THE PHYSIOLOGY OF DRUG RESISTANT MALARIA

为了迅速开发新的更好的治疗方法来对抗耐药疟疾，我们必须充分阐明抗疟疾作用和抗疟疾耐药性的分子机制。此外，迫切需要更快速、更方便的各种抗疟药耐药表型的生化和生理检测方法。通过对疟疾寄生虫生理学和生物物理学进行更详细的单细胞水平分析，这两个问题都得到了有效的解决。因此，我们建议使用现代亚细胞成像和单细胞光度法详细分析活的红细胞内疟疾寄生虫的转运、pH和膜电位现象。具体地说，我们将：1)在恒定灌流和激光共聚焦显微镜下，使用细胞单细胞光度法检测红细胞内活恶性疟原虫的胞液和空泡pH(pHcyt，pHvac)与膜电位和抗疟疾药物耐药性之间的推测关系。2)阐明PHcyt、pHvac和膜电位调节的离子依赖性、抑制剂敏感性和动力学特征。3)利用抗性和敏感寄生虫的遗传杂交后代，检验1，2中的结论。4)确定抗疟疾药物和逆转剂对生物物理参数的影响。5)与恶性疟原虫实验室分离株和间日疟原虫野外分离株进行比较和对比测定。除了确定抗药性寄生虫的离子转运生理学外，这些实验还将着眼于开发有助于快速、“现场兼容”诊断抗药性疟疾的原理。

To rapidly develop new and better therapies to combat drug resistant malaria, we must fully elucidate the molecular mechanisms of antimalarial action and antimalarial drug resistance. In addition, more rapid and convenient biochemical and physiological assays for various antimalarial drug resistance phenotypes are desperately needed. Both of these issues are effectively addressed via more detailed, single-cell level analysis of malarial parasite physiology and biophysics. We thus propose a detailed analysis of transport, pH, and membrane potential phenomena in living intraerythrocytic malarial parasites using modern subcellular imaging and single - cell photometry methods. Specifically, we will: 1) Test putative relationships between cytosolic and vacuolar pH (pHcyt, pHvac) and membrane potentials of living intraerythrocytic P. falciparum and resistance to antimalarial drugs using single-cell photometry of cells under constant perfusion and laser confocal microscopy. 2) Elucidate ion dependencies, inhibitor sensitivities, and kinetic characteristics of pHcyt, pHvac, and membrane potential regulation. 3) Test conclusions from 1,2 using progeny of genetic cross between resistant and sensitive parasites. 4) Define effects of antimalarial drugs and reversal agents on biophysical parameters. 5) Compare and contrast measurement with laboratory strains vs. field isolates of P. falciparum and P. vivax. Along with defining the ion - transport physiology of drug resistant parasites, these experiments will be performed with an eye towards development of principles that will aid in the rapid, "field compatible", diagnosis of drug resistant malaria.