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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

Investigating Chemical Modifications in a Complex Proteome

Crawford, Lisa Ann January 2017 (has links)
Thesis advisor: Eranthie Weerapana / Thesis advisor: Jianmin Gao / Proteins are composed of the 20 naturally occurring amino acids and are further modified by a variety of post-translational modifications (PTMS). Naturally occurring amino acids are diverse in structure and function. Catalytic amino acids, or nucleophilic amino acids, are of particular interest because of their contribution to chemical transformations in the cell. Synthetic covalent modification is a means to further functionalize or diversify proteins. These modifications, or enhancements, allow for improved understanding of protein structure, function and activity. For instance, isotope labeling of amino acid side chains in NMR studies enable investigators to study protein dynamics upon substrate or ligand binding. Fluorescence labeling is particularly useful to investigate protein cellular localization. Covalent modification is a useful tool to investigate the relative level of activity for protein known to be regulated by PTMs. An important feature of covalent modification reactions is site specificity, as this dictates the location, number of modifications, and protein targets. Tyrosine is of particular interest because it is both nucleophilic and aromatic. These characteristics contribute to the existence of tyrosine residues in both the protein surface and hydrophobic cores. Tyrosine is incorporated into proteins at a relatively low frequency. Unlike lysine, which is ubiquitous on protein surfaces, the low number of potential sites for general tyrosine modifications makes it an attractive site for surface bioconjugation modifications. A low number of surface modifications is less likely to perturb native protein function. Bioconjugation reactions give access to functionalizing the surface of proteins with moieties such as fluorophores, PEG, peptides, or drugs. Tyrosine is an attractive target for modifications because it is found in the active sites of a variety of enzymes such as sialidases, glutathione-S transferases, corticosteroid 11-beta-dehydrogensase, DNA topoisomerase, and ferredoxin-NADP+ reductase. Provided here is a survey of the known non-selective and selective synthetic chemical modification reactions for tyrosine. To investigate nucleophilic amino acids, Activity Based Protein Profiling (ABPP) may be implemented to investigate the role of these residues. ABPP utilizes small molecule covalent probes as a tool to selectively target enzymes in their active state. To investigate a protein of interest (POI) (or class of proteins) by ABPP, it is necessary to use a small molecule covalent probe that selectively reacts with the POI over other proteins within the proteome. Due to this requirement, it is necessary to expand the current ABPP probe toolbox to increase the coverage of what proteins in the proteome may be studied. Inspired by findings in the literature, our lab sought to explore the utility of various aryl halides for implementation in ABPP probes to overcome this limitation. This study revealed dichlorotriazine as a biologically relevant and reactive electrophile. A focus was placed on a dichlorotriazine containing probe library (LAS1-LAS20). LAS17 was discovered to be a potent and selective inhibitor of human glutathione S-transferase pi (GSTP1). Further studies revealed GSTP1 as a novel therapeutic target for the treatment of triple negative breast cancer. Other studies revealed several members of the dichlorotriazine library were found to covalently modify purified recombinant human aldolase A (ALDOA) in the presence of a complex cellular background. Additionally, LAS9 was identified as an inhibitor of ALDOA retro aldol condensation activity in vitro. Lastly, the final chapter highlights two collaborations in which tandem mass spectrometry experiments aid in the characterization of experimental data. In the first collaboration, a quantitative cysteine reactivity profiling method was used to characterize the selectivity of a cysteine reactive covalent NRF2-inducing small molecule, MIND4-17. In the second collaboration, analysis of tryptic mass spectrometry data enabled high resolution characterization of peptide sequencing for superfolder green fluorescent protein (sfGFP) expressed from observed internal nonsense suppression. Identification of the misincorporated amino acid facilitated the elucidation of the cross-talk mechanism. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
2

Studies of Human 5S snoRNA Genes

Lin, Su-Yo 06 June 2002 (has links)
The nucleolus of eukaryotic cells contain a number of the intron-coding small nucleolar RNAs (snoRNAs), which functions are related to covalent modification of pre-rRNAs. The snoRNA that from long, phylogenetically conserved sequence complementarity to 28S, 18S, 5.8S and 5S rRNAs are designated as 28S, 18S, 5.8S and 5S snoRNAs, respectively. In the present study, studying on human 5S snoRNAs had been carried out. The human genome encoding candidate 5S snoRNAs were searched using database mining. The transcripts of 5S snoRNA genes were identified by RT-PCR analyses and DNA sequencing. No appreciable diversities of 5S snoRNA genes were observed as evidenced by single strand conformation polymorphism (SSCP) and high resolution agarose gel. Moreover, sequence conservation of 5S snoRNAs reflects a requirement for maintaining their secondary structure on exerting their function. The results of RT-PCR analyses revealed a tissue-specific transcription of 5S snoRNAs. A 5S snoRNA designated as N117 was identified to be highly expressed in normal brain. On the contrary, its expression markly decreased in brain tumor (meningioma). This seems to be associated with the expression of host gene, which encodes a protein similar to synapsin III protein. Consequently, this may implicate that the use of snoRNA as a potential index for the transcription of its host gene.
3

Mass Spectrometry-Based Strategies for Multiplexed Analyses of Protein-Ligand Binding Interactions

DeArmond, Patrick D. January 2011 (has links)
<p>The detection and quantitation of protein-ligand binding interactions is important not only for understanding biological functions but also for the characterization of novel protein ligands. Because protein ligands can range from small molecules to other proteins, general techniques that can detect and quantitate the many classes of protein-ligand interactions are especially attractive. Additionally, the ability to detect and quantify protein-ligand interactions in complex biological mixtures would more accurately represent the protein-ligand interactions that occur in vivo, where differential protein expression and protein complexes can significantly affect a protein's ability to bind to a ligand of interest.</p><p> The work in this dissertation is focused on the development of new methodologies for the detection and measurement of protein-ligand interactions in complex mixtures using multiplex analyses. Methodologies for two types of multiplexed analyses of protein-ligand binding interactions are investigated here. The first type of multiplex analysis involves characterizing the binding of one protein target to many potential ligands, and the second type involves characterizing the binding of one ligand to many proteins. The described methodologies are derived from the SUPREX (stability of unpurified proteins from rates of H/D exchange) and SPROX (stability of proteins from rates of oxidation) techniques, which are chemical modification strategies that measure thermodynamic stabilities of proteins using a relationship between a protein's folding equilibrium and the extent of chemical modification. These two techniques were utilized in the development and application of several different experimental strategies designed to multiplex the analysis of protein-ligand interactions.</p><p> The first strategy that was developed involved a pooled compound approach for making SUPREX-based measurements of multiple ligands binding to a target protein. Screening rates of 6 s/ligand were demonstrated in a high-throughput screening project that involved the screening of two chemical libraries against human cyclophilin A (CypA), a protein commonly overexpressed in types of cancer. This study identified eight novel ligands to CypA with micromolar dissociation constants. Second, an affinity-based protein purification strategy was developed for the detection and quantitation of specific protein-ligand binding interactions in the context of complex protein mixtures. It involved performing SPROX in cell lysates and selecting the protein of interest using immunoprecipitation or affinity tag purification. A third strategy developed here involved a SPROX-based stable isotope labeling method for measuring protein-ligand interactions in multi-protein mixtures. This strategy was used in a proof-of-principle experiment designed to detect and quantify the indirect binding between yeast cyclophilin and calcineurin in a multi-component protein mixture. Finally, a quantitative proteomics platform was developed for the detection and quantitation of protein-ligand binding interactions on the proteomic scale. The platform was used to profile interactions of the proteins in a yeast cell lysate to several ligands, including the bioactive small molecules resveratrol and manassantin A, the cofactor nicotinamide adenine dinucleotide (NAD+), and two proteins, phosphoglycerate kinase (Pgk1) and pyruvate kinase (Pyk1). The above approaches should have broad application for use as discovery tools in the development of new therapeutic agents.</p> / Dissertation
4

Control of Uncoupling Protein-1 (UCP1) by Phosphorylation and the Metabolic Impact of Ectopic UCP1 Expression in Skeletal Muscle of Mice

Adjeitey, Cyril 07 June 2013 (has links)
UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.
5

Control of Uncoupling Protein-1 (UCP1) by Phosphorylation and the Metabolic Impact of Ectopic UCP1 Expression in Skeletal Muscle of Mice

Adjeitey, Cyril January 2013 (has links)
UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.
6

Covalent modification of endogenous proteins for functional analyses and drug discovery based on N-sulfonyl pyridone chemistry / タンパク質の機能解析と薬剤開発を目的としたN-スルホニルピリドン化学による内在性タンパク質の共有結合修飾

Masuda, Marie 26 November 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21425号 / 工博第4535号 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 杉野目 道紀, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
7

Modification Reactivity Analysis of Human Replication Protein A in Biologically Important States

Yoakum, Ryan James 17 May 2016 (has links)
No description available.
8

A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases

Viljanen, Johan January 2008 (has links)
This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification. A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized. A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems. Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.
9

Functionalization of Nanocarbons for Composite, Biomedical and Sensor Applications

Kuznetsov, Oleksandr 24 July 2013 (has links)
New derivatives of carbon nanostructures: nanotubes, nano-onions and nanocrystalline diamonds were obtained through fluorination and subsequent functionalization with sucrose. Chemically modified nanocarbons show high solubility in water, ethanol, DMF and can be used as biomaterials for medical applications. It was demonstrated that sucrose functionalized nanostructures can find applications in nanocomposites due to improved dispersion enabled by polyol functional groups. Additionally, pristine and chemically derivatized carbon nanotubes were studied as nanofillers in epoxy composites. Carbon nanotubes tailored with amino functionalities demonstrated better dispersion and crosslinking with epoxy polymer yielding improved tensile strength and elastic properties of nanocomposites. Reductive functionalization of nanocarbons, also known as Billups reaction, is a powerful method to yield nanomaterials with high degree of surface functionalization. In this method, nanocarbon salts prepared by treatment with lithium or sodium in liquid ammonia react readily with alkyl and aryl halides as well as bromo carboxylic acids. Functionalized materials are soluble in various organic or aqueous solvents. Water soluble nanodiamond derivatives were also synthesized by reductive functionalization of annealed nanodiamonds. Nanodiamond heat pretreatment was necessary to yield surface graphene layers and facilitate electron transfer from reducing agent to the surface of nanoparticles. Other carbon materials such as activated carbon and anthracite coal were also derivatized using reductive functionalization to yield water soluble activated carbon and partially soluble in organic solvents anthracite. It was shown that activated carbon can be effectively functionalized by Billups method. New derivatives of activated carbon can improve water treatment targeting specific impurities and bio active contaminants. It was demonstrated that functionalized carbon nanotubes are suitable for real time radiation measurements. Radiation sensor incorporating derivatized carbon nanotubes is lightweight and reusable. In summary, functionalization of carbon nanomaterials opens new avenues for processing and applications ranging from biomedicine to radiation sensing in space.

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