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You searched for +publisher:"Clemson University" +contributor:("Dr. Kenneth Christensen"). Showing records 1 – 2 of 2 total matches.

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Clemson University

1. Voyton, Charles. Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors.

Degree: PhD, Chemistry, 2018, Clemson University

Members of the class Kinetoplastea including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. cause diseases endemic in rural regions of South America, Sub‐Saharan African and the Eastern Asian continent, effecting hundreds of millions of people and livestock. Existing treatments are associated with high toxicity and rates of resistance, are expensive to produce, and are difficult to administer in rural areas. To develop additional treatment strategies, we must better illuminate the pathways amenable for anti kinetoplastid treatments. One pathway susceptible to drug intervention is glucose metabolism, which in kinetoplasts takes place in glycosomes that are specialized organelles related to mammalian peroxisomes. Disruption of glycosome function is hypothesized to lead to cell death in the pathological bloodstream form of T. brucei as they obtain all cellular ATP via glycosome metabolism. To explore glucose import, and consumption mechanisms in T. brucei we deployed a series of recombinant fluorescent protein biosensors that specifically detect glucose moieties. Biosensors were expressed in T. brucei, and targeted to the cytosol or glycosomes allowing for real time monitoring of intracellular and intraglycosomal glucose concentrations. Using flow cytometry to monitor changes in sensor fluorescence, bloodstream form T. brucei cytosolic and glycosomal glucose were measured as 1.9 ±0.6 mM and 3.5 ± 0.5 mM respectively in response to glucose levels similar to blood (~5mM). Higher glycosomal glucose versus the surrounding cytosol suggests active transport of glucose across the glycosomal membrane, a process that was assumed to occur via passive transport. Monitoring biosensor response in trypanosomes accurately via microscopy is very difficult due to high motility and flagellar undulation. To monitor dynamics in intracellular biosensor response we adapted a microfluidic device which mechanically traps parasites, allowing for continuous imaging of cells under constant perfusion conditions. We found that single trypanosome glucose responses were consistent with bulk glucose measurements, cells also responded in a dose dependent manner when perfused against different glucose concentrations. Microfluidic trapping of T. brucei allows continuous imaging of single cellular dynamics which were previously not possible to image. To identify small molecules that inhibit glucose uptake into parasites we adapted a high throughput screening assay utilizing the fluorescent glucose sensor as a score of glucose uptake inhibition. A pilot screen of 400 compounds identified two novel compounds that inhibit glucose uptake in trypanosome parasites with EC50s of 700nM and 5000nM respectively, one compound exhibited good killing (IC50 5uM) against infectious form parasites. To build upon the success of the pilot screen, 25,000 compounds were analyzed, from this library 57 compounds… Advisors/Committee Members: Dr. George Chumanov, Committee Chair, Dr. Kenneth Christensen, Dr. James Morris, Dr. Daniel Whitehead.

Subjects/Keywords: drug screening; flow cytometry; Fluorescent sensor; genetic biosensor; microfluidics; Trypanosoma brucei

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

APA (6th Edition):

Voyton, C. (2018). Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors. (Doctoral Dissertation). Clemson University. Retrieved from https://tigerprints.clemson.edu/all_dissertations/2099

Chicago Manual of Style (16th Edition):

Voyton, Charles. “Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors.” 2018. Doctoral Dissertation, Clemson University. Accessed November 26, 2020. https://tigerprints.clemson.edu/all_dissertations/2099.

MLA Handbook (7th Edition):

Voyton, Charles. “Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors.” 2018. Web. 26 Nov 2020.

Vancouver:

Voyton C. Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors. [Internet] [Doctoral dissertation]. Clemson University; 2018. [cited 2020 Nov 26]. Available from: https://tigerprints.clemson.edu/all_dissertations/2099.

Council of Science Editors:

Voyton C. Novel Methods for Monitoring Glucose Metabolism in <i>Trypanosoma brucei</i> Using Fluorescent Biosensors. [Doctoral Dissertation]. Clemson University; 2018. Available from: https://tigerprints.clemson.edu/all_dissertations/2099

2. Lin, Sheng. FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI.

Degree: PhD, Chemistry, 2016, Clemson University

Trypanosoma brucei is a unicellular kinetoplastid protozoan that is the causative agent of Human African Trypanosomiasis, or Sleeping Sickness. Combating these protozoan pathogens presents an ongoing challenge due to their efficient immune evasion strategies, lack of effective vaccines, poor drugs and emerging drug resistance, and the general lack of knowledge of their unique biological processes. Among one of the most important biological pathways for kinetoplastid protozoans is the synthesis of ATP from glucose within glycosome organelles. Glycosomes are related to mammalian peroxisomes, but differ in having a fully compartmentalized glycolysis pathway in addition to basic peroxisomal functions. While equivalent processes and organelles have been thoroughly studied through live cell imaging in other organisms, similar studies of T. brucei have only recently made significant progress and many fluorescent-based techniques have not yet been applied in kinetoplastid studies. In order to increase our understanding of trypanosome biology, we have developed quantitative fluorescence-based sensors for sub cellular analysis of metabolic pathways in T. brucei. The pH of the glycosomal compartment was quantified with a probe consisting of a peptide encoding a peroxisomal targeting sequence attached to fluorescein. When incubated with living cells, the probe is internalized within glycosomes and allowed for the quantification of pH through ratiometric analysis of fluorescein emission at 495 nm and 430 nm excitation. Using this probe, we were able to measure the physiological pH (~7.4) of glycosomes under standard culture conditions, as well as discover a pH acidification response (~ 6.8) to nutrient starvation. This starvation response is dependent on Na+ and ATP and regulated independently form the cytosol. The use of transporter and exhcnager inhibitors suggest that V-ATPase and Na+/H+ exchangers are responsible for glycosome pH regulation. We also adapted a ratiometric forster resonance energy transfer (FRET) protein construct (FLIPGlu) consisting of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) FRET pairs flanking a periplasmic binding protein. This protein changes conformation upon binding to glucose, resulting in a measureable change in FRET ratio. FLIPGlu was expressed in T. brucei glycosomes, allowing for the dynamic quantification of glucose. Trypanosomes were found to maintain 230 ± 50 and 530 ± 50 µM glucose in the insect and mammalian life stage respectively. Glucose starvation resulted in a decrease in glycosome glucose levels (~40 ± 10 µM) over 30 minutes but recovers upon reintroduction of glucose. The work presented here fulfills an important need for the implementation of powerful fluorescent techniques to study Trypanosome metabolic pathways. This can greatly advance the understanding of kinetoplastid biology and lead to insights in evolutional divergence of trypanosome biology and therapeutic treatments. While the probes studied here were… Advisors/Committee Members: Dr. Kenneth Christensen, Committee Chair, Dr. Meredith Morris, Dr. Jeffery Anker, Dr. George Chumanov.

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

APA (6th Edition):

Lin, S. (2016). FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI. (Doctoral Dissertation). Clemson University. Retrieved from https://tigerprints.clemson.edu/all_dissertations/1833

Chicago Manual of Style (16th Edition):

Lin, Sheng. “FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI.” 2016. Doctoral Dissertation, Clemson University. Accessed November 26, 2020. https://tigerprints.clemson.edu/all_dissertations/1833.

MLA Handbook (7th Edition):

Lin, Sheng. “FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI.” 2016. Web. 26 Nov 2020.

Vancouver:

Lin S. FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI. [Internet] [Doctoral dissertation]. Clemson University; 2016. [cited 2020 Nov 26]. Available from: https://tigerprints.clemson.edu/all_dissertations/1833.

Council of Science Editors:

Lin S. FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI. [Doctoral Dissertation]. Clemson University; 2016. Available from: https://tigerprints.clemson.edu/all_dissertations/1833

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