-glucuronidase and their effects on the activity of CYP2EI and CYP reductase were not established. AZT caused apoptosis in the placenta and also increased reactive oxygen species and altered XME. The absence of serum enhanced these effects. Alterations in XME expression and activities included a decrease in UGT activity and increases in CYPIA, β-glucuronidase, CYP reductase and glutathione-Ѕ-transferase activity in response to AZT exposure. AZT transport in a perfusion model was bi-directional and reached equilibrium approximately 3 h after addition of AZT into the maternal reservoir. In contrast, transfer of AZT glucuronide (AZT-G) showed significantly greater transport rates out of the fetal compartment resulting in AZT-G concentrations approximately 2-fold higher in the maternal circuit. Transfer of the co-factor UDPGA was significantly greater in the fetal direction and almost complete after 4h of perfusion. The low levels of glucuronidation of AZT catalysed by the human placenta (approximately 2% of a dose) are not due to insufficient transport of the co-factor UDPGA and are unlikely to be significant in terms of maternal whole-body clearance. Therapeutic failure of AZT in protecting the fetus is unlikely to be due to metabolism and clearance performed by the placenta but may be due to placental cytotoxicity. The development of the perfusion model into the first trimester placenta is technically achievable, but was impossible with the tissue available for these studies due to the method used to obtain placentas.Whole document restricted, but available by request, use the feedback form to request access.
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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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Samoshin, Andrey V. "Diastereoselective acylation of trans-2-substituted-cyclohexanols and glycosidase inhibition studies." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/275.
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Part I. The reaction between chiral acyl chlorides and trans -2-substituted-cyclohexanols proceeds diastereoselectively, i.e. produces mixtures of unequal amounts of diastereomers. We found for the first time that addition of pyridine or diisopropylethylamine accelerates the acylation, and unexpectedly for some substituents (RX) may completely invert its diastereoselectivity. These observations have been rationalized in terms of a stereoselective intramolecular assistance by the RX group to the acylation of the neighboring hydroxyl ("bait-and-hook" mechanism). A series of trans -2-substituted-cyclohexanols were synthesized and acylated with a racemic reagent in presence and absence of pyridine. The results showed that the presence of a nucleophilic group on the second carbon allowed for the preferred formation of one of the diastereomers in the absence of pyridine. However, in the presence of pyridine, the diastereoselectivity would inverse, and the reaction would favor the formation of the other diastereomer. To test the intramolecular acyl transfer hypothesis in detail a series of thioglucosides has been synthesized. Part II. The synthesized thioglucosides were tested as inhibitors of fungal glycosidases. Two compounds showed greater than 80% inhibition values in excess of the activity of β-D-glucosidases. More interestingly, the same compounds showed a marked enhancement of α-D-galactosidase activity by as much as 35%.
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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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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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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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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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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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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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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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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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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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Shaker, Nuha. "Examining the Influence and Role of Pharmacogenetics among Children with Autism Spectrum Disorder." TopSCHOLAR®, 2017. https://digitalcommons.wku.edu/theses/2037.
Full textAbstract:
Pharmacogenetics is the study of genomic-guided individualized drug prescription that plays an important role in preventing the severe adverse effects of drugs, decreasing the time and cost of therapeutic choices, and directing healthcare professionals to choose medications that are effective and safe. It is noteworthy that this approach becomes highly beneficial in patients suffering from chronic diseases or disorders, since these conditions may require multiple and long term pharmacological therapies, as in children with autism spectrum disorder (ASD). However, public acceptance is a major challenge when implementation of pharmacogenetics merges into clinical practice. The purpose of this study is a) to investigate, among small cohort group of children with ASD, several genetic variants of enzymes that influence the metabolism of commonly prescribed drugs to treat ASD and b) to inspect the knowledge of, attitude towards and future expectations with regards to pharmacogenetics among parents of children with ASD. A group of 15 school-aged participants with ASD were recruited for the study. Approximately 5 ml of venous blood was drawn for each participant to analyze the genotype of enzymes implicated in drug metabolism via pharmacogenetics testing. Thereafter, the parents of these children attended a training session to help them gain a better understanding of the pharmacogenetics results depicted in the drug panel results. A pre-training and post-training survey was conducted to assess the knowledge of, attitude towards and future expectations of pharmacogenetics among the children’s parents.
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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.
Full textAbstract:
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.