Osteoimmunology of Retarded Bone Regeneration in Periodontitis

多样性行政补充申请（PA-21-071）是书面申请一年 支持西班牙裔后单身女郎实习生，伊丽莎白莱昂女士，根据父母的RO 1赠款，标题， “牙周炎中骨再生的骨免疫学研究”她将进行基础研究， 阐明牙周炎致病性骨吸收的机制。通过拟议的 培训，她不仅将成为熟悉的操作方法，以执行相关的基础研究， 的口腔疾病，而且还掌握了概念的方法，找到知识差距和解决的方法， 存在的问题。提高严谨性和再现性的基本原理，以及 还将学习负责任的研究行为。当然，她将致力于接受在 牙科计划。该培训的目标是支持未来临床医生/科学家的职业发展 在牙科科学的基础和转化研究。 候选人的研究项目背景：我们未发表的数据表明，牙龈卟啉单胞菌的囊泡 能促进破骨细胞的生成，而破骨细胞的生成被从PAD-KO中分离的囊泡显著减弱 P.牙龈炎此外，牙龈卟啉单胞菌囊泡上调RANKL-1细胞中OC-STAMP的表达。 刺激RAW264.7破骨细胞前体细胞。 假设：我们假设由牙龈卟啉单胞菌囊泡表达的PAD可以瓜氨酸化宿主衍生的 波形蛋白，其反过来作用于破骨细胞表达的OC-STAMP并促进破骨细胞生成 候选人的项目目标： 目的1：探讨牙龈卟啉单胞菌囊泡表达的PAD（Pg vesicle-PAD）在牙龈卟啉单胞菌感染中的作用。 在囊泡和破骨细胞的共培养中瓜氨酸化宿主蛋白，包括波形蛋白。 目的2：检测Pg囊泡-PAD介导的瓜氨酸化是否能改变宿主波形蛋白的表达， 波形蛋白对促进RANKL诱导的破骨细胞生成的作用。 将使用以下方法从PAD-KO Pg W83（Potempa博士赠送）和WT Pg W83中分离囊泡： 超浓缩机这些分离的囊泡以及整个细菌细胞将与 破骨细胞前体宿主蛋白的瓜氨酸水平将通过W-印迹和质谱测定。 将使用W-印迹法监测囊泡的PAD表达。最后，可能参与OC- 破骨细胞表达的STAMP是破骨细胞生成中瓜氨酸化波形蛋白的受体， 通过用抗OC-STAMP mAb或抗瓜氨酸化波形蛋白mAb阻断测定。 完成这两个目标后，候选人将掌握细胞培养的方法，W- 印迹、qPCR、流式细胞术和破骨细胞生成测定，以及基本的统计分析和 这些数据增强了她成为研究生候选人资格。

This application for Diversity Administrative Supplement (PA-21-071) is written to request for the one-year support for Hispanic Post Bachelorette Trainee, Ms. Elizabeth Leon, under the parent RO1 grant, titled, “Osteoimmunology of regarded bone regeneration in periodontitis.” She will perform the basic research to elucidate the mechanism underlying the pathogenic bone resorption in periodontitis. Through the proposed training, she will not only become familiar with the operational approach to perform basic research related to the oral diseases, but also master the conceptual approach to find the knowledge gap and the way to solve the existing problem. The fundamentals for increasing the rigor and reproducibility, as well as the principle for responsible research conduct will be also learned. Parallelly, she will be working toward acceptance in the program of dentistry. The goal of this training is to support the career development of a future clinician/scientist in the basic and translational research in the dental sciences. Candidate’s research project background: Our unpublished data demonstrated that P. gingivalis’ vesicles can promote the osteoclastogenesis which was significantly attenuated by the vesicles isolated from PAD-KO P. gingivalis. Furthermore, P. gingivalis’ vesicles up-regulated the expression of OC-STAMP in the RANKL- stimulated RAW264.7 osteoclast precursor cells. Hypothesis: We hypothesized that PAD expressed by vesicles of P. gingivalis can citrullinate the host derived Vimentin which, in turn, acts on OC-STAMP expressed by osteoclasts and promotes osteoclastogenesis Project Objectives for Candidate: Objective 1: To evaluate the role of PAD expressed by vesicles of P. gingivalis (Pg vesicle-PAD) in citrullinating the host proteins, including Vimentin, in the co-culture of vesicles and osteoclasts. Objective 2: To examine whether Pg vesicle-PAD-mediated citrullination of host vimentin can alter Vimentin’s effect on promoting RANKL-induced osteoclastogenesis. The vesicles will be isolated from PAD-KO Pg W83 (gift from Dr. Potempa) and WT Pg W83 using a ultracentrifuge machine. Those isolated vesicles, as well as whole bacterial cells, will be co-cultured with osteoclast precursors. The level of citrullination of host proteins will be determined by W-blot and Mass-Spec. The expression of PAD by vesicles will be monitored using a W-blot. Finally, possible engagement of OC- STAMP expressed by osteoclasts which is the receptor for citrullinated Vimentin in osteoclastogenesis will be determined by blocking with anti-OC-STAMP mAb or anti-citrullinated Vimentin mAb. After completing these two objectives, the candidate will have mastered the methods of cell culture, W- blotting, qPCR, flow cytometry, and osteoclastogenesis assays, as well as basic statistical analysis and interpretation of data which strengthen her credential to be a candidate for graduate program.