subject:(biological chemistry)

University of Washington

1. Trevillian, Bridget Marley. Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors.

Degree: PhD, 2017, University of Washington

URL: http://hdl.handle.net/1773/38596

► Chapter 1: Development of a Small Molecule Regulated Cre Recombinase Our lab has previously developed a chemical genetic method for controlling signaling enzymes with a… (more)

▼ Chapter 1: Development of a Small Molecule Regulated Cre Recombinase Our lab has previously developed a chemical genetic method for controlling signaling enzymes with a small molecule. In this method, the endogenous regulatory domains of an enzyme of interest are replaced with a protein-protein interaction that acts as an artificial regulatory domain. This synthetic enzyme can be controlled by a potent, selective and cell permeable small molecule that disrupts the protein-protein interaction that serves as the artificial regulatory domain. Cre (Causes Recombination) Recombinase is a site-specific tyrosine recombinase from bacteriophage P1. Cre is commonly used to facilitate conditional knock-in, knock-out and inversion studies is mammals8. This chapter describes the development of a Cre Recombinase whose activity can be controlled in cells with the small molecule-regulated switch that we previously reported. Our small molecule-regulated Cre Recombinase allows rapid, reversible, and dose-dependent activation of Cre recombinase and has the potential to be used to conditionally activate or inactivate gene expression in vivo. Applications of our small molecule-regulated Cre Recombinase and efforts to determine the mechanism of small molecule regulation are described. Chapter 2: A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors Protein kinases are a large family of >530 signaling proteins that allow a cell to respond appropriately to a variety of external stimuli (Manning et. al. 2002). Most current research on protein kinases focuses on studying their catalytic activity, but recent evidence has shown that the non-catalytic role of kinases–including scaffolding and DNA binding–is essential to cell survival (Rauch et. al 2011). A major challenge in studying the non-catalytic roles of protein kinases is the lack of selective molecular tools for studying this aspect of kinase function. As a result, the non-catalytic functions of most proteins kinases are not well understood. A major focus of research in the field has been development of inhibitors that selectively bind distinct ATP-binding site conformations of kinases. Specifically, Type I inhibitors (Taylor et al, 2011), which bind catalytically active kinases, and type II inhibitors, which bind to catalytically inactive forms of the ATP-binding site (Ranjitkar et al, 2010; Ranjitkar et al, 2014; Seeliger et al, 2009; Okram et al, 2006). In many kinases, the conformation of the ATP-binding site and regulatory domains are allosterically coupled. Stabilizing different ATP-binding site forms with conformation-selective inhibitors has the potential to divergently modulate the non-catalytic roles of protein kinases in the cell (Rauch et al, 2011). The second half of this thesis describes work analyzing the divergent impacts of conformationally-selective inhibitors on the non-catalytic functions of Src Family Kinases (SFKs) in cells. Advisors/Committee Members: Maly, Dustin J (advisor).

Subjects/Keywords: Biological Chemistry; Chemistry; Chemistry

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APA (6^th Edition):

Trevillian, B. M. (2017). Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/38596

Chicago Manual of Style (16^th Edition):

Trevillian, Bridget Marley. “Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors.” 2017. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/38596.

MLA Handbook (7^th Edition):

Trevillian, Bridget Marley. “Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors.” 2017. Web. 21 Jan 2021.

Vancouver:

Trevillian BM. Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors. [Internet] [Doctoral dissertation]. University of Washington; 2017. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/38596.

Council of Science Editors:

Trevillian BM. Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors. [Doctoral Dissertation]. University of Washington; 2017. Available from: http://hdl.handle.net/1773/38596

University of Washington

2. Lu, Yiching. Structural Studies of the ATPase from the Type II Secretion System.

Degree: PhD, 2014, University of Washington

URL: http://hdl.handle.net/1773/26389

► The type II secretion system (T2SS) in Gram-negative bacteria is a multi-protein machinery that spans both the inner and the outer membranes. The T2SS in… (more)

▼ The type II secretion system (T2SS) in Gram-negative bacteria is a multi-protein machinery that spans both the inner and the outer membranes. The T2SS in Vibrio cholerae, secretes folded proteins including the major virulence factor, cholera toxin, from the periplasm across the outer membrane into the intestinal tract of the host. The T2SS contains 12-15 different proteins, with many of them present in multiple copies. This system can be divided into three major sub-assemblies: the outer membrane complex, the pseudopilus, and the inner membrane complex. The inner membrane complex contains the only ATPase GspE of the T2SS, and is thought to provide the energy for T2SS assembly or secretion. GspE belongs to the Type II/IV secretion ATPase family and has three domains, N1E, N2E and CTE. GspE is tightly associated with the inner membrane complex through its N1E interacting with the cytoplasmic domain of the inner membrane protein GspL (cyto-GspL). Although numerous members of the Type II/IV secretion ATPases have been characterized or crystallized as hexamers, the N1E-truncated GspE crystallized as a 6₁-helical filament. To this date, the oligomeric state of GspE remains unclear. Here, we unravel the crystal structure of the full-length GspE in complex with cyto-GspL from Vibrio vulnificus in the presence of a non-hydrolysable ATP analogue AMPPNP. The structure revealed a novel mutual orientation of N2E and CTE, cyto-GspL rods that extend throughout the crystal, and unexpected interactions of cyto-GspL with AMPPNP and CTE. The GspE in complex with cyto-GspL structure shows no evidence of GspE multimers, therefore, an "assistant hexamer" Hcp1 was fused to GspE for facilitating GspE hexamerization. The N1E-truncated Vibrio cholerae GspE fused to Hcp1 showed a ~20-fold increase in ATPase activity compared to the monomeric form, and crystallized into two different hexamers: a quasi-C6 regular hexamer and an elongated C2 hexamer. Small-angle X-ray scattering studies of the fusion supports the hexameric forms observed in the structures are present in solution, but several conformations are in equilibrium in solution. Based on the analysis of domain arrangements and comparison with homologs from the Type II/IV secretion ATPases, GspE shows considerable conformational flexibility and likely undergoes several different conformations during the secretion process. Advisors/Committee Members: Hol, Wilhelmus G.J. (advisor).

Subjects/Keywords: Biochemistry; biological chemistry

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APA (6^th Edition):

Lu, Y. (2014). Structural Studies of the ATPase from the Type II Secretion System. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/26389

Chicago Manual of Style (16^th Edition):

Lu, Yiching. “Structural Studies of the ATPase from the Type II Secretion System.” 2014. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/26389.

MLA Handbook (7^th Edition):

Lu, Yiching. “Structural Studies of the ATPase from the Type II Secretion System.” 2014. Web. 21 Jan 2021.

Vancouver:

Lu Y. Structural Studies of the ATPase from the Type II Secretion System. [Internet] [Doctoral dissertation]. University of Washington; 2014. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/26389.

Council of Science Editors:

Lu Y. Structural Studies of the ATPase from the Type II Secretion System. [Doctoral Dissertation]. University of Washington; 2014. Available from: http://hdl.handle.net/1773/26389

University of Washington

3. Hsia, Yang. Design of a hyperstable 60-subunit icosahedral nanocage.

Degree: PhD, 2017, University of Washington

URL: http://hdl.handle.net/1773/40491

► The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport1,2. There has… (more)

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APA (6^th Edition):

Hsia, Y. (2017). Design of a hyperstable 60-subunit icosahedral nanocage. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/40491

Chicago Manual of Style (16^th Edition):

Hsia, Yang. “Design of a hyperstable 60-subunit icosahedral nanocage.” 2017. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/40491.

MLA Handbook (7^th Edition):

Hsia, Yang. “Design of a hyperstable 60-subunit icosahedral nanocage.” 2017. Web. 21 Jan 2021.

Vancouver:

Hsia Y. Design of a hyperstable 60-subunit icosahedral nanocage. [Internet] [Doctoral dissertation]. University of Washington; 2017. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/40491.

Council of Science Editors:

Hsia Y. Design of a hyperstable 60-subunit icosahedral nanocage. [Doctoral Dissertation]. University of Washington; 2017. Available from: http://hdl.handle.net/1773/40491

University of Washington

4. Ford, Alexander. Nonparametric Structure Models in Local Protein Conformation Sampling and Design.

Degree: PhD, 2018, University of Washington

URL: http://hdl.handle.net/1773/41742

► Protein design relies of the identification of a sequence that specifically encodes a target conformation as a folded native state. This native states is encoded… (more)

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APA (6^th Edition):

Ford, A. (2018). Nonparametric Structure Models in Local Protein Conformation Sampling and Design. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/41742

Chicago Manual of Style (16^th Edition):

Ford, Alexander. “Nonparametric Structure Models in Local Protein Conformation Sampling and Design.” 2018. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/41742.

MLA Handbook (7^th Edition):

Ford, Alexander. “Nonparametric Structure Models in Local Protein Conformation Sampling and Design.” 2018. Web. 21 Jan 2021.

Vancouver:

Ford A. Nonparametric Structure Models in Local Protein Conformation Sampling and Design. [Internet] [Doctoral dissertation]. University of Washington; 2018. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/41742.

Council of Science Editors:

Ford A. Nonparametric Structure Models in Local Protein Conformation Sampling and Design. [Doctoral Dissertation]. University of Washington; 2018. Available from: http://hdl.handle.net/1773/41742

University of Washington

5. Fong, Kimberly K. Regulation of microtubule nucleation and attachment to spindle pole bodies.

Degree: PhD, 2016, University of Washington

URL: http://hdl.handle.net/1773/35535

► The precise regulation and coordination of the mitotic spindle is vital for accurate chromosomal segregation within a dividing cell. The centrosome is the microtubule organizing… (more)

▼ The precise regulation and coordination of the mitotic spindle is vital for accurate chromosomal segregation within a dividing cell. The centrosome is the microtubule organizing center of the cell, responsible for nucleating and organizing the microtubules of the mitotic spindle. In yeast, the centrosome functional equivalent is called the spindle pole body. The studies presented here aimed to understand how microtubules are nucleated at the spindle pole body and also studied the regulation and mechanical strength of the yeast spindle pole body in vitro. In yeast, the γ-tubulin small complex, a highly conserved heterotetramer essential for microtubule nucleation, is composed of two copies of Tub4, and one copy each of Spc97 and Spc98. In this study, a comprehensive mutagenesis technique coupled with high-throughput sequencing was used to identify regions of Spc97 and Spc98 that were essential for the formation of the γ-tubulin small complex and mutations that influenced the organization of the mitotic spindle. Regions essential for structure and function of the complex were mapped onto the protein sequence for both Spc97 and Spc98 and temperature sensitive mutants identified by the mutagenic screen were isolated and their unique nucleation phenotypes characterized. Improvements were made to historical spindle pole body purification methods to facilitate a variety of in vitro biochemical and biophysical assays that had previously been hindered by technological constraints. Higher spindle pole body yields, purity, and reproducibility enabled a re-evaluation of the yeast spindle pole body cell cycle phosphoproteome. High confidence phosphorylation assignments were collected and compared to previously published data sets to reduce the ambiguity in the phosphoproteome data set while also expanding the data set to analyze the novel phosphorylation profile of the spindle pole body in G1/S. In vivo characterization of the high confidence phosphorylation sites revealed a combination of phosphorylation events in Spc97 that were essential for cell viability. The adaptability of the spindle pole body purification methods also allowed for the purification of genetic mutants of the spindle pole body, which were studied by biophysical assays. The strength of the microtubule attachment to the spindle pole body was probed by a laser trapping technique to determine how mutations in spindle pole body component Spc110 affected the physical integrity of the spindle pole body. It was shown that mutations in Spc110 decreased the force at which the microtubule was pulled from the spindle pole body, providing mechanical evidence for in vivo phenotypes of mitotic spindle failure. Together, these experiments interrogated several aspects of the yeast spindle pole body in vivo and in vitro to better understand the requirements for and regulation of microtubule nucleation. Advisors/Committee Members: Davis, Trisha N. (advisor).

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APA (6^th Edition):

Fong, K. K. (2016). Regulation of microtubule nucleation and attachment to spindle pole bodies. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/35535

Chicago Manual of Style (16^th Edition):

Fong, Kimberly K. “Regulation of microtubule nucleation and attachment to spindle pole bodies.” 2016. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/35535.

MLA Handbook (7^th Edition):

Fong, Kimberly K. “Regulation of microtubule nucleation and attachment to spindle pole bodies.” 2016. Web. 21 Jan 2021.

Vancouver:

Fong KK. Regulation of microtubule nucleation and attachment to spindle pole bodies. [Internet] [Doctoral dissertation]. University of Washington; 2016. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/35535.

Council of Science Editors:

Fong KK. Regulation of microtubule nucleation and attachment to spindle pole bodies. [Doctoral Dissertation]. University of Washington; 2016. Available from: http://hdl.handle.net/1773/35535

University of Washington

6. Menis, Sergey. Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes.

Degree: PhD, 2014, University of Washington

URL: http://hdl.handle.net/1773/25105

► Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) evolved a number of defense strategies to evade protective mechanisms of the immune system. Classical vaccine… (more)

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APA (6^th Edition):

Menis, S. (2014). Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/25105

Chicago Manual of Style (16^th Edition):

Menis, Sergey. “Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes.” 2014. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/25105.

MLA Handbook (7^th Edition):

Menis, Sergey. “Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes.” 2014. Web. 21 Jan 2021.

Vancouver:

Menis S. Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes. [Internet] [Doctoral dissertation]. University of Washington; 2014. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/25105.

Council of Science Editors:

Menis S. Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes. [Doctoral Dissertation]. University of Washington; 2014. Available from: http://hdl.handle.net/1773/25105

University of Michigan

7. Madak, Joseph. Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors.

Degree: PhD, Medicinal Chemistry, 2018, University of Michigan

URL: http://hdl.handle.net/2027.42/144184

► Rapidly growing cells are dependent on sufficient concentrations of nucleotides to sustain proliferation. One enzyme essential for the de novo synthesis of pyrimidine-based nucleotides is… (more)

▼ Rapidly growing cells are dependent on sufficient concentrations of nucleotides to sustain proliferation. One enzyme essential for the de novo synthesis of pyrimidine-based nucleotides is dihydroorotate dehydrogenase (DHODH); a known therapeutic target for many diseases. In cancer, inhibition of DHODH depletes intracellular pyrimidine nucleotide concentrations and halts the cell cycle at S-phase. This metabolic enzyme is necessary for rapidly growing cells and DHODH inhibition has been demonstrated to sensitize resistant cells to current chemotherapy options. Therefore, we pursued a drug discovery project towards developing novel inhibitors of DHODH. A phenotypic screen was utilized to identify hit compounds that may be suitable leads for a drug discovery project. From this, a lead compound was discovered to inhibit cell growth (MIA PaCa-2, BxPC-3) but through an unknown enzyme target. The essential pharmacophore of the lead compound was elucidated via structure activity relationship (SAR) studies and biologically found to be remarkably similar to brequinar, a potent DHODH inhibitor. Cells were rescued from both the lead compound and brequinar by the addition of uridine, a mimic of a downstream byproduct, and both inhibitors have submicromolar IC50 values toward DHODH. For continued optimization, we sought to improve affinity for the DHODH enzyme. We pursued a structure-guided approach toward the development of improved DHODH inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two residues, T63 and Y356, suitable for novel H-bonding interactions were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of two potent quinoline based analogues with an IC50 against DHODH of 10.6 ± 1.1 nM for one and 32.9 ± 4.6 nM for the other. A co-crystal structure between a quinoline analogue and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to a third 1,7-naphthyridine analogue with an IC50 = 53.9 ± 1.7 nM, which forms a novel H-bond with Y356. In conclusion, the data from our SAR investigation supports further preclinical studies of our improved compounds toward selection of a candidate for early stage clinical development. Finally, to fully understand brequinar-based DHODH inhibition, we developed novel brequinar-based probes. We disclose a 16-step convergent synthesis of the first brequinar-PROTAC and a four-step synthesis to the first mitochondrial-directed brequinar probe. Both of these probes possess cytotoxicity that is superior to brequinar in a colony formation assay and are useful for further pharmacology studies. The collective work described in this dissertation furthers the understanding of DHODH inhibition in cancer, identifies novel sites for electrostatic interaction between brequinar-class inhibitors and DHODH, and has… Advisors/Committee Members: Neamati, Nouri (committee member), Showalter, Hollis D (committee member), Nikolovska-Coleska, Zaneta (committee member), Soellner, Matthew Bryan (committee member).

Subjects/Keywords: Medicinal Chemistry; Biological Chemistry; Science

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APA (6^th Edition):

Madak, J. (2018). Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/144184

Chicago Manual of Style (16^th Edition):

Madak, Joseph. “Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors.” 2018. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/144184.

MLA Handbook (7^th Edition):

Madak, Joseph. “Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors.” 2018. Web. 21 Jan 2021.

Vancouver:

Madak J. Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors. [Internet] [Doctoral dissertation]. University of Michigan; 2018. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/144184.

Council of Science Editors:

Madak J. Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors. [Doctoral Dissertation]. University of Michigan; 2018. Available from: http://hdl.handle.net/2027.42/144184

University of Washington

8. Kim, Jae ook. Microtubule attachment and regulation mechanisms of the budding yeast kinetochore.

Degree: 2016, University of Washington

URL: http://hdl.handle.net/1773/37031

► Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 complexes are the main microtubule binding components of… (more)

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APA (6^th Edition):

Kim, J. o. (2016). Microtubule attachment and regulation mechanisms of the budding yeast kinetochore. (Thesis). University of Washington. Retrieved from http://hdl.handle.net/1773/37031

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Kim, Jae ook. “Microtubule attachment and regulation mechanisms of the budding yeast kinetochore.” 2016. Thesis, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/37031.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Kim, Jae ook. “Microtubule attachment and regulation mechanisms of the budding yeast kinetochore.” 2016. Web. 21 Jan 2021.

Vancouver:

Kim Jo. Microtubule attachment and regulation mechanisms of the budding yeast kinetochore. [Internet] [Thesis]. University of Washington; 2016. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/37031.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kim Jo. Microtubule attachment and regulation mechanisms of the budding yeast kinetochore. [Thesis]. University of Washington; 2016. Available from: http://hdl.handle.net/1773/37031

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Michigan

9. Xu, Hao. Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach.

Degree: PhD, Medicinal Chemistry, 2015, University of Michigan

URL: http://hdl.handle.net/2027.42/113378

► Determining the identity of prodrug activating enzyme(s) is key to understanding the mechanistic basis for enhanced cellular delivery, biodistribution, and prodrug stability. In addition, understanding… (more)

▼ Determining the identity of prodrug activating enzyme(s) is key to understanding the mechanistic basis for enhanced cellular delivery, biodistribution, and prodrug stability. In addition, understanding species-specific prodrug sensitivities is critical for evaluating pre-clinical animal models and drug-drug interactions. Competitive Activity Based Protein Profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class within native proteomes, and has proven to be a powerful approach for activity-guided enzyme annotations. Here we describe a modified ABPP approach using direct substrate competition to determine prodrug binding enzymes of the serine hydrolase (SH) class. The ABPP approach was validated by confirming and validating that CES1 is an oseltamivir binding enzyme in intestinal cell homogenates by gel-based fluorophosphonate (FP) competition. Activation was then confirmed with recombinant hCES1. The competitive binding between oseltamivir and the FP ABPP probe was kinetically analyzed on-gel. Addition of WWL50, a mechanism-based specific carbamate inhibitor of CES1, blocked oseltamivir hydrolysis, and demonstrated exceptional selectivity across >50 active human serine hydrolases by SILAC-ABPP utilizing mass spectrometry. A second reported CES1 inhibitor, WWL79, was shown to inhibit the mouse but not human CES1. Further, complete inhibition of the hydrolysis of several additional ethyl ester prodrugs by WWL50 indicates human CES1 as their dominant activating enzyme in Caco-2 and Hep G2. Overall, we have presented a substrate-competitive activity-based protein profiling (scABPP) approach to broadly survey potential prodrug hydrolyzing enzymes, and determined a very specific hCES1 inhibitor (WWL50). The scABPP approach for surveying the SH class of hydrolase enzymes appears to be a promising methodology for new ester prodrug design and preclinical evaluation. Advisors/Committee Members: Amidon, Gordon L. (committee member), Showalter, Hollis D. (committee member), Garcia, George A. (committee member), Mapp, Anna K. (committee member), Martin, Brent Randall (committee member).

Subjects/Keywords: ABPP; Biological Chemistry; Science

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APA (6^th Edition):

Xu, H. (2015). Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/113378

Chicago Manual of Style (16^th Edition):

Xu, Hao. “Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach.” 2015. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/113378.

MLA Handbook (7^th Edition):

Xu, Hao. “Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach.” 2015. Web. 21 Jan 2021.

Vancouver:

Xu H. Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach. [Internet] [Doctoral dissertation]. University of Michigan; 2015. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/113378.

Council of Science Editors:

Xu H. Profiling Ester Prodrug Activation: An Activity Based Protein Profiling (ABPP) Approach. [Doctoral Dissertation]. University of Michigan; 2015. Available from: http://hdl.handle.net/2027.42/113378

University of Michigan

10. Gupta, Nirupama. THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB.

Degree: PhD, Biological Chemistry, 2011, University of Michigan

URL: http://hdl.handle.net/2027.42/89846

► Thiol-based redox regulation of both prokaryotic and eukaryotic systems plays an important role in modulating cellular functions such as gene expression. Transcriptional factors play an… (more)

▼ Thiol-based redox regulation of both prokaryotic and eukaryotic systems plays an important role in modulating cellular functions such as gene expression. Transcriptional factors play an important role in these modulations and exert redox-dependent activity through highly conserved cysteine residues in them. This thesis illustrates thiol-disulfide redox regulation of two such transcriptional regulators: CprK and Rev-erbβ. CprK is a positive transcriptional regulator from a prokaryotic organism, Desulfitobacterium dehalogenans. CprK has been shown to contain a thiol/disulfide redox switch that undergoes reversible inactivation upon oxidation. We have demonstrated that a disulfide bond, formed between Cys11 and Cys200 in vivo, is responsible for oxidative inactivation of CprK. Moreover, we have shown that Cys11 is also required for binding DNA and that Cys11 mutants are unable to bind DNA due to a change in their tertiary structures. Therefore, Cys11 plays dual roles in maintaining the normal activity of CprK and its redox inactivation. Rev-erbβ is a human nuclear receptor and downregulates the expression of target genes in the presence of heme. Our studies with the ligand binding domain of Rev-erbβ (Rev-erbβ LBD) have revealed that its affinity for heme is controlled by a thiol-disulfide redox-switch, where the oxidized LBD exhibits 5-fold lower affinity for heme than the reduced LBD. Oxidation of the protein also triggers a ligand switch in which the reduced Rev-erbβ LBD binds heme via Cys 384/His568 while the oxidized protein coordinates heme primarily via His/neutral residue ligand pair. Gas binding analyses with the heme-Rev-erbβ LBD complexes show a dependence of binding on the redox status of the heme and the protein. The binding constants for CO and H2S are low and below their physiological concentrations. In contrast, NO shows very low binding affinities for heme-Rev-erbβ LBD complexes. Currently, we are characterizing full-length hRev-erbβ and our preliminary results corroborate our earlier findings on the redox-regulation of heme binding and heme ligand switching in the isolated LBD. In addition, full-length hRev-erbβ shows redox regulation of DNA binding. Overall, these findings indicate a potential role for the thiol-disulfide redox-switch in Rev-erbβ in responding to changes in redox poise in the cell under oxidative stress. Advisors/Committee Members: Ragsdale, Stephen W. (committee member), Banerjee, Ruma (committee member), Martens, Jeffrey Randall (committee member), O'Brien, Patrick (committee member), Stuckey, Jeanne A. (committee member).

Subjects/Keywords: Redox Regulation; Biological Chemistry; Science

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APA (6^th Edition):

Gupta, N. (2011). THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/89846

Chicago Manual of Style (16^th Edition):

Gupta, Nirupama. “THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB.” 2011. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/89846.

MLA Handbook (7^th Edition):

Gupta, Nirupama. “THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB.” 2011. Web. 21 Jan 2021.

Vancouver:

Gupta N. THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB. [Internet] [Doctoral dissertation]. University of Michigan; 2011. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/89846.

Council of Science Editors:

Gupta N. THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB. [Doctoral Dissertation]. University of Michigan; 2011. Available from: http://hdl.handle.net/2027.42/89846

University of Washington

11. Johnson, Ethan Thoreau. Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers.

Degree: PhD, 2002, University of Washington

URL: http://hdl.handle.net/1773/9196

► Protein-pigment complexes are integral to many biochemical reactions. The properties of the pigments in these complexes depend in large part on the protein which serves… (more)

▼ Protein-pigment complexes are integral to many biochemical reactions. The properties of the pigments in these complexes depend in large part on the protein which serves as both scaffold and solvent. The relative positions of the pigments determine the electronic coupling of the system while local protein charges and vibrational motions alter the energies of the molecules. Photosynthetic light-harvesting complexes and the photosynthetic reaction center serve as excellent models for understanding protein-pigment interactions. The electronic coupling between the pigments of the Light-Harvesting Complex II from Rhodopseudomonas acidophila is explored by exciton calculations that treat the excitation of the complex as a combination of monomer and charge-transfer transitions. Comparisons of the calculated absorption and circular dichroism spectra to experimental spectra provide a measure of the delocalization of the excitation and the inhomogeneity of the protein environment. The time-dependent relaxation dynamics of the initial, coherently excited superposition state is also described. In the photosynthetic reaction center from Rhodobacter sphaeroides, the effects of ionizable amino acids on the solvation energy of the oxidized primary electron donor are explored by site-directed mutagenesis. Changes in the reduction potential caused by the mutations are measured and compared to theoretical models that treat the solvation by the protein and surrounding water. Changes in the electrostatic environment also affect the electronic coupling of the two bacteriochlorophylls within the primary electron donor. The mutations alter the relative energies of the basis states in which the charge is localized on one bacteriochlorophyll or the other, and shift the spin distribution and the absorption of the oxidized state. A vibronic model that includes both symmetric and antisymmetric vibrational modes is introduced to explain consistently the changes in the spin distribution, reduction potential and absorption spectrum of the oxidized state. The results reveal the importance of the protein's vibrational modes to the electronic coupling between the pigments.

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Johnson, E. T. (2002). Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9196

Chicago Manual of Style (16^th Edition):

Johnson, Ethan Thoreau. “Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers.” 2002. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9196.

MLA Handbook (7^th Edition):

Johnson, Ethan Thoreau. “Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers.” 2002. Web. 21 Jan 2021.

Vancouver:

Johnson ET. Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers. [Internet] [Doctoral dissertation]. University of Washington; 2002. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9196.

Council of Science Editors:

Johnson ET. Electrostatic interactions and exciton coupling in photosynthetic light-harvesting complexes and reaction centers. [Doctoral Dissertation]. University of Washington; 2002. Available from: http://hdl.handle.net/1773/9196

University of Washington

12. Pierce, Sarah B. The role of glycogen synthase kinase 3 in early xenopus development.

Degree: PhD, 1997, University of Washington

URL: http://hdl.handle.net/1773/9204

► Experiments early in this century indicated that dorsoventral axis formation in the Xenopus embryo requires the activity of the Spemann organizer, or gastrula organizer. Cell… (more)

▼ Experiments early in this century indicated that dorsoventral axis formation in the Xenopus embryo requires the activity of the Spemann organizer, or gastrula organizer. Cell and cytoplasmic transplantation studies subsequently defined the earlier acting blastula organizer, or Nieuwkoop center, which induces formation of the Spemann organizer. Several secreted factors, including members of the Wnt family, were found to mimic the Nieuwkoop center when ectopically expressed, but none of these were shown to be endogenously required for dorsoventral axis formation.Here I describe the isolation and characterization of Xenopus glycogen synthase kinase 3 (Xgsk-3), a serine/threonine kinase which is homologous to a component of the intracellular signaling pathway utilized by the Drosophila Wnt homolog Wg. Using a dominant-negative mutant of Xgsk-3, I show that Xgsk-3 is a negative regulator of the dorsal fate. This indicates that Wnt and Wg signal through homologous pathways and that the Wnt pathway is necessary for endogenous axis formation. In addition, dominant-negative Xgsk-3 acts non-cell-autonomously, indicating that a subsequent intercellular signal is required for Spemann organizer induction.The isolation and characterization of, GBP, a protein that binds Xgsk-3, is also described. GBP induces the formation of a secondary axis, suggesting that GBP interferes with the action of Xgsk-3, but it does not inhibit the enzymatic activity of Xgsk-3. However, GBP joins a complex with Xgsk-3, APC and β-catenin. Xgsk-3 regulates dorsoventral axis formation by negatively regulating the stability of β-catenin and APC is thought to participate in this process. I present a model in which GBP induces axis formation by disrupting the function of this complex.It was found that overexpression of Xgsk-3 in the prospective ectoderm resulted in an expansion of the cement gland and other non-neural ectodermal tissues at the expense of lateral ectoderm, without disrupting the borders of the neural plate. This resulted from a greater responsiveness of the ectoderm to cement gland-inducing signals, which could be mimicked by the secreted factor noggin. A model for the role of Xgsk-3 in ectodermal patterning is presented which considers the expression of Wnt family members that could regulate Xgsk-3 during the time of cement gland induction and the endogenous role of noggin as an inhibitor of BMP-4 signaling.

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Pierce, S. B. (1997). The role of glycogen synthase kinase 3 in early xenopus development. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9204

Chicago Manual of Style (16^th Edition):

Pierce, Sarah B. “The role of glycogen synthase kinase 3 in early xenopus development.” 1997. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9204.

MLA Handbook (7^th Edition):

Pierce, Sarah B. “The role of glycogen synthase kinase 3 in early xenopus development.” 1997. Web. 21 Jan 2021.

Vancouver:

Pierce SB. The role of glycogen synthase kinase 3 in early xenopus development. [Internet] [Doctoral dissertation]. University of Washington; 1997. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9204.

Council of Science Editors:

Pierce SB. The role of glycogen synthase kinase 3 in early xenopus development. [Doctoral Dissertation]. University of Washington; 1997. Available from: http://hdl.handle.net/1773/9204

University of Washington

13. Schmiedeskamp, Mia Ruth. NMR studies of the DNA-binding domain of ADR1.

Degree: PhD, 1996, University of Washington

URL: http://hdl.handle.net/1773/9208

► Nuclear magnetic resonance spectroscopy was used to study the DNA-binding domain of yeast transcription factor ADR1. A polypeptide containing the minimal DNA-binding region was overexpressed… (more)

▼ Nuclear magnetic resonance spectroscopy was used to study the DNA-binding domain of yeast transcription factor ADR1. A polypeptide containing the minimal DNA-binding region was overexpressed in E. coli and purified to homogeneity. The construct, ADR1z, contains two Cys₂-His₂ zinc fingers and an N-terminal sequence required for tight DNA binding. Resonance assignments are presented for ADR1z free and bound to specific DNA. The assignment strategy involved traditional NOESY methods, specific amino acid labeling, and triple resonance experiments. Structural data are presented in the form of chemical shift perturbation maps, ¹³C chemical shift index analyses, NOE connectivities, and ^3J_HNα coupling constants. The zinc fingers of ADR1z differ little in structure from single finger peptides and do not interact in the absence of DNA. The zinc fingers do not alter structure upon DNA binding. In contrast, the N-terminal flanking sequence is random coil in the absence of DNA. This region becomes less mobile, less exposed to solvent, and more structured upon DNA binding. The N-terminus does not interact with exposed portions of the zinc fingers in the complex and likely enhances binding by contacting the DNA directly. No fully regular pattern of secondary structure is detected for the bound N-terminus. This region is not helical; it probably adopts a largely extended conformation. The orientation of the N-terminus with respect to the DNA site is deduced from perturbations induced by fixed paramagnetic cobalt. The N-terminus doubles back and runs antiparallel to the zinc fingers, approaching the center of the palindrome. Chemical shift perturbations are used to map the protein-DNA interface. In striking agreement with previous mutagenesis experiments, these data suggest that contacts arise from R115, H118, and R121 of finger 1 and R143 of finger 2. The fingers bind DNA in different orientations, with the entire helix of finger 1 but only the extreme N-terminus of the helix of finger 2 in proximity to DNA. Suboptimal contact by finger 2 likely necessitates the unusual third contact of finger 1 and the participation of the N-terminus in DNA binding.

Subjects/Keywords: Biological chemistry

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

APA (6^th Edition):

Schmiedeskamp, M. R. (1996). NMR studies of the DNA-binding domain of ADR1. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9208

Chicago Manual of Style (16^th Edition):

Schmiedeskamp, Mia Ruth. “NMR studies of the DNA-binding domain of ADR1.” 1996. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9208.

MLA Handbook (7^th Edition):

Schmiedeskamp, Mia Ruth. “NMR studies of the DNA-binding domain of ADR1.” 1996. Web. 21 Jan 2021.

Vancouver:

Schmiedeskamp MR. NMR studies of the DNA-binding domain of ADR1. [Internet] [Doctoral dissertation]. University of Washington; 1996. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9208.

Council of Science Editors:

Schmiedeskamp MR. NMR studies of the DNA-binding domain of ADR1. [Doctoral Dissertation]. University of Washington; 1996. Available from: http://hdl.handle.net/1773/9208

University of Washington

14. Christensen, Devin Eugene. Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase.

Degree: PhD, 2007, University of Washington

URL: http://hdl.handle.net/1773/9222

► My thesis project addresses the need to understand the function of the Breast Cancer Susceptibility Protein, BRCA1, during the process of protein ubiquitination. A significant… (more)

▼ My thesis project addresses the need to understand the function of the Breast Cancer Susceptibility Protein, BRCA1, during the process of protein ubiquitination. A significant number of known cancer predisposing mutations are found within BRCA1's RING domain which, when associated with BARD1, functions as a ubiquitin ligase. Cancer associated RING mutations of BRCA1 eliminate ligase activity in vitro. Substrates of the BRCA1/BARD1 ubiquitin ligase are likely to be involved in important cellular processes related to the function of BRCA1 in tumor suppression, and provide a direct link between BRCA1's ligase function and the development of cancer.A ubiquitin ligase interacts with both a substrate and a ubiquitin conjugating enzyme (E2) to mediate the transfer of ubiquitin from the E2 to the substrate. To properly identify substrates, the E2s that interact and function with BRCA1 must also be determined. Using a "structure-based" yeast two-hybrid strategy, six new BRCA1-E2 interactions were identified for a total of ten E2s that bind to the BRCA1 RING. I found that for BRCA1 the E2 determines the type of ubiquitination product: depending on the E2 present during a ubiquitination assay different ubiquitination products are synthesized on a protein substrate including, mono-ubiquitin, Lys6-, Lys48-, or Lys63-linked poly-ubiquitin chains.I have identified two proteins that interact with BARD1, BABS and ZO-2. I also discovered that BABS is ubiquitinated by BRCA1/BARD1, in vitro , using a subset of the BRCA1-interacting E2s. Ubiquitin transfer is dictated by substrate-E2 interactions, as E2s that transfer ubiquitin to BABS share the common ability to interact with BABS. Though not tested as a substrate, ZO-2 may provide a link between BRCA1 and the tissue specific nature of cancer associated mutations. ZO-2 is a suspected tumor suppressor that has been linked to estrogen signaling and breast tumorigenesis. The identification of BRCA1/BARD1 substrates, and the fate conferred upon them by the ubiquitin tag, will lead to a better understanding of the association between loss-of-function mutations in BRCA1 and breast cancer.

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Christensen, D. E. (2007). Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9222

Chicago Manual of Style (16^th Edition):

Christensen, Devin Eugene. “Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase.” 2007. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9222.

MLA Handbook (7^th Edition):

Christensen, Devin Eugene. “Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase.” 2007. Web. 21 Jan 2021.

Vancouver:

Christensen DE. Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase. [Internet] [Doctoral dissertation]. University of Washington; 2007. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9222.

Council of Science Editors:

Christensen DE. Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase. [Doctoral Dissertation]. University of Washington; 2007. Available from: http://hdl.handle.net/1773/9222

University of Washington

15. Galburt, Eric A. Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase.

Degree: PhD, 2002, University of Washington

URL: http://hdl.handle.net/1773/9223

► Endonucleases are a large class of enzymes that catalyze the formation of site-specific double strand breaks in DNA. Homing endonucleases are expressed from open reading… (more)

▼ Endonucleases are a large class of enzymes that catalyze the formation of site-specific double strand breaks in DNA. Homing endonucleases are expressed from open reading frames within intervening sequences (introns or inteins) and promote the lateral transfer of their host intron to a cognate intronless allele. A novel mechanism of DNA endonucleolytic cleavage, exhibited by the homing endonuclease I-Ppol and characterized by a histidine general base, has been visualized at high resolution by intermediate trapping and studied computationally. The structures of three separate catalytic species were determined by X-ray crystallography and are presented here. The nudged elastic band method was used to calculate the minimum energy path for the catalyzed reaction and the predicted rate is compared to experiment. In addition, the structure of I-Ppol bound to its DNA target site shows that bound DNA is severely bent, resulting in significant deformations of the minor and major grooves near the scissile phosphates. To study the role of conformational changes within the protein catalyst and the DNA substrate, the structures of both the apo enzyme and the L116A mutant protein/DNA complex with severely decreased binding affinity were determined. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the WT and L116A complexes, indicating that binding involves a large distortion of the DNA and a smaller change in protein conformation. The critical function of leucine 116 is also described.T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of a nicked tRNA generated by a bacterial response to infection and facilitate their repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2 haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.

Subjects/Keywords: Biological chemistry

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

APA (6^th Edition):

Galburt, E. A. (2002). Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9223

Chicago Manual of Style (16^th Edition):

Galburt, Eric A. “Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase.” 2002. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9223.

MLA Handbook (7^th Edition):

Galburt, Eric A. “Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase.” 2002. Web. 21 Jan 2021.

Vancouver:

Galburt EA. Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase. [Internet] [Doctoral dissertation]. University of Washington; 2002. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9223.

Council of Science Editors:

Galburt EA. Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase. [Doctoral Dissertation]. University of Washington; 2002. Available from: http://hdl.handle.net/1773/9223

University of Washington

16. Larkin, Michele Keller. The neurogenic genes in Drosophila oogenesis.

Degree: PhD, 1998, University of Washington

URL: http://hdl.handle.net/1773/9229

► The Notch receptor of Drosophila and its homologues in other organisms mediate cell-cell interactions required for the correct partitioning of cell fates within equivalence groups.… (more)

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Larkin, M. K. (1998). The neurogenic genes in Drosophila oogenesis. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9229

Chicago Manual of Style (16^th Edition):

Larkin, Michele Keller. “The neurogenic genes in Drosophila oogenesis.” 1998. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9229.

MLA Handbook (7^th Edition):

Larkin, Michele Keller. “The neurogenic genes in Drosophila oogenesis.” 1998. Web. 21 Jan 2021.

Vancouver:

Larkin MK. The neurogenic genes in Drosophila oogenesis. [Internet] [Doctoral dissertation]. University of Washington; 1998. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9229.

Council of Science Editors:

Larkin MK. The neurogenic genes in Drosophila oogenesis. [Doctoral Dissertation]. University of Washington; 1998. Available from: http://hdl.handle.net/1773/9229

University of Washington

17. Dantas, Gautam. In silico protein evolution by intelligent design: creating new and improved protein structures.

Degree: PhD, 2005, University of Washington

URL: http://hdl.handle.net/1773/9236

► Natural proteins perform a startling diversity of biological functions, but comprise a miniscule fraction of the theoretical sequence-structure space that polypeptides might occupy. The goal… (more)

▼ Natural proteins perform a startling diversity of biological functions, but comprise a miniscule fraction of the theoretical sequence-structure space that polypeptides might occupy. The goal of protein design is to identify new free-energy minima in this sequence-structure landscape so as to expand the functional repertoire of polypeptides beyond that observed in nature. The accurate design of new proteins requires an exacting understanding of the forces that govern protein structure and folding and should allow for the creation of novel molecular machines and therapeutics. This dissertation details the development of a computational protein design method, RosettaDesign, its application to design new and improved protein structures, and the rigorous experimental characterisation and analyses of the designed proteins to evaluate and improve the design process.First, we applied RosettaDesign to computationally redesign the sequence of nine, natural, globular proteins. Experimental characterisation revealed that eight of the redesigned proteins were folded with similar secondary structure to their wild-type counterparts, and six had stabilities equal to or up to 7 kcal/mol greater than the wild-type counterparts. High resolution structures of the two most dramatically stabilised redesigned proteins (human procarboxypeptidase and U1A) showed them to be virtually identical to the template natural counterparts.Second, we extended the capabilities of RosettaDesign to create a protein topology not observed in nature, by iterating between sequence design and structure prediction. We applied this general computational strategy to create a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. We showed that the Top7 protein is folded and extremely stable, and the striking similarity between the x-ray crystal structure and the designed model demonstrated the unprecedented high-resolution accuracy of the design.Third, we showed that the final 49 C-terminal residues of Top7 (named CFr) can be efficiently mistranslated in E. coli. While an overwhelming majority of naturally mistranslated polypeptides are unfolded, the CFr protein folds into an independently stable, obligate, symmetric homo-dimer, with a novel, high-affinity interface. We further stabilised CFr by disulfide-induced covalent circularisation to create an ideal scaffold for novel functional protein design.

Subjects/Keywords: Biological chemistry

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

APA (6^th Edition):

Dantas, G. (2005). In silico protein evolution by intelligent design: creating new and improved protein structures. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9236

Chicago Manual of Style (16^th Edition):

Dantas, Gautam. “In silico protein evolution by intelligent design: creating new and improved protein structures.” 2005. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9236.

MLA Handbook (7^th Edition):

Dantas, Gautam. “In silico protein evolution by intelligent design: creating new and improved protein structures.” 2005. Web. 21 Jan 2021.

Vancouver:

Dantas G. In silico protein evolution by intelligent design: creating new and improved protein structures. [Internet] [Doctoral dissertation]. University of Washington; 2005. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9236.

Council of Science Editors:

Dantas G. In silico protein evolution by intelligent design: creating new and improved protein structures. [Doctoral Dissertation]. University of Washington; 2005. Available from: http://hdl.handle.net/1773/9236

University of Washington

18. Cole, Toby B. (Toby Brian), 1967-. Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3.

Degree: PhD, 2000, University of Washington

URL: http://hdl.handle.net/1773/9250

► This dissertation demonstrates that zinc is taken up into synaptic vesicles by a mechanism that requires the zinc transporter, ZnT3, at the vesicle membrane and… (more)

▼ This dissertation demonstrates that zinc is taken up into synaptic vesicles by a mechanism that requires the zinc transporter, ZnT3, at the vesicle membrane and begins to address the physiological importance of synaptic vesicle zinc. In situ hybridization and immunohistochemistry showed that ZnT3 expression is limited to zinc-containing neurons in the brain, and that ZnT3 protein resides on the membranes of zinc-rich synaptic vesicles in mouse, monkey, and human brain. A genetic approach was used to test whether ZnT3 was essential for vesicular zinc transport, by generating mice that were homozygous for a null mutation in the ZnT3 gene. Histochemically-reactive zinc (i.e., zinc accessible to Timm and TSQ staining reagents) was undetectable in synaptic vesicles in the brains of these ZnT3-deficient mice, indicating that ZnT3 is required for vesicular zinc transport. Upon neuronal activation vesicular zinc is released into the synaptic cleft, where it has been proposed to modulate excitatory and inhibitory neurotransmitter receptors. In addition, synaptically-released zinc has been widely considered to be the source of the zinc that may kill neurons following seizures or ischemic insults. To assess the physiological importance of synaptic vesicle zinc, ZnT3-deficient mice were tested for deficits in sensorimotor functions, learning and memory, sensitivity to seizure-inducing drugs, and neuronal damage. Most of these functions were remarkably normal in the absence of vesicular zinc. ZnT3-deficient mice were more susceptible than wildtype mice to kainic acid-induced seizures, demonstrating that zinc has a net inhibitory effect on seizures. Despite the lack of histochemically reactive zinc in synaptic vesicles in the ZnT3-deficient brain, zinc accumulated in the cytosol of postsynaptic neurons following seizures, and this zinc accumulation was associated with extensive neuronal death. Thus, the histochemically reactive zinc found in synaptic vesicles is not the major source of toxic zinc accumulation following seizures.

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Cole, Toby B. (Toby Brian), 1. (2000). Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9250

Chicago Manual of Style (16^th Edition):

Cole, Toby B. (Toby Brian), 1967-. “Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3.” 2000. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9250.

MLA Handbook (7^th Edition):

Cole, Toby B. (Toby Brian), 1967-. “Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3.” 2000. Web. 21 Jan 2021.

Vancouver:

Cole, Toby B. (Toby Brian) 1. Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3. [Internet] [Doctoral dissertation]. University of Washington; 2000. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9250.

Council of Science Editors:

Cole, Toby B. (Toby Brian) 1. Elimination of zinc from synaptic visicles in the intact mouse brain by targeted disruption of ZnT3. [Doctoral Dissertation]. University of Washington; 2000. Available from: http://hdl.handle.net/1773/9250

University of Washington

19. Holland, Pamela M., 1963-. Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7.

Degree: PhD, 1999, University of Washington

URL: http://hdl.handle.net/1773/9262

► The mitogen activated protein kinase (MAP kinase) cascade has emerged as an evolutionarily conserved element in the transduction of a wide variety of extracellular signals.… (more)

▼ The mitogen activated protein kinase (MAP kinase) cascade has emerged as an evolutionarily conserved element in the transduction of a wide variety of extracellular signals. In mammals, at least three distinct pathways have been identified. These include ERKs, activated by growth factors and other mitogenic stimuli; JNKs, activated by stressful stimuli such as cytokines, UV radiation and protein synthesis inhibitors; and p38 MAP kinases, which are also responsive to stress-related stimuli. Although the existence of distinct MAP kinase pathways has been suggested to provide a basis for signaling specificity, it remains unclear how this might be achieved at a molecular level. One shared characteristic of MAP kinases is their requirement for phosphorylation on both threonine and tyrosine within a TXY motif for activation. These reactions are catalyzed by a family of kinases termed MAP kinase kinases (MKKs). Multiple MKKs have been shown to share specificity for a particular MAP kinase.In order to further understand the activation, localization and substrate specificity of MKKs, a yeast two-hybrid library screen with MKK1 was performed. Results from this screen identified a novel MKK, termed MKK7. MKK7 is activated by stresses, including exposure of cells to osmotic shock or inflammatory cytokines such as interleukin-1. MKK7 directly phosphorylates and activates the MAP kinase JNK. Several stress-activated protein kinases are capable of activating MKK7, indicating that MKK7 may function in multiple stress-mediated signaling pathways. MKK7 is a murine homolog of Drosophila Hemipterous (Hep) and can functionally rescue Hep deficient flies. Loss of function of Hep in Drosophila inhibits dorsal closure, a morphogenetic movement during late embryogenesis.The human homolog of a Drosophila coiled-coil protein, BicaudalD (hBicD) associates specifically with MKK7, and this association is dependent on hBicD phosphorylation. In Drosophila BicD functions to properly localize developmental factors within the oocyte and the developing embryo. One role of hBicD may be to localize MKK7, or components of an MKK7 signaling pathway, to discrete locations within the cell. The properties of MKK7 demonstrate it may be important for normal development and for mediating stress responses, and these events may be partially regulated by its precise cellular localization.

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Holland, Pamela M., 1. (1999). Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9262

Chicago Manual of Style (16^th Edition):

Holland, Pamela M., 1963-. “Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7.” 1999. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9262.

MLA Handbook (7^th Edition):

Holland, Pamela M., 1963-. “Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7.” 1999. Web. 21 Jan 2021.

Vancouver:

Holland, Pamela M. 1. Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7. [Internet] [Doctoral dissertation]. University of Washington; 1999. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9262.

Council of Science Editors:

Holland, Pamela M. 1. Identification, interactions, and specificity of a novel MAP kinase kinase, MKK7. [Doctoral Dissertation]. University of Washington; 1999. Available from: http://hdl.handle.net/1773/9262

University of Washington

20. Robertson, Timothy Allen, 1976-. Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure.

Degree: PhD, 2007, University of Washington

URL: http://hdl.handle.net/1773/9268

► This work outlines the development, validation and application of a series of novel statistical (knowledge-based) potential functions to the prediction of protein/nucleic-acid interactions from structure.… (more)

Subjects/Keywords: Biological chemistry

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APA (6^th Edition):

Robertson, Timothy Allen, 1. (2007). Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/9268

Chicago Manual of Style (16^th Edition):

Robertson, Timothy Allen, 1976-. “Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure.” 2007. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/9268.

MLA Handbook (7^th Edition):

Robertson, Timothy Allen, 1976-. “Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure.” 2007. Web. 21 Jan 2021.

Vancouver:

Robertson, Timothy Allen 1. Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure. [Internet] [Doctoral dissertation]. University of Washington; 2007. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/9268.

Council of Science Editors:

Robertson, Timothy Allen 1. Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure. [Doctoral Dissertation]. University of Washington; 2007. Available from: http://hdl.handle.net/1773/9268

University of Washington

21. Hoyt, Jill Michelle. The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans.

Degree: PhD, 2017, University of Washington

URL: http://hdl.handle.net/1773/38584

► Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling we performed… (more)

Subjects/Keywords:

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APA (6^th Edition):

Hoyt, J. M. (2017). The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/38584

Chicago Manual of Style (16^th Edition):

Hoyt, Jill Michelle. “The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans.” 2017. Doctoral Dissertation, University of Washington. Accessed January 21, 2021. http://hdl.handle.net/1773/38584.

MLA Handbook (7^th Edition):

Hoyt, Jill Michelle. “The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans.” 2017. Web. 21 Jan 2021.

Vancouver:

Hoyt JM. The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans. [Internet] [Doctoral dissertation]. University of Washington; 2017. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/1773/38584.

Council of Science Editors:

Hoyt JM. The SEK-1 p38 MAP Kinase Pathway Regulates Gq Signaling in C. elegans. [Doctoral Dissertation]. University of Washington; 2017. Available from: http://hdl.handle.net/1773/38584

22. Barfield, Matt. The application of dried blood spots in toxicokinetic and pharmacokinetic studies.

Degree: PhD, 2017, University of Lincoln

URL: http://eprints.lincoln.ac.uk/id/eprint/30876/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733788

► Dried Blood Spot (DBS) sampling is a microsampling technique used throughout the World for neonatal screening. The work set out in this thesis shows the… (more)

Subjects/Keywords: 540; C720 Biological Chemistry

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APA (6^th Edition):

Barfield, M. (2017). The application of dried blood spots in toxicokinetic and pharmacokinetic studies. (Doctoral Dissertation). University of Lincoln. Retrieved from http://eprints.lincoln.ac.uk/id/eprint/30876/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733788

Chicago Manual of Style (16^th Edition):

Barfield, Matt. “The application of dried blood spots in toxicokinetic and pharmacokinetic studies.” 2017. Doctoral Dissertation, University of Lincoln. Accessed January 21, 2021. http://eprints.lincoln.ac.uk/id/eprint/30876/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733788.

MLA Handbook (7^th Edition):

Barfield, Matt. “The application of dried blood spots in toxicokinetic and pharmacokinetic studies.” 2017. Web. 21 Jan 2021.

Vancouver:

Barfield M. The application of dried blood spots in toxicokinetic and pharmacokinetic studies. [Internet] [Doctoral dissertation]. University of Lincoln; 2017. [cited 2021 Jan 21]. Available from: http://eprints.lincoln.ac.uk/id/eprint/30876/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733788.

Council of Science Editors:

Barfield M. The application of dried blood spots in toxicokinetic and pharmacokinetic studies. [Doctoral Dissertation]. University of Lincoln; 2017. Available from: http://eprints.lincoln.ac.uk/id/eprint/30876/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733788

23. Arafeh, Sara Ali Jamal. NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants.

Degree: 2018, Marquette University

URL: https://epublications.marquette.edu/theses_open/460

► NADPH-cytochrome p450 oxidoreductase (CYPOR) is a membrane-bound protein in living cells. CYPOR delivers electrons to cytochrome p450 proteins (CYPs) to catalyze metabolism of drugs and… (more)

▼ NADPH-cytochrome p450 oxidoreductase (CYPOR) is a membrane-bound protein in living cells. CYPOR delivers electrons to cytochrome p450 proteins (CYPs) to catalyze metabolism of drugs and synthesis of steroids. Extraction and solubilization of CYPOR from the membrane is typically done with the TritonX-100 detergent. The amount of the solubilized protein by this detergent, however, remains relatively low to structurally analyze CYPOR with NMR spectroscopy. The goal of the first project in this thesis was to optimize the amount of the extracted CYPOR from the E. coli membrane using various detergents and additives. To this aim, non-ionic detergents with variable hydrophobicity (TritonX-100, X-114, and X-405) and binding strength to the extracted protein (TritonX-100, TWEEN20, and Brij35) were evaluated. Besides, the combinations of TritonX-100 with CHAPS or polyamine and alkylamine additives were assessed. None of these detergents and additives extracted more of CYPOR than the typical amount extracted by TritonX-100. Thus, it was concluded that this detergent extracts all of the available and functional CYPOR. The remaining protein is probably in an unusual and aggregated form. Understanding the details of CYPOR dynamics can be achieved by solution NMR spectroscopy. The initial step towards this goal requires NMR signal assignments of crucial residues in the protein. In this contribution, NMR analysis was performed on the soluble form of CYPOR lacking its N-terminal hydrophobic anchor (Δ56). Two dual cysteine mutants of this form of the protein (Q157C/Q517C and Q157C/N271C) were reacted with 13C-methyl-methanethiosulfonate (13C-MMTS). The resulting residue, which is 13C -methylthiocysteine (13C-MTC) gave strong signals in the 1H-13C HSQC and 1H-13C HMQC spectra of the mutants. The new assignment of MTC-271 at 2.46 ppm 1H, 25.42 ppm 13C was established besides the existing assignments of MTC-157 and MTC-517. The NMR spectra of the two mutants were highly resolved, and they lacked the middle peak. This peak was previously reported in the 1H-13C HMQC spectra of several Δ56 CYPOR mutants. It was concluded that this unspecific peak is due to sample preparation rather than the NMR technique. Advisors/Committee Members: Kovrigin, Evgenii, Reiter, Nicholas, Timerghazin, Qadir.

Subjects/Keywords: Biological and Chemical Physics; Chemistry

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APA (6^th Edition):

Arafeh, S. A. J. (2018). NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants. (Thesis). Marquette University. Retrieved from https://epublications.marquette.edu/theses_open/460

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Arafeh, Sara Ali Jamal. “NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants.” 2018. Thesis, Marquette University. Accessed January 21, 2021. https://epublications.marquette.edu/theses_open/460.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Arafeh, Sara Ali Jamal. “NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants.” 2018. Web. 21 Jan 2021.

Vancouver:

Arafeh SAJ. NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants. [Internet] [Thesis]. Marquette University; 2018. [cited 2021 Jan 21]. Available from: https://epublications.marquette.edu/theses_open/460.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Arafeh SAJ. NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants. [Thesis]. Marquette University; 2018. Available from: https://epublications.marquette.edu/theses_open/460

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Kingston University

24. Vieira, Ana. Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment.

Degree: PhD, 2017, Kingston University

URL: http://eprints.kingston.ac.uk/id/eprint/40908/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739717

► Campylobacter jejuni is a foodborne pathogen recognised as the leading cause of human bacterial gastroenteritis. Undercooked poultry products and contaminated water are considered as the… (more)

▼ Campylobacter jejuni is a foodborne pathogen recognised as the leading cause of human bacterial gastroenteritis. Undercooked poultry products and contaminated water are considered as the most important sources of infection. Antimicrobial therapy is warranted only for immunocompromised patients and, although most people recover from this disease, others may develop rare neurodegenerative disorders such as Guillain-Barre Syndrome (GBS). The latter affects the nerves of the body leading to paralysis and requires extensive medical treatment. The wide use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter over the last decade. Investigation of the molecular mechanisms of antibiotic resistance is considered important to control the spreading of resistant bacteria. CmeABC RND-type multidrug efflux (MDR) pump and the tetO gene found on pTet plasmids mediate tetracycline resistance in Campylobacter. CmeABC MDR pump consists of three components: an outer membrane protein CmeA, an inner membrane drug transporter CmeB and a periplasmic protein CmeC. Even though C. jejuni strains G1 and 11168H do not contain the pTet plasmid, the former was shown to be more resistant to tetracycline (Tet). Comparison of the genome of the G1 strain with that of the reference strain, 11168H, revealed a remarkable difference between the nucleotide sequences of their cmeB genes. In addition, it was observed that the transfer of the pTet plasmid from C. jejuni 81-176 to the G1 strain increased the level of Tet resistance above that of the former strain carrying this plasmid. This finding suggests that CmeB of strain G1 has a higher capacity to excrete this drug than its analogue in C. jejuni strains 81-176 and 11168H and thus, the former strain could be considered as an efflux pump variant with increased resistance to antibiotics. In this study we demonstrate that contribution of MDR pumps to antibiotic resistance might be dependent on the sequence variation of CmeB. Although antibiotic resistance is the main function of MDR pumps, these pumps may have other physiological roles, such as in virulence. An important mechanism of bacterial pathogenesis is the survival of Campylobacter inside environmental hosts. As a host of pathogenic microorganisms, the protozoan Acanthamoeba is a good model for the investigation of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The endosymbiotic relationship between this eukaryotic organism and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. Although some studies suggest that Acanthamoeba supports Campylobacter survival in the environment, the type of interaction between these microorganisms needs to be elucidated. Also, the bacterial factors involved in this interaction remain unknown. Using a modified gentamicin protection assay it was found that C. jejuni 81-176 is able to survive and multiply inside…

Subjects/Keywords: 579.3; Biological sciences; Chemistry

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APA (6^th Edition):

Vieira, A. (2017). Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment. (Doctoral Dissertation). Kingston University. Retrieved from http://eprints.kingston.ac.uk/id/eprint/40908/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739717

Chicago Manual of Style (16^th Edition):

Vieira, Ana. “Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment.” 2017. Doctoral Dissertation, Kingston University. Accessed January 21, 2021. http://eprints.kingston.ac.uk/id/eprint/40908/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739717.

MLA Handbook (7^th Edition):

Vieira, Ana. “Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment.” 2017. Web. 21 Jan 2021.

Vancouver:

Vieira A. Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment. [Internet] [Doctoral dissertation]. Kingston University; 2017. [cited 2021 Jan 21]. Available from: http://eprints.kingston.ac.uk/id/eprint/40908/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739717.

Council of Science Editors:

Vieira A. Acanthamoeba as a model for the investigation of the molecular mechanisms of Campylobacter jejuni pathogenesis and survival in the environment. [Doctoral Dissertation]. Kingston University; 2017. Available from: http://eprints.kingston.ac.uk/id/eprint/40908/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739717

University of Michigan

25. Walker, William. Using CRISPR Knockout Screens to Investigate Biological Phenomena.

Degree: PhD, Chemistry, 2020, University of Michigan

URL: http://hdl.handle.net/2027.42/163211

► The discovery of CRISPR has revolutionized the study of life by providing tools for the precise and controlled manipulation of an organisms’ genome. Knocking out,… (more)

▼ The discovery of CRISPR has revolutionized the study of life by providing tools for the precise and controlled manipulation of an organisms’ genome. Knocking out, suppressing, enhancing, or replacing select genes using CRISPR have revealed their unique contributions to an organisms’ survival with an ease that was unachievable with the prior state-of-the-art methods. This innovation has extended to whole-genome screening, previously conducted with small hairpin RNA (shRNA) libraries. CRISPR-Cas9 pooled library screens have been shown to effectively identify genes essential to a number of physiological processes including drug toxicity and viral infection. Herein, we used previously designed CRISPR-Cas9 knockout libraries to perform whole genome screens on cells subjected to oxidative stress to discovery previously unidentified genes that provide protection under these conditions. Conducting these screens revealed previously identified essential genes in the untreated population in accordance with our expectations. Performing the screens in cell populations under oxidative stress revealed several individual genes with putative roles associated with or accessory to stress tolerance but failed to yield a uniform collection of genes clearly identifiable with this physiological response. Recent efforts to improve upon existing CRISPR methods have centered around using the related Cpf1 nuclease. Cpf1 offers several advantages over Cas9, the most important being its ability to process multiple targeting RNAs from a single expression vector and enabling the simultaneous knockout of multiple genes. With this in mind, we designed randomized duplex Cpf1 libraries targeted to the DHHC-family palmitoyltransferases (PAT). The DHHC PATs constitute a large family of structurally related protein acyltransferases for which substrates and the rules that dictate their specification are still largely unknown. These Cpf1 libraries were then used in growth screens with the hope of determining to what extent, if any, that functional redundancy existed in the substrate profiles of DHHC PATs. Initial screens using these libraries revealed no identifiable change in cell growth for any combination of DHHCs. This was also the case for the included essential gene controls, indicating that inefficiency in Cpf1 activity was to blame for the apparent lack of discernable hits generated by the screen. This result was supported by recent literature that found that unmodified Cpf1 significantly underperformed Cas9 when used to perform pooled screens. Although our work with Cas9 and Cpf1 CRISPR screens did not yield the results desired at the outset of this work they did provide us with several considerations that may serve as useful guidelines for others who wish to make use of these powerful genetic tools. Advisors/Committee Members: Bailey, Ryan Castle (committee member), Martin, Brent Randall (committee member), Jenkins, Paul Michael (committee member), Koutmos, Markos (committee member), Koutmou, Kristin (committee member).

Subjects/Keywords: CRISPR; Biological Chemistry; Science

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APA (6^th Edition):

Walker, W. (2020). Using CRISPR Knockout Screens to Investigate Biological Phenomena. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/163211

Chicago Manual of Style (16^th Edition):

Walker, William. “Using CRISPR Knockout Screens to Investigate Biological Phenomena.” 2020. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/163211.

MLA Handbook (7^th Edition):

Walker, William. “Using CRISPR Knockout Screens to Investigate Biological Phenomena.” 2020. Web. 21 Jan 2021.

Vancouver:

Walker W. Using CRISPR Knockout Screens to Investigate Biological Phenomena. [Internet] [Doctoral dissertation]. University of Michigan; 2020. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/163211.

Council of Science Editors:

Walker W. Using CRISPR Knockout Screens to Investigate Biological Phenomena. [Doctoral Dissertation]. University of Michigan; 2020. Available from: http://hdl.handle.net/2027.42/163211

University of Michigan

26. Kaitany, Kipchumba. Substrate Recognition Mechanism of Protein-only RNase P.

Degree: PhD, Biological Chemistry, 2020, University of Michigan

URL: http://hdl.handle.net/2027.42/163075

► Ribonuclease P (RNase P) is the enzyme responsible for catalyzing the removal of the 5’ leader sequence from precursor transfer RNA (pre-tRNA) during the essential… (more)

▼ Ribonuclease P (RNase P) is the enzyme responsible for catalyzing the removal of the 5’ leader sequence from precursor transfer RNA (pre-tRNA) during the essential maturation process of transfer RNA (tRNA). While RNase P was first discovered in lower order organisms as an RNA-based ribozyme, recent work has revealed the existence of a protein-only RNase P (PRORP) within Eukaryotes. The existence of these two types of independently evolved RNase P enzymes, provides the rare opportunity to compare convergent evolutionary strategies of RNA- and protein-based catalysts. Previous work revealed that both the proteinaceous and RNA-based enzymes achieve nucleolytic activity through the same general two-metal ion mechanism. However, how PRORP recognizes tRNA substrates and whether this mechanism is similar to RNA-based RNase P is poorly understood. This work investigates the substrate recognition strategy of PRORP. Mutagenesis and in vitro binding and catalytic activity assays identified several residues within the conserved pentatricopeptide repeat (PPR) domain of Arabidopsis PRORP1 which form binding interactions with pre-tRNA. A crystal structure of the PPR domain bound to tRNA is solved. Residues critical for binding are shown to form interactions with conserved regions of tRNA. This mode of RNA recognition by a PPR domain is novel, differing from the established sequence-specific RNA model. Interestingly, RNA-based RNase P uses a similar mechanism to detect tRNAs and this is additional evidence of convergent evolution between RNA and protein-only forms of RNase P. Overall this work identifies the mechanism of PPR domain pre-tRNA recognition, providing a foundation for the elucidating the function and mechanism of other PPR-motif containing proteins. B Advisors/Committee Members: Fierke, Carol A (committee member), Koutmos, Markos (committee member), Frank, Aaron Terrence (committee member), Freddolino, Peter Louis (committee member), O'Brien, Patrick (committee member), Palfey, Bruce Allan (committee member).

Subjects/Keywords: Biochemistry; Enzymology; Biological Chemistry; Science

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

APA (6^th Edition):

Kaitany, K. (2020). Substrate Recognition Mechanism of Protein-only RNase P. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/163075

Chicago Manual of Style (16^th Edition):

Kaitany, Kipchumba. “Substrate Recognition Mechanism of Protein-only RNase P.” 2020. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/163075.

MLA Handbook (7^th Edition):

Kaitany, Kipchumba. “Substrate Recognition Mechanism of Protein-only RNase P.” 2020. Web. 21 Jan 2021.

Vancouver:

Kaitany K. Substrate Recognition Mechanism of Protein-only RNase P. [Internet] [Doctoral dissertation]. University of Michigan; 2020. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/163075.

Council of Science Editors:

Kaitany K. Substrate Recognition Mechanism of Protein-only RNase P. [Doctoral Dissertation]. University of Michigan; 2020. Available from: http://hdl.handle.net/2027.42/163075

Kingston University

27. Walker, Michael John. Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity.

Degree: PhD, 2016, Kingston University

URL: http://eprints.kingston.ac.uk/id/eprint/37907/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713146

► This commentary reports on work published between 2005 and 2015 forming a record of a varied career building technical competence alongside strategic skills in the… (more)

▼ This commentary reports on work published between 2005 and 2015 forming a record of a varied career building technical competence alongside strategic skills in the analytical chemistry and molecular biology of food. The unifying theme is practice based problem solving in the scientific regulation and enforcement of food safety and authenticity. The work demonstrates advances in sound, forensically robust, measurement science addressing problems arising from food additives, food authenticity and food allergens. In particular the mature discipline that underpins the regulation and enforcement of food additives is shown to be needed for the management of food allergens. The background to food regulation and enforcement is described alongside technical appeals in the official food control system to develop societally meaningful food surveillance, supported by a sustainable UK based official food control infrastructure (Public Analyst service) at the interface between science and the law. For food additives, publication of previously un-collated results informs regulatory practice and demonstrates the value of scientific collaboration between both jurisdictions on the island of Ireland. A definitive strategy is reported for the chemical analysis and risk assessment of ‘jelly mini-cups’ in which gel forming additives have caused choking fatalities and solutions to problems in the analysis of two illegal toxic additives, morpholine and dimethyl yellow are described. For food allergens the portfolio includes the first study to assess in quantitative terms the level of risk to peanut allergic consumers in take-away catering, leading to better training and similar work on coeliac disease and the availability of ‘gluten-free’ food. Systematic assessment of food allergen analysis and a programme of analytical improvement to support allergen risk assessment and risk management are discussed. A narrative account of an allergen related food sabotage incident and the subsequent Crown Court case and previously uncollated reports of court-sanctions for allergen noncompliances, severe incidents and deaths make key policy and practice recommendations for improvement in these areas. Page 7 of 162 In the study of food authenticity a critical review describes the nitrogen content of important species in the food supply chain as a proxy in the quantitative estimation of high value flesh foods in compound products. An exemplar study follows determining previously unavailable nitrogen factor data for farmed salmon and salmon frame mince. A critical survey of the up skilling of the UK Official Food Control System in DNA food authenticity techniques and major historical and contemporary reviews of food fraud (butter and horsemeat) support substantial policy and analytical recommendations. Many threats to our food supply can be assessed and managed only with the assistance of measurement science. Integrating elements of chemico-legal practice including lessons learned from ‘referee analyses’ and metrology in chemistry this commentary concludes…

Subjects/Keywords: 664; Biological sciences; Chemistry

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APA (6^th Edition):

Walker, M. J. (2016). Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity. (Doctoral Dissertation). Kingston University. Retrieved from http://eprints.kingston.ac.uk/id/eprint/37907/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713146

Chicago Manual of Style (16^th Edition):

Walker, Michael John. “Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity.” 2016. Doctoral Dissertation, Kingston University. Accessed January 21, 2021. http://eprints.kingston.ac.uk/id/eprint/37907/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713146.

MLA Handbook (7^th Edition):

Walker, Michael John. “Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity.” 2016. Web. 21 Jan 2021.

Vancouver:

Walker MJ. Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity. [Internet] [Doctoral dissertation]. Kingston University; 2016. [cited 2021 Jan 21]. Available from: http://eprints.kingston.ac.uk/id/eprint/37907/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713146.

Council of Science Editors:

Walker MJ. Safeguarding food : advances in forensic measurement science and the regulation of allergens, additives and authenticity. [Doctoral Dissertation]. Kingston University; 2016. Available from: http://eprints.kingston.ac.uk/id/eprint/37907/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713146

Kingston University

28. Thring, Tamsyn S. A. Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies.

Degree: PhD, 2012, Kingston University

URL: http://eprints.kingston.ac.uk/id/eprint/22395/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548195

► The role of reactive oxygen species and proteinases involved in the destruction of the extra-cellular matrix, microbial infection and inflammation have been shown as key… (more)

▼ The role of reactive oxygen species and proteinases involved in the destruction of the extra-cellular matrix, microbial infection and inflammation have been shown as key factors influencing the skin ageing process. Many herbal extracts and natural compounds have shown promising activity in neutralising free radicals, preventing oxidative stress and acting as inhibitors of proteinases involved in cellular degradation and this evidence provides the platform for this study. The main aim of this study was to develop a panel of assays which would enable the broad screening of a large variety of herbal extracts, individual product ingredients and whole products for their antimicrobial, antioxidant, and antiageing activities. Over 100 herbal extracts from 37 different plant families were screened at several concentrations in 5 main bioassays. Antioxidant activity of each extract was assessed utilising 3 assays. The gallic acid equivalent assay to assess phenolic content, the trolox equivalent antioxidant capacity assay to demonstrate and rank anti-radical activity, and the superoxide dismutase mimetic activity assay to assess for catalytic antioxidant activity. For antiageing evaluation, two assays incorporating 2 ageing associated enzymes; collagenase and elastase were employed. Thin-layer chromatography was used to demonstrate antioxidant activity for extracts not soluble in the three antioxidant assays and, for similar reasons, a new TLC method was developed to demonstrate anti-collagenase activity for commercial products and problematic extracts. A white tea (WT) extract demonstrated the most desirable activity in all assays. Other extracts which performed particularly well include green tea (GT), witch hazel (WH), rose (R), rooibos tea (RB) and mahonia. Several whole commercial products containing WT and WH also demonstrated promising activities. Cell-based assays involving the effects of the 5 extracts and 2 products above and their activity against H[sub]20[sub]2 induced oxidative stress on human dermal fibroblasts were then implemented. The WT again demonstrated powerful protective effects on the cells and prevented the secretion of the inflammatory cytokine interleukin-8. These results demonstrate that plant extracts have the potential to act as antioxidant, anti ageing and anti-inflammatory ingredients in formulations. The factors involved in skin-ageing are also significant contributors in several inflammatory diseases therefore further investigation of active extracts may prove beneficial for future treatments.

Subjects/Keywords: 615.3; Biological sciences; Chemistry

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APA (6^th Edition):

Thring, T. S. A. (2012). Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies. (Doctoral Dissertation). Kingston University. Retrieved from http://eprints.kingston.ac.uk/id/eprint/22395/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548195

Chicago Manual of Style (16^th Edition):

Thring, Tamsyn S A. “Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies.” 2012. Doctoral Dissertation, Kingston University. Accessed January 21, 2021. http://eprints.kingston.ac.uk/id/eprint/22395/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548195.

MLA Handbook (7^th Edition):

Thring, Tamsyn S A. “Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies.” 2012. Web. 21 Jan 2021.

Vancouver:

Thring TSA. Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies. [Internet] [Doctoral dissertation]. Kingston University; 2012. [cited 2021 Jan 21]. Available from: http://eprints.kingston.ac.uk/id/eprint/22395/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548195.

Council of Science Editors:

Thring TSA. Method development and application for the assessment of the antiageing and antioxidant properties of herbal remedies. [Doctoral Dissertation]. Kingston University; 2012. Available from: http://eprints.kingston.ac.uk/id/eprint/22395/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548195

University of Michigan

29. Houghton, Jacob L. Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases.

Degree: PhD, Medicinal Chemistry, 2012, University of Michigan

URL: http://hdl.handle.net/2027.42/96174

► Aminoglycoside (AG) antibiotics have been widely applied to the treatment of bacterial infections since the discovery of streptomycin. Having enjoyed over 60 years of clinical… (more)

▼ Aminoglycoside (AG) antibiotics have been widely applied to the treatment of bacterial infections since the discovery of streptomycin. Having enjoyed over 60 years of clinical success, AGs have encountered problems with bacterial resistance, as do all antibiotics. The covalent chemical modification of the AG structure by AG-modifying enzymes (AMEs) poses a large threat to the future applicability of AGs. Chloramphenicol (CAM), another natural product with excellent antibacterial properties, suffers from a similar resistance problem. The modification of CAM by chloramphenicol acetyltransferase (CAT) renders it inactive. This dissertation focuses on acetyltransferases conferring resistance to antibiotics, discussing progress towards understanding and overcoming a major hurdle in our ability to combat bacterial infections. Our laboratory reported the unusual regio-versatility of the AG N-acetyltransferase (AAC), Eis, from Mycobacterium tuberculosis (Mtb). We sought to understand the order, number, and regio-specificity of the acetylations carried out by Eis by NMR spectroscopy. We found that Eis not only acetylates multiple positions, but that the positions acetylated and order varies based on the particular AG scaffold. Furthermore, Eis is capable of acetylating amines that have never been reported. We also investigated other anti-TB drugs to determine if it was possible that Eis could cause resistance across drug classes. We found that capreomycin (CAP), a cyclic peptide antibacterial agent, could also be acetylated by Eis. Using our knowledge of AMEs, we sought to develop novel AGs with improved/maintained activity and the ability to avoid modification by AMEs. A series of molecules were synthesized and tested against numerous bacterial strains. These studies and the knowledge gained regarding Eis will serve as a guide to the development of novel AGs targeting Mtb and other pathogens. Additionally, we determined the first X-ray crystal structure of CATI with its natural substrate, CAM, bound in the active site, along with a structure of the unbound form of CATI. Comparison to a structure with fusidic acid (FA) bound and CATIII with CAM bound allowed for a deeper understanding of the broader substrate preference of CATI. We hope that the insights provided in our studies may one day aid in the development of novel CAM analogs. Advisors/Committee Members: Garneau-Tsodikova, Sylvie (committee member), Ballou, David P. (committee member), Showalter, Hollis D. (committee member), Sherman, David H. (committee member), Dotson, Garry Dean (committee member).

Subjects/Keywords: Antibacterial Resistance; Biological Chemistry; Chemistry; Science

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

APA (6^th Edition):

Houghton, J. L. (2012). Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/96174

Chicago Manual of Style (16^th Edition):

Houghton, Jacob L. “Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases.” 2012. Doctoral Dissertation, University of Michigan. Accessed January 21, 2021. http://hdl.handle.net/2027.42/96174.

MLA Handbook (7^th Edition):

Houghton, Jacob L. “Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases.” 2012. Web. 21 Jan 2021.

Vancouver:

Houghton JL. Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases. [Internet] [Doctoral dissertation]. University of Michigan; 2012. [cited 2021 Jan 21]. Available from: http://hdl.handle.net/2027.42/96174.

Council of Science Editors:

Houghton JL. Towards Understanding and Overcoming the Antibiotic Resistance Conferred by Acetyltransferases. [Doctoral Dissertation]. University of Michigan; 2012. Available from: http://hdl.handle.net/2027.42/96174

University of Oxford

30. Haslam, Catherine. Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria.

Degree: PhD, 2019, University of Oxford

URL: http://ora.ox.ac.uk/objects/uuid:a9970faa-079c-4d17-bb17-fdc0a61fd66d ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791760

► Potassium (K⁺) efflux systems (Kef) are K⁺/H⁺ antiporters found in most Gram-negative bacteria. Kef systems play a vital role in the protection of bacteria from… (more)

▼ Potassium (K⁺) efflux systems (Kef) are K⁺/H⁺ antiporters found in most Gram-negative bacteria. Kef systems play a vital role in the protection of bacteria from toxic electrophiles by regulating cytoplasmic pH and consequently demonstrate potential as an antibiotic target. Kef is inhibited by glutathione (γ-L-Glu-L-Cys-Gly, GSH) and activated by glutathione S conjugates (GSX). The proposed model of Kef activation in both EcKefC, from Escherichia coli (E. coli), and SdKefQCTDH₆, a construct of Kef from Shewanella denitrificans (S. denitrificans), is a single amino acid switch at the F441 or F448 capping residue, respectively. The aims of this dissertation were to confirm the proposed mechanism of Kef activation and to investigate novel Kef systems in pathogenic bacteria as an antibiotic target. In work to probe the mechanism of Kef activation, p-trifluoromethyl-L-phenylalanine (ptfmF) was incorporated into SdKefQCTDH₆ at position 448 to give SdKefQCTDH₆(F448ptfmF). The conformational change of SdKefQCTDH₆(F448ptfmF) upon adding inhibitory or activating ligands was analysed using ¹⁹F NMR spectroscopy. The data obtained are consistent with the proposed model for Kef activation: activators displace the capping residue, whereas inhibitors, including GSH, do not. This novel ¹⁹F NMR based assay is an efficient method to determine whether a compound is a Kef activator or inhibitor, vital in the design of new Kef ligands. To investigate whether the role played by Kef in E. coli is conserved across Gram negative bacteria, Kef systems were identified in two pathogenic bacteria: Yersinia pestis (YpKefB; with ancillary proteins -G and -P) and Burkholderia pseudomallei (BpKef1 and BpKef2). Dipeptides OXFKEF12-14 were synthesised and tested in E. coli expressing YpKefGBP, BpKef1, or 2 using the Kirby Bauer disc diffusion assay (Figures A and B). In E. coli MJF645 (mutant strain lacking GSH) and Frag 1 (GSH containing strain) expressing YpKefGBP, OXFKEF13 and 14 were active only in the MJF645 transformant, as a zone of growth inhibition/death was observed (Figure A). To rationalise these results, the expression level of YpKefBH₆ (a His-tagged variant of YpKefB) was determined using western blotting and was found to be similar in both MJF645 and Frag 1. Due to its sequence similarity to YpKefBH₆, it can be inferred that the expression level of YpKefB is also comparable in both cell strains, and so the opposing disc assay results must be caused by another factor. GSH may be inhibiting YpKefB, and so the dipeptides may be unable to outcompete it in the ligand binding site. OXFKEF12-14 could also be being degraded by GSH, giving compounds which inhibit YpKefB or are inert. As OXFKEF12-14 may be unstable in the presence of GSH, we wanted to synthesise dipeptides with improved stability with respect to GSH. Novel thioether analogues with aromatic S substituents were synthesised using the thiol-ene reaction: two methyl…

Subjects/Keywords: Organic Chemistry; Biochemistry; Biological Chemistry; Medicinal Chemistry; Chemical Biology

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

APA (6^th Edition):

Haslam, C. (2019). Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria. (Doctoral Dissertation). University of Oxford. Retrieved from http://ora.ox.ac.uk/objects/uuid:a9970faa-079c-4d17-bb17-fdc0a61fd66d ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791760

Chicago Manual of Style (16^th Edition):

Haslam, Catherine. “Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria.” 2019. Doctoral Dissertation, University of Oxford. Accessed January 21, 2021. http://ora.ox.ac.uk/objects/uuid:a9970faa-079c-4d17-bb17-fdc0a61fd66d ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791760.

MLA Handbook (7^th Edition):

Haslam, Catherine. “Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria.” 2019. Web. 21 Jan 2021.

Vancouver:

Haslam C. Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria. [Internet] [Doctoral dissertation]. University of Oxford; 2019. [cited 2021 Jan 21]. Available from: http://ora.ox.ac.uk/objects/uuid:a9970faa-079c-4d17-bb17-fdc0a61fd66d ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791760.

Council of Science Editors:

Haslam C. Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria. [Doctoral Dissertation]. University of Oxford; 2019. Available from: http://ora.ox.ac.uk/objects/uuid:a9970faa-079c-4d17-bb17-fdc0a61fd66d ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791760

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