{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/79341"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/79341","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Characterization of a Seven-Transmembrane Protein and a Putative Ligand with Respect to Behavior and Iron Uptake in Tetrahymena thermophila","abstract":"Ph.D.","abstract_html":"Ph.D.","abstract_has_math":false,"creators":["Zou, Dianxiong"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Hennessey, Todd","Biological Sciences"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-04-04T20:30:34Z","date_published":"2019-04-04T20:30:34Z","updated_at":"2026-07-27T19:05:16Z","subjects":["biology","Molecular","biochemistry"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/79341","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Hennessey, Todd","Biological Sciences"]},{"key":"dc:creator","label":"Author","values":["Zou, Dianxiong"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-04-04T20:30:34Z","2019","2018-11-16 08:56:48"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["biology","Molecular","biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/79341"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Ph.D.","Seven-transmembrane proteins (7TMs), often known as G-protein coupled receptors (GPCRs), are involved in a myriad of cellular processes, including sensory response, synaptic transmission, metabolism, immunity, and locomotion. Because of their physiological importance, 7TMs have been studied in humans, mice, and other closely related animals. The focus on animal 7TMs however, overshadows the fact that their paralogs are ubiquitous in the broader eukaryote lineage, but less attention have been paid to these non-animal receptors. The functioning principles behind these overlooked 7TMs, especially those in protozoans, appear atypical and are not carbon copies of conventional GPCRs. Since many protozoan species are pathogenic or exert significant influence over their ecosystems, it is worthwhile to study their signaling pathways. Tetrahymena thermophila are ciliated protozoans, and one of the few unicellular, eukaryotic model organisms besides fungi and slime molds. A fully sequenced genome as well as an established repertoire of molecular techniques makes Tetrahymena a good platform to study the functions of unknown proteins, such as protozoan sensory receptors. Using gene knockout and overexpression, a putative 7-transmembrane protein called G37p was found to be required for the detection of a chemorepellent compound (called conditioned supernatant factor or CSF) that was secreted by Tetrahymena thermophila. The chemorepellents that we have investigated, like excitatory neurotransmitters, are non-toxic and effective at sub-millimolar concentrations. These depolarizing compounds induce repeated bouts of backward swimming, also known as avoiding reactions, in motile cells such as Tetrahymena. The CSF is a small (less than 1 kilodalton) repellent compound, and its effect is resistant to heat as well as protease and nuclease treatments. External EGTA eliminated the CSF response, suggesting that Ca2+ entry is a critical downstream component for the CSF-induced avoiding reactions. The macronuclear G37 gene knockout mutant (G37KO), which lacks G37p, is able to secrete the CSF, but is both unresponsive to it and over-responsive to a host of other unrelated chemorepellents, such as quinine, lysozyme, and GTP. All of these mutant phenotypes are reversed by the reintroduction and overexpression of the G37 gene in the G37KO background. Prolonged adaptation to the CSF also causes decreased responsiveness to all of the non-CSF repellent stimuli in the wild type and G37 overexpression cells. These results suggest that CSF exposure is able to cross-adapt responding cells to other repellent signals. And that G37KO, with its inability to detect the CSF present in the environment, cannot cross-adapt and thereby retains high basal sensitivity to chemorepellents other than the CSF, To elucidate the chemical makeup and functional significance of the CSF, the CSF was enriched from the cell-free medium by multiple rounds of chromatography. Interestingly, most fractions containing repellent activity also contained iron. Moreover, the most enriched CSF fraction, obtained by thin-layer chromatography in 80% ethanol, appeared to be a hydroxamate and carboxylate molecule that can associate with iron. Iron associating or chelating molecules can be secreted to solubilize the otherwise insoluble extracellular iron. Wild-type cells exhibiting avoidance response to the CSF grew faster (as measured by cell density) than the G37KO mutant did when ferric iron was supplemented. The growth advantage of wild type cells was further enhanced when the enriched CSF was added to ferric iron, suggesting that this may facilitate iron uptake through G37p. In addition to growth, wild type cells possessed higher concentration of intracellular labile iron than G37KO did, but the G37 overexpression mutants had the highest intracellular labile iron concentration overall. The uptake of iron is a double-edged sword, being both vital and dangerous for all cells. This is because iron, which is a co-factor in many life-sustaining metalloenzymes, also generates toxic reactive oxygen species (ROS) when its concentration becomes too high. Real-time PCR showed that wild type cells could downregulate G37 expression to control CSF-mediated iron uptake. However, the G37 overexpression mutants, being unable to regulate G37 expression, appeared harmed by iron overload. Instead of faster growth with iron, the overexpression mutants grew slower with increasing concentrations of supplemented iron, and they virtually ceased growing when the enriched CSF was also added. Consistent with iron toxicity, these mutants exhibited increased peroxide production, but adding catalase (an antioxidant enzyme) to the media could reverse some of the iron-induced growth inhibition. Taken together, these results indicated that the physiological role of G37p was more than chemorepulsion, but it was also the detection and internalization of the ironbound CSF."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Characterization of a Seven-Transmembrane Protein and a Putative Ligand with Respect to Behavior and Iron Uptake in Tetrahymena thermophila"]}]}],"canonical_facts":{"dc:contributor":["Hennessey, Todd","Biological Sciences"],"dc:creator":["Zou, Dianxiong"],"dc:date":["2019-04-04T20:30:34Z","2019","2018-11-16 08:56:48"],"dc:description":["Ph.D.","Seven-transmembrane proteins (7TMs), often known as G-protein coupled receptors (GPCRs), are involved in a myriad of cellular processes, including sensory response, synaptic transmission, metabolism, immunity, and locomotion. Because of their physiological importance, 7TMs have been studied in humans, mice, and other closely related animals. The focus on animal 7TMs however, overshadows the fact that their paralogs are ubiquitous in the broader eukaryote lineage, but less attention have been paid to these non-animal receptors. The functioning principles behind these overlooked 7TMs, especially those in protozoans, appear atypical and are not carbon copies of conventional GPCRs. Since many protozoan species are pathogenic or exert significant influence over their ecosystems, it is worthwhile to study their signaling pathways. Tetrahymena thermophila are ciliated protozoans, and one of the few unicellular, eukaryotic model organisms besides fungi and slime molds. A fully sequenced genome as well as an established repertoire of molecular techniques makes Tetrahymena a good platform to study the functions of unknown proteins, such as protozoan sensory receptors. Using gene knockout and overexpression, a putative 7-transmembrane protein called G37p was found to be required for the detection of a chemorepellent compound (called conditioned supernatant factor or CSF) that was secreted by Tetrahymena thermophila. The chemorepellents that we have investigated, like excitatory neurotransmitters, are non-toxic and effective at sub-millimolar concentrations. These depolarizing compounds induce repeated bouts of backward swimming, also known as avoiding reactions, in motile cells such as Tetrahymena. The CSF is a small (less than 1 kilodalton) repellent compound, and its effect is resistant to heat as well as protease and nuclease treatments. External EGTA eliminated the CSF response, suggesting that Ca2+ entry is a critical downstream component for the CSF-induced avoiding reactions. The macronuclear G37 gene knockout mutant (G37KO), which lacks G37p, is able to secrete the CSF, but is both unresponsive to it and over-responsive to a host of other unrelated chemorepellents, such as quinine, lysozyme, and GTP. All of these mutant phenotypes are reversed by the reintroduction and overexpression of the G37 gene in the G37KO background. Prolonged adaptation to the CSF also causes decreased responsiveness to all of the non-CSF repellent stimuli in the wild type and G37 overexpression cells. These results suggest that CSF exposure is able to cross-adapt responding cells to other repellent signals. And that G37KO, with its inability to detect the CSF present in the environment, cannot cross-adapt and thereby retains high basal sensitivity to chemorepellents other than the CSF, To elucidate the chemical makeup and functional significance of the CSF, the CSF was enriched from the cell-free medium by multiple rounds of chromatography. Interestingly, most fractions containing repellent activity also contained iron. Moreover, the most enriched CSF fraction, obtained by thin-layer chromatography in 80% ethanol, appeared to be a hydroxamate and carboxylate molecule that can associate with iron. Iron associating or chelating molecules can be secreted to solubilize the otherwise insoluble extracellular iron. Wild-type cells exhibiting avoidance response to the CSF grew faster (as measured by cell density) than the G37KO mutant did when ferric iron was supplemented. The growth advantage of wild type cells was further enhanced when the enriched CSF was added to ferric iron, suggesting that this may facilitate iron uptake through G37p. In addition to growth, wild type cells possessed higher concentration of intracellular labile iron than G37KO did, but the G37 overexpression mutants had the highest intracellular labile iron concentration overall. The uptake of iron is a double-edged sword, being both vital and dangerous for all cells. This is because iron, which is a co-factor in many life-sustaining metalloenzymes, also generates toxic reactive oxygen species (ROS) when its concentration becomes too high. Real-time PCR showed that wild type cells could downregulate G37 expression to control CSF-mediated iron uptake. However, the G37 overexpression mutants, being unable to regulate G37 expression, appeared harmed by iron overload. Instead of faster growth with iron, the overexpression mutants grew slower with increasing concentrations of supplemented iron, and they virtually ceased growing when the enriched CSF was also added. Consistent with iron toxicity, these mutants exhibited increased peroxide production, but adding catalase (an antioxidant enzyme) to the media could reverse some of the iron-induced growth inhibition. Taken together, these results indicated that the physiological role of G37p was more than chemorepulsion, but it was also the detection and internalization of the ironbound CSF."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/79341"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["biology","Molecular","biochemistry"],"dc:title":["Characterization of a Seven-Transmembrane Protein and a Putative Ligand with Respect to Behavior and Iron Uptake in Tetrahymena thermophila"],"dc:type":["Text","Dissertation"]},"updated_at":"2026-07-27T19:05:16Z"}