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

Desenvolvimento de culturas de células epiteliais para o estudo de patologias associadas a transportadores de membrana

Tese de mestrado, Biologia (Biologia Molecular e Genética), 2008, Universidade de Lisboa, Faculdade de CiênciasNo presente trabalho estudou-se um modelo de cultura in vitro para o estudo do epitélio respiratório humano e patologias associadas, utilizando culturas de células epiteliais obtidas por nasal brushing. As culturas foram caracterizadas por RT-PCR, observando-se expressão dos genes epiteliais da Citoqueratina 18 (CK18), do regulador da condutância transmembranar da fibrose quística (CFTR) e das subunidades _, _ e _ do canal epitelial de sódio (ENaC). Este ensaio mostrou um aumento da expressão de alguns genes nas células epiteliais polarizadas em relação às células em cultura. Na caracterização imunofenotípica das células não-polarizadas foi observada expressão positiva de ambas as proteínas analisadas, CK18 e CK19. As culturas nas passagens 1 e 2 foram avaliadas quanto à capacidade de formar monocamadas em interface ar-líquido (ALI), apresentando elevados valores de resistência eléctrica trans-epitelial, o que viabiliza a sua utilização no desenvolvimento de modelos in vitro. Devido às limitações da utilização destas células em culturas ALI, foi explorada a utilização de células estaminais mesenquimais diferenciadas em células epiteliais. As células mesenquimais da matriz do cordão umbilical (UC) e do nervo do dente (Nervo) foram caracterizadas imunofenotipicamente, observando-se expressão de ambas as CKs nas células UC e ausência nas Nervo. Na caracterização genotípica concluiu-se que os genes _ e _-enac eram os marcadores ideais de diferenciação epitelial destas células. Para a diferenciação foram desenvolvidos dois protocolos, observando-se ausência de indícios de diferenciação das células Nervo. As células UC apresentaram morfologia epitelióide a partir das duas semanas de diferenciação, com um aumento da expressão de _-enac. Contudo, quando sujeitas a cultura ALI, não ocorreu formação de monocamadas epitelióides. Este trabalho permitiu estabelecer culturas primárias de células epiteliais e abriu uma janela de investigação na área da diferenciação estaminal e na utilização inovadora destas células como fonte de células epiteliais para o estudo do transporte de fármacosIn the present study, epithelial cells from nasal brushing were used in a model of in vitro culture that could allow the study of human respiratory epithelium associated diseases. The cultures were characterized by RT-PCR and expression of epithelial genes was observed, namely for: Cytokeratin 18 (CK18), Cystic fibrosis transmembrane conductance regulator (CFTR) and the _, _ and _ subunits of the epithelial sodium channel (ENaC). An increase of expression was observed in polarized cells (directly isolated) in comparison to the non-polarized cells in culture. In the immunophenotypic characterization of non-polarized cells, expression of CK18 and CK19 was detected. The capability of these cultures, at passages 1 and 2, to form monolayers in air-liquid interface (ALI) was observed, detecting high levels of trans-epithelial electric resistance and showing their potential use for the development of in vitro models. Due to limitations in the use of epithelial cells from nasal brushing, mesenchymal stem cells differentiated into epithelial cells were tested as an alternative for the ALI cultures. The mesenchymal cells of the umbilical cord matrix (UC) and dental nerve cells (Nerve) were characterized immunophenotipically, with expression of both CKs in UC cells, and absence in Nerve cells. The genotypic characterization concluded that the genes _ and _-enac were the best markers for epithelial differentiation of these cells. For the differentiation two protocols were developed and the results showed absence of differentiation signs in Nerve cells. UC cells showed epithelioid morphology after two weeks with an increased expression of _-enac. However, when subjected to ALI cultures no monolayer of epithelioid cells was formed. This study allowed the establishment of primary epithelial cell cultures and opened a window of research in stem cell differentiation and in the use of stem cells as a source of epithelial cells for the study of drug transport studie

Behind bilirubin neurotoxicity: discovering what’s left at the blood-brain barrier

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2013During neonatal life, elevation of unconjugated bilirubin (UCB) levels may lead to minor neurological dysfunction or even to bilirubin encephalopathy (kernicterus). The pathogenesis of this condition involves UCB passage across the blood-brain barrier (BBB), but it is still unknown the role of this barrier in the consequent brain injury. Thus, this thesis intended to investigate the response of human brain microvascular endothelial cells (HBMEC), a simplified in vitro model of the BBB, to UCB, to evaluate the modulation of these effects by therapeutic molecules, and to dissect the neuro-glialvascular alterations in brain parenchyma of neonatal kernicterus cases.First, we observed that HBMEC incubation with UCB induced cell death,cytokine release and oxidative stress. As some of the molecules that the HBMEC produced are known modulators of permeability and angiogenesis, we continued our studies with the evaluation of barrier integrity. Our second study showed that prolonged exposure to high concentrations of UCB caused monolayer fragility and compromised barrier integrity. To complement these studies, we investigated the action of the neuroprotective bile acids, ursodeoxycholic acid (UDCA) and glycoursodeoxycholic acid (GUDCA) against UCB toxicity. The bile acids showed optimal protective abilities in distinct parameters: GUDCA was effective in preventing cell death, while UDCA reduced the production of angiogenic-related molecules and prevented the elevation of permeability. Importantly, the bile acids efficiency was demonstrated in a broad window of opportunity, with both protective and recovery properties. Next, we continued our work by analysing brain regions with great susceptibility to bilirubin, as the cerebellum, hippocampus and basal ganglia, which showed marked neuronal loss. Additionally, the results revealed new players in the neuropathology of kernicterus, including increased vascularization and dysfunction in several BBB components, as astrocytes, pericytes and basement membrane.In conclusion, high levels of UCB compromise endothelial integrity, mainly after prolonged exposure, ultimately leading to BBB breakdown and enhanced UCB passage into the brain. Additionally, our data shows the potential of UDCA and GUDCA as preventive, but also restorative therapeutic molecules against UCB-injury. Moreover, evaluation of kernicterus cases suggests a link between region-specific susceptibility and marked vascular dysfunction. These findings contribute to a better understanding of the neurotoxic steps involved in the irreversible brain damage cause by severe jaundice.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/61646/2009, projetos PEst-OE/SAU/UI4013/2011 e PTDC/SAU-FCF/68819/2006

LDL increases endothelial permeability in an LDLR and cholesterol-dependent way.

<p>(A) HUVECs were plated on top of 0.4 μm pores size transwell inserts and cultured in order to form a confluent and mature monolayer. Cells were either incubated with 100 μg/ml LDL or with the same volume of the control buffer. 24 hours later, cells were washed with serum-free media and the permeability of the monolayer to 70 kDa FITC-dextrans was assessed two hours later, by measurement of the fluorescence at the bottom chamber of the culture system. (B) The same experiment as in (A) with the addition of 2 μg/ml of anti-LDLR or the IgG control at day 7, one hour before the addition of LDL. Fluorescence at the bottom chamber was measured 15 minutes upon the addition of the dextrans. (C) The same experiment as in (B) with the addition of 50 μg/ml of nystatin or the same volume of vehicle at day 8, one hour before the addition of LDL. All the data, except from panel (A), which is a representation of an experiment performed twice with similar results, represent the averages ± standard deviation of at least three independent experiments. Significance values have been calculated using a two-tailed unpaired student <i>t</i> test at the 95% confidence interval (* <i>P</i><0.05).</p

LDL favors the transcytosis of high molecular weight dextrans.

<p>The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> was performed. (A) At the end of the experiment, cells were washed twice with PBS and fixed with PFA. A confocal image showing dextrans inside cells is presented. (B) The number of dextran-containing vesicles per cell, two hours after washing from control and LDL conditions was assessed by widefield fluorescence microscopy. Representative images of each condition are shown and the chart represents the quantification of three independent blind experiments in which 50 cells were analyzed. (C) The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> with addition of 70 kDA dextrans for 15 minutes, followed by three washes and re-incubation with fresh media both at the top and bottom chambers, 15 minutes later a sample from the bottom chamber was collected and analyzed. (D) The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> with the addition of either vehicle or 5 μg/ml of Brefeldin A (BFA). Significance values have been calculated using a two-tailed unpaired student <i>t</i> test at the 95% confidence interval (* <i>P</i><0.05).</p

Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

BACKGROUND: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC). METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. CONCLUSIONS: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period

Nefrectomia Radical com Trombectomia da Veia Cava Laparoscópica num Caso de Feocromocitoma Maligno

Pheochromocytomas with vena cava thrombus are extremely rare, with only a few cases reported in the literature. Radical nephrectomy with adrenalectomy and inferior vena cava (IVC) thrombectomy is the treatment of choice. However, it is a challenging procedure and its surgical approach is yet to be standardized. We present a case of a 49-year-old male incidentally diagnosed with a pheochromocytoma with aggressive local invasion and a level 1 vena cava thrombus. A laparoscopic right radical nephrectomy with right adrenalectomy, IVC thrombectomy and cavorraphy. A detailed revision of the technique is performed and compared with current strategies for pheochromocytoma optimal treatment. Renal and adrenal masses with vena cava thrombus are associated with high morbidity and mortality, particularly in the case of pheochromocytoma. The management is complex but minimally invasive surgery can be performed safely in the context of an experienced multidisciplinary team.Os feocromocitomas com trombo na veia cava são entidades extremamente raras, estando poucos casos descritos na literatura. Apesar da nefrectomia radical com adrenalectomia/suprarrenalectomia e trombectomia da veia cava inferior (VCI) corresponder ao tratamento de escolha, esta é uma técnica cirúrgica desafiante com uma abordagem ainda não padronizada. Apresenta-se um caso de um homem de 49 anos com diagnóstico incidental de um feocromocitoma localmente invasivo, com trombo na VCI nível 1. Foi proposta uma nefrectomia radical direita laparoscópica com adrenalectomia/suprarrenalectomia, trombectomia da VCI e cavorrafia. Neste artigo faz-se uma descrição detalhada da técnica cirúrgica e uma comparação com as estratégias atualmente utilizadas no tratamento do feocromocitoma. Tumores renais e suprarrenais/adrenais com trombo na veia cava estão associados a maior morbilidade e mortalidade, sobretudo no caso do feocromocitoma. Apesar de complexa, a cirurgia minimamente invasiva é uma opção segura no contexto de uma equipa experiente e multidisciplinar

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) modify the distribution of β-catenin in brain endothelial cells.

<p>Cells in mono-culture or co-cultured with astrocytes were fixed and immunostained with an antibody against β-catenin to evaluate its cellular localization (scale bars, 40 and 20 µm, respectively). Disruption of the monolayer with gaps between endothelial cells (*), alterations in protein patterns (arrowheads) with the presence of dot-like staining (yellow arrow), and perinuclear distribution (arrows) are indicated. Representative results from one of two independent experiments are shown.</p