TOXOPLASMA DEVELOPMENT AND CELL CYCLE

描述（改编自申请人的摘要）：专性细胞内 弓形虫是一种重要的条件致病菌， 免疫功能低下的人，如艾滋病患者。 T.弓形虫还导致 严重的新生儿先天缺陷。 这些研究旨在 进一步了解T.弓形虫能够 在人类宿主体内存活并致病 工资支持 K 02奖提供的将使金博士有“受保护的时间”， 集中精力从其他国家引进技术和专业知识， 生物学研究领域，特别是真核细胞周期 调控和信号转导，细胞周期的研究， 弓形虫发育。 在人类宿主中，T.弓形虫存在于 速殖子，这是负责临床上明显的疾病， 缓殖子，是潜伏的包囊形式。 随着免疫功能的减弱， 缓慢生长的缓殖子可以重新激活快速增殖的 速殖子形成导致弓形虫脑炎 鉴于被标记者 速殖子与缓殖子相比的增殖差异， 弓形虫不能在宿主细胞外复制，细胞周期 在弓形虫的发育过程中，监管无疑是至关重要的， 疾病的发病机制。 从对其他真核生物的研究中可以清楚地看到， 对发育或分化信号的反应发生在特定的 细胞周期内的点，并导致协调调节 阶段特异性基因表达和细胞周期进程。 候选 假设T独特方面。弓形虫细胞周期调控 协调导致疾病发病机制和细胞内 生存 目的1：研究T.弓形虫 将集中于核糖核苷酸还原酶（RNR）基因表达进行分析。 目的2：探讨T.弓形虫细胞周期蛋白依赖性激酶 (cdk)将使用免疫荧光、流式细胞术、北方 和Western印迹。 将具有突变cdk的构建体转染入 速殖子，以确定CDK在细胞周期进程中的作用。 最后，在目标3中， 细胞周期调控与T.弓形虫缓殖子发育 将被研究。 细胞周期标志物将用于检查细胞周期 缓殖子发育的依赖性。 细胞周期和缓殖子报告基因 构建体将用于测试细胞周期抑制剂的作用， 对基因表达的分化的潜在诱导剂或抑制剂。 这些将是长期努力的初步实验，以确定 细胞周期调控在弓形虫速殖子-缓殖子中的作用 分化和毒力。

DESCRIPTION (adapted from applicant's abstract): The obligate intracellular parasite Toxoplasma gondii is an important opportunistic pathogen in immunocompromised individuals such as AIDS patients. T. gondii also causes devastating birth defects in neonates. These studies are designed to provide further understanding of the mechanisms by which T. gondii is able to persist and cause disease within its human host. The salary support provided by the K02 award will enable Dr. Kim to have "protected time" to concentrate upon her efforts to adapt technology and expertise from other areas of biological investigation, specifically eukaryotic cell cycle regulation and signal transduction, to the study of cell cycle and Toxoplasma development. Within human hosts, T. gondii exists as tachyzoites, which are responsible for clinically apparent disease, and as bradyzoites, which are latent encysted forms. With waning immune function, slow-growing bradyzoites can reactivate to the rapidly proliferating tachyzoite forms, leading to Toxoplasma encephalitis. Given the marked difference in proliferation of the tachyzoite compared to the bradyzoite and Toxoplasma's inability to replicate outside host cells, cell cycle regulation is undoubtedly critical in Toxoplasma development and pathogenesis of disease. It is clear from studies of other eukaryotes that responses to signals for development or differentiation occur at specific points within the cell cycle and result in a coordinate modulation of stage-specific gene expression and cell cycle progression. The candidate hypothesizes that unique aspects of T. gondii cell cycle regulation coordinate the processes leading to disease pathogenesis and intracellular survival. In Aim 1, regulation of cell cycle gene expression in T. gondii will be analyzed focusing on ribonucleotide reductase (RNR) gene expression. In Aim 2, expression and function of T. gondii cyclin dependent kinases (cdk) will be examined using immunofluorescence, flow cytometry, Northern and Western blotting. Constructs with mutant cdks will be transfected into tachyzoites to determine the role of cdks in cell cycle progression. Finally, in Aim 3, the relationship of cell cycle regulation and T. gondii bradyzoite development will be studied. Cell cycle markers will be used to examine the cell cycle dependence of bradyzoite development. Cell cycle and bradyzoite reporter constructs will be used to test the effects of cell cycle inhibitors and potential inducers or inhibitors of differentiation on gene expression. These will be the initial experiments in a long-term effort to define the role of cell cycle regulation in Toxoplasma tachyzoite-bradyzoite differentiation and virulence.