+publisher:"University of Michigan" +contributor:("Hollenberg, Paul F.")

University of Michigan

1. Hassan, Iman. The Effect of Trichloroethylene on Adverse Birth Outcomes.

Degree: PhD, Toxicology, 2015, University of Michigan

URL: http://hdl.handle.net/2027.42/113631

► Trichloroethylene (TCE) is a chlorinated solvent and a widespread environmental pollutant implicated in adverse reproductive outcomes in humans. TCE toxicity primarily occurs through its biotransformation… (more)

▼ Trichloroethylene (TCE) is a chlorinated solvent and a widespread environmental pollutant implicated in adverse reproductive outcomes in humans. TCE toxicity primarily occurs through its biotransformation to toxic metabolites, including S-(1,2-dichlorovinyl)-L-cysteine (DCVC), which induce oxidative stress and inflammation in the liver and kidney. This thesis used both in vivo and in vitro approaches to test the hypothesis that TCE induces oxidative stress-mediated inflammation in gestational tissues, which contributes to adverse pregnancy outcomes. To investigate TCE-induced oxidative stress and inflammation in gestational tissues, pregnant Wistar rats were exposed daily to 480 mg/kg of TCE from gestational day 6 – 16. Placenta, maternal serum, and amniotic fluid were collected prior to onset of parturition. Exposure to TCE significantly decreased average fetal weights, increased placental 8-hydroxyguanosine, a biomarker of oxidative DNA damage, and increased placental global 5-hydroxymethylcytosine, a marker of DNA methylation changes. A significant increase in interleukin (IL)-6 levels in maternal serum was observed, and immunohistochemistry analysis showed presence of neutrophils in the decidua. These results suggest that TCE exposure in vivo induces systemic inflammation and oxidative stress in the placenta, which can lead to adverse pregnancy outcome. Moreover, DCVC, the bioactive metabolite of TCE, was detected in the amniotic fluid by HPLC/MS/MS, suggesting that the placenta may be capable of metabolizing TCE to DCVC. The effect of DCVC on proinflammatory cytokine release and stimulation of reactive oxygen species (ROS) was tested in human placental cells (HTR-8/SVneo). Results show a direct stimulatory effect of DCVC on release of IL-6. DCVC induced mitochondrial dysfunction and stimulated ROS. DCVC-induced IL-6 release was inhibited by the antioxidants (±)α-tocopherol and deferoxamine, implicating the involvement of ROS in stimulation of IL-6 release. In conclusion, results from the present study show that exposure to TCE stimulates oxidative stress and inflammation in gestational tissues and cells. Because oxidative stress and inflammation have been associated with adverse birth outcomes, these data provide support for a plausible biological explanation for TCE exposure associations with poor pregnancy outcomes. As such, this thesis provides new knowledge about the potential mechanisms by which TCE and other environmental contaminants may contribute to adverse pregnancy outcomes. Advisors/Committee Members: Loch-Caruso, Rita (committee member), Hollenberg, Paul F. (committee member), Lash, Lawrence Harold (committee member), Dolinoy, Dana (committee member), Meeker, John D. (committee member).

Subjects/Keywords: Effect of trichloroethylene on the placenta; Public Health; Health Sciences

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hassan, I. (2015). The Effect of Trichloroethylene on Adverse Birth Outcomes. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/113631

Chicago Manual of Style (16^th Edition):

Hassan, Iman. “The Effect of Trichloroethylene on Adverse Birth Outcomes.” 2015. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/113631.

MLA Handbook (7^th Edition):

Hassan, Iman. “The Effect of Trichloroethylene on Adverse Birth Outcomes.” 2015. Web. 26 Feb 2021.

Vancouver:

Hassan I. The Effect of Trichloroethylene on Adverse Birth Outcomes. [Internet] [Doctoral dissertation]. University of Michigan; 2015. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/113631.

Council of Science Editors:

Hassan I. The Effect of Trichloroethylene on Adverse Birth Outcomes. [Doctoral Dissertation]. University of Michigan; 2015. Available from: http://hdl.handle.net/2027.42/113631

University of Michigan

2. Capper, Cameron P. Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities.

Degree: PhD, Pharmacology, 2016, University of Michigan

URL: http://hdl.handle.net/2027.42/120831

► Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids… (more)

▼ Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids to drive cellular proliferation. Cytochrome P450 17A1 (CYP17A1), an essential enzyme for sex steroid synthesis, represents a clinically established drug target. Inhibiting CYP17A1 decreases androgen and estrogen biosynthesis and thereby blocks the growth of hormone-dependent cancers. The first part of this dissertation characterizes the previously unreported interaction between the FDA approved CYP17A1 inhibitor, abiraterone, and the estrogen receptor (ER). We show for the first time that abiraterone is a weak ER agonist in preclinical models of ER-positive breast cancer cells. Abiraterone induces cellular growth and expression of the ER response gene, GREB1, by binding to ER, and these effects are inhibited with the ER antagonist fulvestrant (ICI 182,780). To further investigate the impact of CYP17A1 expression in breast cancer, we engineered ER-positive MCF-7 cells to express CYP17A1 (MCF-7/CYP17A1). Progesterone treatment induces cell growth and GREB1 expression in these cells but not in the parental MCF-7 cells, which do not express CYP17A1. Tandem mass spectrometry (LC-MS/MS) analysis confirmed that following progesterone treatment, MCF-7/CYP17A1 cells synthesize downstream steroid products that require CYP17A1 activity including 17OH-progesterone, androstenedione, and testosterone. Treatment of these cells with either abiraterone or a novel CYP17A1 inhibitor decreases progesterone metabolite-induced GREB1 expression in a dose-dependent manner. In addition to studies of CYP17A1 in breast cancer, we further hypothesized that characterization of CYP17A1 genetic variants may lead to insights on enzyme structure and function. We therefore utilized a HEK-293T cell-based expression system to characterize the enzymatic properties of two CYP17A1 gene variants, D216H (rs200063521) and G162R (rs141821705). Our results show that the D216H variant selectively alters 16OH-progesterone production, while no effect on 17OH-progesterone synthesis was observed. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to wild-type. Proteasome inhibition with MG132 indicated that this variant is preferentially ubiquitinated and degraded prematurely. Overall, these studies have broadened our understanding of CYP17A1 enzymatic activity in breast cancer, as well as led to new insights into how CYP17A1 structure relates to enzyme function and stability. Advisors/Committee Members: Rae, James M (committee member), Rainey, William (committee member), Osawa, Yoichi (committee member), Auchus, Richard (committee member), Hollenberg, Paul F (committee member), Johnson, Michael D (committee member).

Subjects/Keywords: Cytochrome P450 17A1 (CYP17A1); Breast Cancer; Pharmacy and Pharmacology; Health Sciences

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Capper, C. P. (2016). Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/120831

Chicago Manual of Style (16^th Edition):

Capper, Cameron P. “Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities.” 2016. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/120831.

MLA Handbook (7^th Edition):

Capper, Cameron P. “Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities.” 2016. Web. 26 Feb 2021.

Vancouver:

Capper CP. Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities. [Internet] [Doctoral dissertation]. University of Michigan; 2016. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/120831.

Council of Science Editors:

Capper CP. Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities. [Doctoral Dissertation]. University of Michigan; 2016. Available from: http://hdl.handle.net/2027.42/120831

University of Michigan

3. Teiber, John Francis. Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives.

Degree: PhD, Toxicology, 2000, University of Michigan

URL: http://hdl.handle.net/2027.42/132902

► Human and rat enzymes catalyzing various oxidations of N-nitrosodi- n-propylamine (NDPA) and two of its oxidized derivatives, N-nitroso-beta-hydroxypropylpropylamine (NHPPA) and N-nitroso-beta-oxopropylpropylamine (NOPPA), were identified and… (more)

▼ Human and rat enzymes catalyzing various oxidations of N-nitrosodi- n-propylamine (NDPA) and two of its oxidized derivatives, N-nitroso-beta-hydroxypropylpropylamine (NHPPA) and N-nitroso-beta-oxopropylpropylamine (NOPPA), were identified and their catalytic roles characterized. Assays with cytochrome P450 (P450) specific chemical inhibitors and inhibitory antibodies indicated that P450 2E1 is the major isoform catalyzing the depropylation of NDPA at sub-saturating concentrations in human liver microsomes with possible contributions by 2C 18/19 and other unidentified P450s. The low K_m values for the denitrosation of NDPA by three of the microsomal samples are the same as the K_m values for denitrosation and depropylation for purified-reconstituted rabbit 2E1, concurring with the inhibition studies. Activation of NDPA to a toxic species by human 2E1 was confirmed in transformed human liver epithelial cells (THLE). Intrinsic clearance (I_CL) values calculated for 2B6 and 3A4 and estimated for 2A6, 2C8, 2C9 and 2D6 suggest that they are unlikely to significantly denitrosate or depropylate NDPA at low micromolar concentrations. I_CL values for 2E1 indicate that it will catalyze these reactions at high nanomolar concentrations, with denitrosation occurring at about 10% of depropylation. Purified P450 2B1 (rat) and 2E1 (rabbit) and liver microsomes from phenobarbital and pyridine treated rats, metabolized NOPPA to a DNA methylating species in vitro. Exposure of THLE cells expressing 2E1 to NOPPA resulted in the formation 7-methylguanine (m⁷Gua) DNA adducts and a dose dependent toxicity. Incubation of NHPPA with microsomes from PB, Pyr and non-treated (NT) rats and a human microsomal sample also resulted in M7 Gua formation. NHPPA was oxidized to NOPPA by purified 2B1 and 2E1 and by an NAD⁺-dependent cytosolic dehydrogenase. This oxidation was catalyzed by NT rat liver microsomes at approximately 7 times the rate of rat liver cytosol. Cells derived from hamster lung tissue and THLE cells transfected with rat 2B1 or human 2E1, respectively, oxidized NHPPA to NOPPA in yields approximately 15 fold higher than the yields formed in the respective non-transfected cells. These results suggest that the metabolism of NDPA to a methylating species may largely occur through sequential oxidations, first to NBPPA then to NOPPA, and these oxidations may be predominantly mediated by cytochrome P450s. Advisors/Committee Members: Hollenberg, Paul F. (advisor).

Subjects/Keywords: Carcinogenesis; Cytochrome P450; Derivatives; Metabolism; Nitrosodi-n-propylamine; Oxidized; Tumor Initiation

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Teiber, J. F. (2000). Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/132902

Chicago Manual of Style (16^th Edition):

Teiber, John Francis. “Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives.” 2000. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/132902.

MLA Handbook (7^th Edition):

Teiber, John Francis. “Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives.” 2000. Web. 26 Feb 2021.

Vancouver:

Teiber JF. Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives. [Internet] [Doctoral dissertation]. University of Michigan; 2000. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/132902.

Council of Science Editors:

Teiber JF. Metabolism of N-nitrosodi-n-propylamine and its oxidized derivatives. [Doctoral Dissertation]. University of Michigan; 2000. Available from: http://hdl.handle.net/2027.42/132902

University of Michigan

4. Jushchyshyn, Monica Irene. The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide.

Degree: PhD, Pharmacology, 2003, University of Michigan

URL: http://hdl.handle.net/2027.42/123616

► Human cytochrome P450 2B6 (2B6) is an enzyme that plays a role in the biotransformation of many xenobiotics. However, little is known about this enzyme;… (more)

▼ Human cytochrome P450 2B6 (2B6) is an enzyme that plays a role in the biotransformation of many xenobiotics. However, little is known about this enzyme; both mechanistically and structurally. This dissertation describes the characterization of phencyclidine (PCP) as a mechanism-based inactivator for 2B6, the metabolism of PCP by 2B6 and other 2B isoforms, and the investigation of the identity of the PCP-modified peptide. PCP satisfied all of the requirements for a mechanism-based inactivator of reconstituted 2B6. PCP inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of 2B6 in a time-, concentration-, and NADPH-dependent manner. The K_I was 10 muM and the k_inact was 0.01 min^-1. Spectral analysis of the heme moiety demonstrated the heme was not modified during inactivation. Inactivation was not reversible by dialysis. 7-Ethoxycoumarin, an alternate substrate, protected 2B6 from inactivation. The exogenous nucleophiles, glutathione (GSH) and cyanide, did not protect 2B6 from PCP inactivation, demonstrating that the reactive intermediate remained within the active site. HPLC analysis of 2B6 inactivated in the presence of [³H]-PCP showed that PCP binding was specific for the P450 and not the other proteins in the reaction mixture. The stoichiometry of binding was 5.5:1 (PCP/P450) and 1:1 in the presence of GSH. This stoichiometry was further supported using electrospray ionization – liquid chromatography – mass spectrometry (ESI-LC-MS). The metabolism of PCP by P450s 2B1 (rat), 2B4 (rabbit) and 2B6 is described in this thesis. ESI-LC-MS/MS was used to tentatively identify three major metabolites. Two GSH-PCP metabolite conjugates were also detected. One conjugate contained a GSH and oxygen adducted to the phenyl or cyclohexyl ring and the other conjugate contained a GSH adducted to the piperidine ring. An anti-PCP antibody was used for a Western blot analysis of peptides from a PCP-inactivated 2B6 digestion. The antibody cross-reacted with the peptide M₃₄₇-K₃₈₄. ESI-LC-MS/MS or matrix assisted laser desorption ionization-MS was used in further attempts to identify the PCP-adducted peptide or amino acid. No such adduct could be detected from PCP-inactivated P450 2B1 or 2B6 digestions. Advisors/Committee Members: Hollenberg, Paul F. (advisor).

Subjects/Keywords: Covalently Modified; Cytochrome P450 2b6; Human; Identity; Investigations; Mechanism-based Inactivation; Peptide; Phencyclidine

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Jushchyshyn, M. I. (2003). The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/123616

Chicago Manual of Style (16^th Edition):

Jushchyshyn, Monica Irene. “The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide.” 2003. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/123616.

MLA Handbook (7^th Edition):

Jushchyshyn, Monica Irene. “The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide.” 2003. Web. 26 Feb 2021.

Vancouver:

Jushchyshyn MI. The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide. [Internet] [Doctoral dissertation]. University of Michigan; 2003. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/123616.

Council of Science Editors:

Jushchyshyn MI. The mechanism -based inactivation of human cytochrome P450 2B6 by phencyclidine and investigations of the identity of the covalently modified peptide. [Doctoral Dissertation]. University of Michigan; 2003. Available from: http://hdl.handle.net/2027.42/123616

University of Michigan

5. Shebley, Mohamad. Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies.

Degree: PhD, Pharmacology, 2007, University of Michigan

URL: http://hdl.handle.net/2027.42/126819

► This dissertation presents studies performed to elucidate the structure-function relationships of cytochrome P450 (P450) 2B6, the human 2B homologue responsible for the metabolism of several… (more)

▼ This dissertation presents studies performed to elucidate the structure-function relationships of cytochrome P450 (P450) 2B6, the human 2B homologue responsible for the metabolism of several drugs, carcinogens, and some environmental chemicals. Phencyclidine (PCP), a psychomimetic drug of abuse, was shown to be metabolized by P450 2B6 and its metabolism results in mechanism-based inactivation of the enzyme. This study shows that P450 2B6 metabolizes phencyclidine via pathways different from the rat 2B1 and rabbit 2B4 homologues. Reactive intermediates of PCP were chemically trapped with glutathione (GSH) and N-acetylcysteine (NAC) during the metabolism and inactivation, and the resulting conjugates were identified. It appears that P450 2B6 favors oxygenation pathways that lead to the formation of a hydroxylated iminium or an epoxide intermediate of PCP, while a dehydrogenation pathway was favored by P450s 2B1 and 2B4, leading to the formation of the 2,3-dihydropyridinium metabolite of PCP. Studies with the P450 2B6 (K262R) mutant showed that this single residue mutation resulted in loss of inactivation of the enzyme by PCP, apparently because the mutant enzyme does not metabolize PCP to a reactive intermediate similar to that formed by the wild type enzyme. However, the K262R mutant retained the ability to reversibly bind PCP and metabolize it to all of the stable products. Mechanistic studies demonstrated that inactivation of P450 2B6 by PCP significantly decreased its benzphetamine binding affinity. The rate of benzphetamine binding was decreased by approximately 15 fold due to the inactivation. Analysis of benzphetamine metabolism showed that the rates of formation of two of the metabolites by the inactivated enzyme were significantly decreased, suggesting that benzphetamine binds with a different orientation in the active site of the inactivated protein. Furthermore, following inactivation by PCP the overall rate of the first electron transfer was decreased approximately 6 fold, with a concomitant increase in the uncoupling of the electron transfer from substrate turnover to increased hydrogen peroxide production. These studies show that the covalent modification of the P450 2B6 apo-protein by PCP may lead to dramatic changes in the chemistry of the enzyme catalysis. Advisors/Committee Members: Hollenberg, Paul F. (advisor).

Subjects/Keywords: Based; Benzphetamine; Cytochrome P450 2b6; Enzyme Catalysis; Human; Inactivation; Mechanism; Mechanistic; Metabolism; Phencyclidine; Structural; Studies

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Shebley, M. (2007). Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/126819

Chicago Manual of Style (16^th Edition):

Shebley, Mohamad. “Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies.” 2007. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/126819.

MLA Handbook (7^th Edition):

Shebley, Mohamad. “Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies.” 2007. Web. 26 Feb 2021.

Vancouver:

Shebley M. Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies. [Internet] [Doctoral dissertation]. University of Michigan; 2007. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/126819.

Council of Science Editors:

Shebley M. Mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine: Metabolism, structural, and mechanistic studies. [Doctoral Dissertation]. University of Michigan; 2007. Available from: http://hdl.handle.net/2027.42/126819

University of Michigan

6. Moreno, Rosa Luz. The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation.

Degree: PhD, Pharmacology, 2001, University of Michigan

URL: http://hdl.handle.net/2027.42/123702

► The goal of this study was to evaluate the possible use of two naturally occurring isothiocynates, benzyl- (BITC) and phenethyl-isothiocynate (PEITC), as probes for P450… (more)

▼ The goal of this study was to evaluate the possible use of two naturally occurring isothiocynates, benzyl- (BITC) and phenethyl-isothiocynate (PEITC), as probes for P450 2E1 active site studies. The use of chemical probes to target amino acids within the active site is a useful approach towards understanding the general structure of the active site and the function of specific active site residues in catalysis. Compounds that become metabolically activated to reactive intermediates leading to enzyme inactivation through covalent modification of active site residues are most useful. Because of their reactivity and due to their dependence on metabolic activation, these compounds are generally referred to as mechanism-based inactivators. BITC and PEITC are found in cruciferous vegetables and have been frequently studied for their antitumorigenicity. This work could have important implications for the interpretation of results from in vivo studies on the antitumorigenic activity of various isothiocyanates. BITC and PEITC inactivated cytochrome P450 2E1 in a mechanism-based manner. Inactivation was time- and concentration-dependent and demonstrated a requirement for metabolic activation of the isothiocyanate compound. Mass analysis of the inactivated protein confirmed the covalent binding of an isocyanate intermediate to the apoprotein. Attempts to characterize the site of modification within the P450 2E1 active site indicated that more than one peptide might be modified by the reactive intermediate. However, results with the P4502E1 T303A mutant point strongly to a role for this highly conserved residue in the inactivation process. This mutant enzyme was not inactivated by BITC and showed a metabolic shift producing more non-inactivating metabolites than P450 2E1. The differential metabolism of BITC by this mutant suggests that the docking orientation of BITC within the active site of P450 2E1T303A differs from that in the wild type enzyme. While BITC interacts differentially with these two enzymes it was demonstrated that PEITC inactivates P450 2E1T303A in a mechanism-based manner and with similar kinetics to those observed with P450 2E1. Advisors/Committee Members: Hollenberg, Paul F. (advisor).

Subjects/Keywords: Cytochrome P450 2e1; Implications; Inactivation; Isothiocyanates; Naturally; Occurring; Process; Rabbit; Role; T303a; Threonine 303

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Moreno, R. L. (2001). The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/123702

Chicago Manual of Style (16^th Edition):

Moreno, Rosa Luz. “The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation.” 2001. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/123702.

MLA Handbook (7^th Edition):

Moreno, Rosa Luz. “The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation.” 2001. Web. 26 Feb 2021.

Vancouver:

Moreno RL. The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation. [Internet] [Doctoral dissertation]. University of Michigan; 2001. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/123702.

Council of Science Editors:

Moreno RL. The inactivation of rabbit cytochrome P450 2E1 and P450 2E1 T303A by naturally occurring isothiocyanates: Implications for the role of threonine 303 in the process of inactivation. [Doctoral Dissertation]. University of Michigan; 2001. Available from: http://hdl.handle.net/2027.42/123702

University of Michigan

7. Blobaum, Anna L. The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site.

Degree: PhD, Pure Sciences, 2004, University of Michigan

URL: http://hdl.handle.net/2027.42/124631

► Rabbit cytochromes P450 2E1 and P450 2E1 T303A were inactivated by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) in a time-, concentration- and NADPH-dependent… (more)

▼ Rabbit cytochromes P450 2E1 and P450 2E1 T303A were inactivated by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) in a time-, concentration- and NADPH-dependent manner with K_I values in the millimolar range. Losses in the enzymatic activity, the P450 CO spectrum and native P450 heme were accompanied by the formation of two different tBA- or tBMP-modified heme products as detected by ESI-LC-MS analysis (m/z of 661 or 705 Da, respectively). Only the tBA-inactivated P450 2E1 revealed a tBA adduct to the apoprotein. Surprisingly, losses in the activity, the CO spectrum and native heme of the tBA-inactivated T303A mutant were completely restored following dialysis, suggesting a reversible heme alkylation. Investigations into the mechanism for the novel reversible inactivation of P450 2E1 T303A by tBA demonstrated that the activity and CO spectral losses were restored following overnight incubation. The reversibility was time-dependent, required an intact P450 enzyme, and was independent of NADPH and tBA. ESI-LC-MS/MS analysis under non-denaturing conditions of a pre-acidified tBA-inactivated T303A sample yielded two tBA adducts (m/z 661 Da) that were absent in non-acidified samples. In contrast, both non- and pre-acidified tBA-inactivated wild-type 2E1 samples were able to form the two tBA adducts, suggesting that the T303A mutant was deficient in the delivery of protons to the enzyme active site. Substrate binding analysis of tBA and tBMP with the P450s 2E1 yielded K_S values in the micromolar range. Spectral examination of the tBA-inactivated T303A mutant revealed the formation of a novel, reversible acetylene-iron intermediate with an absorption maximum at 485 nm. The artificial oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of P450 2E1 by tBA, but only tBHP supported the inactivation of 2E1 T303A, suggesting a disruption in proton delivery in this enzyme. The tBHP- and CHP-supported inactivations resulted in the formation of tBA-adducted heme products and were completely irreversible with dialysis. In support of these data, models of the P450s 2E1 with tBA bound in the active site suggest a role for the conserved T303 residue in proton delivery and in the inactivation of the 2E1 P450s by small acetylenic compounds. Advisors/Committee Members: Hollenberg, Paul F. (advisor).

Subjects/Keywords: 2e1; Acetylenes; Active; Based; Butyl; Characterization; Conserved; Cytochromes P450; Inactivation; Mechanism; Novel; Proton Delivery; Reversible; Role; Site; T303; T303a; Tert

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Blobaum, A. L. (2004). The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/124631

Chicago Manual of Style (16^th Edition):

Blobaum, Anna L. “The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site.” 2004. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/124631.

MLA Handbook (7^th Edition):

Blobaum, Anna L. “The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site.” 2004. Web. 26 Feb 2021.

Vancouver:

Blobaum AL. The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site. [Internet] [Doctoral dissertation]. University of Michigan; 2004. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/124631.

Council of Science Editors:

Blobaum AL. The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site. [Doctoral Dissertation]. University of Michigan; 2004. Available from: http://hdl.handle.net/2027.42/124631

8. Nahar, Muna S. Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus.

Degree: PhD, Toxicology, 2014, University of Michigan

URL: http://hdl.handle.net/2027.42/107192

► The ubiquitous monomer, bisphenol A (BPA), is an endocrine active compound used in the production of polycarbonate plastics and epoxy resin. Environmental biomonitoring and epidemiology… (more)

▼ The ubiquitous monomer, bisphenol A (BPA), is an endocrine active compound used in the production of polycarbonate plastics and epoxy resin. Environmental biomonitoring and epidemiology studies report continuous exposure in humans that are associated with different adverse health outcomes. The overall goal of this work was to characterize BPA concentrations, toxicokinetic and toxicodynamic profiles that influence tissue-specific BPA biotransformation via xenobiotic metabolizing enzymes (XME), and BPA dependent changes in biotransformation using healthy 1st-2nd trimester clinical specimens obtained from a fetal biobank. First, we reported a wide range of BPA concentrations in N=50 fetal liver specimens (total BPA range: below limit of quantification - 96.8 ng/g). Both concentrations and metabolic profiles varied across age with significant reduction in BPA-specific XME gene expression of UGT2B15, SULT1A1, and STS in fetal versus adult livers. Next, we examined matched fetal liver, kidney, and placenta in N=12 subjects and observed significant tissue-dependent differences in BPA concentrations, XME expression profiles, and global DNA methylation. Fetal livers exhibited higher BPA concentrations compared to matched tissues; however, XME expression profiles suggest an increased likelihood of BPA-glucuronide deconjugation and BPA-sulfate conjugation across the fetal compartments. With organ-specific differences in the epigenome, only placental global methylation measurements were associated with BPA. Finally, we investigated BPA’s role in pathway specific biological outcomes and regulation in fetal liver. In particular, we identified 14 different XME candidate genes that were down regulated with higher BPA concentrations. In summary, results suggest that the 1st-2nd trimester human fetus is exposed to a considerable amount of BPA in utero, especially of the active free BPA form. XME expression profiles reveal an altered capacity for BPA biotransformation in the fetus compared to adults, with distinct metabolic profiles across different tissues. Interestingly, higher BPA concentrations in fetal liver were associated with reduced expression of novel XME candidate genes mediated by epigenetic mechanisms. These findings indicate that environmentally relevant concentrations of BPA, even across a short window of development, result in detectable changes in the host’s toxicological defense system. Advisors/Committee Members: Dolinoy, Dana (committee member), Hollenberg, Paul F. (committee member), Park, Sung Kyun (committee member), Harris, Craig (committee member).

Subjects/Keywords: Bisphenol a Metabolism in the Human Fetus; Molecular, Cellular and Developmental Biology; Pediatrics; Pharmacy and Pharmacology; Public Health; Health Sciences

…polycarbonatefree polypropylene tubing at -80 °C, prior to shipment to the University of Michigan 16…

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APA (6^th Edition):

Nahar, M. S. (2014). Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/107192

Chicago Manual of Style (16^th Edition):

Nahar, Muna S. “Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus.” 2014. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/107192.

MLA Handbook (7^th Edition):

Nahar, Muna S. “Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus.” 2014. Web. 26 Feb 2021.

Vancouver:

Nahar MS. Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus. [Internet] [Doctoral dissertation]. University of Michigan; 2014. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/107192.

Council of Science Editors:

Nahar MS. Human Bisphenol a Biomonitoring and Biotransformation Programming in the Developing Fetus. [Doctoral Dissertation]. University of Michigan; 2014. Available from: http://hdl.handle.net/2027.42/107192

9. Jenkins, Gary J. Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase.

Degree: PhD, Pharmacology, 2008, University of Michigan

URL: http://hdl.handle.net/2027.42/58432

► Guanabenz, a clinically used anti-hypertensive agent, inhibits the P450-like enzyme neuronal NO-synthase (nNOS) and enhances its ubiquitination and degradation. To better understand the molecular trigger… (more)

▼ Guanabenz, a clinically used anti-hypertensive agent, inhibits the P450-like enzyme neuronal NO-synthase (nNOS) and enhances its ubiquitination and degradation. To better understand the molecular trigger for nNOS ubiquitination and degradation, we characterized the mechanism of guanabenz inhibition of nNOS and identified the site of ubiquitin attachment to the enzyme. Using purified nNOS and an in vitro system, we found that guanabenz treatment leads to the oxidation of tetrahydrobiopterin by nNOS-derived superoxide. Tetrahydrobiopterin is a known cofactor for NO synthesis by nNOS, binding near the heme and stabilizing the active dimeric structure of the enzyme. Tetrahydrobiopterin was found to reverse the guanabenz-mediated inhibition of nNOS in vitro. Similarly, administration of tetrahydrobiopterin to rats prevented both nNOS inhibition and loss of enzyme after guanabenz treatment, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. To investigate if the loss of tetrahydrobiopterin was sufficient for eliciting the enhanced turnover of nNOS, we depleted tetrahydrobiopterin in cells by inhibiting GTP cyclohydrolase I with 2,4-diamino-6-hydroxypyrimidine. A 75% decrease in tetrahydrobiopterin levels led to a 2-fold increase in the amount of nNOS-ubiquitin conjugates detected. Consistent with our cellular observations, in vitro ubiquitination and degradation of nNOS by reticulocyte lysate proteins was decreased when tetrahydrobiopterin was added. Thus, tetrahydrobiopterin may serve as an endogenous regulator of nNOS protein levels. Through mutagenesis studies, we were able to localize the ubiquitination site to the calmodulin binding region of nNOS (residues 720-756). Peptide mapping studies using capillary flow liquid chromatography interfaced with a linear ion trap mass spectrometer identified residue 754 as a site for ubiquitin attachment. Furthermore, using methylated ubiquitin and purified nNOS, we determined that mono-ubiquitination of nNOS is sufficient for proteasomal degradation in vitro. Thus, it is possible that alterations of the heme active site structure, in this case through oxidation of tetrahydrobiopterin, are recognized by cellular factors that direct the ubiquitination of a lysine residue in the calmodulin binding region, resulting in the selective proteasomal degradation of nNOS. Advisors/Committee Members: Osawa, Yoichi (committee member), Hollenberg, Paul F. (committee member), Pratt, William B. (committee member), Richardson, Rudy J. (committee member).

Subjects/Keywords: Neuronal Nitric Oxide Synthase; Ubiquitin; Ubiquitination; Post-translational Modification; Peptide Mapping; Mass Spectrometry; Health Sciences; Science

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APA (6^th Edition):

Jenkins, G. J. (2008). Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/58432

Chicago Manual of Style (16^th Edition):

Jenkins, Gary J. “Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase.” 2008. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/58432.

MLA Handbook (7^th Edition):

Jenkins, Gary J. “Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase.” 2008. Web. 26 Feb 2021.

Vancouver:

Jenkins GJ. Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase. [Internet] [Doctoral dissertation]. University of Michigan; 2008. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/58432.

Council of Science Editors:

Jenkins GJ. Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase. [Doctoral Dissertation]. University of Michigan; 2008. Available from: http://hdl.handle.net/2027.42/58432

10. Gerlach, Deidra L. Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites.

Degree: PhD, Chemistry, 2013, University of Michigan

URL: http://hdl.handle.net/2027.42/102331

► Iron-sulfur clusters and hemes are found throughout biological systems in a variety of unique and important enzymatic systems. Only one type of enzyme contains both… (more)

▼ Iron-sulfur clusters and hemes are found throughout biological systems in a variety of unique and important enzymatic systems. Only one type of enzyme contains both a heme and an iron-sulfur cluster at the active site of the enzyme, sulfite and nitrite reductase (SIR/NIR). The assimilatory variety of these enzymes is capable of both sulfite and nitrite reduction to produce sulfide and ammonia, respectively. As these enzymes function at ambient temperature and pressure and perform a six-electron reduction without substrate dissociation, they provide an attractive blueprint for biologically inspired multi-electron catalysts. The enzyme active site contains a [4Fe-4S] cluster which supplies electrons to a catalytic heme center through an axially coordinated sulfide bridge. The goal of this research is to synthesize complexes biologically inspired by SIR/NIR for the purpose of reductive catalysis. The three key structural features for the first generation complex design include (i) a metalloporphyrin, for use as a catalytic site, (ii) a [4Fe-4S] cluster, for use as an electron reservoir, and (iii) a bridging ligand or atom to link the catalytic and electron storage components. Two designs were formulated using different forms of connectivity for the bridging component: a) axial coordination of an iron-sulfur cluster via a bridging ligand to a heme complex, and b) covalent ligation of the iron-sulfur cluster directly to the functionalized porphyrin ligand. The axially bound model developed here was optimized to accomplish the optimal binding of the heme and [4Fe-4S] cluster by modification of the electron density of the heme iron by the addition of electron withdrawing groups to the porphyrin, and by evaluation of different types of organic bridges to connect these components. The covalently bound model utilizes a singly functionalized porphyrin ligand intended for direct binding to an iron-sulfur cluster for electron transfer from the cluster to the heme iron via the porphyrin ligand. The covalent bound model is likely the more viable route for further progress. In the process of synthesizing and testing different types of functionalized [4Fe-4S] clusters, an interesting, sulfide-bridged cluster was discovered. The properties of the sulfide-bridged double cubane cluster with pyridylthiolate ligands were then investigated. Advisors/Committee Members: Lehnert, Nicolai (committee member), Hollenberg, Paul F. (committee member), Lim, Mi Hee (committee member), Szymczak, Nathaniel (committee member).

Subjects/Keywords: Bioinorganic Chemistry; Iron-sulfur Clusters; Metalloporphyrins; Chemistry; Science

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APA (6^th Edition):

Gerlach, D. L. (2013). Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/102331

Chicago Manual of Style (16^th Edition):

Gerlach, Deidra L. “Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites.” 2013. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/102331.

MLA Handbook (7^th Edition):

Gerlach, Deidra L. “Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites.” 2013. Web. 26 Feb 2021.

Vancouver:

Gerlach DL. Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites. [Internet] [Doctoral dissertation]. University of Michigan; 2013. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/102331.

Council of Science Editors:

Gerlach DL. Synthesis and Characterization of Functionalized [4Fe-4S] Cubane Clusters and Linkage to Metalloporphryrins as Catalytic Sites. [Doctoral Dissertation]. University of Michigan; 2013. Available from: http://hdl.handle.net/2027.42/102331

11. Carolan, James Patrick. Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles.

Degree: PhD, Chemical Biology, 2015, University of Michigan

URL: http://hdl.handle.net/2027.42/113323

► The process of transcription underlies the expression of all gene profiles. Normal expression is caused by carefully coordinating the assembly of transcriptional proteins at specific… (more)

▼ The process of transcription underlies the expression of all gene profiles. Normal expression is caused by carefully coordinating the assembly of transcriptional proteins at specific genes, a procedure mediated through transcription factors that bind specific sequences of DNA. The glucocorticoid receptor (GR) is a transcription factor that influences the expression of genes involved in inflammation pathways. Drug-like molecules targeting GR are capable of stimulating GR to bind DNA; however, these molecules are unable to exert complete control over the members of the transcriptional complex recruited by GR. As such, we are limited in our control of GR activity. My dissertation focuses on addressing this with new GR ligands designed to recruit specific transcriptional proteins to produce novel, desired expression profiles. We first conjugated a GR molecule to a ligand of the protein FKBP. This bifunctional ligand was capable of localizing GR to DNA, recruiting FKBP-fusions to the GR-regulated gene, and producing transcriptional activity dictated by the recruitment. This was achieved with the recruitment of both a transcriptionally activating protein and a repressing protein, demonstrating the adaptability of this system to toggle the output of a gene of interest. The design of our system benefits from its inherent modularity; expanding to new targets is simply achieved through synthetic conjugation to an alternative ligand. In a first application of this strategy, we conjugated an agonistic GR ligand to a selective inhibitor of the transcriptional protein BRD4. Recruitment of BRD4 to GR resulted in the suppression of transcription at select genes, and this selectivity drives a novel profile of activated and suppressed genes. In a second application, we conjugated a GR antagonist to the BRD4 inhibitor, allowing forthe further production of novel transcriptional profiles with potential pharmacological utility. This dissertation also includes a study aimed at introducing undergraduate students to the scientific principles of modern chemical biology research. To enhance student learning, we have developed a new guided-inquiry experimental module for biochemistry laboratory courses. This has been well received, and assessment metrics indicate that the incorporation into the University of Michigan’s biochemistry course has raised student cognitive abilities in analysis and application. Advisors/Committee Members: Mapp, Anna K. (committee member), Hollenberg, Paul F. (committee member), Nikolovska-Coleska, Zaneta (committee member), Soellner, Matthew Bryan (committee member).

Subjects/Keywords: Bifunctional Ligands; Glucocorticoids; Nuclear Receptors; Guided-Inquiry; Biological Chemistry; Chemistry; Science

…University of Michigan. In Chapter 4, efforts to rationally redesign portions of this course’s…

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APA (6^th Edition):

Carolan, J. P. (2015). Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/113323

Chicago Manual of Style (16^th Edition):

Carolan, James Patrick. “Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles.” 2015. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/113323.

MLA Handbook (7^th Edition):

Carolan, James Patrick. “Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles.” 2015. Web. 26 Feb 2021.

Vancouver:

Carolan JP. Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles. [Internet] [Doctoral dissertation]. University of Michigan; 2015. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/113323.

Council of Science Editors:

Carolan JP. Design and Characterization of Bifunctional Glucocorticoid Ligands Capable of Producing Novel Transcriptional Profiles. [Doctoral Dissertation]. University of Michigan; 2015. Available from: http://hdl.handle.net/2027.42/113323

12. Remy, Matthew Sean. Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane.

Degree: PhD, Chemistry, 2011, University of Michigan

URL: http://hdl.handle.net/2027.42/86311

► Abstract Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane by Matthew Sean Remy Chair: Melanie S. Sanford One of the… (more)

▼ Abstract Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane by Matthew Sean Remy Chair: Melanie S. Sanford One of the challenges of developing a homogeneous catalyst for oxidative oligomerization of methane (OOM) is promoting a C–C coupling reaction from the product of alkane C–H activation. Heterolytic C–H activation of methane almost exclusively generates a monoalkyl-metal species. In order to make a new C–C bond, it is proposed that a polymethyl intermediate must be generated. Our studies showed that aryl disproportionation is far more favorable than methyl disproportionation. Careful tuning of ancillary ligands at monomethyl-palladium(II) complexes using density functional theory (DFT) calculations facilitated optimization of thermodynamics for methyl disproportionation. This allowed the first observed disproportionation reaction to form a dimethyl-palladium(II) complex. Heating dimethyl complex (tBu-bpy)PdII(CH3)2 (tBu-bpy = 4,4-ditertbutyl-2,2-bipyridine) to 100 °C produced a mixture of methane and ethane over 24 h. One-electron oxidants and 1,4-benzoquinone were found to be effective promoters of ethane formation from (tBu-bpy)PdII(CH3)2 at 25 °C. Mechanistic study of one-electron oxidation uncovered an oxidatively-induced methyl transfer reaction which produced ethane from [(tBu-bpy)PdIV(CH3)3(solvent)]+. Subsequently, one-electron oxidation of complexes of the general formula (tBu-bpy)PdII(CH3)X was developed as an effective method for generation of ethane from monomethyl-palladium(II) complexes. Platinum complexes, (N–N)PtII(CH3)2, similarly undergo one-electron to produce [(N–N)PtIV(CH3)3(solvent)]+. However, these platinum(IV) products are generally stable to reductive elimination of ethane. When the N–N ligand is designed to have steric interactions with axial ligands of platinum(IV), ethane reductive elimination becomes favorable, occurring cleanly over 8.5 hours in acetone and over 1 h in dichloromethane. This document describes experimental evidence for the generation of polymethyl palladium and platinum complexes from model palladium(II) and platinum(II) products of C–H activation subsequent ethane elimination from the polymethyl complexes. In an unrelated project, N-insertion into palladium-carbon bond was observed when palladium(II) complexes of bidentate C–N ligands were reacted with an iminoiodinane oxidant. The reaction was proposed to occur through an imido-palladium(IV) intermediate. Advisors/Committee Members: Sanford, Melaine S. (committee member), Banaszak Holl, Mark M. (committee member), Hollenberg, Paul F. (committee member), Lehnert, Nicolai (committee member).

Subjects/Keywords: One-electron Oxidation of Palladium and Platinum; Methyl Group Transfer; Gas to Liquids; Chemistry; Science

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APA (6^th Edition):

Remy, M. S. (2011). Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/86311

Chicago Manual of Style (16^th Edition):

Remy, Matthew Sean. “Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane.” 2011. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/86311.

MLA Handbook (7^th Edition):

Remy, Matthew Sean. “Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane.” 2011. Web. 26 Feb 2021.

Vancouver:

Remy MS. Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane. [Internet] [Doctoral dissertation]. University of Michigan; 2011. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/86311.

Council of Science Editors:

Remy MS. Group 10 Methyl Transfer Reactions toward Catalyst Development for Oxidative Oligomerization of Methane. [Doctoral Dissertation]. University of Michigan; 2011. Available from: http://hdl.handle.net/2027.42/86311

13. Walker, Vyvyca J. The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2.

Degree: PhD, Pharmacology, 2014, University of Michigan

URL: http://hdl.handle.net/2027.42/107249

► Cytochrome P450s (CYPs) are a superfamily of monooxygenases catalyzing the metabolism of various endogenous and exogenous compounds. CYP2J2 is highly expressed in the cardiovascular system… (more)

▼ Cytochrome P450s (CYPs) are a superfamily of monooxygenases catalyzing the metabolism of various endogenous and exogenous compounds. CYP2J2 is highly expressed in the cardiovascular system and catalyzes the metabolism of arachidonic acid to give four vasoactive epoxyeicosatrienoic acids (EETs). Similar to the endogenous cannabinoid system, CYP2J2 is also expressed in the gastrointestinal tract, liver, kidney, and the brain. Therefore, we investigated the ability of CYP2J2 to metabolize anandamide (AEA), an endogenous cannabinoid, and JWH-018, a synthetic cannabinoid, thereby contributing to the regulation of the ECS. We determined that purified CYP2J2 metabolizes AEA to give the 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid-ethanolamides (EET-EAs) and 20-hydroxyeicosatrienoic acid-ethanolamide (HETE-EA) and characterized the metabolism in detail. Since AEA plays a role in energy balance, we investigated AEA metabolism by rat liver microsomes from rats bred to be susceptible or resistant to diet-induced obesity and determined that both diet and obesity have significant effects on AEA metabolism. Approaches for increasing the stability of the EET-EAs may aid in understanding their biological actions in vivo. Therefore, we tested several inhibitors of soluble epoxide hydrolase (sEH), the enzyme primarily responsible for EET hydrolysis, as inhibitors of the hydrolysis of EET-EAs. Our results indicate that the compounds tested were not efficacious and that new inhibitors specific for the EET-EAs’ epoxide hydrolases need to be developed. Finally, we determined that JWH-018 is a substrate for CYP2J2 and characterized its metabolism in detail. JWH-018 is a drug of abuse that causes several adverse cardiovascular side effects, but relatively little is known about the metabolism or the pharmacology of this compound. Utilizing an animal model, we determined that JWH-018 causes a significant increase in blood pressure that appears to be only partially mediated by activation of the cannabinoid 1 receptor. Additional studies are required to fully understand the involvement of CYP2J2 in the ECS; however, because CYP2J2 regulates the metabolic fates of at least two ligands for the ECS, it is possible that CYP2J2 may play an important role in the ECS. Advisors/Committee Members: Hollenberg, Paul F. (committee member), Peters-Golden, Marc (committee member), Osawa, Yoichi (committee member), Smith, William L. (committee member).

Subjects/Keywords: Metabolism; Pharmacy and Pharmacology; Health Sciences

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APA (6^th Edition):

Walker, V. J. (2014). The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/107249

Chicago Manual of Style (16^th Edition):

Walker, Vyvyca J. “The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2.” 2014. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/107249.

MLA Handbook (7^th Edition):

Walker, Vyvyca J. “The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2.” 2014. Web. 26 Feb 2021.

Vancouver:

Walker VJ. The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2. [Internet] [Doctoral dissertation]. University of Michigan; 2014. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/107249.

Council of Science Editors:

Walker VJ. The Oxidation of the Endogenous Cannabinoid Anandamide and the Synthetic Cannabinoid JWH-018, a Drug of Abuse, by Human Cytochrome P450 2J2. [Doctoral Dissertation]. University of Michigan; 2014. Available from: http://hdl.handle.net/2027.42/107249

14. Calinski, Diane M. Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants.

Degree: PhD, Pharmacology, 2013, University of Michigan

URL: http://hdl.handle.net/2027.42/99932

► The oxazaphosphorines, cyclophosphamide (CPA) and ifosfamide (IFO), are commonly used cancer therapeutics. Hallmarks of oxazaphosphorine-treatment include variable patient response and severe adverse drug reactions. These… (more)

▼ The oxazaphosphorines, cyclophosphamide (CPA) and ifosfamide (IFO), are commonly used cancer therapeutics. Hallmarks of oxazaphosphorine-treatment include variable patient response and severe adverse drug reactions. These deleterious effects are related to the metabolism of the oxazaphosphorines. Improved understanding of CPA and IFO metabolism is important for the optimization of treatment regimens utilizing these drugs. This dissertation has focused on advancing our knowledge of the two primary metabolic pathways for oxazaphosphorine metabolism, hydroxylation and N-dechloroethylation, by the human cytochrome P450s (P450s) (CYP2B6, CYP2C9, CYP2C19 and CYP3A4). Hydroxylation of the oxazaphosphorines activates the drugs, whereas N-dechloroethylation results in inactivation and the formation of toxic metabolites. We demonstrated that CYP2B6 is 38-fold more efficient than the other P450s for the activation of CPA and that only CYP3A4 catalyzed the inactivation of CPA. For IFO, CYP3A4 was 2.8-fold more efficient at activation of IFO than the other P450s and multiple P450s catalyzed the inactivation of IFO. As the P450s are highly polymorphic enzymes, and polymorphic variants may have altered catalytic activities, we assessed commonly expressed variants for alterations in the rate of oxazaphosphorine metabolism. We observed statistically significant differences in the activation of both of the drugs by some of the polymorphic variants when compared to the wild type enzymes. We also evaluated the ability to favor the activation pathway of oxazaphosphorine metabolism in two ways; specific P450 inhibition in human liver microsomes (HLM) and deuterium labeling of the oxazaphosphorines. Inhibition of P450s in HLM was effective for favoring the activation of CPA, but not IFO. Deuterium labeling of CPA and IFO, resulted in inhibition of inactivation for both drugs but it was more evident with IFO, likely because IFO undergoes inactivation more readily than CPA. Thus, different methods are better for increasing the ratio of activation to inactivation for of each the drugs. Taken together, the work compiled in this thesis has enhanced our mechanistic understanding of the metabolism of the oxazaphosphorines. Our hope is that this information will encourage future investigations focused on the improvement of current cancer chemotherapies and the use of pharmacogenomics to design enhanced chemotherapeutic regimens. Advisors/Committee Members: Hollenberg, Paul F. (committee member), Waskell, Lucy (committee member), Rae, James M. (committee member), Osawa, Yoichi (committee member).

Subjects/Keywords: P450; Polymorphic Variants; Cyclophosphamide; Ifosfamide; Pharmacy and Pharmacology; Health Sciences

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APA (6^th Edition):

Calinski, D. M. (2013). Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/99932

Chicago Manual of Style (16^th Edition):

Calinski, Diane M. “Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants.” 2013. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/99932.

MLA Handbook (7^th Edition):

Calinski, Diane M. “Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants.” 2013. Web. 26 Feb 2021.

Vancouver:

Calinski DM. Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants. [Internet] [Doctoral dissertation]. University of Michigan; 2013. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/99932.

Council of Science Editors:

Calinski DM. Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants. [Doctoral Dissertation]. University of Michigan; 2013. Available from: http://hdl.handle.net/2027.42/99932

15. Clapp, Kelly M. Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase.

Degree: PhD, Pharmacology, 2012, University of Michigan

URL: http://hdl.handle.net/2027.42/91503

► Nitric oxide synthase (NOS), a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes.… (more)

▼ Nitric oxide synthase (NOS), a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our lab has found that certain drugs, such as guanabenz, inactivate neuronal NOS (nNOS) and lead to the ubiquitination of the nNOS. To better understand the molecular trigger for nNOS ubiquitination, we characterized the proteins that are involved in nNOS ubiquitination, examined a mutant nNOS that can serve as a model for inactivated nNOS, and identified the sites of ubiquitin attachment to nNOS. Using an in vitro model for ubiquitination containing Fraction II, the DE52-retained fraction of reticulocyte lysates that contains all ubiquitinating enzymes and the proteasome, we found that CHIP (C-terminus of Hsp70-interacting protein) and Hsp70 play a major role in promoting the ubiquitination of nNOS, whereas Hsp90, in concert with calmodulin, protects nNOS from ubiquitination. We found a C331A nNOS mutant that is highly susceptible to CHIP-dependent ubiquitination in cells and in vitro systems. Substrates and other ligands stabilize the C331A nNOS against ubiquitination, suggesting that subtle alterations to the active site cleft are sufficient for triggering the ubiquitination. The C331A nNOS is ubiquitinated in an Hsp70/CHIP-dependent manner, similar to the wildtype enzyme. With the use of an in vitro system containing purified proteins, including E1 ubiquitin-activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase CHIP, we recapitulated the ubiquitination of nNOS seen in cells. We identified the sites of ubiquitination on nNOS through LC-MS/MS analysis of the ubiquitinated nNOS obtained from the in vitro purified system. Of the twelve sites that were identified, nine are located in the oxygenase domain, two in the calmodulin-binding region, and one in the reductase domain of the enzyme. These data are consistent with studies showing that the oxygenase domain and the calmodulin-binding domain play a major role in regulating the ubiquitination of nNOS. Thus, alterations to the heme active site structure, whether by drug-mediated inactivation or the destabilizing C331A mutation, leads to ubiquitination by Hsp70 and CHIP in the calmodulin-binding region and/or the oxygenase domain of nNOS. Advisors/Committee Members: Osawa, Yoichi (committee member), Hollenberg, Paul F. (committee member), Pratt, William B. (committee member), Sun, Yi (committee member).

Subjects/Keywords: Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase; Neuronal Nitric Oxide Synthase; Hsp70- and CHIP-mediated Ubiquitination; Pharmacy and Pharmacology; Health Sciences

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APA (6^th Edition):

Clapp, K. M. (2012). Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/91503

Chicago Manual of Style (16^th Edition):

Clapp, Kelly M. “Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase.” 2012. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/91503.

MLA Handbook (7^th Edition):

Clapp, Kelly M. “Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase.” 2012. Web. 26 Feb 2021.

Vancouver:

Clapp KM. Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase. [Internet] [Doctoral dissertation]. University of Michigan; 2012. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/91503.

Council of Science Editors:

Clapp KM. Chaperone-dependent Ubiquitination of Neuronal Nitric Oxide Synthase. [Doctoral Dissertation]. University of Michigan; 2012. Available from: http://hdl.handle.net/2027.42/91503

University of Michigan

16. Dhaini, Hassan R. Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma.

Degree: PhD, Toxicology, 2002, University of Michigan

URL: http://hdl.handle.net/2027.42/131733

► Osteosarcoma is a primary malignancy of bone with a tendeney for early pulmonary metastasis. There is a need to identify patients who may benefit from… (more)

▼ Osteosarcoma is a primary malignancy of bone with a tendeney for early pulmonary metastasis. There is a need to identify patients who may benefit from more aggressive pre-operative therapy or perhaps require less aggressive chemotherapy. A reliable biomarker predicting clinical outcome at initiation of treatment has not yet been identified. We hypothesized that one or more drug-metabolizing enzymes, mainly cytochromes P450 are related to drug resistance and/or poor clinical outcome in osteosarcoma. To test the hypothesis, tissue microarray blocks containing 18 osteogenic and 109 soft tissue sarcoma primary tumors were used to identify the expression of P450s 1A1/2, 1B1, 2B6, 2D6, and 3A4/5 as well as metallothionein, MDR1, UGT2B7 and mdm-2 by enzyme-linked avidin-biotin-complexed immunocytochemistry. CYP1A1/2, CYP1B1, CYP3A4/5, MT, UGT2B7, MDR-1 and mdm-2 were detected in 59%, 64%, 76%, 87%, 63%, 90% and 94% of soft tissue tumors, and in 83%, 67%, 83%, 72%, 78%, 72% and 11% of osteosarcomas, respectively. CYP2B6 and CYP2D6 immunoreactivities were seen in 3% and 20% of soft tissue tumors, respectively, and were not detected in osteosarcomas. In addition, osteosarcomas were heterogenous with respect to the expression CYP3A4/5, an enzyme involved in the metabolic activation and detoxification of ifosfamide, etoposide, and doxorubicin, which are used in the treatment of osteosarcoma. This observation was extended by developing a quantitative immunofluorescence technique to assess the levels of CYP3A4/5 in 18 archival primary osteosarcoma sections. Expression of CYP3A4/5 was found to be significantly higher in primary biopsies of patients that developed distant metastatic disease compared to biopsies from metastatic disease-free patients (p ≤ 0.0004). Results from clonogenic assays using two human osteosarcoma cell lines showed that cells expressing CYP3A4 are more resistant to etoposide compared to non-expressing cells. No difference in resistance was evident among the cell lines when exposed to ifosfamide or doxorubicin or etoposide in the presence of ketoconazole, a potent inhibitor of CYP3A4. This thesis provides evidence that selective P450 isoenzymes are expressed in sarcomas. Moreover, increased levels of CYP3A4/5 expression appears to predict poor clinical outcome in osteosarcomas, and may confer protection through mechanisms of resistance other than regular detoxification pathways. Advisors/Committee Members: Hollenberg, Paul F. (advisor), Harris, Craig (advisor).

Subjects/Keywords: Cyp3a4/5; Cytochrome P450; Factor; New; Osteosarcoma; Prognostic

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APA (6^th Edition):

Dhaini, H. R. (2002). Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/131733

Chicago Manual of Style (16^th Edition):

Dhaini, Hassan R. “Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma.” 2002. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/131733.

MLA Handbook (7^th Edition):

Dhaini, Hassan R. “Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma.” 2002. Web. 26 Feb 2021.

Vancouver:

Dhaini HR. Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma. [Internet] [Doctoral dissertation]. University of Michigan; 2002. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/131733.

Council of Science Editors:

Dhaini HR. Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma. [Doctoral Dissertation]. University of Michigan; 2002. Available from: http://hdl.handle.net/2027.42/131733

17. Kenaan, Cesar. Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase.

Degree: PhD, Chemical Biology, 2012, University of Michigan

URL: http://hdl.handle.net/2027.42/94027

► A requirement for cytochrome P450 (CYP or P450)-mediated drug metabolism is the association of P450s with cytochrome P450 reductase (CPR). Although P450s form a 1:1… (more)

▼ A requirement for cytochrome P450 (CYP or P450)-mediated drug metabolism is the association of P450s with cytochrome P450 reductase (CPR). Although P450s form a 1:1 complex with CPR, they exist in excess over CPR in the endoplasmic reticulum (ER). Very little is known about the effect of less than stoichiometric amounts of CPR relative to P450 in the ER on the interaction of P450s with CPR and substrate metabolism. Equally little is known about the mechanism of interaction of P450s with CPR since much of our knowledge regarding the specific residues that mediate this interaction stems from a limited number of mutagenesis studies. In this thesis we developed methodology to directly probe the CYP2B4-CPR binding interface and demonstrated novel roles for residues V267 and L270 of CYP2B4 in binding CPR. We harnessed this knowledge to engineer a CYP2B4 with greater rates of reduction and substrate metabolism. We also found that CYP2E1, an inducible P450 isoform, significantly inhibited the catalytic activity of CYP2B4 in a concentration-dependent manner. We proposed a preliminary model to explain the inhibitory behavior of CYP2E1 toward CYP2B4 that was based on two key findings: 1) direct CYP2B4-CYP2E1 interactions alone do not lead to inhibition of CYP2B4 activity in the presence of saturating concentrations of substrate and 2) CYP2E1 has a higher affinity for CPR in the presence of CYP2B4. In this model we suggested that CYP2E1 and CYP2B4 associate to form a CYP2B4-CYP2E1 complex that interacts with the functional site of CPR with a higher affinity than CYP2E1 alone, and this complex may allow CYP2E1 to compete with CYP2B4 for CPR. Taken together, the work presented in this thesis establishes a new approach to the identification of amino acid residues that mediate redox-partner recognition and demonstrates how these residues can be used to enhance P450 activity. Additionally, these reports provide us with valuable insights into the potential for protein-protein interactions in the P450 system to confound in vitro – in vivo drug metabolism extrapolations and may play an important role in improving our ability to predict in vivo drug clearance and drug-drug interactions from in vitro data. Advisors/Committee Members: Hollenberg, Paul F. (committee member), Ballou, David P. (committee member), Dotson, Garry Dean (committee member), Saper, Mark A. (committee member), Smith, William L. (committee member).

Subjects/Keywords: Cytochrome P450 Interactions; Biological Chemistry; Science

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APA (6^th Edition):

Kenaan, C. (2012). Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/94027

Chicago Manual of Style (16^th Edition):

Kenaan, Cesar. “Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase.” 2012. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/94027.

MLA Handbook (7^th Edition):

Kenaan, Cesar. “Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase.” 2012. Web. 26 Feb 2021.

Vancouver:

Kenaan C. Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase. [Internet] [Doctoral dissertation]. University of Michigan; 2012. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/94027.

Council of Science Editors:

Kenaan C. Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase. [Doctoral Dissertation]. University of Michigan; 2012. Available from: http://hdl.handle.net/2027.42/94027

18. Lofgren, Michael W. Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity.

Degree: PhD, Biological Chemistry, 2013, University of Michigan

URL: http://hdl.handle.net/2027.42/99939

► The presence of cofactors in enzymes broadens the range of chemical transformations catalyzed in Nature. Adenosylcobalamin (AdoCbl) or coenzyme-B12, is a biologically active derivative of… (more)

Subjects/Keywords: B12 Trafficking Proteins; Allostery; Biological Chemistry; Science

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APA (6^th Edition):

Lofgren, M. W. (2013). Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/99939

Chicago Manual of Style (16^th Edition):

Lofgren, Michael W. “Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity.” 2013. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/99939.

MLA Handbook (7^th Edition):

Lofgren, Michael W. “Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity.” 2013. Web. 26 Feb 2021.

Vancouver:

Lofgren MW. Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity. [Internet] [Doctoral dissertation]. University of Michigan; 2013. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/99939.

Council of Science Editors:

Lofgren MW. Auxiliary Proteins and Allosteric Control of the Mitochondrial Branch of B12 Trafficking, Assembly, and Reactivity. [Doctoral Dissertation]. University of Michigan; 2013. Available from: http://hdl.handle.net/2027.42/99939

19. Haak, Andrew J. Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma.

Degree: PhD, Pharmacology, 2015, University of Michigan

URL: http://hdl.handle.net/2027.42/111506

► Rho GTPases regulate multiple biological functions; most notably, they stimulate formation of F-actin stress fiber formations. Through their modulation of the actin cytoskeleton Rho GTPases… (more)

▼ Rho GTPases regulate multiple biological functions; most notably, they stimulate formation of F-actin stress fiber formations. Through their modulation of the actin cytoskeleton Rho GTPases also activate gene transcription through myocardin-related transcription factors (MRTF) and the serum response factor (SRF). Recent evidence suggests MRTF/SRF-regulated gene transcription represent a novel target for the treatment of systemic sclerosis and melanoma. The work of this thesis details mechanisms of Rho GTPase and MRTF/SRF activation in these diseases, and provides in vitro and in vivo efficacy studies for a small-molecule inhibitor of the MRTF pathway developed in our lab, CCG-203971. Tissue fibrosis disorders, including systemic sclerosis are characterized by activated myofibroblasts within the affected tissue. These myofibroblasts are stimulated by multiple microenvironment factors. Transforming growth factor beta (TGF-beta) signaling involves cross talk between multiple mediators including Rho GTPase. However, the mechanism by which TGF-beta activates Rho GTPase and subsequent MRTF/SRF-regulated transcription is poorly understood. Based on the evidence here we outline a mechanism where TGF-beta stimulates expression of connective tissue growth factor (CTGF) and endothelin-1 (ET-1), which then mediate the activation of the Rho/MRTF pathway. MRTF/SRF transcription is a convergent mechanism downstream of multiple receptors that stimulate fibrosis. We tested our MRTF pathway inhibitor, CCG-203971 in models of systemic sclerosis and showed inhibition of multiple markers of fibrosis in vitro, as well as efficacy in a mouse model of dermal fibrosis in vivo. Melanoma is the most deadly form of skin cancer, with the majority of deaths attributed to metastasis. Rho GTPases have been implicated in cancer metastasis for many years, however recently it has been shown that MRTF/SRF-regulated gene transcription is essential for melanoma metastasis. Here we show that in highly invasive and metastatic melanoma cells, overexpression of RhoC leading to constitutively active MRTF. CCG-203971 treatment in these cells reduced cellular migration and invasion in vitro and blocked lung colonization in a mouse model of melanoma metastasis in vivo. Advisors/Committee Members: Neubig, Richard Robert (committee member), Hollenberg, Paul F. (committee member), Lawlor, Elizabeth (committee member), Larsen, Scott D. (committee member), Tesmer, John (committee member).

Subjects/Keywords: Rho/MRTF Signaling; Biological Chemistry; Science

…Eliza Tsou, Phillip Campbell, Jeffrey Ruth, and Asif Amin from the University of Michigan… …Lawlor’s lab by Melanie Krook and Merlin Airik from the University of Michigan, Department of… …the remaining experiments at both The University of Michigan Department of Pharmacology and…

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APA (6^th Edition):

Haak, A. J. (2015). Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/111506

Chicago Manual of Style (16^th Edition):

Haak, Andrew J. “Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma.” 2015. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/111506.

MLA Handbook (7^th Edition):

Haak, Andrew J. “Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma.” 2015. Web. 26 Feb 2021.

Vancouver:

Haak AJ. Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma. [Internet] [Doctoral dissertation]. University of Michigan; 2015. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/111506.

Council of Science Editors:

Haak AJ. Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma. [Doctoral Dissertation]. University of Michigan; 2015. Available from: http://hdl.handle.net/2027.42/111506

University of Michigan

20. Malhotra, Shefali. Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin.

Degree: PhD, Pharmacology, 2002, University of Michigan

URL: http://hdl.handle.net/2027.42/132511

► Grapefruit juice, a common citrus juice purchased by one in every five American households, increases the systemic exposure of a number of drugs that are… (more)

▼ Grapefruit juice, a common citrus juice purchased by one in every five American households, increases the systemic exposure of a number of drugs that are substrates for a major drug metabolizing enzyme, cytochrome P450 (CYP) 3A4. This grapefruit juice effect is mediated in part by inhibition of intestinal CYP3A4 catalytic activity as well as by a reduction in CYP3A4 immunoreactive protein in the epithelial cells (enterocytes) lining the small intestine. Although several CYP3A4 inhibitors in the juice, primarily furanocoumarins (FCs), have been implicated as the causative agents, there is controversy in the literature as to which FC(s) is (are) the major active component(s). A series of in vivo studies in humans and in vitro studies utilizing cDNA-expressed enzyme and human intestinal enzyme systems (microsomes and CYP3A4-expressing Caco-2 cells) indicated that 6^',7^'-dihydroxybergamottin (DHB), an abundant FC in grapefruit juice, accounts for much of the juice's effect. DHB acted by multiple mechanisms: reversible inhibition, mechanism-based inactivation, and reduction of CYP3A4 protein. DHB was therefore chosen as a representative active component to further examine the mechanism(s) underlying enterocyte CYP3A4 protein loss following grapefruit juice ingestion. DHB inactivated purified CYP3A4 in a time-, concentration-, and NADPH-dependent manner. Although this mechanism-based inactivation was unaccompanied by heme modification or destruction, it was irreversible, implying covalent binding to the apoprotein. Moreover, in CYP3A4-expressing Caco-2 cells, DHB accelerated CYP3A4 protein degradation while having no effect on the rate of CYP3A4 synthesis. DHB decreased the half-life of CYP3A4 from 14 to 3 hours. This accelerated degradation involved a proteasome-dependent, but likely an ubiquitin- and hsp90-independent, pathway. In conclusion, DHB, a major active component of grapefruit juice, exerts its effect on intestinal CYP3A4 by multiple mechanisms, including mechanism-based inactivation, followed by accelerated intracellular degradation of CYP3A4 protein via the proteasome. This characterization of the molecular mechanisms underlying the grapefruit juice effect provides insight into how FCs could be used in the future to selectively ablate intestinal CYP3A4 and thereby improve the oral bioavailability of some drugs. Advisors/Committee Members: Hollenberg, Paul F. (advisor), Watkins, Paul B. (advisor).

Subjects/Keywords: Cytochrome P450; Dihydroxybergamottin-6',7'; Effect; Grapefruit Juice; Mechanisms; Role; Ubiquitin; Underlying

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Malhotra, S. (2002). Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/132511

Chicago Manual of Style (16^th Edition):

Malhotra, Shefali. “Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin.” 2002. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/132511.

MLA Handbook (7^th Edition):

Malhotra, Shefali. “Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin.” 2002. Web. 26 Feb 2021.

Vancouver:

Malhotra S. Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin. [Internet] [Doctoral dissertation]. University of Michigan; 2002. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/132511.

Council of Science Editors:

Malhotra S. Mechanisms underlying the grapefruit juice effect: Role of 6',7'-dihydroxybergamottin. [Doctoral Dissertation]. University of Michigan; 2002. Available from: http://hdl.handle.net/2027.42/132511

21. Sikora, Matthew Joseph. Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors.

Degree: PhD, Pharmacology, 2011, University of Michigan

URL: http://hdl.handle.net/2027.42/86575

► The treatment of estrogen-receptor positive breast cancer is based on endocrine therapy, with aromatase inhibitors (AIs) serving as the current front-line therapy in post-menopausal women.… (more)

▼ The treatment of estrogen-receptor positive breast cancer is based on endocrine therapy, with aromatase inhibitors (AIs) serving as the current front-line therapy in post-menopausal women. Despite prolonging disease-free survival, ~20% of patients receiving adjuvant AIS will relapse within 10 years of treatment initiation. Mechanisms of resistance to AI therapy remain unclear, and there is currently no way to predict which patients will benefit from AI therapy. We hypothesize that androgen metabolites generated independent of aromatase may induce breast cancer growth in the absence of primary estrogens. Thus the generation of alternative estrogens from precursor androgens represents a potential mechanism of resistance to AIs. This dissertation examines the ro9les of alternative pathways of testosterone metabolism that may generate estrogen-like steroids independent of aromatase. We show that the androgen metabolite 3βAdiol induces the proliferation of breast cancer cells through direct activation of ERα. In the absence of conventional estrogens during AI therapy, 3βAdiol may be an important mediator of estrogen-dependent breast cancer growth. Another pathway of testosterone metabolism, mediated by cytochrome P450 2B6, likely represents a mechanism of the clearance of testosterone by hydroxylation. Our observations with 3βAdiol led to the hypothesis that breast cancer cells maintained long-term in low estrogen concentrations may mimic conditions in patients on AI therapy. These models demonstrated that low concentrations of estrogens such as 3βAdiol promote novel estrogen-independent phenotypes. To improve studies of the role of steroidogenic enzymes from clinical trials sample sets, we also developed improved methods for genotype assessment from formalin-fixed paraffin-embedded tissues. These studies proved a basis for further clinical investigation into the tumo estrogen environment. Alternative estrogens represent a potential mechanism of AI resistance that can be targeted with novel therapeutic strategies. The advances made in this dissertation suggest that low estrogen concentrations promote unique mechanisms of AI resistance. Further, the adaptive response to low estrogen concentrations may confer resistance to targeted therapies. Identifying mechanisms of resistance, based in part on biomarkers that can be developed using the models described herein, will allow patients to be treated with therapies specifically targeted to their tumor, thereby improving clinical outcomes. Advisors/Committee Members: Rae, James M. (committee member), Carey, Thomas E. (committee member), Hollenberg, Paul F. (committee member), Johnson, Michael D. (committee member), Pratt, William B. (committee member), Ruddon Jr, Raymond W. (committee member).

Subjects/Keywords: Breast Cancer; Estrogens; Cytochrome P450; Oncology and Hematology; Pharmacy and Pharmacology; Health Sciences

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APA (6^th Edition):

Sikora, M. J. (2011). Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/86575

Chicago Manual of Style (16^th Edition):

Sikora, Matthew Joseph. “Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors.” 2011. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/86575.

MLA Handbook (7^th Edition):

Sikora, Matthew Joseph. “Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors.” 2011. Web. 26 Feb 2021.

Vancouver:

Sikora MJ. Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors. [Internet] [Doctoral dissertation]. University of Michigan; 2011. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/86575.

Council of Science Editors:

Sikora MJ. Alternative Androgen Metabolism in Resistance to Aromatase Inhibitors. [Doctoral Dissertation]. University of Michigan; 2011. Available from: http://hdl.handle.net/2027.42/86575

University of Michigan

22. Hein, Nichole DeEtta. Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity.

Degree: PhD, Toxicology, 2009, University of Michigan

URL: http://hdl.handle.net/2027.42/63785

► Organophosphorus (OP) compounds, used in insecticides, pharmaceuticals, and weapons of biochemical warfare inhibit serine hydrolases. Exposure to OP compounds has shown that a phosphylation of… (more)

▼ Organophosphorus (OP) compounds, used in insecticides, pharmaceuticals, and weapons of biochemical warfare inhibit serine hydrolases. Exposure to OP compounds has shown that a phosphylation of certain serine esterases results in two distinct types of toxicities: an acute cholinergic toxicity associated with inhibition of acetylcholinesterase (AChE), and a more chronic toxicity associated with the inhibition and aging of neuropathy target esterase (NTE). OP induced delayed neurotoxicity (OPIDN) occurs when a threshold of NTE is inhibited and aged, and is characterized by axonopathies in the peripheral and central nervous systems 1-4 weeks after exposure. An accurate in vivo model of OPIDN is difficult to develop, due to interspecies variations of inhibitor sensitivity and metabolism. Understanding the mechanism of inhibition and aging of serine esterases by OP compounds and correlating this with pathological axonopathies are important for research on OPIDN. Fluorinated aminophosphonates (FAP) are a group of OP compounds that were hypothesized to inhibit serine esterases through a scission in a chemically stable carbon-phosphorus bond. Through the use of surface enhanced laser desorption/absorption time of flight mass spectrometry, the FAP compounds were shown to covalently phosphorylate the active site serine of butyrylcholinesterase and subsequently age through dealkylation. To begin modeling OPIDN, correlations were found in the bimolecular rate constants of inhibition of AChE and NTE using hen brain, mouse brain, and human recombinant enzymes. Furthermore, correlations in relative inhibitory potentials were found that could predict the neuropathic potential of OP compounds. Finally, two point mutations in NTE were found in patients with a hereditary spastic paraplegia that had clinical presentations similar to OPIDN. Through site-directed mutagenesis, these mutations were created in the catalytic domain of NTE and found to have altered enzymological properties, including reduced kinetic rates of substrate hydrolysis, inhibition, and aging. This research reveals that the mechanism of inhibition by OP compounds can be elucidated using mass spectrometry. Additionally, associations of kinetic values between rodents, hens, and humans may lead to further modeling of OPIDN. In conclusion, alterations in the enzymological properties of NTE may be associated with pathology presented in patients with and associated motor neuron disease. Advisors/Committee Members: Richardson, Rudy J. (committee member), Hollenberg, Paul F. (committee member), Mancuso, Peter (committee member), Stuckey, Jeanne A. (committee member).

Subjects/Keywords: Neuropathy Target Esterase; Organophosphorus; Acetylcholinesterase; Motor Neuron Disease; Mass Spectrometry; Health Sciences; Science

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APA (6^th Edition):

Hein, N. D. (2009). Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/63785

Chicago Manual of Style (16^th Edition):

Hein, Nichole DeEtta. “Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity.” 2009. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/63785.

MLA Handbook (7^th Edition):

Hein, Nichole DeEtta. “Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity.” 2009. Web. 26 Feb 2021.

Vancouver:

Hein ND. Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity. [Internet] [Doctoral dissertation]. University of Michigan; 2009. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/63785.

Council of Science Editors:

Hein ND. Mechanisms of Prediction and Potential Causation of Organophosphate Induced Delayed Neurotoxicity. [Doctoral Dissertation]. University of Michigan; 2009. Available from: http://hdl.handle.net/2027.42/63785

University of Michigan

23. Bumpus, Namandj‚ N. The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6.

Degree: PhD, Pharmacology, 2008, University of Michigan

URL: http://hdl.handle.net/2027.42/58482

► A reconstituted monooxygenase system containing purified P450 2B6 and NADPH-cytochrome P450-reductase (reductase), was used to investigate the catalytic properties of a naturally occurring mutation in… (more)

▼ A reconstituted monooxygenase system containing purified P450 2B6 and NADPH-cytochrome P450-reductase (reductase), was used to investigate the catalytic properties of a naturally occurring mutation in P450 2B6 (2B6.4) in which a lysine is changed to an arginine. To probe this, several structurally unrelated mechanism-based inactivators of wild-type P450 2B6 were used. P450 2B6.4 was not inactivated by two of these compounds, 17-alpha-ethynylestradiol (17EE) and efavirenz. Subsequent studies were performed to determine which step(s) in the P450 catalytic cycle might be compromised in the mutant enzyme leading to the changes in the ability of the two compounds to act as mechanism-based inactivators. Studies on the binding of substrates and reductase to the P450s revealed similarities in the abilities of both enzymes to associate with substrates and the reductase. The reaction stoichiometries for the metabolism of efavirenz and 17EE indicated that the mutant enzyme was more uncoupled than the wild-type enzyme. However, the addition of cytochrome b5, a P450 redox partner, to the reconstitution mixture resulted in increased coupling of the P450 2B6.4-catalyzed reactions. In addition, in the presence of cytochrome b5, the mutant enzyme was now inactivated by both 17EE and efavirenz. These data lead us to suggest that one of the oxygen intermediates formed during the catalytic cycle, either the oxyferrous P450 and/or the iron-peroxo intermediate, may be less stable when formed by P450 2B6.4 compared to the same intermediates formed by the wild-type enzyme. In order to determine the site(s) of interaction between P450 2B6 and reductase, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6 to reductase. Cross-linked peptides were then identified by reconstituting the peptides in 18O-water following proteolysis, based on the principle that the cross-linked peptides would be expected to incorporate twice as many 18O atoms as non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides lead to the identification of a cross-linked peptide candidate. De novo sequencing of the peptide suggests that it is a complex between the C-helix of the P450 and the connecting domain of reductase. Advisors/Committee Members: Hollenberg, Paul F. (committee member), Osawa, Yoichi (committee member), Pratt, William B. (committee member), Waskell, Lucy (committee member).

Subjects/Keywords: Cytochrome P450 2B6 Genetic Polymorphism; Mechanism-based Inactivation; Pharmacy and Pharmacology; Health Sciences

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Bumpus, N. N. (2008). The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/58482

Chicago Manual of Style (16^th Edition):

Bumpus, Namandj‚ N. “The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6.” 2008. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/58482.

MLA Handbook (7^th Edition):

Bumpus, Namandj‚ N. “The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6.” 2008. Web. 26 Feb 2021.

Vancouver:

Bumpus NN. The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6. [Internet] [Doctoral dissertation]. University of Michigan; 2008. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/58482.

Council of Science Editors:

Bumpus NN. The Effects of a Naturally Occurring Genetic Polymorphism on the Catalytic Properties of Human Cytochrome P450 2B6. [Doctoral Dissertation]. University of Michigan; 2008. Available from: http://hdl.handle.net/2027.42/58482

University of Michigan

24. Li, Shengying. Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites.

Degree: PhD, Medicinal Chemistry, 2009, University of Michigan

URL: http://hdl.handle.net/2027.42/64678

► The superfamily of cytochrome P450 monooxygenases is involved in diverse oxidative processes including xenobiotic catabolism, steroid synthesis, and biosynthetic tailoring of diverse natural products. During… (more)

▼ The superfamily of cytochrome P450 monooxygenases is involved in diverse oxidative processes including xenobiotic catabolism, steroid synthesis, and biosynthetic tailoring of diverse natural products. During the past decade, the synthetic potential of biosynthetic P450 enzymes from microorganisms has gained special attention due to their non-membrane bound nature, considerable catalytic efficiency, and high regio- and stereoselectivity. However, current barriers to their application in synthetic chemistry include their instability, inherent dependence on separate redox partners, and narrow substrate spectra. As these hurdles have been gradually overcome, it is likely that these biosynthetic P450s will find expanded use in the production of chemicals, fragrances, pharmaceutical compounds, biofuels, and application in bioremediation. My dissertation research has focused on the bacterial cytochrome P450 PikC from the pikromycin macrolide antibiotic biosynthetic pathway in Streptomyces venezuelae. The inherent substrate flexibility and hydroxylation pattern of PikC suggests its unique oxidative mechanism and synthetic potential. Starting from the rystal structures of PikC, we not only elucidated the structural basis for its substrate flexibility, but also discovered a unique desosamine sugar anchoring functionality of this enzyme. These observations directly inspired a substrate engineering strategy that utilizes the desosamine anchor to deliver diverse structures into the PikC active site for selective oxidation. Using this approach, the substrate spectrum of PikC has been significantly broadened. Specifically, by using an engineered PikCD50N-RhFRED with self-sufficiency and significantly higher catalytic efficiency, a series of carbocyclic rings linked to the desosamine glycoside were effectively hydroxylated in a regioselective manner. Associated analysis of co-crystal structures of PikC with selected unnatural desosaminyl substrates provided significant insights into the mechanism of its oxidative selectivity control. Taken together, these results offer an applicable enzymatic solution of a central challenge in synthetic chemistry - the selective oxidation of an unactivated sp3 C-H bond. Moreover, a number of other biosynthetic P450 enzymes, two O-methyltransferases, an FAD-dependent oxidase, and a type III polyketide synthase were also studied during the course of my dissertation research. Together, these studies provide new insights into biosynthesis of secondary metabolites and how these enzymes can be adapted for biotechnological use. Advisors/Committee Members: Sherman, David H. (committee member), Garneau-Tsodikova, Sylvie (committee member), Gestwicki, Jason E. (committee member), Hollenberg, Paul F. (committee member), Woodard, Ronald W. (committee member).

Subjects/Keywords: P450; Biosynthesis; Secondary Metabolites; PikC; Bioengineering; Pikromycin; Biological Chemistry; Science

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Li, S. (2009). Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/64678

Chicago Manual of Style (16^th Edition):

Li, Shengying. “Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites.” 2009. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/64678.

MLA Handbook (7^th Edition):

Li, Shengying. “Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites.” 2009. Web. 26 Feb 2021.

Vancouver:

Li S. Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites. [Internet] [Doctoral dissertation]. University of Michigan; 2009. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/64678.

Council of Science Editors:

Li S. Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites. [Doctoral Dissertation]. University of Michigan; 2009. Available from: http://hdl.handle.net/2027.42/64678

University of Michigan

25. Snider, Natasha Tasheva. Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes.

Degree: PhD, Pharmacology, 2009, University of Michigan

URL: http://hdl.handle.net/2027.42/62204

► Arachidonoyl ethanolamide (anandamide), an endogenous derivative of arachidonic acid, activates the same molecular targets as the main psychoactive constituent of the cannabis (marijuana) plant and… (more)

▼ Arachidonoyl ethanolamide (anandamide), an endogenous derivative of arachidonic acid, activates the same molecular targets as the main psychoactive constituent of the cannabis (marijuana) plant and it is therefore classified as an endogenous cannabinoid. Anandamide belongs to a large and therapeutically important signaling system, the endocannabinoid system, which also includes the two known cannabinoid receptors, CB1 and CB2, the endocannabinoid synthetic and degrading enzymes, and the associated signaling pathways regulated by the CB1 and CB2 receptors. The various components of this system represent novel pharmacological targets for the treatment of many neurological, neuropsychiatric, inflammatory and metabolic disorders. Therefore, it is critical to gain a thorough understanding of all the enzymes that could potentially exert control over the anandamide tone in vivo. The cytochrome P450 monooxygenases (P450s) are one such group of enzymes. The central hypothesis of this thesis was that P450s metabolize anandamide to various oxygenated products which may have physiological and pharmacological significance. By using human liver, kidney and brain tissue preparations, in addition to purified P450 enzymes in the reconstituted system, it was determined that anandamide is metabolized by P450s 2D6, 3A4, and 4F2 to form various hydroxylated and epoxygenated products. Epoxide hydrolase and P450 2D6 further metabolized the primary epoxides of anandamide to form several di-oxygenated derivatives. Pharmacological studies revealed that the epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective CB2 receptor agonist which has the potential to modulate immune cell function. Activation of CB2 by 5,6-EET-EA may lead to important anti-inflammatory effects under certain pathological situations affecting many organs, such as the liver during ischemia/reperfusion injury or fibrosis, and the brain during stroke or neurodegeneration. The results from this work may also lead to important insights into the mechanisms of action of two novel classes of drug candidates which have the potential to indirectly increase endogenous 5,6-EET-EA levels. Collectively, these findings uncover an endocannabinoid bioactivation pathway and establish the cytochrome P450 enzymes as important players in endocannabinoid signaling. Advisors/Committee Members: Hollenberg, Paul F. (committee member), Osawa, Yoichi (committee member), Pratt, William B. (committee member), Smith, William L. (committee member), Sunahara, Roger (committee member), Traynor, John R. (committee member).

Subjects/Keywords: Cytochrome P450; Anandamide; Endocannabinoid; Metabolism; Eicosanoid; Oxidation; Pharmacy and Pharmacology; Health Sciences

Record Details Similar Records Cite « Share

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Snider, N. T. (2009). Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/62204

Chicago Manual of Style (16^th Edition):

Snider, Natasha Tasheva. “Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes.” 2009. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/62204.

MLA Handbook (7^th Edition):

Snider, Natasha Tasheva. “Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes.” 2009. Web. 26 Feb 2021.

Vancouver:

Snider NT. Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes. [Internet] [Doctoral dissertation]. University of Michigan; 2009. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/62204.

Council of Science Editors:

Snider NT. Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide (Anandamide) by Cytochrome P450 Enzymes. [Doctoral Dissertation]. University of Michigan; 2009. Available from: http://hdl.handle.net/2027.42/62204

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