Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis

项目摘要/摘要 这是对R01AI156382奖的这一多样性补充，该奖项的标题是“在动质体内发展一种多重分析”。 寄生虫识别糖酵解的探针“将支持克莱姆森大学的Sabrina Pizarro的培训 来自卡托巴印第安人民族的遗传学和生物化学系研究生 科学界代表性不足的群体。这个项目的目标有两个。首先，该项目将支持 Sabrina致力于开发和验证用于非洲锥虫的新分子工具， 布氏锥虫。Sabrina将产生前环状(Pf)和血流型(BF)布氏支原体寄生虫， 研制了一种基于pH-2的蛋白质pH生物传感器。PHluorin 2是一种比率pH变异体，它显示一个pH (酸)依赖的兴奋，可以很容易地通过流式细胞仪和显微镜进行监测。Pf和BF寄生虫 表达这些传感器将被用来解析酸化的细胞机制或亚细胞细胞器。 那里面有糖酵解，糖小体。PH值感应细胞系将起到额外的作用，在 父建议书中描述的工作。PHLORIN2线将用作辅助屏幕，使我们能够 通过扰乱细胞或糖体的pH来区分影响糖酵解酶活性的小分子 这些抑制物更直接地抑制了该途径中的酶。该项目的第二个目标是支持 萨布丽娜的职业发展，因为她努力实现她的职业目标。通过与PI的交互 和共同导师，以及参与各种职业发展机会，我们预计 塞布丽娜将做好充分的准备，成功地在她的职业道路上迈出下一步。

Project Summary/Abstract This Diversity Supplement to the award R01AI156382, "Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis” will support the training of Sabrina Pizarro, a Clemson University graduate student in the Department of Genetics and Biochemistry that is from the Catawba Indian Nation, an under-represented group in the sciences. The goal of this project is two-fold. First, the project will support Sabrina’s research effort to develop and validate new molecular tools for use in the African trypanosome, Trypanosoma brucei. Sabrina will generate procyclic form (PF) and bloodstream form (BF) T. brucei parasites that express a protein pH biosensor based on pHluorin2. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid) dependent excitation that can be readily monitored by flow cytometry and microscopy. PF and BF parasites expressing these sensors will be used to resolve cellular mechanisms of acidification or the subcellular organelle that houses glycolysis, the glycosome. The pH sensing cell lines will serve an additional purpose, playing a role in the work described in the parent proposal. The pHluorin2 lines will be used as a counterscreen, allowing us to discriminate small molecules that influence glycolytic enzyme activity by disrupting cellular or glycosomal pH from those inhibitors that more directly inhibit enzymes in the pathway. The second goal of the project is to support Sabrina’s professional development as she works to achieve her career goals. Through interactions with the PI and co-mentor, along with participation in a variety of professional development opportunities, we anticipate that Sabrina will be fully positioned to successfully pursue the next step in her career path.