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- Untangling sex differences in glia-to-neuron communication in chronic alcohol exposurePublication . Pacheco, Raquel; Canedo, Teresa; Rodrigues, Ana Margarida; Moreira, Joana; Relvas, João B.; Vieira, Cristina P.; Socodato, Renato; Sumavielle, Teresa; Summavielle, TeresaExcessive alcohol consumption is global health challenge with profound neurobiological consequences. Our laboratory has shown that alcohol exposure induces reactive astrocytic changes, affecting gene expression, activity, and proliferation, while also altering microglial morphology and immune responses [1]. This study aims to characterise sex-specific effects of chronic alcohol consumption on the prefrontal cortex (PFC), focusing on glial cell morphology, synaptic density, and behaviour. Adult mice underwent a voluntary drinking model using an intermittent ‘every-other-day’ (EOD) paradigm with 15% (v/v) ethanol for three weeks. Behavioural tests for anxiety, depression, and memory were conducted before sacrifice. Brains were processed for glial cell analysis using immunohistochemistry and confocal microscopy. Synaptosomes were isolated for proteomics and analysis used STRING and Panther. Preliminary data reveal sex-dependent glial and synaptic adaptations. Males showed increased astrocyte volume in the ventromedial PFC (vmPFC) and hyper-ramification in the ventrolateral PFC (vlPFC), while females showed reductions in astrocyte size and complexity. Microglia also displayed sex-specific changes: males had decreased microglial volume in the vlPFC, while females exhibited increased microglial size. Inhibitory synapse density was elevated in males, while females showed an increase in excitatory synapses. These changes correlated with behavioural differences—males displayed heightened anxiety, whereas females exhibited reduced anxiety. Proteomic analysis further supports sex-dependent molecular adaptations: male-enriched proteins were linked to myelination and glial development, while female-specific proteins were associated with mRNA processing, RNA transport, and axonogenesis. Chronic alcohol exposure induces sex-specific neuroimmune and synaptic alterations, potentially contributing to differential susceptibility to alcohol use disorders (AUD).
- Association between placental weight and birthweight, adjusted for parity, sex and PAPP-A levels: a retrospective cohort studyPublication . Santos, Inês; Moreira, Rui; Gonçalves, Inês Sarmento; Dias, Cláudia Camila; Ramalho, CarlaPlacental weight has been studied as an indicator of placental function, correlating with birthweight and perinatal outcomes. The fetal-to-placental ratio (FPR) reflects the adaptation to hypoxemia and fetal growth needs. This study aims to assess the association between placental weight and birthweight, adjusting for parity, fetal sex and PAPP-A. A retrospective cohort study was conducted at a tertiary university hospital. We included singleton pregnancies undergoing first-trimester screening and delivering at the same hospital between May 2013 and September 2024. Clinical data were gathered from electronic databases (SClínico®, ObsCare®, ASTRAIA®). Categorical data were analyzed using Chi-squared tests. Continuous variables were assessed through ANOVA or T test. Pearson or Spearman correlation was applied as appropriate. For significance, a p-value<0.05 was assumed. All data was processed with IBM SPSS v29.0. Results: Among 16492 singleton pregnancies, 54.9% women were nulliparous and 8.9% newborns were small for gestational age. Placental weight was positively correlated with birthweight (r=0.517,p<0.001), which increased by 1.77g per gram of placental weight, explaining 26.7% of the birthweight variation. Preterm deliveries (6.8%) had lower mean placental weight (493.1g versus 579.2g) and birthweight (900g difference, p<0.001) compared to term group. Mean male fetuses’ birthweight was 121.3g higher than female (p<0.001). Additionally, “female fetuses” and “nulliparity” were associated with decreased placental weight (p<0.001). On average, the FPR was 6:1, with male fetuses exhibiting greater FPR (p<0.001) as well as term deliveries (5.7) compared to preterm (4.8), p<0.001. FPR was also correlated with Apgar score at 5min (Apgar<7:5.0 vs. Apgar≥7:5.7, p<0.001) and PAPP-A levels (p<0.001). However, parity (p=0.121) showed no significant association with FPR. Placental weight correlates with birthweight. FPR reflects placental efficiency and perinatal outcome, influenced by fetal sex and gestational age.
- Breastfeeding and malocclusions: Characterization of a pediatric dentistry population enrolled in a birth cohort studyPublication . Marques, Sofia Cameron; Baptista, Manuel; Magalhães, Inês; Morais, Juliana; Ferreira, Ana Filipa; Pestana, Rebeca; Jerónimo, Maria Luís; Pinto, Carla; Sousa, Marta; Pires, Inês Falcão; Deuster, Denise; Zaura, Egija; Areias, Cristina; Maia, Benedita SampaioBreastfeeding plays an important role in the development of the craniofacial complex (1,2). However, the role of breastfeeding duration, non-nutritive sucking habits, and the timing of solid food introduction on the development of malocclusions is still in debate (25). Therefore, this study aimed to investigate these associations in a birth cohort. In the ongoing OralBioBorn birth cohort, occlusion of three-year-old children was assessed in maxiumum intercuspidation, examining the left and right sides, followed by a frontal assessment. In addition, the information on breastfeeding type and duration, solid food introduction, and non-nutritive sucking habits was collected through a parental questionnaire. Preliminary data from 39 children revealed a high prevalence of open bite (33.3%), crossbite (20.5%), and overbite (10.3%). Scissor bite, high/narrow palate, and crowding were observed in only one child each. The relationship between these occlusal characteristics and breastfeeding duration, non-nutritive sucking habits, and solid food introduction will be further explored. The preliminary findings suggest that malocclusions are frequent at an early age, reinforcing the need to investigate their potential early-life determinants. Future work will involve a more in-depth analysis of the associations between occlusal development and early feeding practices, considering additional factors such as genetic predisposition and environmental influences. Longitudinal follow-up of this cohort will provide valuable insights into the long-term impact of breastfeeding and oral habits on craniofacial growth, ultimately contributing to early preventive strategies.
- Association between maternal cardiometabolic risk factors and oral health of infantsPublication . Pestana, Rebeca Saad; Baptista, Manuel; Magalhães, Inês; Morais, Juliana; Ferreira, Ana Filipa; Marques, Sofia Cameron; Jerónimo, Maria Luís; Pinto, Carla; Sousa, Marta; Falcão-Pires, Inês; Duister, Denise; Zaura, Egija; Azevedo, Maria João; Maia, Benedita SampaioMaternal health conditions during pregnancy influence infant health1-3. Cardiometabolic risk factors (CRFs), such as obesity, hypertension, and gestational diabetes, increase maternal and neonatal complications due to chronic inflammation, which disrupts immune and endocrine adaptation4,5. While the effects of CRFs on infant development are well studied, their impact on oral health remains unclear. Therefore, this study aims to assess the association between maternal CRFs and oral health of infants at three years of age. The prospective OralBioBorn cohort follows pregnant women (healthy and with CRFs) and their children up to 36 months postpartum. At age three, oral hygiene habits, caries experience using ICDAS, and Quigley-Hein plaque index (PI) of children were assessed. PI was evaluated on buccal and lingual surfaces. Preliminary data from 36 children (26 from healthy mothers, 10 from mothers with CRFs) showed no cavitated, missing, or filled teeth due to caries, nor PUFA Index lesions, in either group. The PI did not differ significantly between children of healthy mothers and those of mothers with CRFs (0.17±0.32 vs. 0.49±0.72, p>0.05, Mann-Whitney U test). The lack of associations between maternal CRFs and child oral health may reflect multiple factors, including the small sample size, the young age of the children, and the possibility that maternal CRFs do not exert a strong early influence on oral health. Future work will focus on evaluating a larger cohort and considering additional determinants, such as oral hygiene and dietary habits, which may play a more prominent role in shaping early oral health outcomes. This will provide a deeper understanding of potential mediating factors influencing early oral health trajectories
- Early-life exposure to non-nutritive sweeteners: Impact on adipose tissue morphology and metabolic functionPublication . Oliveira-Barbosa, Margarida; Bracchi, Isabella; Keating, Elisa; Negrão, RitaNon-sugar sweeteners (NSS) are sugar alternatives widely incorporated into food and beverages (1), providing sweetness with negligible caloric contribution (2). Over the past two decades, their consumption has increased among pregnant women (3,4). In 2023, WHO discouraged NSS intake highlighting the need for studies regarding exposure during critical windows of development (5), such as pregnancy and childhood (6). The MHSWEET project explores the role of NSS consumption on fetal programming of metabolic dysfunction. This branch of the MHSWEET aims to evaluate metabolic function of adipose tissue of adult mice exposed in utero to Rebaudioside A (RebA), the main sweetener component of the NSS Stevia. It also seeks to determine if this early-life exposure affects offspring’s susceptibility to metabolic dysfunction induced by a high-fat diet (HFD). Female Sprague-Dawley rats (G0) ingested RebA in drinking water (4mg steviol eq/kg body weight/day – EFSA’s ADI, n=8), or regular water (C, n=8), before mating until weaning (13 weeks). After weaning, offspring (G1) were fed a standard diet (STD) until 8 months of age, when they were administered STD or HFD until 10 months of age, creating 4 groups of study: C/STD, C/HFD, RebA/STD and RebA/HFD. Mesenteric adipose tissue morphology (H&E staining), as well as lipid and mitochondrial metabolic pathways (RT-PCR) will be assessed. The results so far showed that RebA exposure increased body weight in female offspring from 30 weeks of age onwards compared to controls (p=0.0165). Blind histological and RT-PCR analyses are ongoing to search for: a) adipocyte hypertrophy potentially induced by RebA exposure or HFD, mainly in RebA exposed offspring and b) alterations in lipid and mitochondrial metabolic pathways that may explain the observed changes in body weight and any changes in adipocyte morphology. This study will be crucial to assess health implications of NSS consumption during vulnerable stages of life.
- Peripheral metabolism in rat with olanzapine subchronic treatment supplemented with acetyl-L carnitinePublication . Amorim, Manuela; Silva, Ana Isabel; Alves, Cecília Juliana; Monteiro, Pedro R.; Magalhães, Ana; Summavielle, TeresaOlanzapine (Olz) have been reported to promote higher risk for metabolic side-effects, such as obesity, dyslipidemia, and diabetes. Moreover/On the other hand, acetyl-L-carnitine (ALC) has been reported to improve homeostasis. This study aims to explore if ALC could improve metabolic homeostasis due to Olz effects on a rodent model. We investigated the effects of subchronic administration of Olz and with the addition of ALC on body weight gain and glucose metabolism in Wistar rats. Mid-term Olz exposure in rats significantly increases blood glucose levels in fasting and after the overload of glucose compared to the sali-ne-treated group (p<0.05). However, in rats mid-term treated with olanzapine plus acetyl-L-carnitine had no significant differences in fasting blood glucose levels compared to the saline-treated group. The findings suggest that ALC added to Olz treatment appeared to be effective to improve glucose metabolism during olanzapine subchronic treatment.
- Deciphering neuroimmune interactions in alcohol intake in mouse model of intermittent access in male and femalePublication . Pacheco, Raquel; Canedo, Teresa; Rodrigues, Ana M.; Moreira, Joana; Relvas, João B.; Socodato, Renato; Summavielle, Teresa; Summavielle, TeresaExcessive alcohol consumption continues to pose a significant global health challenge, with detrimental effects on millions of individuals. Our laboratory has shown that alcohol exposure triggers reactive changes in astrocytes, including alterations in gene expression, activity, and proliferation, while also affecting microglial morphology and immune responses. We are to characterizing the effects of chronic alcohol consumption using a wellestablished voluntary alcohol drinking model in adult mice, to investigate the impact of chronic alcohol exposure on the prefrontal cortex (PFC), focusing on glial cell morphology, synaptic density, and behaviour. Mice are exposed to intermittent “every-other-day” (EOD) access to alcohol 15% (v/v) for 3 weeks, and behaviourally tested for anxiety, depression and memory, before sacrifice at 21 days of alcohol, or at 7 days of withdrawal. Brains were processed for glial cell analysis. Our preliminary findings revealed sex-specific responses following chronic alcohol exposure. Male mice exhibited increased astrocyte volume in the ventromedial PFC (vmPFC) and hyper-ramification in the ventrolateral PFC (vlPFC), whereas females displayed reductions in astrocyte size and complexity. Microglia morphology also differed between sexes, with females showing increased cell volume and males displaying reduced microglial volume in the vlPFC. These results suggest distinctive immune and synaptic responses to ethanol in males and females. Of note, we observed heightened inhibitory synapse density in the male PFC, while females exhibited increased excitatory synapse density. We are now conducting a proteomic analysis of PFC synaptosomes to identify important molecular targets in the crosstalk between neuros and glial cells. With this work we expect to clarify the complex interplay between chronic ethanol exposure, sex, and PFC function, find also new targets for innovative therapeutic approaches.
- Early-life exposure to non-nutritive sweeteners: effects on hepatic FGF21 pathways and mitochondrial functionPublication . Brandão, Sofia; Bracchi, Isabella; Felipe, Stela Mirla; Magalhães, José; Keating, Elisa; Negrão, RitaTo combat obesity, diets and food products containing non-nutritive sweeteners (NNS), which provide sweetness without caloric intake, have gained popularity, even among pregnant women who reported intentional NNS consumption during gestation. In 2023, the World Health Organization warned about NNS consumption, as their long-term effects may be associated with an increased risk of type 2 diabetes, cardiovascular diseases, and mortality. Importantly, the environment during early life can induce permanent changes in metabolic health in adult life, suggesting that fetal exposure to NNS may induce long-term metabolic dysfunction in offspring. Fibroblast growth factor 21 (FGF21) is a key regulator of metabolic homeostasis. So, this branch of the MHSWEET project aims to study the effects of Rebaudioside A (RebA, the major sweetener component of stevia) exposure during the perigestational period on hepatic lipid metabolism of adult offspring and the involvement of FGF21. Female Sprague-Dawley rats were administered RebA in the drinking water or water as control from 4 weeks before mating until weaning. Food and beverage consumption, morphometric parameters, fasting glucose, oral glucose tolerance, and insulin sensitivity were measured before sacrifice at 10 months of age. Functionality of G1 hepatic mitochondria (OROBOROS® Oxygraph-2k system), hepatic triglyceride levels (Oil-Red-O staining), genes involved in hepatic lipid metabolic pathways and inflammation (RT-PCR) will be evaluated. The results obtained so far, showed that RebA exposure increased body weight in adult female offspring and induced dysglycemia in adult male offspring. The remaining studies are still being conducted. Results seem to support the hypothesis that intrauterine exposure to RebA may program offspring to a dysmetabolic state. These results will be useful to improve knowledge, and support public health policies regarding NNS use throughout life.
- Proteomics characterization of human uterine samples during reproductive ageingPublication . Dias, Bruna; Formoso, Inês; Guedes-Martins, Luís; Almeida, Henrique; Silva, Elisabete; Silva, André M.N.; Matos, LilianaNowadays, postponing motherhood has become more common in modern societies. Such decision rises major social and health concerns, as advanced maternal age associates with low fertility, pregnancy complications and a greater need for assisted reproductive techniques. Female reproductive ageing is associated with alterations that are particularly evident in the ovaries. Yet, given the pivotal contribution of the uterus to embryo adhesion, implantation, and fetal development, we believe that age-dependent uterine changes impair fertility. Evidence in mice already reported age-associated uterine changes. Also, albumin carbonylation was found increased in uterine samples from older term-pregnant women. Moreover, preliminary data show changes in composition and oxidation status of uterine extracellular matrix proteins with higher maternal age. We hypothesize that the uterine proteome changes during reproductive ageing, impairing tissue structure and function. This study aims to identify age-related alterations in uterine protein composition through a complete proteomics approach. Uterine samples were collected during c-section from termpregnant women and immediately fixed for paraffin-embedding or frozen for molecular analysis. Six samples were selected: three from younger and three from reproductively aged women. Paraffin-embedded samples were histologically analyzed after H+E staining. Protein lysates were obtained after sample homogenization and total protein content was determined and were then separated by SDS-PAGE, stained with Coomassie Blue, and will be subjected to a MS based proteomics study. Histological analysis of the samples has proven their uterine origin. SDS-PAGE results confirmed the successful protein extraction. The identification of uterine proteins differentially expressed according to maternal age will allow the development of therapeutic approaches, aiming to attenuate age-associated female fertility decline.
- Obesity and physical loading during manual liftingPublication . Colim, Ana; Arezes, Pedro; Flores, Paulo; Monteiro, Pedro; Mesquita, InêsManual lifting greatly increases the risk of developing work-related musculoskeletal disorders (WRMSD). Muscle loading related to manual tasks may be significantly influenced by workers’ body conditions, being obesity one of them. In the present study, different tasks of manual lifting were performed by 14 participants with different obesity levels, defined in terms of individuals’ fat mass percentage. Surface electromyography (EMG) data were collected bilaterally from 3 muscles recruited during this type of tasks. EMG data normalization was based on the percentage of maximum contraction during each task (MCT). Furthermore, the participants reported physical loading by using the Rated Perceived Exertion scale (RPE). These techniques sought to analyze the physical loading during manual lifting tasks. The obtained results indicate that obesity level influenced the MCT percentage, which in turn increases the muscle loading during manual lifting tasks. However, obesity level does not seem to influence the RPE values. In this context, further studies based on biomechanical approaches are required to provide a more complete understanding of the obesity effects on musculoskeletal loading during manual lifting.
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