+publisher:"Cornell University" +contributor:("Sondermann, Holger")

Cornell University

1. Heberle, Frederick. Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior.

Degree: PhD, Biophysics, 2011, Cornell University

URL: http://hdl.handle.net/1813/33521

► The spatial organization of lipids and proteins in biological membranes is of considerable interest. Multiple lines of evidence support the existence of specialized lipid microdomains… (more)

Subjects/Keywords: fret; rafts; membranes

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APA (6^th Edition):

Heberle, F. (2011). Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/33521

Chicago Manual of Style (16^th Edition):

Heberle, Frederick. “Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior.” 2011. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/33521.

MLA Handbook (7^th Edition):

Heberle, Frederick. “Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior.” 2011. Web. 27 Feb 2021.

Vancouver:

Heberle F. Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior. [Internet] [Doctoral dissertation]. Cornell University; 2011. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/33521.

Council of Science Editors:

Heberle F. Fxc3X96Rster Resonance Energy Transfer In Lipid Bilayers: Theory And Applications For Examining Phase Behavior. [Doctoral Dissertation]. Cornell University; 2011. Available from: http://hdl.handle.net/1813/33521

Cornell University

2. Gottlieb, Colin. Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3.

Degree: PhD, Pharmacology, 2015, Cornell University

URL: http://hdl.handle.net/1813/40896

► Palmitoylation refers to the post-translational attachment of a 16-carbon fatty acid to proteins via a thioester bond at cysteine residues. To date, several hundred palmitoyl… (more)

▼ Palmitoylation refers to the post-translational attachment of a 16-carbon fatty acid to proteins via a thioester bond at cysteine residues. To date, several hundred palmitoyl proteins have been identified in cells, including many proteins active in the pathogenesis of disease. DHHC palmitoyltransferases are integral membrane proteins that catalyze the addition of palmitate to intracellular substrate proteins. Little is known about the structure of DHHC proteins and how conserved elements contribute to the catalytic mechanism of these enzymes. No crystal structure of a DHHC protein has been reported, and the sequence of the DHHC protein catalytic domain contains no predicted secondary structural elements. This work focuses on understanding the palmitoylation, structure, and function of the catalytic cysteine rich domain (DHHCCRD) of DHHC3. DHHC proteins employ a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate and subsequently transfers that palmitate to a substrate protein. Mutation of the cysteine in the DHHC motif to serine (DHHS) blocks autoacylation and transfer activity in vitro, suggesting that the DHHC cysteine is the lone site of palmitoylation in the autoacylated intermediate. I developed a custom mass spectrometry method that directly identified two novel palmitoylation sites in the iii catalytic DHHC-CRD of DHHC3. A subsequent indirect mass spectrometry analysis identified additional palmitoylation sites in this domain. Mutation of identified palmitoylation sites and other conserved cysteines in DHHC3 dramatically reduced enzyme activity and altered the tertiary structure of the DHHC-CRD. These biochemical characteristics were shown to be the result of destabilized zinc binding in the DHHC-CRD. The removal of palmitate resulted in no dose dependent impact on the activity or structure of DHHC3 as revealed by limited proteolysis assays. As the cysteines mutated in this study are highly conserved, and have been associated with similar biochemical characteristics when mutated in other DHHC proteins, I propose that zinc binding is a conserved structural feature of the catalytic domain of DHHC proteins. iv Advisors/Committee Members: Linder,Maurine E. (chair), Sondermann,Holger (committee member), Sevier,Carolyn S (committee member).

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APA (6^th Edition):

Gottlieb, C. (2015). Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/40896

Chicago Manual of Style (16^th Edition):

Gottlieb, Colin. “Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3.” 2015. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/40896.

MLA Handbook (7^th Edition):

Gottlieb, Colin. “Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3.” 2015. Web. 27 Feb 2021.

Vancouver:

Gottlieb C. Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3. [Internet] [Doctoral dissertation]. Cornell University; 2015. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/40896.

Council of Science Editors:

Gottlieb C. Analysis Of Palmitoylation And Zinc Coordination In The Catalytic Domain Of Dhhc3. [Doctoral Dissertation]. Cornell University; 2015. Available from: http://hdl.handle.net/1813/40896

Cornell University

3. Congleton, Johanna. Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells.

Degree: PhD, Environmental Toxicology, 2012, Cornell University

URL: http://hdl.handle.net/1813/29336

► All-trans retinoic acid (ATRA) is successful in treating acute promyelocytic leukemia but has shown limited success with other cancers. Identifying signaling pathways that confer ATRA… (more)

▼ All-trans retinoic acid (ATRA) is successful in treating acute promyelocytic leukemia but has shown limited success with other cancers. Identifying signaling pathways that confer ATRA responsiveness and compounds that enhance the effects of the drug could expand its therapeutic efficacy. The leukocyte antigen CD38 is an ectoenzyme and receptor that propels MAPK signaling and ATRA-induced differentiation when overexpressed. CD38 enzymatic activity regulates calcium flux and NAD metabolism while its receptor functions drive phosphorylation of ERK, c-Cbl, and p85 PI3K. We found that crippling enzymatic activity with inhibitors or a point mutation (CD38 E226Q) did not affect ATRA induction, while a cytosolic truncation (CD38 [DELTA]11-20) that prevented CD38 membrane expression crippled ATRA-induced differentiation. This indicated that CD38 receptor function is important for ATRA induction. In contrast, we found CD38 [DELTA]11-20 cells responded to the monocytic inducer 1,25dihydroxyvitamin D3 similar to wild-type cells, suggesting CD38 signaling is important for granulocytic but not monocytic maturation. We also found that CD38 may sustain ERK phosphorylation to maintain a cellular precommitment memory associated with ATRA exposure. Since CD38 receptor functions appeared necessary for ATRA induction we identified CD38 interaction partners. These included SLP-76, Vav1, and Lyn, and we found that this complex was interrupted by inhibiting Lyn. Lyn inhibition also blocked ATRA- and CD38-stimulated phosphorylation of c-Cbl and p85 PI3K, suggesting some aspects of CD38 signaling are dependent on Lyn kinase activity. Finally we found that the Lyn inhibitors PP2 and dasatinib enhanced ATRA-induced differentiation. Co-treatment with ATRA plus either inhibitor upregulated total Lyn and c-RafpS259, and increased associations between Lyn/c-Raf and cRaf/ERK. This was consistent with increased c-Raf C-terminus phosphorylation that may be regulated by ERK-propelled feedback signaling within a KSR1 scaffold. PP2 and dasatinib had different effects on Lyn activity suggesting that Lyn acts as an adaptor/scaffold independent of its kinase function. These results provide valuable insight as to how ATRA-regulated molecules including CD38, Vav1, SLP-76, Lyn, and the Raf/MEK/ERK axis may enhance the differentiation of acute myelogenous leukemia cells. This report also identifies new therapeutic targets and compounds that may be used in combination with ATRA to improve its clinical efficacy. Advisors/Committee Members: Yen, Andrew (chair), Sondermann, Holger (committee member), Lin, Hening (committee member), Bloom, Stephen Earl (committee member).

Subjects/Keywords: All-trans retinoic acid; cd38; Src family kinases

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APA (6^th Edition):

Congleton, J. (2012). Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/29336

Chicago Manual of Style (16^th Edition):

Congleton, Johanna. “Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells.” 2012. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/29336.

MLA Handbook (7^th Edition):

Congleton, Johanna. “Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells.” 2012. Web. 27 Feb 2021.

Vancouver:

Congleton J. Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells. [Internet] [Doctoral dissertation]. Cornell University; 2012. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/29336.

Council of Science Editors:

Congleton J. Signal Regulation By Cd38, Lyn, And Mapk-Associated Molecules During All-Trans Retinoic Acid-Induced Differentiation Of Acute Myelogenous Leukemia Cells. [Doctoral Dissertation]. Cornell University; 2012. Available from: http://hdl.handle.net/1813/29336

Cornell University

4. Patel, Vaidehi. Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis.

Degree: PhD, Microbiology, 2018, Cornell University

URL: http://hdl.handle.net/1813/64848

► The cell wall is an essential organelle for many bacteria since it provides mechanical strength and prevents bacterial cell lysis due to internal turgor pressure.… (more)

▼ The cell wall is an essential organelle for many bacteria since it provides mechanical strength and prevents bacterial cell lysis due to internal turgor pressure. Additionally, the cell wall is the first line of defense against various external stresses. The mesh of peptidoglycan is a major component of bacterial cell wall that surrounds the outer membrane. The cell wall of the gram-positive rod-shaped bacterium Bacillus subtilis consists of a thick peptidoglycan (PG) layer and phosphate containing anionic polymers called teichoic acids. To maintain the rod shape, B. subtilis elongates its lateral cell wall first, followed by cell division. Both tasks are carried out by distinct but coordinated machineries termed as elongasome (also termed as the Rod complex) and divisome, respectively. The synthesis of peptidoglycan is a dynamic process that is affected by various factors such as the availability of nutrients and external stresses. I have characterized the physiological role of an essential gluconeogenic factor GlmR (previously known as YvcK) in B. subtilis. Homologs of GlmR present in bacteria from different phyla and preliminary observations hint towards a conserved role of GlmR in different organisms. Except the phenotypic observations, for a long-time the function of GlmR has been a mystery. In my dissertation, I show that GlmR functions at the interface of the central carbon metabolism and the peptidoglycan biosynthesis pathway. I have discovered that under gluconeogenic growth condition, GlmR plays an essential role in diverting carbon from the central carbon metabolism to the peptidoglycan biosynthesis, putatively by activation of GlmS- the first enzyme in PG precursor biosynthesis. Additionally, I have studied a B. subtilis signaling nucleotide, cyclic diadenosine monophosphate (c-di-AMP), and an extracytoplasmic function (ECF) σ-factor, σM, in peptidoglycan homeostasis in response to beta-lactam stress. GlmR as well as c-di-AMP are known to be important for virulence in many pathogenic bacteria. GlmR is essential for establishing infection in Listeria monocytogenes and Mycobacterium tuberculosis. Many pathogens use c-di-AMP to modulate the host immune system to facilitate the infection. Taken together, this dissertation adds to the current understanding of the effects of nutrient availability and stress on peptidoglycan homeostasis in B. subtilis which can be further applied to understand bacterial virulence. Advisors/Committee Members: Helmann, John D. (chair), Alani, Eric E. (committee member), Sondermann, Holger (committee member).

Subjects/Keywords: Beta-lactam; Gluconeogenesis; moonlighting enzyme; Genetics; Microbiology; Molecular biology; Metabolism; Bacillus subtilis; Peptidoglycan

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APA (6^th Edition):

Patel, V. (2018). Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/64848

Chicago Manual of Style (16^th Edition):

Patel, Vaidehi. “Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis.” 2018. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/64848.

MLA Handbook (7^th Edition):

Patel, Vaidehi. “Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis.” 2018. Web. 27 Feb 2021.

Vancouver:

Patel V. Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis. [Internet] [Doctoral dissertation]. Cornell University; 2018. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/64848.

Council of Science Editors:

Patel V. Characterizing the role of central carbon metabolism and cell wall stress responses in Bacillus subtilis cell wall synthesis. [Doctoral Dissertation]. Cornell University; 2018. Available from: http://hdl.handle.net/1813/64848

Cornell University

5. Clarke, Benjamin. Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking.

Degree: PhD, Molecular and Cell Biology, 2014, Cornell University

URL: http://hdl.handle.net/1813/37157

► Lysophospholipid acyltransferases (LPATs) catalyze the addition of an acyl chain to a lysophospholipid to form a phospholipid, dramatically altering lipid structure and behavior. These changes… (more)

▼ Lysophospholipid acyltransferases (LPATs) catalyze the addition of an acyl chain to a lysophospholipid to form a phospholipid, dramatically altering lipid structure and behavior. These changes influence membrane curvature, the processes of vesicle and membrane tubule biogenesis, and the structure and trafficking dynamics of secretory organelles. At the beginning of my studies, I investigated the synergistic and antagonistic relationships between the activities of LPATs and phospholipases in regulating membrane trafficking. I went on to identify, by performing an overexpression screen, the human LPAT isoforms that are most important for regulating secretory membrane trafficking. I chose one of these enzymes for further characterization, the human membrane bound O-acyltransferase (MBOAT) gene family member lysophosphatidylcholine acyltransferase 3 (LPCAT3). Using selective membrane permeabilization immunostaining, I determined the topological orientation of LPCAT3's 11 transmembrane domains and luminal active site. I also found that a C-terminal K(x)KXX motif was necessary for LPCAT3 localization to the endoplasmic reticulum (ER). I observed the influence of LPCAT3 activity on the structure and dynamics of the early secretory system, through overexpression which reduced soluble protein secretion to 22% of control and caused relocalization of COPI and COPII vesicle markers. Additionally, LPCAT3 knockdown had profound effects on retrograde trafficking from the Golgi and ER-Golgi intermediate complex (ERGIC) to the ER. Knockdown slowed trafficking of the p58 receptor (ERGIC-53) and Brefeldin A induced recycling of Golgi phosphoprotein of 130 kDa to the ER. Slowed retrograde transport was accompanied by increased membrane tubulation. These effects of knockdown were reflected in the localization of ERGIC-53 and markers of COPII and COPI vesicles, which were mislocalized in knockdown cells. However, the alteration of LPCAT3 expression levels, through knockdown or overexpression, did not disrupt the morphology of the Golgi complex, trans-Golgi network (TGN), adaptor protein 1 (AP-1)/clathrin-coated vesicles, or endosomes. These results suggest that LPCAT3 activity is important for efficient COPI vesiculation, the production of retrograde membrane tubules, and the fusion of retrograde membrane tubules with the ER. These findings support a novel role for LPAT activity in the regulation of COPI function, membrane tubulation, and retrograde trafficking to the ER. Advisors/Committee Members: Brown, William J (chair), Sondermann, Holger (committee member), Feigenson, Gerald W (committee member).

Subjects/Keywords: LPACT3; retrograde transport; COPI vesicle

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APA (6^th Edition):

Clarke, B. (2014). Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/37157

Chicago Manual of Style (16^th Edition):

Clarke, Benjamin. “Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking.” 2014. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/37157.

MLA Handbook (7^th Edition):

Clarke, Benjamin. “Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking.” 2014. Web. 27 Feb 2021.

Vancouver:

Clarke B. Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking. [Internet] [Doctoral dissertation]. Cornell University; 2014. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/37157.

Council of Science Editors:

Clarke B. Characterization Of Human Lysophospholipid Acyltransferases In The Regulation Of Membrane Trafficking. [Doctoral Dissertation]. Cornell University; 2014. Available from: http://hdl.handle.net/1813/37157

Cornell University

6. Li, Li. Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions.

Degree: PhD, Biophysics, 2011, Cornell University

URL: http://hdl.handle.net/1813/29396

► DNA and RNA are very important biological molecules. Both are highly negativelycharged. Positively-charged ions can bind to them through electrostatic interactions. The interaction between DNA… (more)

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APA (6^th Edition):

Li, L. (2011). Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/29396

Chicago Manual of Style (16^th Edition):

Li, Li. “Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions.” 2011. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/29396.

MLA Handbook (7^th Edition):

Li, Li. “Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions.” 2011. Web. 27 Feb 2021.

Vancouver:

Li L. Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions. [Internet] [Doctoral dissertation]. Cornell University; 2011. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/29396.

Council of Science Editors:

Li L. Understanding The Mechanism Of Ion-Mediated Inter-Dna And Inter-Rna Interactions. [Doctoral Dissertation]. Cornell University; 2011. Available from: http://hdl.handle.net/1813/29396

Cornell University

7. Wang, Chenyue. Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin.

Degree: PhD, Chemistry and Chemical Biology, 2015, Cornell University

URL: http://hdl.handle.net/1813/39316

► The heterotrimeric G protein, transducin, and its cognate effector, cyclic GMP (cGMP) phosphodiesterase (PDE) 6 in the vertebrate rod photoreceptors play a pivotal role in… (more)

▼ The heterotrimeric G protein, transducin, and its cognate effector, cyclic GMP (cGMP) phosphodiesterase (PDE) 6 in the vertebrate rod photoreceptors play a pivotal role in visual signal transduction. Rhodopsin, upon activation by light, catalyzes the exchange of GTP for the bound GDP on the [alpha]-subunit of the heterotrimeric G-protein, transducin. Upon GTP binding, transducin dissociates into the GTP-bound alpha subunit ([alpha]T-GTP) and the accessory beta-gamma complex ([beta][gamma]). [alpha]T-GTP activates PDE by removing the inhibition of its catalytic activity by its regulatory gamma subunit (PDE[gamma]). The activated PDE is able to hydrolyze cGMP, a second messenger that is essential for regulating transmembrane ion channels. Due to the hydrolysis of cGMP, an ionic gradient is generated across the cell membrane that enables the photoreceptor to transduce visual signals to the secondary visual nervous cells. Although the photo-transduction signaling pathway has been studied for a few decades, the structural basis by which [alpha]T- GTP stimulates its effector, the PDE, still remains unclear. To investigate the molecular basis of PDE activation by [alpha]T-GTP, we generated a number of point mutants of chimeric [alpha]T and characterized them based on their ability to stimulate PDE. Using techniques that include environmentally-sensitive fluorescence emission and fluorescence polarization from extrinsic probes, we examined the binding affinity of each [alpha]T mutant for PDE, and identified the [alpha]G/[alpha]4 loop as a previously unappreciated region in [alpha]T that is required for full stimulation of PDE activity. We then examined the Switch regions in [alpha]T in detail to determine if they impact PDE function. GTP binding to the [alpha] subunit of heterotrimeric G protein (G[alpha]) results in a conformational change in three regions of G[alpha], designated Switch I, II, and III, respectively. In G[alpha]-GTP, an ionic interaction network that consists of five conserved residues establishes a link between Switch II and III. Mutating a conserved glutamate residue (E241) in this network to alanine yields a dominant-negative [alpha]T mutant, which is capable of binding to PDE but unable to stimulate it. This finding suggests that the [alpha]3 helix, where the glutamate residue is located, is likely to be an important structural element that translates effector binding to effector activation. With the previous discoveries from our lab and other research groups, the results of this study identifies a potential PDE-interacting surface on [alpha]T, which is comprised of the [alpha]3 helix, the [alpha]3/[beta]5 loop, Switch II, Switch III, the [alpha]G/[alpha]4 loop, the [alpha]4 helix and the [alpha]4/[beta]6 loop. Advisors/Committee Members: Cerione, Richard A (chair), Crane, Brian (committee member), Sondermann, Holger (committee member).

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APA (6^th Edition):

Wang, C. (2015). Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/39316

Chicago Manual of Style (16^th Edition):

Wang, Chenyue. “Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin.” 2015. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/39316.

MLA Handbook (7^th Edition):

Wang, Chenyue. “Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin.” 2015. Web. 27 Feb 2021.

Vancouver:

Wang C. Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin. [Internet] [Doctoral dissertation]. Cornell University; 2015. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/39316.

Council of Science Editors:

Wang C. Mechanism Of Retinal Cyclic Gmp Phosphodiesterase Activation By The Alpha Subunit Of Transducin. [Doctoral Dissertation]. Cornell University; 2015. Available from: http://hdl.handle.net/1813/39316

Cornell University

8. Burrowes, Paul Denyse. IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1.

Degree: M.S., Biochemistry, Molecular and Cell Biology, Biochemistry, Molecular and Cell Biology, 2019, Cornell University

URL: http://hdl.handle.net/1813/67730

► The ubiquitination of proteins at the plasma membrane (PM) serves as an endocytosis signal in the downregulation of PM protein levels and cellular responses to… (more)

Subjects/Keywords: Ubiquitin; adapter; Art1; NEDD4; Rsp5; E3 Ligase; Biochemistry

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APA (6^th Edition):

Burrowes, P. D. (2019). IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1. (Masters Thesis). Cornell University. Retrieved from http://hdl.handle.net/1813/67730

Chicago Manual of Style (16^th Edition):

Burrowes, Paul Denyse. “IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1.” 2019. Masters Thesis, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/67730.

MLA Handbook (7^th Edition):

Burrowes, Paul Denyse. “IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1.” 2019. Web. 27 Feb 2021.

Vancouver:

Burrowes PD. IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1. [Internet] [Masters thesis]. Cornell University; 2019. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/67730.

Council of Science Editors:

Burrowes PD. IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1. [Masters Thesis]. Cornell University; 2019. Available from: http://hdl.handle.net/1813/67730

Cornell University

9. Krasteva, Petya. Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors.

Degree: PhD, Molecular and Cell Biology, 2011, Cornell University

URL: http://hdl.handle.net/1813/33598

► In recent years a novel, nucleotide-based small molecule, c-di-GMP, has emerged in the spotlight of scientific investigation as a second messenger unique to the bacterial… (more)

▼ In recent years a novel, nucleotide-based small molecule, c-di-GMP, has emerged in the spotlight of scientific investigation as a second messenger unique to the bacterial world. The discovery that its intracellular levels strictly regulate cell adhesion and persistence of bacterial biofilms on one hand, and motility and virulence of planktonic cells on the other, has related this RNA molecule to a variety of disease states including both chronic and acute bacterial infections. Interestingly, intracellular signaling mechanisms involving c-di-GMP appear to be spatially restricted, yet cellular targets for this nucleotide remain mostly unknown. Here we set out to identify and provide comprehensive structure-function analyses of putative or known c-di-GMP receptors. By using structural biology methods we would first determine the atomic resolution structures and conformational states of appropriate targets and then use these molecular blueprints to guide our research into their mechanistic role in the big picture of intracellular signaling networks. We identified VpsT of V.cholerae as a novel c-di-GMP receptor and solved the crystal structures of the nucleotide-free and c-di-GMP-bound states. Our studies identified two biologically relevant dimerization interfaces and the potential formation of higher order oligomeric species assembling upon nucleotide recognition. VpsT defines a novel class of response regulators that utilize a characteristic structural feature to dimerize upon a signaling input regardless of concurrent phosphorylation events. The relative orientation of the DNA-binding domains of VpsT favors a model in which gene regulation is likely accompanied by major changes in DNA architecture. We showed that VpsT is a master regulator of biofilm formation, integrating c-di-GMP signaling events to inversely control exopolysaccharide production and flagellar motility. In a separate study, we provide a complete mechanistic analysis of the structure and function of LapD, a transmembrane c-di-GMP receptor in P. fluorescens which directly translates intracellular c-di-GMP levels in a signal for biofilm dispersal or initiation. We solved three novel crystal structures capturing distinct intermediates in the inside-out signaling process. Most importantly, our structural and functional analyses helped us identify homologous systems in a number of free-living and pathogenic species, likely controlling biofilm formation or toxin expression in a largely similar manner. Advisors/Committee Members: Sondermann, Holger (chair), Crane, Brian (committee member), Hay, Anthony G. (committee member).

Subjects/Keywords: Bacteria; biofilm; persistence; second messenger; c-di-GMP; receptors; structure; mechanism

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APA (6^th Edition):

Krasteva, P. (2011). Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/33598

Chicago Manual of Style (16^th Edition):

Krasteva, Petya. “Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors.” 2011. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/33598.

MLA Handbook (7^th Edition):

Krasteva, Petya. “Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors.” 2011. Web. 27 Feb 2021.

Vancouver:

Krasteva P. Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors. [Internet] [Doctoral dissertation]. Cornell University; 2011. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/33598.

Council of Science Editors:

Krasteva P. Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors. [Doctoral Dissertation]. Cornell University; 2011. Available from: http://hdl.handle.net/1813/33598

Cornell University

10. Li, Yunxing. ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC.

Degree: PhD, Biophysics, 2018, Cornell University

URL: http://hdl.handle.net/1813/59294

► Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of… (more)

Subjects/Keywords: Cancer metabolism; Biophysics; Glutaminase; mitochondrion

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APA (6^th Edition):

Li, Y. (2018). ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/59294

Chicago Manual of Style (16^th Edition):

Li, Yunxing. “ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC.” 2018. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/59294.

MLA Handbook (7^th Edition):

Li, Yunxing. “ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC.” 2018. Web. 27 Feb 2021.

Vancouver:

Li Y. ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC. [Internet] [Doctoral dissertation]. Cornell University; 2018. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/59294.

Council of Science Editors:

Li Y. ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC. [Doctoral Dissertation]. Cornell University; 2018. Available from: http://hdl.handle.net/1813/59294

Cornell University

11. Chatterjee, Debashree. Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis.

Degree: PhD, Chemistry and Chemical Biology, 2013, Cornell University

URL: http://hdl.handle.net/1813/34224

► Bacteria have developed multiple strategies to adapt to diverse ecological niches and hostile environments. One such strategy involves formation and maintenance of multicellular communities known… (more)

▼ Bacteria have developed multiple strategies to adapt to diverse ecological niches and hostile environments. One such strategy involves formation and maintenance of multicellular communities known as biofilms. In these microbial aggregates, sessile bacterial cells are encased in an extracellular matrix. It has now been established that c-di-GMP, a ubiquitous bacterial second messenger, is a central regulator of this developmental process in bacteria. It exerts its effects on transcriptional, translational and post-translational levels. While diguanylate cyclases and phosphodiesterases with conserved GGDEF and EAL (and HD-GYP) domains are responsible for the production and degradation of the dinucleotide, respectively, the receptors form a more diverse group with degenerate, catalytically inactive GGDEFEAL domain-containing proteins representing a major subfamily. One such protein, LapD from Pseudomonas fluorescens, uses an inside-out signaling mechanism to relay intracellular c-di-GMP concentration to control the localization of an outer-membrane anchored large adhesin protein LapA at the cell surface, by sequestering a periplasmic cysteine protease, LapG. When free, LapG cleaves the N-terminus of LapA, releasing it from the cell surface and ultimately leading to biofilm dispersal. Based on our structure-function analysis, here we propose a mechanism for the c-di-GMP-mediated, regulation of periplasmic proteolysis by LapD. We first elucidate the molecular basis of signal recognition and relay by P. fluorescens LapD and identify orthologous systems in multiple other bacteria including many pathogens such as Legionella pneumophila. This is followed by our work on L. pneumophila LapG, which provides us with the first atomic models of a bacterial protease of the DUF920 family and we are able to identify a highly conserved Ca2+-binding motif integral to its function. We then characterize the LapD-ortholog CdgS9 from L. pneumophila which confirms a common molecular mechanism. The crystal structure of the periplasmic output domain module reveals novel conformations and sheds new light on the mode of activation of the receptor. We finally report the structure of a complex between CdgS9output and P. fluorescens LapG which led to the discovery of the pharmacologically relevant binding interface between the output domain and LapG. Advisors/Committee Members: Cerione, Richard A (chair), Sondermann, Holger (committee member), Lin, Hening (committee member).

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APA (6^th Edition):

Chatterjee, D. (2013). Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/34224

Chicago Manual of Style (16^th Edition):

Chatterjee, Debashree. “Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis.” 2013. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/34224.

MLA Handbook (7^th Edition):

Chatterjee, Debashree. “Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis.” 2013. Web. 27 Feb 2021.

Vancouver:

Chatterjee D. Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis. [Internet] [Doctoral dissertation]. Cornell University; 2013. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/34224.

Council of Science Editors:

Chatterjee D. Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis. [Doctoral Dissertation]. Cornell University; 2013. Available from: http://hdl.handle.net/1813/34224

Cornell University

12. Konyakhina, Tatyana. Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior.

Degree: PhD, Biophysics, 2015, Cornell University

URL: http://hdl.handle.net/1813/39426

► Nonrandom mixing of lipids and proteins in the plasma membrane plays an important role in many cellular processes, including immune signaling, protein sorting, endocytosis, and… (more)

▼ Nonrandom mixing of lipids and proteins in the plasma membrane plays an important role in many cellular processes, including immune signaling, protein sorting, endocytosis, and virus entry and exit, yet the mechanisms governing the coalescence of these small domains are still not completely understood. Functional domains ("lipid rafts") that arise from nonrandom mixing of membrane components are believed to be important in governing the spatial organization of lipids and proteins. Model systems comprising chemically simplified lipid mixtures have played a central role in studies aimed at elucidating the mechanisms responsible for forming and regulating the size and lifetime of membrane domains. In this study we report the first solved phase diagram for the four-component mixture DSPC/DOPC/POPC/Chol (distearoylphosphatidylcholine/ dioleoylphosphatidylcholine/1-palmitoyl,2-oleoylphosphatidylcholine/cholesterol), which exhibits a domain size transition from nanoscopic to macroscopic, including a regime of spatially modulated domains within the region of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. I also describe a novel FRET method used to determine boundaries. This phase diagram clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has iii nanoscopic phase domains of Ld + Lo. Together, our studies show that cells could control domain size and morphology by merely changing lipid composition. Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. In this work we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoylsn-glycero-3phosphocholine/ cholesterol). These mixtures show macroscopic Ld + Lo phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. In addition, modulated phases are observed within the Ld + Lo liquid-liquid immiscibility region upon addition of fourth component POPC. We find mixtures of BSM/SDPC/POPC/Chol to exhibit nanoscopic Ld + Lo domains over a very large volume of composition space. In this work we have successfully accomplished the following goals: 1) solved the first ternary phase diagram containing a PUFA lipid; 2) solved the first phase diagram for a fourcomponent lipid bilayer mixture, including developing a new FRET method for accurate location of boundaries; and 3) synthesized a novel phosphorescent lipid-probe analog. iv Advisors/Committee Members: Feigenson, Gerald W (chair), Zipfel, Warren R. (committee member), Sondermann, Holger (committee member).

Subjects/Keywords: phase diagrams; lipids; lipid rafts

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APA (6^th Edition):

Konyakhina, T. (2015). Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/39426

Chicago Manual of Style (16^th Edition):

Konyakhina, Tatyana. “Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior.” 2015. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/39426.

MLA Handbook (7^th Edition):

Konyakhina, Tatyana. “Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior.” 2015. Web. 27 Feb 2021.

Vancouver:

Konyakhina T. Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior. [Internet] [Doctoral dissertation]. Cornell University; 2015. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/39426.

Council of Science Editors:

Konyakhina T. Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior. [Doctoral Dissertation]. Cornell University; 2015. Available from: http://hdl.handle.net/1813/39426

Cornell University

13. Wasilko, David Jonathan. STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION.

Degree: PhD, Biochemistry, Molecular and Cell Biology, 2018, Cornell University

URL: http://hdl.handle.net/1813/64858

► Legionella pneumophila, a gram-negative intracellular pathogen, is the causative agent of a severe form of pneumonia termed Legionnaires’ disease. Following engulfment by alveolar macrophages, Legionella… (more)

▼ Legionella pneumophila, a gram-negative intracellular pathogen, is the causative agent of a severe form of pneumonia termed Legionnaires’ disease. Following engulfment by alveolar macrophages, Legionella secretes ~330 effector proteins into the host cell. These proteins facilitate the formation of the Legionella-Containing Vacuole (LCV), a replicative niche that allows Legionella to escape phagosomal degradation and subsequently permits intracellular bacterial replication. A growing number of effectors have been reported to alter host ubiquitin signaling, and further characterization of these effectors is crucial for understanding how host defenses are eluded. Here, I present work that identifies the underlying mechanisms that govern E2 binding and ubiquitin transfer to a Legionella-encoded E3 ligase SidC. We have shown that the insertion (INS) domain of SidC is important for binding of ubiquitin-loaded E2. To define the key residues critical for this interaction, we solved the crystal structure of the SidC paralog SdcA in complex with an E2. We also crystallized and solved the structure of the catalytic domain of SidC in complex with its preferred E2 covalently conjugated to ubiquitin. This ternary structure reveals the molecular interactions that lead to the transfer of ubiquitin from E2 to SidC. This study furthers our understanding of the mechanism by which this novel E3 ligase carries out its function. The SidE family of effectors catalyzes a form of ubiquitination that functions independently of the canonical E1, E2, and E3 enzymatic cascade. However, the mechanism by which this reaction is carried out remained elusive. Here, we present the crystal structure of a fragment of the SidE family member SdeA that retains full ubiquitination activity. Our structure reveals that the ubiquitination catalytic module contains two distinct domains, a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the conversion of ubiquitin to ADP-ribose-ubiquitin (ADPR-Ub) and the ligation of ADPR-Ub to substrates are two independent activities of SdeA. Furthermore, our crystal structures of a homologous PDE domain from the L. pneumophila effector SdeD in complex with both Ub and ADPR-Ub reveals an intriguing mechanism of how SdeA processes ADPR-Ub to release AMP and conjugates PR-Ub to serine residues of substrates. Advisors/Committee Members: Mao, Yuxin (chair), Sondermann, Holger (committee member), Brown, William J. (committee member).

Subjects/Keywords: Ubiquitin; Molecular biology; Legionella; E3 Ligase; Biochemistry; Phosphoribosyl Ubiquitination

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APA (6^th Edition):

Wasilko, D. J. (2018). STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/64858

Chicago Manual of Style (16^th Edition):

Wasilko, David Jonathan. “STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION.” 2018. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/64858.

MLA Handbook (7^th Edition):

Wasilko, David Jonathan. “STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION.” 2018. Web. 27 Feb 2021.

Vancouver:

Wasilko DJ. STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION. [Internet] [Doctoral dissertation]. Cornell University; 2018. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/64858.

Council of Science Editors:

Wasilko DJ. STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION. [Doctoral Dissertation]. Cornell University; 2018. Available from: http://hdl.handle.net/1813/64858

Cornell University

14. Goh, Shih Lin. Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling.

Degree: PhD, Biochemistry, 2013, Cornell University

URL: http://hdl.handle.net/1813/34075

► The plasma membrane is a multicomponent mixture of lipids and proteins. Functional domains ("lipid rafts") that arise from nonrandom mixing of membrane components are believed… (more)

▼ The plasma membrane is a multicomponent mixture of lipids and proteins. Functional domains ("lipid rafts") that arise from nonrandom mixing of membrane components are believed to be important in governing the spatial organization of lipids and proteins. Proper compartmentalization of lipids and proteins is vital to facilitate cellular processes, such as signaling, endocytosis and trafficking. While evidence has shown that lipids play an integral role in protein-mediated processes, the chemical complexity of the plasma membrane and the dynamic nature of intermolecular interactions pose challenges for systematic investigations that aim to determine the interplay between lipids and proteins. Model membrane mixtures provide chemically simplified systems for studies of both lipid-lipid, and lipid-protein interactions. In four-component lipid mixtures that model the outer leaflet of the plasma membrane, we observed a nanoscopic-to-macroscopic transition of domain size and morphology by tuning lipid composition. Using fluorescence microscopy imaging of giant unilamellar vesicles (GUVs), we found that this nano-to-macro transition exhibits a regime of patterned fluid domains within the liquid coexistence region of this four-component system. Temperature-dependent FRET and microscopy studies strongly suggest that the patterned domains are thermodynamically stable, lending support for the existence of nanoscopic domains with possibly complex morphology in cellular plasma membranes. Together, our studies allude to a possible mechanism for cells to control domain size and morphology by merely changing lipid composition. The importance of lipids in facilitating cellular processes is evident from examining protein-mediated membrane remodeling events. Using in vitro liposome deformation and liposome binding assays, we examined the activation mechanism of pacsin-1, an F-BAR domain protein enriched in neurons, by dynamin-1 PRD (prolinerich domain). While key basic residues in the PRD were vital to the activation of pacsin-1, we found that pacsin-1's membrane sculpting potential depends on membrane properties such as curvature and bending rigidity. In separate in vitro investigations of HIV-1 Gag-membrane associations, we found that lipid composition strongly affects Gag membrane affinity. These results highlighted the complex nature of protein-mediated membrane remodeling processes, which requires understanding both protein function and lipid phase behavior. Advisors/Committee Members: Feigenson, Gerald W (chair), Sondermann, Holger (coChair), Baird, Barbara Ann (committee member), Brown, William J (committee member).

Subjects/Keywords: model membranes; BAR domain; patterned phases

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APA (6^th Edition):

Goh, S. L. (2013). Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/34075

Chicago Manual of Style (16^th Edition):

Goh, Shih Lin. “Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling.” 2013. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/34075.

MLA Handbook (7^th Edition):

Goh, Shih Lin. “Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling.” 2013. Web. 27 Feb 2021.

Vancouver:

Goh SL. Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling. [Internet] [Doctoral dissertation]. Cornell University; 2013. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/34075.

Council of Science Editors:

Goh SL. Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling. [Doctoral Dissertation]. Cornell University; 2013. Available from: http://hdl.handle.net/1813/34075

Cornell University

15. Meng, Hsien-Wei. Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display.

Degree: PhD, Veterinary Medicine, 2014, Cornell University

URL: http://hdl.handle.net/1813/36083

► SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of… (more)

Subjects/Keywords: Aptamer; Cell-SELEX; Yeast Surface Display

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APA (6^th Edition):

Meng, H. (2014). Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/36083

Chicago Manual of Style (16^th Edition):

Meng, Hsien-Wei. “Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display.” 2014. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/36083.

MLA Handbook (7^th Edition):

Meng, Hsien-Wei. “Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display.” 2014. Web. 27 Feb 2021.

Vancouver:

Meng H. Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display. [Internet] [Doctoral dissertation]. Cornell University; 2014. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/36083.

Council of Science Editors:

Meng H. Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display. [Doctoral Dissertation]. Cornell University; 2014. Available from: http://hdl.handle.net/1813/36083

Cornell University

16. Zhang, Jingwen. Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival.

Degree: PhD, Pharmacology, 2013, Cornell University

URL: http://hdl.handle.net/1813/34284

► Tissue transglutaminase (tTG) is a dual functional acyl transferase/GTPase that has important roles in many cellular processes, as well as in the development of various… (more)

▼ Tissue transglutaminase (tTG) is a dual functional acyl transferase/GTPase that has important roles in many cellular processes, as well as in the development of various disease states. Here we show that tTG expression is frequently up-regulated in high-grade brain tumors or Glioblastoma (GBM), and that tTG is essential for the growth and survival of these highly aggressive cancer cells. We discovered a novel mechanism through which tTG mediates the transformed phenotypes of GBMs, which involves its ability to enhance the signaling activity of the cell surface receptor tyrosine kinase and proto-oncogene, the epidermal growth factor receptor (EGFR). In particular, tTG can associate with and functionally inactivate the E3 ubiquitin ligase c-Cbl, a negative regulator of EGFR, thus delaying its downregulation and extending its signaling half-life. Interestingly, the ability of tTG to influence c-Cbl function is independent of its crosslinking activity, but dependent on a specific conformation that tTG adopts. X-ray crystallographic studies showed that tTG can exist in a nucleotide-bound, crosslinking inactive closed conformation, or a nucleotide free, crosslinking competent, open conformation. Previous findings from my first study and by others showed that ectopically expressed forms of tTG that adopt a closed conformation in cells, promote cell growth and survival, whereas tTG mutants that adopt an open conformation trigger cell death. Thus, we set out to better understand the molecular mechanisms that regulate the transition of tTG between these two different conformational states. Two pairs of intramolecular hydrogen bonds formed between the C-terminal end of tTG and its catalytic core domain were identified and shown to play an important role in maintaining tTG in its closed conformation. Disrupting these interactions causes tTG to adopt a constitutively open configuration and trigger cell death. Collectively, these new insights into the regulation and function of tTG have not only led to its identification as an important player in brain tumor progression, but also raise the possibility that developing strategies that cause tTG to adopt an open conformation could be beneficial for the treatment of human cancers. Advisors/Committee Members: Cerione, Richard A (chair), Baird, Barbara Ann (committee member), Sondermann, Holger (committee member), Linder, Maurine E. (committee member).

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APA (6^th Edition):

Zhang, J. (2013). Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/34284

Chicago Manual of Style (16^th Edition):

Zhang, Jingwen. “Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival.” 2013. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/34284.

MLA Handbook (7^th Edition):

Zhang, Jingwen. “Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival.” 2013. Web. 27 Feb 2021.

Vancouver:

Zhang J. Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival. [Internet] [Doctoral dissertation]. Cornell University; 2013. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/34284.

Council of Science Editors:

Zhang J. Mechanistic Studies On Tissue Transglutaminase And Its Roles In Cell Growth And Survival. [Doctoral Dissertation]. Cornell University; 2013. Available from: http://hdl.handle.net/1813/34284

Cornell University

17. Toulabi, Leila. Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase.

Degree: PhD, Molecular and Cell Biology, 2014, Cornell University

URL: http://hdl.handle.net/1813/36111

► The bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, which is associated with intracellular replication of the bacteria in macrophages of the… (more)

▼ The bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, which is associated with intracellular replication of the bacteria in macrophages of the human innate immune system. L. pneumophila enters host cells through phagocytosis. Once inside the host cells L. pneumophila manipulates vesicular trafficking pathways and establishes Legionella-containing vacuoles (LCV) that serve two purposes: to provide a safe niche for intracellular bacterial replication and to prevent bacterial degradation by the host's bactericidal lysosomal compartments. pneumophila uses a type IV secretion system called L. "Intracellular multiplication/Defective for organelle trafficking" (Icm/Dot), a key virulence factor, to inject almost 300 secreted effector proteins into its host cells. The secreted effector proteins are believed to play important roles in LCV biogenesis and intracellular multiplication of L. pneumophila. Several of these effector proteins are capable of interacting with host phosphoinositides (PIs). However, there has been no report to date of a L. pneumophila-coded PI- metabolizing enzyme. Given the indispensable role of PIs in vesicle trafficking, the main goal of my research was to identify, characterize, and study the role of L. pneumophila PI phosphatases in exploiting host PIs and subverting host cell vesicular trafficking during infection. I was able to show that L. pneumophila encodes an effector protein that we named SidP, which functions as a PI-3-phosphatase specifically hydrolyzing PI(3)P and PI(3,5)P2 in vitro. The enzymatic activity of SidP rescues the growth phenotype of a yeast strain defective in PI(3)P phosphatase activity. My crystal structure of a SidP ortholog from Legionella longbeachae reveals that this unique PI-3-phosphatase is comprised of three distinct domains: a large catalytic domain, an appendage domain inserted into the N-terminal portion of the catalytic domain, and a C-terminal -helical domain. SidP has a small catalytic pocket that likely provides substrate specificity by limiting the accessibility of bulky PIs with multiple phosphate groups. A unique conformation of the conserved arginine residue in the catalytic motif of SidP and the presence of a hydrophobic loop that covers the catalytic motif may participate in regulating the activity of SidP. Together, my identification of a unique family of Legionella PI phosphatases highlights a common scheme of exploiting host PI lipids in intracellular bacterial pathogen infections. Advisors/Committee Members: Mao, Yuxin (chair), Vogt, Volker M (committee member), Sondermann, Holger (committee member), Fromme, Joseph Chris (committee member).

Subjects/Keywords: membrane trafficking; lipid signaling; Legionella; bacterial infection; PI(3)P; phagocytosis; Phosphatase; Structure

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APA (6^th Edition):

Toulabi, L. (2014). Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/36111

Chicago Manual of Style (16^th Edition):

Toulabi, Leila. “Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase.” 2014. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/36111.

MLA Handbook (7^th Edition):

Toulabi, Leila. “Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase.” 2014. Web. 27 Feb 2021.

Vancouver:

Toulabi L. Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase. [Internet] [Doctoral dissertation]. Cornell University; 2014. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/36111.

Council of Science Editors:

Toulabi L. Identification And Structural Characterization Of A Legionella Phosphoinositide Phosphatase. [Doctoral Dissertation]. Cornell University; 2014. Available from: http://hdl.handle.net/1813/36111

Cornell University

18. Byrnes, Laura. Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin.

Degree: PhD, Biophysics, 2013, Cornell University

URL: http://hdl.handle.net/1813/34301

► The membrane of the endoplasmic reticulum (ER) is the site of numerous complex activities essential to the survival of eukaryotic cells. In the membrane sheets… (more)

▼ The membrane of the endoplasmic reticulum (ER) is the site of numerous complex activities essential to the survival of eukaryotic cells. In the membrane sheets of the rough ER (rER), integral membrane proteins are synthesized, folded, modified, and transported throughout the cell. In the smooth ER (sER) tubules, lipids are synthesized and processed with a variety of functionalities, and trafficked to specific membrane compartments. The sER is also the site of calcium storage and controlled release, an important function for several cell types including neurons and muscle. The sER requires energy and the action of numerous proteins to maintain its highly curved, tubular shape and its reticular, interconnected structure. The recent discovery of a family of proteins called atlastins has helped answer some of the questions surrounding the tubular, reticular nature of the sER. Atlastins comprise a group of ER resident proteins that have been shown to facilitate the fusion of membrane tubules within this organelle. They are part of the dynamin superfamily of proteins, which use the energy stored in GTP to sculpt membranes throughout the cell. However, the exact molecular mechanism for how atlastin mediates membrane fusion remained mysterious. In this study, we have taken apart and analyzed atlastin-1, one of three isoforms of atlastin in humans. This isoform is found primarily in the central nervous system and is mutated in the neurodegenerative disorders Hereditary Spastic Paraplegia and Hereditary Sensory Neuropathy. This study has resulted in a collection of three-dimensional, high-resolution structures of atlastin-1's catalytic core fragment, comprising its GTPase and middle domains bound to various forms of the guanine nucleotide. These structures revealed key information about the catalytic mechanism of atlastin-1, as well as interesting and important conformational changes that occur within the structure. Using these structures, as well as information from small-angle X-ray scattering (SAXS), sizeexclusion chromatography coupled to multi-angle light scattering (SEC-MALS), Förster resonance energy transfer (FRET), and enzymatic activity assays, we have put together a general working model for atlastin membrane fusion and have gathered important information about its mechanism that may be used to target the protein for the treatment of disease. Advisors/Committee Members: Sondermann, Holger (chair), Fetcho, Joseph R. (committee member), Brown, William J (committee member), Feigenson, Gerald W (committee member).

Subjects/Keywords: atlastin; dynamin; membrane fusion

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APA (6^th Edition):

Byrnes, L. (2013). Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/34301

Chicago Manual of Style (16^th Edition):

Byrnes, Laura. “Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin.” 2013. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/34301.

MLA Handbook (7^th Edition):

Byrnes, Laura. “Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin.” 2013. Web. 27 Feb 2021.

Vancouver:

Byrnes L. Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin. [Internet] [Doctoral dissertation]. Cornell University; 2013. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/34301.

Council of Science Editors:

Byrnes L. Molecular Basis For Nucleotide-Dependent Conformational Changes Of The Large Gtpase Atlastin. [Doctoral Dissertation]. Cornell University; 2013. Available from: http://hdl.handle.net/1813/34301

19. Pinello, Jennifer Fricke. HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE.

Degree: PhD, Immunology and Infectious Disease, 2017, Cornell University

URL: http://hdl.handle.net/1813/47721

► A crucial step in the life cycle of eukaryotes is sexual reproduction. For productive sex to occur, the first cellular steps that must be taken… (more)

▼ A crucial step in the life cycle of eukaryotes is sexual reproduction. For productive sex to occur, the first cellular steps that must be taken are gamete recognition, adhesion, and membrane fusion. However, little is known about the proteins governing these initial events. Helpful in this regard, are studies on HAP2/GCS1, an ancient and highly conserved gamete membrane protein necessary for successful fertilization in a wide range of eukaryotic species. Efforts have already recognized the necessity of HAP2-mediated membrane fusion in the male gametes of multiple species, but they have come short of describing its mechanism of fusion, structure, or potential origins. Here, we investigated the expression, genetic necessity, and fusogenic capacity of HAP2 during the sexual cycle of the model single-celled ciliate species, Tetrahymena thermophila. Unlike in other species, we found that HAP2 is expressed in all seven mating types of T. thermophila, and fusion and fertility is only prevented when HAP2 is deleted from both cells of a mating pair. Along the way, we also functionally examined a hypothetical HAP2 accessory protein, ZFR1, probed the life cycle of a ciliated fish parasite for the presence of a conjugation stage, and accidentally found instances of unexpected parthenogenesis in T. thermophila. Perhaps most importantly, we developed a flow-cytometry-based assay for quantifying sexual cell fusion events in an in vivo model of eukaryotic fertilization, identified a predicted high-confidence structural homology between HAP2 and class II viral fusogens, and tested the functional extent of this homology through targeted deletions and biophysical tests of the HAP2 fusion loop. In addition to providing indications of a viral-like fusogenic mechanism for HAP2, these findings that show a structural and functional similarity between a viral and gamete fusogen harken back to early theories that a “selfish” genetic element promoting membrane fusion as a means for intergenomic transmission led to the origin of eukaryotic sex. Advisors/Committee Members: Clark, Theodore G. (chair), Whittaker, Gary R (committee member), Sondermann, Holger (committee member), Holowka, David Allan (committee member).

Subjects/Keywords: gamete; HAP2; membrane fusion; sex; Tetrahymena; Evolution & development; Cellular biology; Microbiology

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APA (6^th Edition):

Pinello, J. F. (2017). HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/47721

Chicago Manual of Style (16^th Edition):

Pinello, Jennifer Fricke. “HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE.” 2017. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/47721.

MLA Handbook (7^th Edition):

Pinello, Jennifer Fricke. “HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE.” 2017. Web. 27 Feb 2021.

Vancouver:

Pinello JF. HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE. [Internet] [Doctoral dissertation]. Cornell University; 2017. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/47721.

Council of Science Editors:

Pinello JF. HAP2-MEDIATED CELL FUSION IN A SEXUAL CILIATE. [Doctoral Dissertation]. Cornell University; 2017. Available from: http://hdl.handle.net/1813/47721

Cornell University

20. Wang, Qi. Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators.

Degree: PhD, Biophysics, 2011, Cornell University

URL: http://hdl.handle.net/1813/33600

► Protein domains are the basic functional modules that maintain cell functions at a molecule level. Previous studies have mainly focused on the functions of isolated… (more)

▼ Protein domains are the basic functional modules that maintain cell functions at a molecule level. Previous studies have mainly focused on the functions of isolated protein domains. The general objective of this thesis is to understand functions and regulations of multi-domain containing proteins. The study is based on two protein classes: naturally occurred BAR domain-containing proteins and artificially engineered calcium indicators. BAR domain super-family proteins BAR (Bin/Amphiphysin/Rvs) domain super-family proteins are peripheral membrane proteins that regulate membrane curvatures during the membrane remodeling events such as endocytosis, vesicular trafficking and cell growth. Via multiple biophysical approaches, I systematically studied BAR domain functions in Sorting Nexin 9, Endophilin and Pacsins at the presence of other protein domains. Two major findings are presented in this thesis. First I show that the diverse membrane sculpture activity of BAR domains is encoded in their unique molecular structures, and is influenced by membrane properties. Second, I show that this function diversity is highly regulated by other protein domains. Some protein domains have synergetic effects and play important roles in regulating cellular membrane remodeling. This work is significant in that it provides the molecular basis for the functional diversity of BAR domains and established the regulatory mechanism of BAR domain mediated-membrane deformation process. Genetically Encoded Calcium Indicators Genetically encoded calcium indicator GCAMP is an artificially designed multi-domain containing protein that can be endogenously expressed in cells to monitor calcium signals. The molecular mechanisms of its signal response and fast kinetics are poorly understood. Using fluorescent spectrometry and site-directed mutagenesis, I show that the calcium-dependent brightness of GCAMP is due to the different protonation states of the chromophore. Structural characterization of GCAMP reveals that the calmodulin domain regulates chromophore protonation states via a sophisticated water-mediated hydrogen bond network. This finding provided a general scheme for designing GCAMP-like sensors. Furthermore, I show that distinct electron properties of the protonated and deprotonated chromophore can be applied to design color switchable fluorescent proteins. This finding provides a novel approach to design the ratio metric pH sensors with an improved sensitivity. Advisors/Committee Members: Sondermann, Holger (chair), Sethna, James Patarasp (committee member), Feigenson, Gerald W (committee member), Collins, Ruth N. (committee member).

Subjects/Keywords: bar; f-bar; calcium indicators; GECIs; pacsin; sorting nexin; gcamp; mkate; crystal structure; em

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APA (6^th Edition):

Wang, Q. (2011). Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/33600

Chicago Manual of Style (16^th Edition):

Wang, Qi. “Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators.” 2011. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/33600.

MLA Handbook (7^th Edition):

Wang, Qi. “Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators.” 2011. Web. 27 Feb 2021.

Vancouver:

Wang Q. Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators. [Internet] [Doctoral dissertation]. Cornell University; 2011. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/33600.

Council of Science Editors:

Wang Q. Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators. [Doctoral Dissertation]. Cornell University; 2011. Available from: http://hdl.handle.net/1813/33600

Cornell University

21. Nadworny, Alyson. Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit.

Degree: PhD, Pharmacology, 2012, Cornell University

URL: http://hdl.handle.net/1813/31394

► Owing in large part to the low regenerative capacity of the heart, cardiovascular disease (CVD) remains a leading cause of morbidity and mortality worldwide. Compelling… (more)

▼ Owing in large part to the low regenerative capacity of the heart, cardiovascular disease (CVD) remains a leading cause of morbidity and mortality worldwide. Compelling evidence suggests the presence of resident cardiac precursor cells (CPCs) in the neonatal and, to a lesser extent, in the adult myocardium. These CPCs are identified based on specific marker expression including c-kit, a type III receptor tyrosine kinase. Here we report the isolation and characterization of neonatal c-kit+ CPCs from a bacterial artificial chromosome (BAC) transgenic mouse in which enhanced green fluorescent protein (EGFP) is driven by the kit locus. We demonstrate that c-kit expression identifies CPCs capable of expanding and differentiating into all three cardiac lineages. In addition, we establish that this mesodermally derived cell population comprises a mixture of precursors and more mature cells in the early stages of lineage commitment. Progressing from the identification of CPCs, cell-based therapy has emerged as a strategy for cardiac repair. However, the pathological environment of the infarcted heart, attributed in part to the production of reactive oxygen species (ROS), impedes transplanted cell survival. In spite of well-known pathological roles, ROS also participate in normal cell development and recent research indicates that redox state is a critical regulator of precursor cell function. Because the NADPH oxidases (Nox) are a major source of ROS in the heart, a significant amount of research has been conducted to elucidate roles for these enzymes in cardiomyogenesis and heart failure. However, little research has been directed at the function of Nox enzymes in CPCs. As a successful cell-based therapy requires both the survival of transplanted cells and their differentiation into cardiomyocytes, understanding the mechanisms of ROS signaling in CPCs is critical. Therefore, utilizing genetic tools to manipulate the Nox enzymes of neonatal c-kit+ CPCs, I demonstrate that specific cardiac Nox homologues are critical modulators of c-kit+ cell precursor and differentiation status. I speculate that these and other redox genes identified by messenger RNA (mRNA) arrays will provide novel targets for improving the regenerative capacity of c-kit+ CPCs, which can later be integrated into cell-based therapies for myocardial infarction (MI). Advisors/Committee Members: Davisson, Robin L (chair), Tumbar, Tudorita (committee member), Kotlikoff, Michael I. (committee member), Stuhlmann, Heidi (committee member), Sondermann, Holger (committee member).

Subjects/Keywords: Redox; Stem Cells; Cardiac Differentiation

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APA (6^th Edition):

Nadworny, A. (2012). Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/31394

Chicago Manual of Style (16^th Edition):

Nadworny, Alyson. “Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit.” 2012. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/31394.

MLA Handbook (7^th Edition):

Nadworny, Alyson. “Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit.” 2012. Web. 27 Feb 2021.

Vancouver:

Nadworny A. Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit. [Internet] [Doctoral dissertation]. Cornell University; 2012. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/31394.

Council of Science Editors:

Nadworny A. Examining The Role Of Redox Biology In A Population Of Neonatal Cardiac Precursor Cells Marked By The Type Iii Receptor Tyrosine Kinase, C-Kit. [Doctoral Dissertation]. Cornell University; 2012. Available from: http://hdl.handle.net/1813/31394

Cornell University

22. O'Donnell, John P. Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin.

Degree: PhD, Pharmacology, 2017, Cornell University

URL: http://hdl.handle.net/1813/59027

► Homotypic membrane fusion of the smooth endoplasmic reticulum (ER) is catalyzed by atlastin (ATL), a member of the dynamin superfamily. Maintaining proper membrane architecture and… (more)

▼ Homotypic membrane fusion of the smooth endoplasmic reticulum (ER) is catalyzed by atlastin (ATL), a member of the dynamin superfamily. Maintaining proper membrane architecture and dynamics in this highly compartmentalized organelle is accomplished in part through ATL-mediated fusion of ER tubules to form a highly interconnected reticular system of three-way junctions. Mechanistically, ATL couples the energy from GTP hydrolysis to a series of conformational changes and homodimerization events that result in membrane fusion. Higher eukaryotes express three ATL isoforms. Two of these isoforms, human ATL1 and ATL3 have been implicated in neurological disorders, namely Hereditary Spastic Paraplegia (HSP) and Hereditary Sensory Neuropathy (HSN) where axon function is impaired. Disease causing mutations in atl1 and atl3 are dominant-negative and cause the exchange of reticular ER for long unbranched membrane tubules. The underlying mechanistic differences between human isoforms and disease-associated mutations are poorly understood. Here we identify and give order to discrete temporal steps in the catalytic cycle for ATL1 and ATL3. A comparative study demonstrates that isoforms sample the same series of events but do so at different timescales. We also establish a conserved mechanism for the displacement of the catalytic magnesium ion post GTP hydrolysis. Investigation into mechanisms of disease pathogenesis yields structural and functional defects of an atypical HSP variant. This HSP mutant, ATL1-F151S, is impaired in its GTP hydrolysis cycle but is capable of sampling the high affinity homodimer when bound to a transition state analog. These findings along with additional structural information from mutant proteins define a mechanism for allosteric coupling where F151 is the central residue in a hydrophobic interaction network connecting the active-site to an inter-domain interface responsible for nucleotide loading. Advisors/Committee Members: Sondermann, Holger (chair), Lin, Hening (committee member), Cerione, Richard A. (committee member), Sevier, Carolyn S. (committee member).

Subjects/Keywords: membrane fusion; allosteric regulation; dynamin-related proteins; enzyme mechanism; enzyme structure; GTPase; Biochemistry; Biophysics

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APA (6^th Edition):

O'Donnell, J. P. (2017). Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/59027

Chicago Manual of Style (16^th Edition):

O'Donnell, John P. “Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin.” 2017. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/59027.

MLA Handbook (7^th Edition):

O'Donnell, John P. “Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin.” 2017. Web. 27 Feb 2021.

Vancouver:

O'Donnell JP. Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin. [Internet] [Doctoral dissertation]. Cornell University; 2017. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/59027.

Council of Science Editors:

O'Donnell JP. Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin. [Doctoral Dissertation]. Cornell University; 2017. Available from: http://hdl.handle.net/1813/59027

23. Reinholt, Sarah Jessica. HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS.

Degree: PhD, Biological and Environmental Engineering, 2017, Cornell University

URL: http://hdl.handle.net/1813/47718

► Precision medicine is the idea where diagnostics and therapeutics are catered to each individual patient to provide personalized care that is optimally effective. For this… (more)

▼ Precision medicine is the idea where diagnostics and therapeutics are catered to each individual patient to provide personalized care that is optimally effective. For this to be achieved, technologies must exist that extensively examine samples and provide a highly detailed diagnosis for each patient, and processes must exist that can produce personalized drugs that specifically target the patient’s illness. Aptamers are short single stranded nucleic acids that bind to their targets with high affinity and specificity. Aptamers could make a substantial impact toward the goal of precision medicine. However, one of the main challenges preventing aptamers from reaching their potential is the efficient discovery of new high-affinity aptamers. Currently, aptamer selections are very time consuming and expensive, and often do not result in the discovery of a high-quality aptamer. The ability to reliably select aptamers with high affinity and specificity is paramount to the widespread use of aptamers. Consequently, there is great interest in improving selection technology to obtain high-quality aptamers much more rapidly. Toward this effort, we have developed a Microplate-based Enrichment Device Used for the Selection of Aptamers (MEDUSA) that uses affinity microcolumn chromatography. Its versatile 96-well microplate-based design allows this device to be compatible with downstream plate-based processing in aptamer selections, and it lends itself to automation using existing microplate-based liquid-handling systems. MEDUSA is also reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We have demonstrated its use in high-throughput aptamer selections, characterization and optimization of the aptamer selection process, and characterization of previously selected aptamers. More specifically, MEDUSA was used to perform 96 simultaneous tests that determined the optimal target loading on resin to maximize aptamer enrichment for three target proteins, GFP, HSF, and NELF-E. These tests also verified the specificity of aptamers to these three proteins, as well as the non-specific binding of two suspected background binding aptamers. MEDUSA was also used to performed novel RNA aptamer selections to 19 different targets simultaneously. For these selections, a new, more efficient selection strategy was tested that greatly reduced the selection time and reagent consumption. Through the use of MEDUSA, aptamer selections can be optimized and performed in a high-throughput manner, and the success rate of novel aptamer discovery can be drastically improved. In addition to the improvement of novel aptamer discovery, developing valuable applications that use aptamers is of equal importance. An area of study in which aptamers could be of great benefit is cancer. Cancer cells are extremely diverse and contain genetic mutations that allow them to escape the regulatory processes necessary for the healthy function of tissues and organs. Moreover, there are numerous mechanisms for malignancy each with… Advisors/Committee Members: Craighead, Harold G (chair), Sondermann, Holger (committee member), Kirby, Brian (committee member).

Subjects/Keywords: Biomedical engineering

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APA (6^th Edition):

Reinholt, S. J. (2017). HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/47718

Chicago Manual of Style (16^th Edition):

Reinholt, Sarah Jessica. “HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS.” 2017. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/47718.

MLA Handbook (7^th Edition):

Reinholt, Sarah Jessica. “HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS.” 2017. Web. 27 Feb 2021.

Vancouver:

Reinholt SJ. HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS. [Internet] [Doctoral dissertation]. Cornell University; 2017. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/47718.

Council of Science Editors:

Reinholt SJ. HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS. [Doctoral Dissertation]. Cornell University; 2017. Available from: http://hdl.handle.net/1813/47718

24. Acevedo, Lucila Andrea. NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT.

Degree: PhD, Biophysics, 2018, Cornell University

URL: http://hdl.handle.net/1813/59553

► Protein-Protein interactions are fundamental for varied biological processes. These interactions are governed by different forces between the proteins, which result in a wide range of… (more)

▼ Protein-Protein interactions are fundamental for varied biological processes. These interactions are governed by different forces between the proteins, which result in a wide range of interaction strengths. Nuclear Magnetic Resonance (NMR) is an atomistic technique commonly used to understand protein-protein interaction mechanisms, especially weak interactions (KD>µM). In this work, two different biological systems are studied by NMR. The first system that was studied is related to two proteins involved in cytoskeleton dynamics and cell migration, and the second is related to an enzyme-substrate interaction that controls a gene transcription regulation circuit. In the first case, a novel mechanism for protein-protein interaction between the Ena/VASP Homology 1 (EVH1) domain of the actin polymerize regulator Vasodilator-Stimulated phosphoprotein (VASP) and the cytoskeleton protein Zyxin was determined. This interaction is important for cellular shape, cell motility and stress fiber repair. This study presented a model for a mechanism of action where phosphorylation can act as a stoichiometric switch and can explain the importance of the phosphorylation cycle in the localization of VASP by Zyxin. The second protein-protein interaction focuses on the prolyl isomerase enzyme LRT2 (lateral rootless2) acting on the repressor protein OsIAA11. This interaction is critical for lateral root initiation in rice. The mechanism of action for this process is through the regulation of the cis-trans isomerization of the well conserved 104Trp-Pro105 peptide bond in OsIAA11. OsIAA11 plays a role in repressing gene expression in a manner dependent on the amount of the phytohormone Auxin. At high Auxin concentration, OsIAA11 is specifically degraded in the cis conformation of the 104W-P105 peptide bond. Thus, studies of LRT2 catalysis of OsIAA11 isomerization are essential to understand this fundamental biological process. In this dissertation, the microscopic equilibrium and rate constants of the LRT2 enzymatic cycle acting on OsIAA11 were determined by NMR lineshape analysis and validated by independent NMR exchange measurements. We found that LRT2 is optimally tuned for supplying maximum OsIAA11 in the cis isomer into a slower degradation pathway. Moreover, these studies predict that the catalyzed exchange rate is insensitive to concentrations of OsIAA11 at cellular conditions but scales lineally to the LRT2 concentration. Advisors/Committee Members: Nicholson, Linda K. (chair), Sondermann, Holger (committee member), Oswald, Robert Edward (committee member).

Subjects/Keywords: Auxin circuit; Cyclophilins; PPIASE; Protein-protein interaction; VASP-Zyxin; NMR; Biochemistry; Biophysics

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APA (6^th Edition):

Acevedo, L. A. (2018). NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/59553

Chicago Manual of Style (16^th Edition):

Acevedo, Lucila Andrea. “NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT.” 2018. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/59553.

MLA Handbook (7^th Edition):

Acevedo, Lucila Andrea. “NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT.” 2018. Web. 27 Feb 2021.

Vancouver:

Acevedo LA. NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT. [Internet] [Doctoral dissertation]. Cornell University; 2018. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/59553.

Council of Science Editors:

Acevedo LA. NMR AS A TOOL TO QUANTITATIVELY CHARACTERIZE MOLECULAR MECHANISMS GOVERNING ACTIN DYNAMICS AND RICE LATERAL ROOT DEVELOPMENT. [Doctoral Dissertation]. Cornell University; 2018. Available from: http://hdl.handle.net/1813/59553

25. Nazarova, Evgeniya. Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis.

Degree: PhD, Microbiology, 2017, Cornell University

URL: http://hdl.handle.net/1813/56718

► Mycobacterium tuberculosis (Mtb) as a causative agent of human tuberculosis has successfully adapted to survive within the host for decades, and the bacterium’s ability to… (more)

Subjects/Keywords: cholesterol; fatty acids; LucA; macrophages; mce; tuberculosis; Biochemistry; Microbiology

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APA (6^th Edition):

Nazarova, E. (2017). Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/56718

Chicago Manual of Style (16^th Edition):

Nazarova, Evgeniya. “Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis.” 2017. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/56718.

MLA Handbook (7^th Edition):

Nazarova, Evgeniya. “Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis.” 2017. Web. 27 Feb 2021.

Vancouver:

Nazarova E. Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis. [Internet] [Doctoral dissertation]. Cornell University; 2017. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/56718.

Council of Science Editors:

Nazarova E. Assimilation of fatty acids and cholesterol in Mycobacterium tuberculosis. [Doctoral Dissertation]. Cornell University; 2017. Available from: http://hdl.handle.net/1813/56718

Cornell University

26. Ohouo, Patrice. Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae.

Degree: PhD, Molecular and Cell Biology, 2013, Cornell University

URL: http://hdl.handle.net/1813/34334

Subjects/Keywords: DNA damage; Replication checkpoint; Signaling; DNA repair; Mec1; Rad53; Slx4; Rtt107; Dpb11; Rad9

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

APA (6^th Edition):

Ohouo, P. (2013). Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/34334

Chicago Manual of Style (16^th Edition):

Ohouo, Patrice. “Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae.” 2013. Doctoral Dissertation, Cornell University. Accessed February 27, 2021. http://hdl.handle.net/1813/34334.

MLA Handbook (7^th Edition):

Ohouo, Patrice. “Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae.” 2013. Web. 27 Feb 2021.

Vancouver:

Ohouo P. Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae. [Internet] [Doctoral dissertation]. Cornell University; 2013. [cited 2021 Feb 27]. Available from: http://hdl.handle.net/1813/34334.

Council of Science Editors:

Ohouo P. Coordination Of Dna Damage Checkpoint Signaling And Repair In Saccharomyces Cerevisiae. [Doctoral Dissertation]. Cornell University; 2013. Available from: http://hdl.handle.net/1813/34334

Open Access Theses and Dissertations