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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Characterization of the Thioesterase Domain of the Bacillaene Pathway

Horsman, Mark 17 May 2018 (has links)
The goals of this work are to advance the understanding of the mechanisms of thioesterase loading-substrate and release-nucleophile selectivity and to develop an analytical methodology and modelling theory as tools for quantitative measures of thioesterase activity. The first aspect of this work is a bioinformatic examination of examples of thioesterases that catalyze different forms of release at different rates of reaction dependent on the type of thioester substrate presented to it. In addition, the type I thioesterase domain from the Type I modular polyketide synthase pathway responsible for the production of bacillaene was cloned, expressed, and purified for kinetic characterization as a model trans-AT type thioesterase. N-Acetylcysteamine and acyl carrier protein-bound acyl substrates were synthesized as substrates for hydrolysis catalysed by the bacillaene thioesterase. The rate of reaction was indirectly observed with a widely-adopted visual spectroscopy assay facilitated by the thiol indicator 5,5'-dithio-bis-[2-nitrobenzoic acid]. Variants of the bacillaene thioesterase coded from two different bacterial isolates with 57 % amino acid identity (73 % similarity) both exhibited rapid declining activity under the conditions used for Ellman’s, indicative of inhibition. This matches rare literature examples of other thioesterase domains under the same spectroscopic assay conditions. We demonstrate that initial rate of reaction data is insufficient to describe substrate selectivity in these systems and develop a time-dependent model capable of accounting for irreversible substrate inhibition during catalysis. This model was sufficient to reproduce the thioesterase activity observed between the bacillaene thioesterase and methyl valeric-SNAC but is insufficient to model the responses observed during the hydrolysis of trans-2-methyl-2-pentenoyl-SNAC or cis-3-methyl-2-pentenoyl-SNAC. Finally, we detailed synthetic and assay conditions for the production, cleavage, and detection of the ACP-tethered substrate equivalents. This contributes towards the development of complex thioesterase assays that better estimate type I bacterial thioesterase activity during polyketide synthesis.
2

Characterization of cDNA and Genomic Clones for a Palmitoyl-acyl Carrier Protein Thioesterase and an Osmotin-Like PR5 Protein in Gossypium Hirsutum.

Yoder, David W. 05 1900 (has links)
Putative cotton cDNA clones and cognate genomic clones for a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE) and an osmotin-like pathogenesis-related 5 (PR5) protein have been isolated and characterized. PATE is a class B fatty acid thioesterase with specificity for saturated long-chain fatty acids such as palmitate, and is implicated as a key enzyme to be targeted for regulation of fatty acid synthesis in order to alter cotton seed oil profiles. A nearly full-length 1.7-kb cDNA clone was isolated using a hybridization probe derived from an Arabidopsis PATE cDNA clone designated TE 3-2. A 17-kb genomic segment encompassing the PATE gene was also isolated, which has six exons and five introns with high sequence identity with other FatB cDNA/gene sequences. The deduced PATE preprotein amino acid sequence of 413 residues has putative signal sequences for targeting to the chloroplast stroma. PR5 proteins called osmotins are made in response to fungal pathogen stress or osmotic stress (water deprivation or salt exposure). Osmotins may actually form pores in fungal membranes, leading to osmotic rupture and destruction of the fungal cells. A cotton osmotin-like PR5 cDNA insert of 1,052 base-pairs was isolated and shown to encode a preprotein of 242 amino acids and is predicted to be secreted to the extracellular matrix as a neutral isoform. The deduced amino acid sequence has 16 cysteine residues that are highly conserved in osmotin-like proteins and are important in stabilizing the three-dimensional structure seen in thaumatin, zeamatin, and PR5-d. The intronless cognate cotton genomic clone has two putative ethylene response elements (GCC boxes) found in other PR5 gene promoter regions, as well as several tentative promoter/enhancer elements possibly involved in spatial/temporal gene expression.
3

Probing the Stereospecificity and Chemospecificity of Polyketide Thioesterases

Argyropoulos, Panos 06 May 2014 (has links)
Macrocyclization is a synthetically challenging step in the total synthesis of natural products. The success of chemical approaches such as the Corey-Nicolaou, Yamaguchi and Keck macrolactonization is heavily based on the confirmation and stereochemistry of the substrate. While there have been some advances in computational modeling, it has been difficult to predict whether the above-mentioned reactions will work. We have begun characterizing polyketide thioesterase catalytic activity and substrate tolerance to find more efficient and dependable routes towards macrolactonization and macrolactamization.
4

Synthese und biologische Evaluierung von Inhibitoren der Acyl-Protein-Thioesterase 1

Deck, Patrick Hugo. Unknown Date (has links) (PDF)
Universiẗat, Diss., 2002--Dortmund.
5

Probing the Stereospecificity and Chemospecificity of Polyketide Thioesterases

Argyropoulos, Panos January 2014 (has links)
Macrocyclization is a synthetically challenging step in the total synthesis of natural products. The success of chemical approaches such as the Corey-Nicolaou, Yamaguchi and Keck macrolactonization is heavily based on the confirmation and stereochemistry of the substrate. While there have been some advances in computational modeling, it has been difficult to predict whether the above-mentioned reactions will work. We have begun characterizing polyketide thioesterase catalytic activity and substrate tolerance to find more efficient and dependable routes towards macrolactonization and macrolactamization.
6

Characterizing the Macrocyclization Activity of Fungal Polyketide Synthase Thioesterases

Wirz, Monica Hélène 12 January 2012 (has links)
Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.
7

Biosynthesis of medium chain fatty acids by cell free fractions in adenocarcinomas and normal mouse mammary tissue

Kendra, Albert 03 June 2011 (has links)
Ball State University LibrariesLibrary services and resources for knowledge buildingMasters ThesesThere is no abstract available for this thesis.
8

Characterizing the Macrocyclization Activity of Fungal Polyketide Synthase Thioesterases

Wirz, Monica Hélène 12 January 2012 (has links)
Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.
9

Gene Delivery of Rat Thioesterase II in Hepatocytes

Lin, Hsiu-Chu 31 July 2003 (has links)
Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.
10

Characterizing the Macrocyclization Activity of Fungal Polyketide Synthase Thioesterases

Wirz, Monica Hélène 12 January 2012 (has links)
Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

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