Osteoimmunology of Retarded Bone Regeneration in Periodontitis

这份申请书是为了请求对非洲裔美国人单身后培训生的一年支持， 女士Roodelyne Pierrelus在"研究补充，以促进健康相关的多样性"计划下， 研究（PA-21 - 071）"。本研究管理补充材料旨在补充现行的 RO1赠款项目（PI，Kawai），标题为"母RO1赠款，标题为"骨免疫学 牙周炎的再生。"她从诺瓦东南大学心理学院毕业后， (NSU)2019年5月10日，她开始在Kawai实验室从事评估P的影响的研究。 gingivalis特有的脂质磷酸甘油二氢神经酰胺（PGDHC）以及E. coli LPS和Pg LPS对 血小板聚集她在上述研究项目中表现出色， 因提交给IADR/AADR会议的摘要而两次获得IADR Bloc旅行奖（2020年3月，以及 2021年7月）。在未来的一年里，她将把血小板在牙周炎中的作用的研究范围扩大到 涉及破骨细胞生物学。当然，她将努力争取接受在研究生课程的MD/博士 或等同物。因此，我们要求资助她一年的全职研究经验。 背景资料：我们发现活化的血小板可以部分促进破骨细胞的生成 通过在其表面表达的Semaphorin 4D（Sema4D）的表达。然而， Sema4D与抗Sema4D-mAb仅部分抑制血小板介导的 破骨细胞生成我们了解到，波形蛋白，OC-STAMP的假定配体也在细胞上表达。 活化的血小板表面。 假设：我们假设活化血小板上表达的波形蛋白可以作用于OC-STAMP 由破骨细胞前体表达并促进其RANKL诱导的破骨细胞生成。 候选人的项目目标： 目的1：探讨活化血小板表面表达的波形蛋白（Vimentin）在促进血小板活化中的作用。 RANKL诱导的破骨细胞生成。活化血小板将与RANKL-破骨细胞共培养 在存在或不存在抗-OC-STAMP-mAb、抗-波形蛋白-多克隆Ab或对照的情况下， mAb/Ab.破骨细胞生成将通过q-PCR、TRAP染色和陷窝形成测定来确定。使用 生物层干涉法（BLI），OC-STAMP与活化的血小板或重组体之间的结合亲和力 将监测波形蛋白。我们还将分析血小板衍生的波形蛋白是否被瓜氨酸化。如果是则 将评价波形蛋白-瓜氨酸对OC-STAMP介导的破骨细胞生成的影响。 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、BLI和破骨细胞生成试验，以及基本统计分析和解释 这些数据加强了她成为MD/PhD研究生课程候选人的资格。

This application is written to request for the one-year support for African American Post Bachelorette Trainee, Ms. Roodelyne Pierrelus under the program of “Research Supplements to Promote Diversity in Health-Related Research (PA-21-071).” This Research Administrative Supplements is requested to supplement the active RO1 grant project (PI, Kawai) entitled “parent RO1 grant, titled, “Osteoimmunology of regarded bone regeneration in periodontitis.” After she graduated from College of Psychology, Nova Southeastern University (NSU), on May 10, 2019, she started working in Kawai Laboratory on the study that evaluate the effects of P. gingivalis’ unique lipid, phosphoglycerol dihydroceramide (PGDHC), as well as E. coli LPS and Pg LPS on the platelet aggregation. She demonstrated outstanding performance in the above noted research project, and received IADR Bloc travel award twice for the abstract submitted to IADR/AADR meeting (March 2020, as well as July 2021). In the coming year, she will expand her research scope of platelet’s role in periodontitis to involve osteoclast biology. Parallelly, she will be working toward acceptance in graduate program of MD/PhD or equivalent. As such, we request funding to support her full-time research experience for one year. Background information: We discovered that activated platelets can promote the osteoclastogenesis in part by the expression of Semaphorin 4D (Sema4D) expressed on its surface. However, the neutralization of Sema4D with anti-Sema4D-mAb only partially inhibited the platelet-mediated upregulation of osteoclastogenesis. We learned that vimentin, the putative ligand for OC-STAMP is also expressed on the cell surface of activated platelets. Hypothesis: We hypothesized that vimentin expressed on activated platelets can act on OC-STAMP expressed by osteoclast precursors and promote their RANKL-induced osteoclastogenesis. Project Objectives for Candidate: Objective 1: To establish the role of Vimentin expressed on the activated platelets in the promotion of RANKL-induced osteoclastogenesis. Activated platelets will be co-cultured with RANKL- osteoclast precursors in the presence or absence of anti-OC-STAMP-mAb, anti-Vimentin-polyclonal Ab or control mAb/Ab. Osteoclastogenesis will be determined by q-PCR, TRAP staining and pit-formation assay. Using a biolayer interferometry (BLI), the binding affinity between OC-STAMP and activated platelet or recombinant vimentin will be monitored. We will also analyze whether platelet-derived vimentin is citrullinated. If so, the impact of Vimentin-citrullination on OC-STAMP-mediated osteoclastogenesis will be evaluated. After completing these two objectives, the candidate will have mastered the methods of cell culture, W- blotting, qPCR, BLI, and osteoclastogenesis assays, as well as basic statistical analysis and interpretation of data which strengthen her credential to be a candidate for graduate program of MD/PhD.