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1
McIntosh, Cecilia A., Karin Bartoszuk, and Scott Kirkby. "Professional Development Provided by the School of Graduate Studies: Enhancing Mentoring and the Graduate Student Experience." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/363.
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East Tennessee State University has taken several approaches to offering professional development for graduate students over the past several years. This includes graduate student research grants, thesis and dissertation awards, teaching awards, awards for service for the public good, Graduate Student Success Specialist service, Thesis/Dissertation/Capstone Boot Camp, Add-on Fellowships, GA Fee Scholarship, Thesis/Dissertation Scholarships, formal courses (GRAD), graduate student research magazine, and workshops. These will be briefly described, including funding sources supporting the initiatives. A mention of professional development for faculty will also be presented. There will be ample time for discussion of strategies and sharing of ideas by participants.
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Lee, Mo-lan Monica, and 李慕蘭. "Graduate profile and employer's expectations: case study of a Hong Kong secondary school." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1995. http://hub.hku.hk/bib/B31958023.
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Schnädelbach, Holger Martin. "Mixed reality architecture." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/16045/.
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This thesis develops and investigates Mixed Reality Architectures (MRA), dynamic shared architectural topologies, which span physical and virtual spaces. A theoretical framework is developed to describe the field of possible architectures. As the result of a first pilot study, this is then extended with the concept of the Mixed Reality Architectural Cell (MRACell). MRACells consist of one physical and one virtual space, linked by a two-way video and audio connection. The video of a real physical space is rendered on an MRACell, which can move within the virtual environment. A projector and screen in the real space renders an image of the virtual environment from the point of view of that MRACell. Inhabitants can move their MRACell in relation to all others within the shared virtual environment, allowing ad hoc as well as planned remote social interaction. In this sense MRACells can be described as novel architectural interfaces extending real physical space, via a shared virtual environment to link to other real spaces. An in-depth study lasting one year and involving six office-based MRACells, used video recordings, the analysis of event logs, diaries and an interview survey. This produced a series of ethnographic vignettes describing social interaction within MRA in detail. The study found that the MRA was effective at supporting remote social interaction between users. Usage patterns appeared to be motivated by awareness and communication or conversely privacy requirements. This usage maintained and strengthened social ties. Social interaction was both visible to others and part of the everyday activities at the respective office spaces. It resulted from the virtual adjacencies introduced by MRA that allowed the ‘spatial’ integration of remote locations. However, the virtual spatial framework making this possible, introduced new topological limitations on the number of concurrent connections that were available. Overall, it was found that the dynamic architectural topology directly affected social interaction, while social interaction itself re-shaped the topology. These findings are of direct relevance to current developments, which aim to use communications media to overcome the spatial dispersion of work groups in modern organizations. Finally, the differences in use that were observed between groups of inhabitants suggest that spatial cognition in Mixed Reality is affected both by the interface technology and by the social practices surrounding it. In response, it is suggested that in order to investigate the new generation of mixed physical and virtual technologies, cognitive science should take into account their affordances as ‘virtual extensions’ to both our bodies and to our environment.
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Fernando, J. Roshan C. "Theoretical studies on quinone reactivity a thesis presented to the faculty of the Graduate School, Tennessee Technological University /." Click to access online, 2009. http://proquest.umi.com/pqdweb?index=50&did=1908035911&SrchMode=1&sid=1&Fmt=6&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1265041952&clientId=28564.
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Moyo, Awelani Lena. "Between self and author : an autoethnographic approach towards the crafting of reflexive compositions in post graduate drama studies." Thesis, Rhodes University, 2009. http://hdl.handle.net/10962/d1002375.
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This thesis explores the merits of reflexivity in the processes of creating a performance and of performing research in Drama Studies. In it, I make a case for the validity of autobiographical material as an aid to generating such reflexivity. Through an autoethnographic case study of my work entitled Compositions (a series of performance projects) in which I focus on the theme of migration, I provide an indepth account of my experiences, focusing specifically on the interrelated concerns of body, space and journey in my ritualistic performance. My examination explores the dynamic effects of liminality within identity politics, through which I foreground several issues of concern which I have encountered as an emerging scholar and theatremaker working within an academic institution. I propose that the process of studying drama in a University ultimately requires one to continually negotiate a range of subject positions, whilst finding connections between these various identities that one may take up during the course of one’s studies. By developing an awareness of the overlapping of such identities and inhabiting the spaces in-between subject positions, I demonstrate how taking into account one’s personal lived experience can help illuminate one’s understanding of both the work of art and the research report, as well as the broader contexts in which such practice-based work exists. I illustrate how such an understanding has ultimately maximised the knowledge and learning that I have gathered, and has contributed to the crucial project of developing my authorial voice in writing and performance, which is central to the aims of the Master of Arts degree in Drama.
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Parker, Judith. "Racial Socialization and African American Students' Academic Motivation and Self-Efficacy and Likelihood Attending Graduate School." ScholarWorks, 2016. https://scholarworks.waldenu.edu/dissertations/2169.
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Enrollment by African American students in U.S. colleges and universities has increased since 2009, but graduation and retention remains low for this group. Within the African American community, education is considered a central aspect of social empowerment; however, the effect of discrimination, bias, and stereotyping are key when considering achievement indices within a cultural framework. The coping mechanisms that African Americans have developed to combat discrimination, bias, and stereotype threat may contribute to poor performance in college. The purpose of this study was to examine the relationship between racial socialization and African American students' academic motivation, academic self-efficacy, and likelihood of attending graduate school. Drawing from self-determination theory, it was hypothesized that the variables have a positive relationship. A quantitative survey design was used. One hundred-three African American undergraduate students were recruited via convenience sampling to participate in an online survey. Results from a multiple regression analyses indicated no significant relationship between the variables. The findings from this study are not consistent with previous findings that indicate racial socialization has an impact on African Americans' academic performance and motivations, implying the need for future research to include multiple variables that might impact outcomes such as family functioning, access to educational resources, or peer association. Greater numbers of African Americans enrolled in undergraduate programs may, in turn, increase the pool of participants eligible to attend graduate programs. Future research can help parents, researchers, and practitioners better understand the diverse and unique needs of African American youths.
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Sepulveda, Celia Anna. "Consuming merit: Social mobility and class contradictions of working class and lower class women in graduate school." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/280742.
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This study utilizes a multi-method approach to analyzing the experience of working class and lower class women's experience in graduate school. A quantitative analysis is used to determine the number of working class and lower class females in graduate school using parents' education as a proxy. Most first-generation females in graduate school were found in Research I universities in the field of Education. A qualitative analysis includes semi-structured interviews of 34 women from two Research I institutions in the Southwest in the fields of Education, Psychology, Health Sciences and Biology. Data consists of the women's definitions of social class, values and experiences as well as their perceptions of graduate school culture and their mobility process during their graduate school experience. The women in this study revealed a contemporary definition of social class unlike academic Marxist and other sociological definitions. Their experiences of graduate student culture reveal a direct conflict with their social class values. Finally, their mobility experience in graduate school reveals contradictory feelings of pride and hiding their accomplishments from family.
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Senevirathna, H. R. Wasana U. "Theoretical studies of hydrogen abstraction from hydrofluoropropanes a thesis presented to the faculty of the Graduate School, Tennessee Technological University /." Click to access online, 2009. http://proquest.umi.com/pqdweb?index=0&did=2000396231&SrchMode=1&sid=2&Fmt=6&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1279222401&clientId=28564.
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Stone, Linda Shapiro. "Women who have a graduate school education who have chosen to make mothering the major focus of their time : a descriptive study." W&M ScholarWorks, 1987. https://scholarworks.wm.edu/etd/1539618902.
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This is a descriptive study of 33 graduate school educated women who have chosen to stay home with their children. The study includes a discussion of five major areas: personality characteristics, decision-making process, level of job satisfaction, peer and family relationships, and self-image.;The Methodology for this study involved four data gathering procedures: the demographic data form, the structural interview, the California Psychological Inventory (CPI), and the Bems Sex-Role Inventory (BSRI). Participants were obtained by sending fliers home with children at five preschools in the Richmond, Virginia Metropolitan area.;Demographic Data. The mean age for the group was 34. All were part of an intact two parent family. Fourteen graduate majors were represented. Ninety percent had held jobs which were directly related to their advanced degree. Seventy-six percent are actively involved in a career-related activity.;Personality Characteristics. This high functioning group has a composite profile that shows the ability to achieve independently and they prefer their own judgement. They have strength intellectually. The composite personality is someone who has successfully combined some of the best parts of traditional masculine and feminine qualities.;Decision-making Process. The reasons for choosing to stay home related to feeling that their family was their main priority and they didn't want someone else raising their children.;Job Satisfaction. Neither level of status nor dissatisfaction with their last job was the primary reason for choosing to stay home at the time.;Peer and Family Relationships. Support systems were extremely important to this group. Husbands were also very involved in decision-making, child care, and emotional support.;Self-image. These women generally feel good about their choices and believe this is the right role for them at this time. They are aware of what they have given up, but believe they and their children have gained much more than they could ever give up.
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Michell, Theodore William Henry. "The psychasthenia of deep space : evaluating the 'reassertion of space in critical social theory'." Thesis, University College London (University of London), 2002. http://discovery.ucl.ac.uk/4325/.
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The aim of this work is to question the notion of space that underlies the claimed ‘spatial turn’ in geographical and social theory. Section 1 examines this theoretical literature, drawing heavily on Soja as the self declared taxonomist of the genre, and also seeks parallels with more populist texts on cities and space, to suggest, following Williams, that there is a new ‘structure of feeling’ towards space. Section 1 introduces two foundational concepts. The first, derived from Soja’s misunderstanding of Borges’ story The Aleph, argues for an ‘alephic vision’, an imposition of a de-materialized and revelatory understanding of space. This is related to the second, an ‘ecstatic vision’, which describes the tendency, illustrated through the work of Koolhaas and recent exhibitions on the experience of cities, to treat spatial and material experience in hyperbolic and hallucinatory terms. Section 2 offers a series of theoretical reconstructions which seek to draw out parallels between the work of key theorists of what I term the ‘respatialization’ literature (Harvey, Giddens, Foucault and Lefebvre) and the work of Hillier et al in the Space Syntax school. A series of empirical studies demonstrate that the approach to the material realm offered by Space Syntax is not only theoretically compatible but can also help to explain ‘real world’ phenomena. However, the elision with wider theoretical positions points to the need for a reworking of elements of Space Syntax, and steps towards this goal are offered in section 3. In the final ‘speculative epilogue’ I reopen the philosophical debates about the nature of space, deliberately suppressed from the beginning, and suggest that perhaps the apparent theoretical and empirical versatility of Space Syntax, based upon a configurational approach to space as a complex relational system, may offer an alternative approach to these enduring metaphysical debates.
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Vaughan, Laura Sophia. "Clustering, segregation and the 'ghetto' : the spatialisation of Jewish settlement in Manchester and Leeds in the 19th century." Thesis, University College London (University of London), 1999. http://discovery.ucl.ac.uk/661/.
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This thesis deals with the urban phenomenon of minority clusters, which are invariably referred to as 'ghettos'. A review of the literature on 'ghettos' suggests that the clustering of identifiable minorities is commonly associated with segregation - be it physical, economic, social or linguistic - although it is the physical segregation which tends to be most frequently noticed. Moreover, one type of segregation, such as physical - is believed to lead to another type, such as economic. Through studying Jewish settlement in Leeds and Manchester in the 19th century, two key questions are addressed in this thesis: The first is whether there is a link between spatial clustering and spatial segregation and the second is whether spatial clustering is linked to other forms of segregation, such as economic, occupational and social. Another two questions arise from the analysis: whether Jewish settlement patterns are distinctive in their own right, and whether it is possible to identify a pattern in the process of formation of Jewish settlement that may have broader implications for immigrant/minority settlement in general. The techniques and theories of 'Space Syntax' are used here to analyse the settlements in question by using detailed street-level mapping of census data on the entire Jewish population of Manchester and Leeds and of all non-Jewish individuals in the key Jewish districts of each of the cities (the key Jewish districts are generally referred to as 'ghettos'). This enables a multi-level socio-spatial comparison to be made: between Jewish families and their immediate neighbours; between Jewish families and the population of the city as a whole; and between the initial and secondary stages of Jewish settlement. In order to investigate questions relating specifically to immigrant settlement, non-Jewish people born outside of Britain are also considered as a separate group, although they are not the main subject. The analysis suggests that spatial clustering does not necessarily lead to spatial segregation and that spatial clustering may also be associated with some types of segregation, such as occupational but not with others, such as economic. It also suggests that Jewish settlement patterns are distinctive and that they are identifiable for a longer period than expected after immigration, when compared with other immigrants. This thesis also sheds light on the process of the formation of Jewish settlement, proposing a pattern whereby after establishing a core of settlement, streets already established become more densely populated, whilst new streets are settled more slowly. Analysis of the key districts of Jewish settlement also suggests that certain areas of cities are especially prone to settlement by the disadvantaged, due to characteristics that make such areas firstly, tend to be economically unsuccessful due to their spatial segregation and secondly, less attractive to those who have the means to move elsewhere and that such areas are not so much defined by their immigrant constituents, but by their long-standing inhabitants that cannot move elsewhere.
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Arjunon, Sivakkumar. "P-version refinement studies in the boundary element method a dissertation presented to the faculty of the Graduate School, Tennessee Technological University /." Click to access online, 2009. http://proquest.umi.com/pqdweb?index=19&sid=2&srchmode=1&vinst=PROD&fmt=6&startpage=-1&clientid=28564&vname=PQD&RQT=309&did=1786737301&scaling=FULL&ts=1250860988&vtype=PQD&rqt=309&TS=1250861000&clientId=28564.
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Thomas, Kim G. "Selected Students’, Parents’, and Graduate Student Tutors’ Experiences and Perceptions in a Community of Interest Summer Literacy Camp." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3709.
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In this qualitative case study, I examined a local summer literacy camp in which
graduate student tutors tutored elementary and middle school students in reading and
writing. I focused the study on the primary stakeholders in the summer literacy camp:
�������������������������������������� ts, and the course instructor/camp director because
their voices are limited in the current literature. In this Community of Interest Summer
Literacy Camp, the graduate student tutors moved from a position of fear and trepidation
to a position of empowerment in which they hoped to make changes in their classrooms,
schools, and communities. The tutees learned to appreciate the tutoring program and
some tutees began to understand tutoring could be an enrichment experience rather than
only a remedial experience. There was limited parental participation in the tutoring
program and that may have hindered a richer experience in which parents learned
strategies to help their child/children excel in reading and writing.
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Hawkins, Luquanda Neekey. "LET THEM SPEAK!: VOICES OF URBAN BLACK HIGH SCHOOL GRADUATES IN SAN BERNARDINO CALIFORNIA." CSUSB ScholarWorks, 2019. https://scholarworks.lib.csusb.edu/etd/882.
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This qualitative study examined Urban Black high school students’ pathways to academic success, with particular attention to their perspectives about achieving academic success despite stereotype threats and impeding social factors, the support systems they rely upon, and the coping mechanisms they employ when encountering challenges. Study data suggested that the way urban Black high school students achieved academic success despite stereotype threats and impeding social factors is to identify and focus on their goals, circumventing what obstacles they could and cognitively and emotionally coping with what they could not. Students primarily relied upon family members, community members, school staff, and college-bound programs as support mechanisms. In response to challenge, they employed coping mechanisms of cognitive, emotional, and behavioral strategies that enhanced their focus on the goal, reduced their stress, helped them avoid distraction, and promoted their achievement of the goal. These strategies indicated that the students possessed grit and a growth mindset. The relatively few instances of racism and stereotyping identified in this study indicate the need for more study conducted using different methods to reveal the more widespread and insidious instances of racism and stereotyping within school systems. Longitudinal research also may be helpful for revealing the challenges, racism, and stereotypes students experience as they happen, along with the sensemaking, coping strategies, and support mechanisms they employ to persist through to completion.
Keywords: African American, Urban, high school students, racism, stereotype threat, success factors
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Conroy-Dalton, Ruth Alison. "Spatial navigation in immersive virtual environments." Thesis, University College London (University of London), 2001. http://discovery.ucl.ac.uk/1111/.
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Chase, Steve. "Activist Training in the Academy: Developing a Master's Program in Environmental Advocacy and Organizing at Antioch New England Graduate School." [Yellow Springs, Ohio] : Antioch University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=antioch1163790650.
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Thesis (Ph. D.)--Antioch University New England, 2006.<br>Title from PDF t.p. (viewed Apr. 12, 2007). Advisor: Heidi Watts. "A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy [in] Environmental Studies at Antioch New England Graduate School 2006"--The title page. Keywords: environmental advocacy, activist training, social movements, curriculum action research, master's curriculum, environmental studies, popular education, critical pedagogy, education for citizenship. Includes bibliographical references (p. 345-357).
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Kollipara, Sireesha. "Theoretical studies of rates of electron transfer between cytochrome b₅ reductase and cytochrome b₅ a thesis presented to the faculty of the Graduate School, Tennessee Technological University /." Click to access online, 2008. http://proquest.umi.com/pqdweb?index=0&did=1674959791&SrchMode=1&sid=2&Fmt=6&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1268413954&clientId=28564.
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Kannan, Pravin. "Physicochemical studies of expandable polystyrene beads and foam as applicable in lost foam castings a dissertation presented to the faculty of the Graduate School, Tennessee Technological University /." Click to access online, 2009. http://proquest.umi.com/pqdweb?index=18&sid=1&srchmode=1&vinst=PROD&fmt=6&startpage=-1&clientid=28564&vname=PQD&RQT=309&did=1756870151&scaling=FULL&ts=1250798704&vtype=PQD&rqt=309&TS=1250798717&clientId=28564.
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Tse, Wai-yuen Patrick, and 謝偉源. "An evaluation of the non-graduate teacher qualifications assessment scheme in Hong Kong: an attempt to find outif it is an effective way to provide local primary schools withappropriately qualified teachers." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1996. http://hub.hku.hk/bib/B31959076.
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Bidwell, Joseph, Cerrone Foster, Anna Hiatt, Cecelia A. McIntosh, and Rebecca Pyles. "Progress on Implementing Transformative Approaches in Southern Appalachia." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/354.
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Over the last year the Faculty of Biological Sciences at East Tennessee State University have been evaluating core coursework for biology majors and working towards transforming teaching approaches to enhance the undergraduate learning experience and improve overall learning outcomes among our graduates. Our institution serves a large population of low-income and first generation college students living and working in Southern Appalachian region. The most important motivation behind transformation is to provide highly-qualified graduates who can contribute to the economic development and growth of the area. In focusing on traineeship and skills development of graduates, we adopted an action plan focused on students, faculty, and curriculum development. Under the leadership of the chair, productive and positive discourse on curricular goals among faculty has shaped well-defined core concepts and competencies we envision for our graduates. Several faculty are using research-based methods to improve the core curriculum and to infuse best teaching practices in the classroom. The strengths of our program lie in having demonstrated success in providing students with authentic research experiences in upper-level courses. We are working towards infusing these ideas and skills-development into the introductory core curriculum in hopes of increasing the number of undergraduates who pursue professional graduate degrees. Our hopes are that investing in transformative changes in the classrooms and providing high-quality educational opportunities will increase the time our students devote to STEM career development and ultimately result in large-scale economic development on the Southern Appalachian community.
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Devaiah, Shivakumar P., and Cecelia A. McIntosh. "Towards Understanding of Glucosyltransferase Specifi city in Citrus Paradisi." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/350.
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Flavonoids are a broad class of low molecular weight, secondary plant phenolics characterized by the fl avan nucleus. Widely distributed in plants, food and traditional herbal medicines, more than 6000 fl avonoids have been identifi ed up to date. They are present mainly as glycosides whose phenolic hydrogen or hydrogens are substituted to sugar moiety. An increasing number of fl avonoids have attracted much attention in relation to their biological activities, including anti-viral, anti-infl ammatory, anti-bacterial, and vasodilatory activities. Present work is to understand the structure and function of a fl avonol specifi c glucosyltransferase from Citrus paradisi. The study is one of the many steps towards custom designing of the protein. We employed homology modeling, site-directed mutagenesis and yeast expression system to generate mutants of glucosyltransferase and study their substrate specifi city, regiospecifi city and kinetic properties.
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McIntosh, Cecilia A. "Building and Sustaining Research at East Tennessee State University." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/366.
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Carter, Lisa, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Mutagenesis of a Flavonol- 3-O-Glucosyltransferase and the Effect on Enzyme Function." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/349.
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Flavonoids are an important group of secondary metabolites found in plants and have a wide variety of properties. Some play a role in fl ower pigmentation, while others have antimicrobial properties. Glucosylation is an important modifi cation of fl avonoids and is mediated by glucosyltransferases. In this process, the enzyme transfers glucose from UDP-glucose to a specifi c position on the fl avonoid. Previous study from the lab characterized a glucosyltransferase from C. paradisi that is fl avonol specifi c. In this study an attempt has been made to study the structure and function of this fl avonol specifi c glucosyltransferase using site directed mutagenesis. The glutamine residue at position 87 of the Cp-3-O-GT enzyme was changed to isoleucine, the analogous residue in the 3-O-glucosyltransferase of Clitoria ternatea. Similarly, the histidine at position 154 was changed to tyrosine. We hypothesize that these mutations will change substrate specifi city. The glutamate at position 88 was changed to an aspartic acid. We hypothesize that this will change the regiospecifi city of the enzyme, as aspartic acid is the analogous residue found in some 7-O-glucosyltransferases. Finally, we introduced a double mutation with glutamine 87 becoming isoleucine and glutamate 88 becoming aspartic acid, with the hypothesis that both regiospecifi city and substrate specifi city will be changed.
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Kandel, Sangam, Sarah Khaja, Shiva K. Devaiah, and Cecelia A. McIntosh. "Effect of Mutant P145T on the Enzyme Activity of Glucosyltransferase from Citrus paradisi." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/353.
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Flavonoids are the C-15 phenolic compounds containing two phenyl rings and a heterocyclic ring. The majority of the flavonoids accumulated in grapefruit are flavonol, flavanone, flavone, dihydroflavonol, and chalcone glycosides. Most flavonoids are present in glucosylated form and the glucosylation is mediated by a class of enzymes called glucosyltransferases that transfer glucose from a high energy sugar donor to the acceptor aglycone at a particular position. A clone encoding a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) from Citrus paradisi has been previously characterized in our lab. The study of structure and function of flavonoid GTs is an important aspect of our research that contributes to the synthesis of novel glucosides by changing the glucosylation patterns of GTs. Our study focuses on the structural and functional analysis of Cp-3-O-GT through site directed mutation and analysis of mutated enzyme in terms of substrate specificity and regiospecificity. Multiple sequence alignment and homology modeling was used to identify candidate areas for mutation. For this study, Cp-3-O-GT was modeled with a flavonoid 3- O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT – P145T mutant to test the hypothesis that that mutation of key amino acid residues (proline) in Cp-3-O-GT by position specific amino acids of VvGT (threonine) could alter substrate specificity or regiospecificity of Cp-3-O-GT. Initial screening results suggested that the mutant P145T glucosylates flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group for glucosylation. HPLC was performed to identify the reaction products. Early results indicate that the P145T mutant glucosylates naringenin at 7-OH position forming naringenin-7-O-glucoside and this is being confirmed. Product identification with other substrates is also being conducted. Results are being used to revisit and refine the structure model.
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Birchfield, Aaron, and Cecelia A. McIntosh. "Crystallization of a Flavonol-Specific 3-O-Glucosyltrasnferase found in Citrus paradisi." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/365.
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Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified in part by a class of enzymes called glycosyltransferases. This class of enzymes is of substantial interest to this lab due to their unique structural and functional properties. Glycosides of flavonoids produced by glycosyltransferases have emerged in recent years as a critical part of plant metabolism, thus impacting every aspect of their growth, cultivation, production, and utilization. One such glycosyltransferase, found in Duncan Grapefruits (Citrus paradisi), was previously identified, recombinantly expressed, and shown through biochemical characterization to exclusively glycosylate the flavonol class of flavonoids. The structural basis that accounts for a glycosyltransferase's selectivity has been determined by protein crystallization in other labs, yet no structural basis currently exists for the specificity exhibited by this flavonol-specific glycosyltransferase. Currently, the WT enzyme and two mutants were expressed in E. coli, where they underwent site-directed mutagenesis to insert thrombin cleavage tags for removal of protein purification vectors, with the goal of transforming into yeast for adequate protein production. Subsequent purification and crystallization screens will allow for formation and acquisition of glycosyltransferase crystals, whose x-ray diffraction patterns will be decoded, thus revealing the enzyme's complete structure. We hypothesize that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, we hypothesize that a thrombin- cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.
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McIntosh, Cecilia A. "Structure and Function of Flavonoid Glucosyltransferases: Using a Specific Grapefruit Enzyme as a Model." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/355.
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Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.
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McIntosh, Cecilia A. "Structure and Function of Flavonoid Glucosyltransferases: Using a specific Grapefruit Enzyme as a Model." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/369.
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Wamucho, Anye, Deborah Hayford, and Cecelia A. McIntosh. "Heterologous Expression of Grapefruit Clones PGT3 and PGT9 in Yeast and Screening of Recombinant Protein for Activity." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/336.
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The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.
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McIntosh, Cecilia A. "Supporting Students’ Writing and Degree Completion: Boot Camps, Write-Ins and Writing Retreats." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/370.
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Devaiah, Shivakumar P., and Cecelia A. McIntosh. "Site-Directed Mutational Analysis of Flavonol 3-0-Glucosyltransferases from Citrus paradisi." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/340.
Full textAbstract:
Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.
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Kandel, Sangam, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Biochemical Characterization of a Cp-3-O-GT Mutant P145T and Study of the Tags Effect on GT Activity." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/361.
Full textAbstract:
Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-Oglucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3-O-GT in particular.
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Spaulding, Nathan, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Effect of the Mutation D344P on the Regio and/or Stereospecificity of Cp3-O-Gt." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/362.
Full textAbstract:
Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate, reactions catalyzed by glucosyltransferases. Citrus paradise contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modelled against an anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the regio and/or steriospecificity of Cp3-OGT. An initial screening assay has been performed using radioactive UDP-glucose as a sugar donor. Early results indicated that the mutant D344P has particular affinity for flavonols and for diosmetin, a flavone. Kinetic assays are being performed to confirm these results. Studies of time course, enzyme concentration, HPLC product analysis, pH optimum and reaction kinetics will be performed to further complete D344P protein characterization.
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Adepoju, Olusegun A., Devaiah K. Shiva, and Cecelia A. McIntosh. "Selected Point Mutations of a Flavonoid 3-O-Glucosyltransferase from Citrus paradisi (Grapefruit) and Effect on Substrate and Regiospecificity." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/343.
Full textAbstract:
Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3GTs from grape and grapefruit were modeled against F7GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modeling. Enzyme activity of the mutant F3-GT proteins will be analyzed for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant enzymes to test for change in substrate specificity.
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Devaiah, Shivakumar P., and Cecelia A. McIntosh. "Expression and Biochemical Characterization of Two Glucosyltransferases from Citrus paradisi." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/335.
Full textAbstract:
Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. The enzymes that lead to glucoside formation are known as glucosyltransferases (GTs), and characteristically accomplish this task by transferring a UDP-activated glucose to a corresponding acceptor molecule. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides and limonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. The research focus of our lab is to establish the function of putative secondary product glucosyltransferase clones identified from Citrus paradisi. In the present study, we report on the activity and biochemical characterization of two clones, PGT 7 (Flavonol-3-O-GT) and PGT8 (Limonoid GT) which were expressed in Pichia pastoris.
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Adepoju, Olusegun A., Devaiah K. Shiva, and Cecelia A. McIntosh. "Selected Point Mutation of a Flavonoid 3-0-Glucosyltransferase from Citrus paradisi (Grapefruit) and its Effect on Substrate and Regiospecificity." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/339.
Full textAbstract:
Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity as well as regiospecificity. Several F3GT's characterized from other plants also had the ability to glucosylate anthocyanidins, however the grapefruit F3GT did not. This research is designed to test the hypothesis that specific amino acid residues impart the substrate specificity and regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modelling. Enzyme activity of the mutant F3-GT proteins will be tested with flavonols for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant F3-GT enzyme to test for change in substrate specificity. The result from this study will add to our knowledge of GTs.
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Devaiah, Shivakumar P., Cheng Zhang, and Cecelia A. McIntosh. "Structure and Functional Analysis of Glucosyltransferase from Citrus paradisi." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/344.
Full textAbstract:
Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site-directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3-O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting L41M, N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3-O-GT.
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Kandel, Sangam, Sarah Khaja, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Structural and Functional Analysis of Grapefruit C-3OGT Mutant P145T." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/356.
Full textAbstract:
Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases are the enzymes that mediate glucosylation by transferring glucose from a high energy sugar donor to the acceptor substrates. Our study focuses on the structural and functional analysis of a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) clone from Citrus paradisi that has been characterized previously in our lab. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-OGT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at 7-OH position forming prunin. Product identification with other substrates is in progress. Results are being used to revisit and refine the structure model. Structural and functional analysis of flavonoid GTs may contribute to custom design of GTs for the synthesis of novel glucosides by changing glucosylation patterns.
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King, Kathleen, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "The Effect of R382W Mutation on C. paradisi Flavonol-Specific 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/358.
Full textAbstract:
Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. This process can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and anthocyanidins. Homology modeling using the crystallized structure of the V. vinifera GT predicted sites of amino acids that could influence substrate binding site. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This change is hypothesized to cause a shift in substrate specificity of the Cp3OGT to accept anthocyanidins as well as flavonols. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and transformed into yeast for expression. Western blot determined the optimal protein induction period for the cells, after which the cells were broken to extract the recombinant mutant protein. Purification of the R382W 3GT allowed for enzyme analysis to be performed by measuring the incorporation of radioactive glucose into the reaction product. HPLC will be used to identify reaction products. An enzyme kinetics study will show the extent of any biochemical change in function as a result of this mutation; results will then be incorporated into a refined protein model.
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Spaulding, Nathan, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Affect of Mutation D344P on the Regio- and/or Substrate Specificity of CP3-OGT." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/360.
Full textAbstract:
Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against a anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the regio and/or steriospecificity of Cp3-OGT. An initial screening assay has been performed using radioactive UDP- glucose as a sugar donor. Early results indicated that the mutant D344P has particular affinity for flavonols and for diosometin, a flavone. Kinetic assays are being performed to confirm these results. Studies of time course, enzyme concentration, HPLC product analysis, pH optimum and reaction kinetics will be performed to further complete D344P protein characterization.
40
McIntosh, Cecilia A. "Position-Specific Flavonoid Glucosyltransferases: Structure and Functional Analysis of Grapefruit Flavonol-Specific 3-O-GT." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/367.
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Kandel, Sangam, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Biochemical Characterization of a Cp-3-O-GT Mutant P145T and Study of the Tag Effect on GT Activity." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/359.
Full textAbstract:
Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-O-glucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GTP145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3- O-GT in particular.
42
Devaiah, Shivakumar P., and Cecelia A. McIntosh. "Substrate Specificity and Kinetic Properties of Flavonol-3-O-Glucosyltransferase From Citrus Paradisi." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/342.
Full textAbstract:
Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3- O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3- O-GT.
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Adepoju, Olusegun A., Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Investigating Potentially Key Residues Which Imparts the Substrate and Regiospecifi city of aFlavonol-Specifi c 3-O-Glucosyltransferase from Grapefruit." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/348.
Full textAbstract:
Most naturally-occurring fl avonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. Citrus paradisi fl avonol-specifi c glucosyltransferase (Cp-F3-O-GT) is recognized for its rigid substrate and regiospecifi city. In this work, homology modeling, site-directed mutagenesis, and biochemical analyses of the recombinant mutant Cp-F3-O-GT proteins were used to investigate potential amino acid residues that might be responsible for the enzymes strict regiospecifi city while also investigating its substrate specifi city. The single point mutations of three amino acid residues within the grapefruit F3-O-GT identifi ed through sequence alignment and homology modeling were performed. Analyses of the enzyme activity of the recombinant mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzymes inactive with fl avonol substrates at 6% and 12% respectively relative to wild-type. However, the mutation of glycine 392 to glutamate (G392E) remained active and glucosylated the fl avonol acceptors quercein (Km app= 11 μM; Vmax = 5.7 pKat/μg) relative to the wild-type (Km app= 93 μM; Vmax = 41.7 pKat/μg), and kaempferol (Km app= 7 μM; Vmax = 3.8 pKat/μg) relative to the wild-type (Km app = 39 μM; Vmax = 4.2 pKat/ μg). The mutant enzyme also did not show broadened acceptor substrate specifi city as it also favored fl avonols as the preferred acceptor substrate. The optimum pH of the mutant enzyme was 8.0 similar to the wild-type F3-O-GT. Activity of the mutant enzyme was stimulated by NaCl and KCl, but inhibited by Cu2+, Zn2+, Fe2+ as well as UDP with an apparent Ki of 10μM. Product identifi cation to determine glucosylation position is being investigated for a possible change in regiospecifi city.
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Sathanantham, Preethi, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Structure-Function Investigations of Site-Directed Mutants of Citrus paradisi Flavonol-Specific 3 O Glucosyltransferase (Cp3OGT) – Impact of Mutations of Serine, Histidine, and Glutamine." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/357.
Full textAbstract:
Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.
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Williams, Bruce E., and Cecelia A. McIntosh. "Putative Glucosyltransferase 11 from Citrus paradisi: Cloning, Recombinant Expression in Yeast, and Substrate Screening." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/338.
Full textAbstract:
Plant secondary products, which include the flavonoids, have a variety of roles in plant systems. Their roles include biosignalling, UV protection, antifeedant activity, pollinator attraction, stress response, and many others. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin. Glucosyltransferases catalyze glucosylation reactions. Putative plant secondary product glucosyltransferases may be identified by the loosely conserved “PSPG box” amino acid sequence; however, with current knowledge, biochemical characterization is the only way to determine with certainty the function of these enzymes. The hypothesis tested here is that PGT11 is a plant secondary product glucosyltransferase. Recombinant PGT11 has been expressed in yeast using the pPICZ A vector. To investigate the hypothesis, the enzyme will be screened for glucosylation activity with various flavonoid and phenolic substrates.
46
Spaulding, Nathan, Shivakumar Devaiah, and Cecelia A. McIntosh. "Determination of the Substrate Specificity of the Mutant D344P of Citrus paradisi Flavonol-Specific 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/364.
Full textAbstract:
Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against an anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quickscreening assay using radioactive UDP-glucose as a sugar donor suggested there may have been expansion of substrate acceptance. Confirming time course assays did not support this. Additionally, results of these assays show that D344P protein has decreased activity with flavonols as compared to wild type Cp3-O-GT. with no expansion of substrate specificity. Models suggest that a change in protein conformation has resulted in decreased activity.
47
Loftis, Peri, and Cecelia A. McIntosh. "Recombination and Screening of Putative Glucosyltransferase Clone 4 in Pichia pastoris." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/334.
Full textAbstract:
Flavonoids are a group of plant secondary metabolites that are vital to the cell systems of plants. The intake of these chemicals is advantageous to animals for their antioxidant properties that affect the function of immune and inflammatory cells. The bitter taste of grapefruit (Citrus paradise) and other citrus species is caused by the accumulation of glycosylated flavonoids. Glucosyltransferases (GTs) are enzymes that add glucose moieties to a carbon or hydroxyl group of natural products. The function of a putative secondary product GT clone was tested. In previous research, putative GT 4 was cloned into a pCD1 modified pET expression system, heterologously expressed in E.coli, and screened for activity with only a few substrates, and little GT activity was found. Issues of protein localized to inclusion bodies in bacteria are being addressed. PGT 4 is being heterologously expressed in yeast (Pichia pastoris) to allow for protein production and analysis. PGT 4 will be screened for GT activity with different flavonoid subclass representatives and simple phenolics. PGT 4’s significant impact on the biochemical regulation of Citrus paradise will be elucidated with its characterization and determination of PGT 4’s structure and function.
48
Loftis, Peri, and Cecelia A. McIntosh. "Recombination and Screening of Putative Grapefruit Glucosyltransferase 4 in Pichia pastoris." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/337.
Full textAbstract:
Flavonoids are a group of plant secondary metabolites that are vital to the cell systems of plants. The intake of these chemicals is advantageous to animals for their antioxidant properties that affect the function of immune and inflammatory cells. The bitter taste of grapefruit (Citrus paradisi) and other citrus species is caused by the accumulation of glycosylated flavonoids. Glucosyltransferases (GTs) are enzymes that add glucose moieties to a carbon or hydroxyl group of natural products. The function of a putative secondary product GT clone was tested. In previous research, putative GT 4 was cloned into a pCD1 modified pET expression system, heterologously expressed in E.coli, and screened for activity with a few substrates; little GT activity was found. Issues of protein localized to inclusion bodies in bacteria were addressed. PGT 4 is being heterologously expressed in yeast (Pichia pastoris) to allow for protein production and analysis. PGT 4 was screened for GT activity with different flavonoid subclass representatives and simple phenolics.
49
Sathanantham, Preethi, Shiva K. Devaiah, and Cecelia A. McIntosh. "Structure-Function Analysis of Grapefruit Glucosyltransferase Protein – Identification of Key Amino Acid Residues for its Rigid Substrate Specificity." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/352.
Full textAbstract:
Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.
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Byrd, Rebekah J., and Danica Hays. "Evaluating a Safe Space Training for School Counselors and Trainees Using a Randomized Control Group Design." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/880.
Full textAbstract:
School counselors need to advocate and act as an ally for all students. Safe Space, a training designed to facilitate competency for working with and serving LGBTQ youth (i.e., LGBTQ competency), has received increased attention in the field of school counseling. However, limited empirical support exists for training interventions such as Safe Space, with only one study to date examining its effectiveness for graduate psychology students (see Finkel, Storaasli, Bandele, & Schaefer, 2003). This study used a randomized pretest-posttest control group design to evaluate and examine the impact of Safe Space training on competency levels of a sample of school counselors/school counselor trainees and to explore the relationship between LGBTQ competency and awareness of sexism and heterosexism.