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

Administration of the antioxidant n-acetyl-cysteine in pregnant mice has long-term positive effects on metabolic and behavioral endpoints of male and female offspring prenatally exposed to a high-fat diet

A growing body of evidence suggests the consumption of high-fat diet (HFD) during pregnancy to model maternal obesity and the associated increase in oxidative stress (OS), might act as powerful prenatal stressors, leading to adult stress-related metabolic or behavioral disorders. We hypothesized that administration of antioxidants throughout gestation might counteract the negative effects of prenatal exposure to metabolic challenges (maternal HFD feeding during pregnancy) on the developing fetus. In this study, female C57BL/6J mice were fed HFD for 13 weeks (from 5-weeks of age until delivery) and were exposed to the N-acetyl-cysteine (NAC) antioxidant from 10-weeks of age until right before delivery. Body weight of the offspring was assessed following birth, up to weaning and at adulthood. The metabolic, neuroendocrine and emotional profile of the adult offspring was tested at 3-months of age. Prenatal HFD increased mother’s body weight and offspring’s weight at the time of weaning, when administered in conjunction with NAC. In females, NAC administration reduced high levels of leptin resulting from prenatal HFD. Prenatal NAC administration also resulted in greater glucose tolerance and insulin sensitivity while increasing adiponectin levels, as well as increasing exploratory behavior, an effect accompanied by reduced plasma corticosterone levels in response to restraint stress. Analysis of glutathione levels in the hypothalamus and in brown adipose tissue indicates that, while HFD administration to pregnant dams led to reduced levels of glutathione in the offspring, as in the male hypothalamus, NAC was able to revert this effect and to increase glutathione levels both in the periphery (Brown Adipose Tissue, both males and females) and in the central nervous system (males). Overall, results from this study indicate that the body redox milieu should be tightly regulated during fetal life and that buffering OS during pregnancy can have important long-term consequences on metabolic and behavioral endpoints

Antioxidant N-acetyl-cysteine administration in pregnant mice has long-term positive effects on metabolic and behavioural endpoints of male and female offspring prenatally exposed to a high-fat-diet

Consumption of a high-fat-diet (HFD) during pregnancy acts as powerful prenatal stressor leading to adult stress-related metabolic or behavioural disorders. We hypothesized that administration of antioxidants throughout gestation might counteract the negative effects of maternal HFD feeding during pregnancy on the developing foetus. Female C57BL/6J mice were fed HFD for 13 weeks and exposed to the N-acetyl-cysteine (NAC) antioxidant from 10-weeks of age until right before delivery. Body weight of the offspring was assessed from birth until adulthood. At this age, the metabolic, neuroendocrine and emotional profiles were also tested. Prenatal HFD increased mother and offspring body weight when administered in conjunction with NAC. In female offspring, NAC reduced the increase in leptin levels resulting from prenatal HFD. NAC administration resulted also in greater glucose tolerance, insulin sensitivity and increased adiponectin levels. Exploratory behaviour was increased in NAC offspring, an effect accompanied by reduced stress-induced plasma corticosterone levels. Moreover, prenatal HFD administration led to reduced glutathione levels in the hypothalamus of males while NAC was able to revert this effect both in the brain (males) as well as in the brown adipose tissue (both males and females). Overall, results from this study indicate that the body redox milieu should be tightly regulated during foetal life and that buffering oxidative stress during pregnancy can have important long-term consequences on metabolic and behavioural endpoints. Support: H2020 AwE (N. 633589)

Temperature response of soil respiration largely unaltered with experimental warming

© 2016, National Academy of Sciences. All rights reserved. The respiratory release of carbon dioxide (CO2) from soil is a major yet poorly understood flux in the global carbon cycle. Climatic warming is hypothesized to increase rates of soil respiration, potentially fueling further increases in global temperatures. However, despite considerable scientific attention in recent decades, the overall response of soil respiration to anticipated climatic warming remains unclear. We synthesize the largest global dataset to date of soil respiration, moisture, and temperature measurements, totaling \u3e 3,800 observations representing 27 temperature manipulation studies, spanning nine biomes and over 2 decades of warming. Our analysis reveals no significant differences in the temperature sensitivity of soil respiration between control and warmed plots in all biomes, with the exception of deserts and boreal forests. Thus, our data provide limited evidence of acclimation of soil respiration to experimental warming in several major biome types, contrary to the results from multiple single-site studies. Moreover, across all nondesert biomes, respiration rates with and without experimental warming follow a Gaussian response, increasing with soil temperature up to a threshold of ∼25 °C, above which respiration rates decrease with further increases in temperature. This consistent decrease in temperature sensitivity at higher temperatures demonstrates that rising global temperatures may result in regionally variable responses in soil respiration, with colder climates being considerably more responsive to increased ambient temperatures compared with warmer regions. Our analysis adds a unique cross-biome perspective on the temperature response of soil respiration, information critical to improving our mechanistic understanding of how soil carbon dynamics change with climatic warming

Temperature response of soil respiration largely unaltered with experimental warming

The respiratory release of carbon dioxide (CO2) from soil is a major yet poorly understood flux in the global carbon cycle. Climatic warming is hypothesized to increase rates of soil respiration, potentially fueling further increases in global temperatures. However, despite considerable scientific attention in recent decades, the overall response of soil respiration to anticipated climatic warming remains unclear. We synthesize the largest global dataset to date of soil respiration, moisture, and temperature measurements, totaling >3,800 observations representing 27 temperature manipulation studies, spanning nine biomes and over 2 decades of warming. Our analysis reveals no significant differences in the temperature sensitivity of soil respiration between control and warmed plots in all biomes, with the exception of deserts and boreal forests. Thus, our data provide limited evidence of acclimation of soil respiration to experimental warming in several major biome types, contrary to the results from multiple single-site studies. Moreover, across all nondesert biomes, respiration rates with and without experimental warming follow a Gaussian response, increasing with soil temperature up to a threshold of ∼25 °C, above which respiration rates decrease with further increases in temperature. This consistent decrease in temperature sensitivity at higher temperatures demonstrates that rising global temperatures may result in regionally variable responses in soil respiration, with colder climates being considerably more responsive to increased ambient temperatures compared with warmer regions. Our analysis adds a unique cross-biome perspective on the temperature response of soil respiration, information critical to improving our mechanistic understanding of how soil carbon dynamics change with climatic warming