Identification of neuronal alterations induced by Shank3 mutations using iPS cells from Phelan-McDermid Patients' Fibroblasts

A família de proteínas Shank é o principal conjunto de proteinas de suporte e está localizada na densidade pós-sináptica das sinapses excitatórias. Existem 3 genes na família Shank, Shank1, Shank2 e Shank3 e são caracterizados por múltiplos domínios repetidos de anquirina próximo ao N-terminal seguido pelos domínios Src homologo 3 e PDZ, uma região longa rica em prolina e um domínio de motivo α estéril próximo ao C-terminal. Shank proteínas conectam duas subunidades de receptors glutamatérgicos, recetores NMDA e recetores metabotrópicos de glutamato do tipo-I (mGluRs). O domínio PDZ da Shank conecta-se ao C-terminal do GKAP e este, liga-se, ao complexo recetor PSD-95-NMDA. Por outro lado, a proteína Homer interage com o domínio rico em prolina para confirmar a associação entre a proteína Shank com o mGluR tipo-I. A proteína específica em estudo, Shank3, é haploinsuficiente em pacientes com sindrome Phelan-McDermid devido à deleções no braço comprido do cromossoma 22 levando à danos intelectuais, ausência ou atraso no discurso, comportamentos semelhantes ao autismo, hipotonia e características dismórficas. Neste trabalho, investigamos o papel da Shank3 na função sináptica para compreender a relação entre alterações nesta proteína e as características neurológicas presente em Pacientes com síndrome Phelan-McDermid. Foram utilizados dois modelos diferentes, ratinhos knockout Shank3 e hiPSC de pacientes com PMS. Ratinhos geneticamente modificados são ferramentas uteis no estudo de genes e na compreensão dos mecanismos que experiências in vitro não são capazes de reproduzir, mas de maneira a compreender melhor as patologias humanas, decidimos trabalhar também com células humanas. Os fibroblastos dos pacientes com síndrome Phelan-McDermid fora reprogramados em hiPS cells, diferenciados em neurónios e comparados com os neurónios obtidos a partir de doadores saudavéis e da mesma idade. A reprogramação em iPSC foi realizada por infecção de lentivirus com quatro genes de reprogramação OCT4, c-MYC, SOX2 e KFL4 para posteriormente serem diferenciados em neurónios, com cada passo sendo positivamente confirmado através de marcadores neuronais. Através dos neurónios diferenciados, analisamos a expressão de proteínas sinápticas. Pacientes com haploinsuficiencia na proteína Shank3 apresentam níveis elevados de proteína mGluR5 e decrescidos de proteína Homer sugerindo que a haploinsuficiencia leva a desregulação do complexo mGluR5-Homer-Shank3 conduzindo também, a defeitos na maturação sináptica. Assim, a expressão da proteína mGluR5 está alterada nos pacientes com PMS podendo estar relacionada com defeitos encontrados na diferenciação neuronal e maturação sináptica observados nos neurónios de pacientes. Conclusivamente, iPS cells representam um modelo fundamental no estudo da proteína Shank3 e a sua influência no sindrome de Phelan-McDermid.

A simplified four-branch model for the analytical study of the out-of-plane performance of regular stone URM walls

During the last years, several studies have been made aiming to assess the out-of-plane seismic response of unreinforced stone masonry structures. This fact led to the development of a wide variety of models and approaches, ranging from simple kinematic based analytical models up to complex numerical simulations. Nevertheless, for the sake of simplicity, the out-of-plane seismic response of a masonry wall pier may be obtained by means of a simple single-degree-of-freedom system while still providing good results. In fact, despite the assumptions associated with such a simple formulation, it is also true that the epistemic uncertainty inherent with the selection of appropriate input parameters in more complex models may render them truly ineffective. In this framework, this paper focuses on the study of the out-of-plane bending of unreinforced stone masonry walls (cantilevers) by proposing a simplified analytical approach based on the construction of a linearized four-branch model, which is used to characterize the linear and nonlinear response of such structural elements through an overturning moment-rotation relationship. The formulation of the four-branch model is presented and described in detail and the meaningful parameters used for its construction are obtained from a set of experimental laboratory tests performed on six full-scale unreinforced regular sacco stone masonry specimens. Moreover, a parametric analysis aiming to evaluate the effect of these parameters’ variation on the final configuration of the model is presented and critically discussed. Finally, the results obtained from the application of the developed four-branch model on real unreinforced regular sacco stone masonry walls are thoroughly analysed and the main conclusions obtained from its application are summarized.

Experimental characterization of the out-of-plane performance of regular stone masonry walls, including test setups and axial load influence

Stone masonry is one of the oldest and most worldwide used building techniques. Nevertheless, the structural response of masonry structures is complex and the effective knowledge about their mechanical behaviour is still limited. This fact is particularly notorious when dealing with the description of their out-of-plane behaviour under horizontal loadings, as is the case of the earthquake action. In this context, this paper describes an experimental program, conducted in laboratory environment, aiming at characterizing the out-of-plane behaviour of traditional unreinforced stone masonry walls. In the scope of this campaign, six full-scale sacco stone masonry specimens were fully characterised regarding their most important mechanic, geometric and dynamic features and were tested resorting to two different loading techniques under three distinct vertical pre-compression states; three of the specimens were subjected to an out-of-plane surface load by means of a system of airbags and the remaining were subjected to an out-of-plane horizontal line-load at the top. From the experiments it was possible to observe that both test setups were able to globally mobilize the out-of-plane response of the walls, which presented substantial displacement capacity, with ratios of ultimate displacement to the wall thickness ranging between 26 and 45 %, as well as good energy dissipation capacity. Finally, very interesting results were also obtained from a simple analytical model used herein to compute a set of experimental-based ratios, namely between the maximum stability displacement and the wall thickness for which a mean value of about 60 % was found.

Simulation of masonry out-of-plane failure modes by multi-body dynamics

The seismic assessment of the local failure modes in existing masonry buildings is currently based on the identification of the so-called local mechanisms, often associated with the out-of-plane wall behavior, whose stability is evaluated by static force-based approaches and, more recently, by some displacement-based proposals. Local mechanisms consist of kinematic chains of masonry portions, often regarded as rigid bodies, with geometric nonlinearity and concentrated nonlinearity in predefined contact regions (unilateral no-tension behavior, possible sliding with friction). In this work, the dynamic behavior of local mechanisms is simulated through multi-body dynamics, to obtain the nonlinear response with efficient time history analyses that directly take into account the characteristics of the ground motion. The amplification/filtering effects of the structure are considered within the input motion. The proposed approach is validated with experimental results of two full-scale shaking-table tests on stone masonry buildings: a sacco-stone masonry façade tested at Laboratório Nacional de Engenharia Civil and a two-storey double-leaf masonry building tested at European Centre for Training and Research in Earthquake Engineering (EUCENTRE).