Characterization of O-GlcNAc signalling in human placenta

The aim of this thesis is to investigate the role of the recently discovered post-translational modification, O-GlcNAcylation, in the regulation of human placenta physiology. The rationale for this research work arises from the increasing evidence showing that O-GlcNAc signalling is a key mediator of cellular functions involved in a wide range of chronic diseases. However, there is a paucity of studies investigating its role in human placenta. Abnormal placenta physiology is considered as an underlying cause of poor pregnancy outcomes including miscarriage, preeclampsia and intrauterine growth alterations, the latter associated with increased risk for the development of chronic disorders later in life. Despite its importance, some of the molecular mechanisms controlling placental formation and function are still poorly understood. In this thesis, the BeWo cell line, a validated model to study in vitro mechanisms occurring in human trophoblast, and term placental biopsies, were employed. A transient OGT-depleted BeWo cell line was generated using a small interfering RNA (siRNA) methodology, and was used to identify the role of O-GlcNAc transferase (OGT) and its biochemical mark O-GlcNAcylation during the formation of syncytiotrophoblast, a crucial structure controlling the majority of placental functions. In addition, an antibody-based enrichment method, followed by proteomic analysis, was used to identify O-GlcNAc-modified proteins in BeWo extracts. Term placental biopsies were used to investigate correlations between OGT and feto-maternal parameters. Overall, the results obtained in this research work support a critical role for OGT in the control of syncytiotrophoblast formation via mechanisms involving PKA-MAPKs signalling. Importantly, the proteomic analysis showed that O-GlcNAc-modified proteins belong to a wide range of functional classes, in particular, transcription and chromatin remodelling factors. Finally, maternal body mass index (BMI) and depression target OGT signalling in term placentas with consequences on the activity of regulators of fetal growth and mechanisms of placental response to stress. Taken together, this thesis uncovered novel roles for placental OGT in controlling a wide range of placental mechanisms that are crucial to human reproduction success and identified O-GlcNAc signalling as the molecular link between maternal environment and in utero fetal programming of human diseases