Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Radiomic Texture Analysis Mapping Predicts Areas of True Functional MRI Activity.

Individual analysis of functional Magnetic Resonance Imaging (fMRI) scans requires user-adjustment of the statistical threshold in order to maximize true functional activity and eliminate false positives. In this study, we propose a novel technique that uses radiomic texture analysis (TA) features associated with heterogeneity to predict areas of true functional activity. Scans of 15 right-handed healthy volunteers were analyzed using SPM8. The resulting functional maps were thresholded to optimize visualization of language areas, resulting in 116 regions of interests (ROIs). A board-certified neuroradiologist classified different ROIs into Expected (E) and Non-Expected (NE) based on their anatomical locations. TA was performed using the mean Echo-Planner Imaging (EPI) volume, and 20 rotation-invariant texture features were obtained for each ROI. Using forward stepwise logistic regression, we built a predictive model that discriminated between E and NE areas of functional activity, with a cross-validation AUC and success rate of 79.84% and 80.19% respectively (specificity/sensitivity of 78.34%/82.61%). This study found that radiomic TA of fMRI scans may allow for determination of areas of true functional activity, and thus eliminate clinician bias.

Procéduralisme épistémique et légitimité démocratique : une défense de l'égalité politique

Ce mémoire explore la relation qui lie démocratie et légitimité politique, dans une perspective épistémique. La démocratie, dans son acception la plus générale, confère à chacun la possibilité de faire valoir les intérêts qu'il estime être les siens et ceux de sa communauté, en particulier à l’occasion d’un scrutin. Cette procédure décisionnelle qu’est le vote consacre ainsi en quelque sorte la liberté et l’égalité dont profitent chacun des citoyens, et confère une certaine légitimité au processus décisionnel. Cela dit, si le vote n’est pas encadré par des considérations épistémiques, rien ne garantit que le résultat politique qui en découlera sera souhaitable tant pour les individus que pour la collectivité: il est tout à fait permis d’imaginer que des politiques discriminatoires, économiquement néfastes ou simplement inefficaces voient ainsi le jour, et prennent effet au détriment de tous. En réponse à ce problème, différentes théories démocratiques ont vu le jour et se sont succédé, afin de tenter de lier davantage le processus démocratique à l’atteinte d’objectifs politiques bénéfiques pour la collectivité. Au nombre d’entre elles, la démocratie délibérative a proposé de substituer la seule confrontation d’intérêts de la démocratie agrégative par une recherche collective du bien commun, canalisée autour de procédures délibératives appelées à légitimer sur des bases plus solides l’exercice démocratique. À sa suite, la démocratie épistémique s’est inspirée des instances délibératives en mettant davantage l’accent sur la qualité des résultats obtenus que sur les procédures elles-mêmes. Au final, un même dilemme hante chaque fois les différentes théories : est-il préférable de construire les instances décisionnelles en se concentrant prioritairement sur les critères procéduraux eux-mêmes, au risque de voir de mauvaises décisions filtrer malgré tout au travers du processus sans pouvoir rien y faire, ou devons-nous avoir d’entrée de jeu une conception plus substantielle de ce qui constitue une bonne décision, au risque cette fois de sacrifier la liberté de choix qui est supposé caractériser un régime démocratique? La thèse que nous défendrons dans ce mémoire est que le concept d’égalité politique peut servir à dénouer ce dilemme, en prenant aussi bien la forme d’un critère procédural que celle d’un objectif politique préétabli. L’égalité politique devient en ce sens une source normative forte de légitimité politique. En nous appuyant sur le procéduralisme épistémique de David Estlund, nous espérons avoir démontré au terme de ce mémoire que l’atteinte d’une égalité politique substantielle par le moyen de procédures égalitaires n’est pas une tautologie hermétique, mais plutôt un mécanisme réflexif améliorant tantôt la robustesse des procédures décisionnelles, tantôt l’atteinte d’une égalité tangible dans les rapports entre citoyens.

La distribution ‘juste’ de la signature savante dans les collaborations de recherche multidisciplinaire en sciences de la santé

L’auteur qui appose son nom à une publication universitaire sera reconnu pour sa contribution à la recherche et devra également en assumer la responsabilité. Il existe divers types d’agencements pouvant être utilisés afin de nommer les auteurs et souligner l’ampleur de leur contribution à ladite recherche. Par exemple, les auteurs peuvent être nommés en ordre décroissant selon l’importance de leurs contributions, ce qui permet d’allouer davantage de mérite et de responsabilité aux premiers auteurs (à l’instar des sciences de la santé) ou bien les individus peuvent être nommés en ordre alphabétique, donnant une reconnaissance égale à tous (tel qu’on le note dans certains domaines des sciences sociales). On observe aussi des pratiques émergeant de certaines disciplines ou des champs de recherche (tel que la notion d’auteur correspondant, ou directeur de recherche nommé à la fin de la liste d’auteurs). En science de la santé, lorsque la recherche est de nature multidisciplinaire, il existe différentes normes et pratiques concernant la distribution et l’ordre de la signature savante, ce qui peut donner lieu à des désaccords, voire à des conflits au sein des équipes de recherche. Même si les chercheurs s’entendent pour dire que la signature savante devrait être distribué de façon ‘juste’, il n’y a pas de consensus sur ce que l’on qualifie de ‘juste’ dans le contexte des équipes de recherche multidisciplinaire. Dans cette thèse, nous proposons un cadre éthique pour la distribution juste de la signature savante dans les équipes multidisciplinaires en sciences de la santé. Nous présentons une critique de la documentation sur la distribution de la signature savante en recherche. Nous analysons les enjeux qui peuvent entraver ou compliquer une distribution juste de la signature savante tels que les déséquilibres de pouvoir, les conflits d’intérêts et la diversité de cultures disciplinaires. Nous constatons que les normes internationales sont trop vagues; par conséquent, elles n’aident pas les chercheurs à gérer la complexité des enjeux concernant la distribution de la signature savante. Cette limitation devient particulièrement importante en santé mondiale lorsque les chercheurs provenant de pays développés collaborent avec des chercheurs provenant de pays en voie de développement. Afin de créer un cadre conceptuel flexible en mesure de s’adapter à la diversité des types de recherche multidisciplinaire, nous proposons une approche influencée par le Contractualisme de T.M. Scanlon. Cette approche utilise le respect mutuel et la force normative de la raison comme fondation, afin de justifier l’application de principes éthiques. Nous avons ainsi développé quatre principes pour la distribution juste de la signature savante en recherche: le mérite, la juste reconnaissance, la transparence et la collégialité. Enfin, nous proposons un processus qui intègre une taxonomie basée sur la contribution, afin de délimiter les rôles de chacun dans le projet de recherche. Les contributions peuvent alors être mieux comparées et évaluées pour déterminer l’ordre de la signature savante dans les équipes de recherche multidisciplinaire en science de la santé.

Is ultrasound perfusion imaging capable of detecting mismatch? A proof-of-concept study in acute stroke patients.

In this study, we compared contrast-enhanced ultrasound perfusion imaging with magnetic resonance perfusion-weighted imaging or perfusion computed tomography for detecting normo-, hypo-, and nonperfused brain areas in acute middle cerebral artery stroke. We performed high mechanical index contrast-enhanced ultrasound perfusion imaging in 30 patients. Time-to-peak intensity of 10 ischemic regions of interests was compared to four standardized nonischemic regions of interests of the same patient. A time-to-peak >3 s (ultrasound perfusion imaging) or >4 s (perfusion computed tomography and magnetic resonance perfusion) defined hypoperfusion. In 16 patients, 98 of 160 ultrasound perfusion imaging regions of interests of the ischemic hemisphere were classified as normal, and 52 as hypoperfused or nonperfused. Ten regions of interests were excluded due to artifacts. There was a significant correlation of the ultrasound perfusion imaging and magnetic resonance perfusion or perfusion computed tomography (Pearson`s chi-squared test 79.119, p < 0.001) (OR 0.1065, 95% CI 0.06-0.18). No perfusion in ultrasound perfusion imaging (18 regions of interests) correlated highly with diffusion restriction on magnetic resonance imaging (Pearson's chi-squared test 42.307, p < 0.001). Analysis of receiver operating characteristics proved a high sensitivity of ultrasound perfusion imaging in the diagnosis of hypoperfused area under the curve, (AUC = 0.917; p < 0.001) and nonperfused (AUC = 0.830; p < 0.001) tissue in comparison with perfusion computed tomography and magnetic resonance perfusion. We present a proof of concept in determining normo-, hypo-, and nonperfused tissue in acute stroke by advanced contrast-enhanced ultrasound perfusion imaging.

Report on the possibility of revision of the Bustamante code or the Code of private international law /

Mode of access: Internet.

Annual report - American School of Classical Studies at Athens.

At head of title, [1881- ]: Archaeological Institute of America.

American journal of archaeology : the journal of the Archaeological Institute of America.

Imprint varies: Boston, MA : Archaeological Institute of America,

Down our way ; stories of southern and western character,

A visible sign.--The end of society.--An adventure of a lady of quality.--A part of the new South.--A conflict of rights.--The Morningstar elopement.--The wife of a carpenter.--A gentlewoman.--The blue blazers.

Principles of private international law /

Includes index.

A selection of cases and other readings on the law of nations, chiefly as it is interpreted and applied by British and American courts,

Includes lists of readings.

Modern American law; a systematic and comprehensive commentary on the fundamental principles of American law and procedure, accompanied by leading illustrative cases and legal forms, with a rev. ed. of Blackstone's Commentaries;

v. I. The modern democracy, the citizen and the law - Legal ethics - Law : its origin, nature, development - Courts : federal and state - Law of contracts -- v. 2. Law of torts -- v. 3. Criminal Law - Law of criminal procedure - Law of persons and domestic relations -- v. 4. Personal property and bailments - Law of liens and pledges - Law of agency - Law of sales of personal property -- v. 5. Law of real property -- v. 6. Law of descent and distribution, wills and administration, guardian and ward - Law of landlord and tenant - Law of irrigation and water rights - Law of mines and mining -- v. 7. Equity - Law of trusts - Law of quasi-contacts - Law of estoppel -- v. 8. Law of negotiable instruments - Law of suretyship and guaranty - Law of mortgages : real and chattel - Interpretation of statutes -- v. 9. Law of private corporations - Law of partnership - law of banks, banking and trust companies - Law of receivers -- v. 10. Pleadings in civil actions at common law and under modern statutes - Practice in civil actions - Law of equity pleading - Law of evidence - Laws of attachment and garnishments - Law of judgments and executions - Law of extraordinary remedies - Law of habeas corpus -- v. 11. Constitutional law : definitions and general principles - Organization and powers of the United States Government - Constitutional guaranties of fundamental rights - Eminent domain - Taxation - Naturalization -- v. 12. Conflict of laws - International law - Law of interstate commerce - Law of bankruptcy - Law of patents - Law of copyright - Law of trademarks - Unfair competition and good-will -- v. 13. Law of public service companies, especially common carriers - Law of municipal corporations - Law of public officers and elections - Parliamentary law -- v. 14. Law of damages - Law of insurance - Admiralty law - Medical jurisprudence - Forms -- v. 15. Blackstone's Commentaries.

Romanian street beggars in Stockholm. A conflict between global city aspirations and informal livelihoods.

This thesis is looking into the issue of Romanian street beggars in Stockholm, Sweden. In the last couple of years there was an increase in the number of beggars in the Swedish capital, while most of them seem to come from Romania. This lead quickly to a national debate. The aim of the paper is to shed some light on this issue and see why it is a problem and for whom, looking at three main stakeholders: the state, the activists and the beggars. The theoretical framework for this project is mainly based on the concept of the conflict of rationalities, with Stockholm as an aspiring global city and those which are begging as livelihood strategy. The data is collected mainly through semi-structured interviews, informal interviews and personal observations. Also, the literature review provides context and brings similar examples for a better grasp of the situation in Sweden. The results show that Stockholm sends a branded image of itself to the world, while the new type of migrant is in conflict with this view. The activists try to make the life better for the beggars which are virtually not doing anything illegal. The Swedish state opened discussions with the Romanian state, but did nothing to integrate the newcomers. In this case, the beggars share similar experience in the city.

Governança corporativa e custo de agência: uma visão do mercado brasileiro

Esse trabalho investiga empiricamente a relação entre custo de agência e as medidas de monitoramento interno disponíveis aos investidores brasileiros nas empresas nacionais, utilizando amostras de companhias abertas entre os anos de 2010 e 2014, totalizando 134 empresas analisadas e 536 observações. Para medir tal relação, foram utilizadas, como variáveis de monitoramento interno, informações sobre a remuneração variável dos executivos, entre elas o uso de outorgas de opções de compra de ações, a composição do conselho de administração, dando ênfase à representatividade dos conselheiros independentes e à dualidade entre Chairman e CEO, e o percentual do capital social das companhias que está sob propriedade dos executivos. Como proxy para custo de agência, foram utilizados os indicadores Asset Turnover Ratio e General & Administrative Expenses (G&A) como percentual da Receita Líquida. Neste contexto, foram estabelecidas duas hipóteses de pesquisa e estimados modelos de regressão em painel controlados por efeitos fixos de tempo e empresa, empregando como variável dependente as variáveis proxy do custo de agência e utilizando as variáveis endividamento e tamanho das empresas como variáveis de controle. Os resultados dos modelos demonstram que, na amostra selecionada, há uma relação positiva e significativa entre o percentual da remuneração variável e as proxies de custo de agência, comportamento este contrário ao esperado originalmente. Conclui-se assim que as empresas que apresentam uma maior composição variável no total remunerado ao executivo, incorrem em um maior custo de agência, o que leva à conclusão de que tais ferramentas não são boas estratégias de alinhamento de interesses entre executivos e acionistas. As demais variáveis de monitoramento interno não apresentaram significância.