S-adenosylhomocysteine promotes endothelial dysfunction and activation : a role for hypomethylation in vascular disease

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2015omocysteine has been established as a risk factor for cardiovascular disease (CVD) by mechanisms incompletely defined. S-Adenosylhomocysteine (SAH) is the metabolic precursor of homocysteine that accumulates in the setting of hyperhomocysteinemia and is a negative regulator of most cell methyltransferases. This thesis project investigated whether methylation imbalance, caused by excess SAH, disrupts endothelium homeostasis and favors the establishment of a pro-atherogenic phenotype. To experimentally address this possibility, studies were conducted in human endothelial cells, in which SAH accumulation was induced using either a pharmacologic or a siRNA approach. As the major regulator of vascular homeostasis, the endothelium exerts a number of vasoprotective effects that are largely mediated by nitric oxide (NO), the most potent endogenous vasodilator. Decreased NO bioavailability is a principal manifestation of underlying endothelial dysfunction, an early marker of atherosclerosis and CVD. To determine whether excess SAH alters NO bioavailability, the expression and activity of endothelial nitric oxide synthase (eNOS), and NO production were monitored in cells. These experiments showed that excess SAH increased the levels of eNOS mRNA but caused a decrease in eNOS protein and activity, to decrease cellular production of NO. Another important feature of endothelial dysfunction is oxidative stress. Studies in endothelial cells revealed that a hypomethylating environment, induced by excess SAH, impairs, not only NO production, but also the cellular redox state. Glutathione peroxidase-1 (GPx-1) is a selenoprotein and a major cellular antioxidant. A link between homocysteine-associated suppression of GPx-1 and endothelial dysfunction had been reported previously; however, the causal molecular mechanisms remained unresolved. The experiments presented here demonstrate a specific mechanism by which SAH-mediated hypomethylation suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of a subset of selenoproteins (including GPx-1) is dependent on a specific methylation of the selenocysteine-tRNA (Sec-tRNA). Thus, SAH accumulation was found to inhibit the formation of this methylated isoform of Sec-tRNA resulting in decreased GPx-1 expression, as well as alterations in the expression of other selenoproteins, to promote oxidative stress and a pro-inflammatory activation state in endothelial cells. The observation that Sec-tRNA methylation is decreased by excess SAH, suggests that other RNA species may also be targets for SAH-mediated hypomethylation. Therefore, the effect of SAH on methylation modifications was determined in total and size-fractionated RNA samples from our cell model. Additionally, to confirm these observations, RNA methylation was analyzed in tissue samples from a hyperhomocysteinemic mouse model, where SAH accumulation results from a genetic disorder affecting homocysteine metabolism. Conditions of excess SAH altered the content of some RNA methylation modifications, suggesting that specific RNA methyltransferases may be more susceptible to inhibition by SAH. The activation of endothelial cells that occurs during atherogenesis is characterized by the up-regulation of adhesion molecules, which by recruiting circulating leukocytes favor their transendothelial migration. In a series of studies, the physiological relevance of SAH-induced endothelial cell activation was demonstrated by determining that these SAH-activated cells promoted leukocyte adhesion and migration. Further, the role of DNA hypomethylation on the SAH-induced up-regulation of adhesion molecules was examined. ICAM-1 (intercellular adhesion molecule 1) was found to be up-regulated by SAH accumulation as well as by a DNA methyltransferase inhibitor, suggesting that its expression may be regulated by DNA methylation. Analysis of its promoter methylation; however, showed that it was demethylated in untreated cells, suggesting that it may be regulated by factors other than DNA promoter methylation in response to excess SAH. To understand better the factors involved in the pro-inflammatory activation of endothelial cells, the role of NFkB (nuclear factor kappa B) in SAH-induced responses was examined. These studies establish a role for NFkB in the endothelial cell response to SAH and further link these responses to a suppression of the epigenetic regulator EZH2 (enhancer of zeste homolog 2). EZH2 is a methyltransferase that regulates gene expression by mediating a repressive histone methylation. These results identify EZH2 as a new target of SAH regulation important in inflammatory responses, demonstrating that EZH2 suppression and NFkB activation mediated by excess SAH accumulation may contribute to its adverse effects in the vasculature. Overall, these studies implicate SAH as a key modulator of epigenetic mechanisms by compromising RNA, DNA, and histone methylation. More importantly, our results clearly present SAH as a key player in the disruption of endothelial homeostasis, supporting a role for SAH as an important mediator of homocysteine-associated vascular disease.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/73021/201

A durabilidade dos geossintéticos: estigma ou fator de sustentabilidade

Este artigo pretende mostrar que, paralelamente ao sucesso da utilização dos geossintéticos em inúmeras aplicações, estes tiveram sempre de superar grandes desafios/estigmas para se impor no mercado, muitos deles relacionados com a sua durabilidade: numa 1ª fase era questionado se a sua durabilidade seria suficiente, tendo em consideração a vida útil das obras geotécnico-ambientais em que eram (e são) aplicados e mais recentemente, questionando a sua durabilidade excessiva, pondo em causa a sua sustentabilidade. Assim, pretende-se mostrar que a durabilidade dos geossintéticos é e sempre foi um fator de sustentabilidade e desmistificar esse estigma

Albufeiras na ilha da Madeira: (imprescindível) impermeabilização com geossintéticos

Na ilha da Madeira, existem vários reservatórios de água para diferentes fins, entre eles as albufeiras das barragens de Pico da Urze e da Portela. Estas infraestruturas têm em comum a particularidade de as características das formações geológicas vulcânicas ocorrentes e os materiais de construção das barragens não garantirem a estanquidade necessária para a retenção da água, tornando imprescindível o uso de geossintéticos na impermeabilização do corpo das barragens e das suas albufeiras. Na Barragem do Pico da Urze foi utilizado um geocompósito de impermeabilização (geomembrana de PVC termoacoplada a um geotêxtil) e na Barragem da Portela foi usada uma geomembrana de PEAD. A utilização destes materiais colocou alguns desafios ao nível da conceção e construção dos seus sistemas de impermeabilização. Neste artigo, apresentam-se os principais desafios enfrentados durante as fases de projeto e de construção dos sistemas de impermeabilização, que o Laboratório Nacional de Engenharia Civil acompanhou no âmbito de assessorias técnicas prestadas aos donos de obra

Commingled and Disarticulated Human Remains related to 1755 Lisbon Earthquake: Height Estimation from incomplete and complete femoral bones

Introduction: In Forensic Medicine, the estimation of the stature often has a crucial role in the reconstructive phase of disjointed populations. The femur, being the longest bone in the human body, is usually the most reliable source in height estimation. However, in these populations, intact femurs are hardly ever found, making it necessary to use femur fragments for the same purpose. Aim: This investigation aims to estimate the stature of the catastrophic population concerning the earthquake that occurred in Lisbon, in 1755. Materials and Methods: The study was conducted on 8 whole femurs and 21 fragments, which were measured and weighted. These measurements were applied in a regression formula, obtained from the gathered research, in order to estimate the stature of the population. Results: The results showed that, for the whole femur, the correspondent height varies between 147.96 cm and 168.82 cm. For the fragments, the obtained estimates vary between 151,96 cm and 174.96 cm. Conclusions: The methods used proved to be reliable in estimating the length of the femur, as well as in deducting the height of individuals through this bone, allowing the study of these parameter’s evolution in generations.</p

Glutaryl-CoA dehydrogenase misfolding in glutaric acidemia type 1

Glutaric acidemia type 1 (GA1) is a neurotoxic metabolic disorder due to glutaryl-CoA dehydrogenase (GCDH) deficiency. The high number of missense variants associated with the disease and their impact on GCDH activity suggest that disturbed protein conformation can affect the biochemical phenotype. We aimed to elucidate the molecular basis of protein loss of function in GA1 by performing a parallel analysis in a large panel of GCDH missense variants using different biochemical and biophysical methodologies. Thirteen GCDH variants were investigated in regard to protein stability, hydrophobicity, oligomerization, aggregation, and activity. An altered oligomerization, loss of protein stability and solubility, as well as an augmented susceptibility to aggregation were observed. GA1 variants led to a loss of enzymatic activity, particularly when present at the N-terminal domain. The reduced cellular activity was associated with loss of tetramerization. Our results also suggest a correlation between variant sequence location and cellular protein stability (p < 0.05), with a more pronounced loss of protein observed with variant proximity to the N-terminus. The broad panel of variant-mediated conformational changes of the GCDH protein supports the classification of GA1 as a protein-misfolding disorder. This work supports research toward new therapeutic strategies that target this molecular disease phenotype

Cytotoxicity and genotoxicity assessment of the extract and lectins from Moringa oleifera Lam. Seeds / Avaliação da citotoxicidade e genotoxicidade do extrato e lectinas das sementes de Moringa oleifera Lam

Moringa oleifera seeds are used globally as a treatment for water and contain the lectins cMoL and WSMoL, which display coagulant activity. In this study, we sought to determine the cytotoxicity and genotoxicity of the M. oleifera seed extract (SE), prepared with the same procedure that people use for treating water, as well as cMoL and WSMoL, in human peripheral blood mononuclear cells (PBMCs). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, while genotoxicity was evaluated using the comet assay, with cell nucleoids categorized in classes ranging from 0 (without damage) to 4 (maximum damage). The PBMCs treated with SE, cMoL, and WSMoL displayed viability higher than 60% in treatments with concentrations up to 100 µg/mL. In addition, SE and cMoL displayed low genotoxicity owing to the detection of nucleoids in class 1. However, the number of nucleoids in all classes increased when 50 and 100 µg/mL of WSMoL was administered, reaching a damage frequency of 50.0%. Although M. oleifera SE, cMoL, and WSMoL were not cytotoxic to PBMCs after 24 h of exposure, dose-dependent genotoxic effects were observed, especially with WSMoL. These findings indicate that caution must be exercised when selecting a lectin/extract concentration for water treatment

Single Nucleotides Polymorphisms in COX2 Gene and their Association with Signs and Symptoms of Teething – A Pilot Study

Objective: To investigate the association between single nucleotide polymorphisms in the COX2 gene (rs689466 and rs5275) and local and systemic signs and symptoms of teething. Material and Methods: Forty-four pairs of mothers-babies/toddlers were included. Erupted primary teeth were evaluated during clinical examination. Local and systemic signs and symptoms of teething were obtained from mothers\u27 reporting via anamnesis. Samples of buccal cells were retrieved for DNA genotyping using real-time PCR. The T-test, Chi-square test, logistic regression, and haplotype analyses were applied. Results: Almost all mothers (95.5%) reported at least one local or systemic sign and symptom of teething. The most common was increased salivation (79.5%), diarrhea (72.3%), and fever (70.5%). The mean number of signs and symptoms per child was higher in boys than girls (mean = 5.1; SD= 1.5; p=0.008). Sleep disturbance (p=0.03) and loss of appetite (p=0.05) were more reported in boys. The rs689466 and rs5275 were not associated with signs and symptoms of teething (p&gt;0.05). Conclusion: The single nucleotide polymorphisms in the COX2 gene (rs689466 and rs5275) were not associated with local and systemic signs and symptoms of teething

Investigating the association between dental age and polymorphisms in genes encoding estrogen receptors

Background: Genetic polymorphisms have been shown to influence several physiological traits, including dental and craniofacial characteristics. Understanding the clinical relevance of genetic polymorphisms in dental practice is crucial to personalize treatment plans and improve treatment outcomes. Objective: to evaluate the association between dental age and genetic polymorphisms in genes encoding estrogen receptors alpha and beta (ESR1 and ESR2, respectively) in a sample of Brazilian children. Methodology: This retrospective cross-sectional study was performed with children undergoing orthodontic treatment. Patients with syndromes, congenital anomalies, craniofacial deformities, under hormonal or systemic treatment, and with a previous history of facial trauma were excluded. Panoramic radiographs were used to assess dental age according to the Demirjian, Goldstein, and Tanner method. A delta [dental age-chronological age (DA-CA)] was obtained, which shows whether the patient tends to have a normal, delayed (negative values), or advanced (positive values) dental age. DNA isolated from buccal cells was used to genotype four genetic polymorphisms: rs9340799 (A&gt;G) and rs2234693 (C&gt;T), located in ESR1; and rs1256049 (C&gt;T) and rs4986938 (C&gt;T), located in ESR2. A statistical analysis was performed and values of p&lt;0.05 indicated statistical difference. Results: A total of 79 patients were included, 44 (55.70%) girls and 35 (44.30%) boys. The Demirjian, Goldstein, and Tanner method, in general, overestimated patients’ age by 0.75 years. There was no difference in the delta of dental age between the sexes (p&gt;0.05). Genetic polymorphisms in ESR1 and ESR2 were not associated with dental age (p&gt;0.05). Conclusion: The studied genetic polymorphisms in ESR1 and ESR2 were not associated with dental age in Brazilian childre

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra-and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new ther-apeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery

Does Suppression Levels of Testosterone Have an Impact in The Craniofacial Growth? A Systematic Review in Animal Studies/ A supressão de testosterona impacta o crescimento craniofacial? Uma revisão sistemática de estudos com animais

Sexual hormonal disturbances in humans alter the growth bone. Suppression testosterone is performed in animals for evaluated their effects on craniofacial complex. The aim of this study is to investigate, through of a systematic review from animal studies, the effects of testosterone suppression on the craniofacial complex development. Seven databases, including Open Grey literature, were searched since inception to March 01, 2021, following strategy MEDLINE for terms conducted the search. The study design PICOS was used to establish the eligibility criteria: P - Animals; I - Suppression of testosterone production; C - Animals with normal levels of testosterone; O - Effect in craniofacial growth/development; S - In vivo studies. Relevant data were collected and inserted in characteristics of studies table. Risk of bias was assessed using SYRCLE’s risk of bias tool. Ten studies were included in the systematic review. Two were classified with low risk of bias and eight with unclear. The mandible in experiment group was significantly smaller than control group. The trabecular bone mineral density of the mandible was decrease after testosterone suppression. There was an increase in the number of osteoclasts in the experimental groups. All cephalometric measurements of the maxilla, except in one study, were reduced in orchiectomized rats. The expression of androgen receptor was significantly reduced in head condyle of the experimental group. Testosterone suppression decreases the growth of craniofacial complex bones through imbalance of the bone turnover due to the increase in the number of osteoclasts