Mechanism of Superoxide Mediated Regulation of Particle Uptake and Exocytosis by a GPI-anchored Superoxide Dismutase C in Dictyostelium

Dictyostelium discoideum is a simple model organism that can be used to study endocytic pathways such as phagocytosis and macropinocytosis because of its homology to cells of the mammalian innate immune system, namely macrophages and neutrophils. Consequently, Dictyostelium can also be used to study the process of exocytosis. In our laboratory, we generated Dictyostelium cells lacking superoxide dismutase SodC. Our data suggest that cells that lack SodC are defective in macropinocytosis and exocytosis when compared to wild type cells. In this study I describe a regulatory mechanism of macropinocytosis by SodC via regulation of RasG, which in turn controls PI3K activation and thus macropinocytosis. Our results show that proper metabolism of superoxide is critical for efficient particle uptake, for the proper trafficking of internalized particles, and a timely exocytosis of fluid uptake in Dictyostelium cells.

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Establishing a baseline of psychometric estimates of measurement of intercultural competence of Ukrainian faculty: Setting the stage for domestic and global intercultural understanding

Post-Soviet Ukraine is in a time of upheaval and transition. Internal relations between pro-Western and pro-Russian supporters have deteriorated in the light of recent political events of Euro Revolution, Russia's occupation of the Crimean peninsula, and the militant confrontations in the southeastern regions of the country. In the light of these developments, intercultural competence is greatly needed to alleviate domestic tensions and enable effective intercultural communication with the representatives of different cultures within the country and beyond its borders.^ This study established a baseline of psychometric estimates of intercultural competence of Ukrainian higher education faculty. A sample of 276 professors of different academic majors from one university in Western Ukraine participated in the research. The Global Perspective Inventory (GPI; Merrill, Braskamp, & Braskamp, 2012) was chosen as a research instrument to measure intercultural competence of the faculty members. The GPI takes into account cognitive, intrapersonal, and interpersonal domains, each of which contains two scales reflective of theories of cultural development and intercultural communication – Cognitive-Knowing, Cognitive-Knowledge, Intrapersonal-Identity, Intrapersonal-Affect, Interpersonal-Social Responsibility, and Interpersonal-Social Interaction. Because the research instrument has neither been previously used as a measure of intercultural competence, nor administered in Ukraine, it was cross-validated using a Table of Specification (Newman, Lim, & Pineda, 2013) and two sets of factor analyses. As a result, a modified version of the GPI was created for use in Ukraine.^ Multiple linear regression analyses were used to test relationships between the participants' GPI scores on intercultural competence, and several independent variables that consisted of academic discipline, intercultural experience, and how long the participants taught at the university. The analyses determined a positive relationship between the scores on three out of six scales of the original version and two out of five scales of the modified version of the GPI and all the independent variables simultaneously. The relationship between the faculty responses on the six scales of both GPI versions and the independent variables controlling for each other produced mixed results. A unique role of intercultural professional development in predicting intercultural competence was discussed.^

Undergraduate Students’ Perceptions of Study Abroad and Their Level of Achievement of Global Learning Outcomes

This study expanded on current research on study abroad and global learning, using the Global Perspective Inventory (GPI), and conducted at Florida International University (FIU) in Miami, FL. The GPI assesses the holistic development of a global perspective in higher education within three domains and their respective FIU-determined equivalents: cognitive (global awareness), intrapersonal (global perspective), and interpersonal (global engagement). The main purpose of this study was to assess FIU’s undergraduate students’ perceptions of study abroad on their level of achievement of global awareness, global perspective, and global engagement. The secondary purpose was to determine how the students described their study abroad experience and achievement of global learning. The research design for this study consisted of parallel mixed methods. The quantitative component was an ex post facto with hypothesis design, using a pretest/posttest nonequivalent group methodology. FIU undergraduates (N=147) who studied abroad for one semester or more completed the GPI pre- and post-tests. Descriptive statistics and paired t-tests were conducted to compare the means. The interviews included 10 students, and were analyzed through Structural coding, Saldaña’s In Vivo coding, and Value coding. Quantitative analyses indicated positive changes in the students’ global awareness and global perspective. These analyses also showed that the FIU students achieved higher post-test means on all the domains of the GPI compared to other studies. Qualitative analyses showed that the students’ experiences incorporated all three global learning outcomes, most notably global awareness and perspective.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Establishing a Baseline Measurement of Intercultural Competence of Ukrainian Faculty: Setting the Stage for Domestic and Global Intercultural Understanding

Post-Soviet Ukraine is in a time of upheaval and transition. Internal relations between pro-Western and pro-Russian supporters have deteriorated in the light of recent political events of Euro Revolution, Russia’s occupation of the Crimean peninsula, and the militant confrontations in the southeastern regions of the country. In the light of these developments, intercultural competence is greatly needed to alleviate domestic tensions and enable effective intercultural communication with the representatives of different cultures within the country and beyond its borders. This study established a baseline of psychometric estimates of intercultural competence of Ukrainian higher education faculty. A sample of 276 professors of different academic majors from one university in Western Ukraine participated in the research. The Global Perspective Inventory (GPI; Merrill, Braskamp, & Braskamp, 2012) was chosen as a research instrument to measure intercultural competence of the faculty members. The GPI takes into account cognitive, intrapersonal, and interpersonal domains, each of which contains two scales reflective of theories of cultural development and intercultural communication – Cognitive-Knowing, Cognitive-Knowledge, Intrapersonal-Identity, Intrapersonal-Affect, Interpersonal-Social Responsibility, and Interpersonal-Social Interaction. Because the research instrument has neither been previously used as a measure of intercultural competence, nor administered in Ukraine, it was cross-validated using a Table of Specification (Newman, Lim, & Pineda, 2013) and two sets of factor analyses. As a result, a modified version of the GPI was created for use in Ukraine. Multiple linear regression analyses were used to test relationships between the participants’ GPI scores on intercultural competence, and several independent variables that consisted of academic discipline, intercultural experience, and how long the participants taught at the university. The analyses determined a positive relationship between the scores on three out of six scales of the original version and two out of five scales of the modified version of the GPI and all the independent variables simultaneously. The relationship between the faculty responses on the six scales of both GPI versions and the independent variables controlling for each other produced mixed results. A unique role of intercultural professional development in predicting intercultural competence was discussed.