Caveolae ��� from ultrastructure to molecular mechanisms

Almost 50 years after the first sighting of small pits that covered the surface of mammalian cells, investigators are now getting to grips with the detailed workings of these enigmatic structures that we now know as caveolae.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

Supervis��o como um ���TEAR���

Tomando como ponto de partida e de chegada a met��fora do ���tear��� utilizada por uma das supervisoras entrevistadas neste estudo descrevem-se estrat��gias e pr��ticas de supervis��o do ponto de vista das supervisoras e das respectivas supervisandas. Descrevem-se estrat��gias de ���scaffolding��� (colocar andaimes) numa perspectiva individual e sist��mica, envolvendo todos os adultos em processos de aprendizagem para a melhoria da qualidade pedag��gica. Estas estrat��gias s��o descritas usando a met��fora inicial do ���tear���. Fornece-se suporte te��rico para o uso destas met��foras.

Presta����o de servi��os de assessoria e fiscaliza����o da obra de "reabilita����o das coberturas, acessibilidades e seguran��a da antiga escola Veiga Beir��o - Pal��cio Valadares, em Lisboa"

O presente relat��rio diz respeito a um est��gio de natureza profissional realizado com o objectivo de constituir Trabalho Final de Mestrado para obten����o do grau de Mestre em Engenharia Civil no perfil de Edifica����es, do Instituto Superior de Engenharia de Lisboa. O est��gio subordinado ao tema ���Presta����o de servi��os de Assessoria, Fiscaliza����o e Coordena����o de Seguran��a da Obra de REABILITA����O DAS COBERTURAS, ACESSIBILIDADES E SEGURAN��A DA ANTIGA ESCOLA VEIGA BEIR��O ��� PAL��CIO VALADARES, EM LISBOA���, decorreu na Pengest ��� Planeamento, Engenharia e Gest��o, S.A., empresa vocacionada para a presta����o de servi��os de Assessoria, Gest��o, Coordena����o e Fiscaliza����o de Obras, sector no qual possui vasta e diversificada experi��ncia, e reconhecidos m��rito e compet��ncia. O objectivo da empreitada �� o de recupera����o deste im��vel, interven����o necess��ria �� instala����o no local da Exposi����o ���Rep��blica e Ensino��� enquadrada no ��mbito das Comemora����es do Centen��rio da Rep��blica. A empreitada foi adjudicada ao Empreiteiro Teixeira Duarte, S.A, pelo valor de ���998.072,76. O prazo �� de 4,5 meses prevendo-se a conclus��o para 18 de Mar��o de 2010. Embora a proposta de est��gio tenha decorrido no in��cio de 2010, no ��mbito do tema que viria a ser proposto e de forma a integrar a Estagi��ria numa empresa em pleno funcionamento, o est��gio teve in��cio aquando do in��cio da empreitada, em Novembro de 2009. Exceptuando os trabalhos de montagem da grua-torre e dos andaimes para a cobertura provis��ria, que decorreram antes da consigna����o, foi acompanhado todo o processo de Fiscaliza����o. A Estagi��ria participou na an��lise do Plano de Trabalhos, nas vistorias ��s frac����es aut��nomas e edif��cios adjacentes, nas visitas �� obra e na elabora����o de relat��rios mensais. Acompanhou os procedimentos da Pengest no Controlo do Planeamento, da Qualidade e Financeiro. Em rela����o �� Coordena����o de Seguran��a em obra, por ser um tema t��o vasto e espec��fico, teve acesso aos relat��rios, n��o tendo efectivamente participado nas actividades desenvolvidas sobre esta mat��ria. Sob a co-orienta����o da Eng.�� Isabel Vicente, respons��vel da Fiscaliza����o e orienta����o do Eng.�� Jos�� Pedro Fernandes do ISEL, a estagi��ria desenvolveu este est��gio de forma proveitosa, sempre com o aux��lio de uma equipa experiente em Gest��o e Fiscaliza����o.

Constru����o de um rob�� trepador com locomo����o atrav��s de rodas e ades��o atrav��s de meios magn��ticos

Mestrado em Engenharia Electrot��cnica e de Computadores

A survey of technologies and applications for climbing robots locomotion and adhesion

The interest in the development of climbing robots has grown rapidly in the last years. Climbing robots are useful devices that can be adopted in a variety of applications, such as maintenance and inspection in the process and construction industries. These systems are mainly adopted in places where direct access by a human operator is very expensive, because of the need for scaffolding, or very dangerous, due to the presence of an hostile environment. The main motivations are to increase the operation efficiency, by eliminating the costly assembly of scaffolding, or to protect human health and safety in hazardous tasks. Several climbing robots have already been developed, and other are under development, for applications ranging from cleaning to inspection of difficult to reach constructions. A wall climbing robot should not only be light, but also have large payload, so that it may reduce excessive adhesion forces and carry instrumentations during navigation. These machines should be capable of travelling over different types of surfaces, with different inclinations, such as floors, walls, or ceilings, and to walk between such surfaces (Elliot et al. (2006); Sattar et al. (2002)). Furthermore, they should be able of adapting and reconfiguring for various environment conditions and to be self-contained. Up to now, considerable research was devoted to these machines and various types of experimental models were already proposed (according to Chen et al. (2006), over 200 prototypes aimed at such applications had been developed in the world by the year 2006). However, we have to notice that the application of climbing robots is still limited. Apart from a couple successful industrialized products, most are only prototypes and few of them can be found in common use due to unsatisfactory performance in on-site tests (regarding aspects such as their speed, cost and reliability). Chen et al. (2006) present the main design problems affecting the system performance of climbing robots and also suggest solutions to these problems. The major two issues in the design of wall climbing robots are their locomotion and adhesion methods. With respect to the locomotion type, four types are often considered: the crawler, the wheeled, the legged and the propulsion robots. Although the crawler type is able to move relatively faster, it is not adequate to be applied in rough environments. On the other hand, the legged type easily copes with obstacles found in the environment, whereas generally its speed is lower and requires complex control systems. Regarding the adhesion to the surface, the robots should be able to produce a secure gripping force using a light-weight mechanism. The adhesion method is generally classified into four groups: suction force, magnetic, gripping to the surface and thrust force type. Nevertheless, recently new methods for assuring the adhesion, based in biological findings, were proposed. The vacuum type principle is light and easy to control though it presents the problem of supplying compressed air. An alternative, with costs in terms of weight, is the adoption of a vacuum pump. The magnetic type principle implies heavy actuators and is used only for ferromagnetic surfaces. The thrust force type robots make use of the forces developed by thrusters to adhere to the surfaces, but are used in very restricted and specific applications. Bearing these facts in mind, this chapter presents a survey of different applications and technologies adopted for the implementation of climbing robots locomotion and adhesion to surfaces, focusing on the new technologies that are recently being developed to fulfill these objectives. The chapter is organized as follows. Section two presents several applications of climbing robots. Sections three and four present the main locomotion principles, and the main "conventional" technologies for adhering to surfaces, respectively. Section five describes recent biological inspired technologies for robot adhesion to surfaces. Section six introduces several new architectures for climbing robots. Finally, section seven outlines the main conclusions.

Climbing robots: a survey of technologies and applications

The interest in the development of climbing robots is growing rapidly. Motivations are typically to increase the operation efficiency by obviating the costly assembly of scaffolding or to protect human health and safety in hazardous tasks. Climbing robots are starting to be developed for applications ranging from cleaning to inspection of difficult to reach constructions. These robots should be capable of travelling on different types of surfaces, with varying inclinations, such as floors, walls, ceilings, and to walk between such surfaces. Furthermore, these machines should be capable of adapting and reconfiguring for various environment conditions and to be self-contained. Regarding the adhesion to the surface, they should be able to produce a secure gripping force using a light-weight mechanism. This paper presents a survey of different applications and technologies proposed for the implementation of climbing robots.

An engine for generating XSLT from examples

XSLT is a powerful and widely used language for transforming XML documents. However its power and complexity can be overwhelming for novice or infrequent users, many of which simply give up on using this language. On the other hand, many XSLT programs of practical use are simple enough to be automatically inferred from examples of source and target documents. An inferred XSLT program is seldom adequate for production usage but can be used as a skeleton of the final program, or at least as scaffolding in the process of coding it. It should be noted that the authors do not claim that XSLT programs, in general, can be inferred from examples. The aim of Vishnu - the XSLT generator engine described in this paper ��� is to produce XSLT programs for processing documents similar to the given examples and with enough readability to be easily understood by a programmer not familiar with the language. The architecture of Vishnu is composed by a graphical editor and a programming engine. In this paper we focus on the editor as a GWT web application where the programmer loads and edits document examples and pairs their content using graphical primitives. The programming engine receives the data collected by the editor and produces an XSLT program.

An example-based generator of XSLT programs

XSLT is a powerful and widely used language for transforming XML documents. However, its power and complexity can be overwhelming for novice or infrequent users, many of whom simply give up on using this language. On the other hand, many XSLT programs of practical use are simple enough to be automatically inferred from examples of source and target documents. An inferred XSLT program is seldom adequate for production usage but can be used as a skeleton of the final program, or at least as scaffolding in the process of coding it. It should be noted that the authors do not claim that XSLT programs, in general, can be inferred from examples. The aim of Vishnu���the XSLT generator engine described in this chapter���is to produce XSLT programs for processing documents similar to the given examples and with enough readability to be easily understood by a programmer not familiar with the language. The architecture of Vishnu is composed by a graphical editor and a programming engine. In this chapter, the authors focus on the editor as a GWT Web application where the programmer loads and edits document examples and pairs their content using graphical primitives. The programming engine receives the data collected by the editor and produces an XSLT program.

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells.

Cyclooxyganase-2 (COX-2), a rate-limiting enzyme in the prostaglandin synthesis pathway, is overexpressed in many cancers and contributes to cancer progression through tumor cell-autonomous and paracrine effects. Regular use of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors (COXIBs) reduces the risk of cancer development and progression, in particular of the colon. The COXIB celecoxib is approved for adjunct therapy in patients with Familial adenomatous polyposis at high risk for colorectal cancer (CRC) formation. Long-term use of COXIBs, however, is associated with potentially severe cardiovascular complications, which hampers their broader use as preventive anticancer agents. In an effort to better understand the tumor-suppressive mechanisms of COXIBs, we identified MAGUK with Inverted domain structure-1 (MAGI1), a scaffolding protein implicated in the stabilization of adherens junctions, as a gene upregulated by COXIB in CRC cells and acting as tumor suppressor. Overexpression of MAGI1 in CRC cell lines SW480 and HCT116 induced an epithelial-like morphology; stabilized E-cadherin and β-catenin localization at cell-cell junctions; enhanced actin stress fiber and focal adhesion formation; increased cell adhesion to matrix proteins and suppressed Wnt signaling, anchorage-independent growth, migration and invasion in vitro. Conversely, MAGI1 silencing decreased E-cadherin and β-catenin localization at cell-cell junctions; disrupted actin stress fiber and focal adhesion formation; and enhanced Wnt signaling, anchorage-independent growth, migration and invasion in vitro. MAGI1 overexpression suppressed SW480 and HCT116 subcutaneous primary tumor growth, attenuated primary tumor growth and spontaneous lung metastasis in an orthotopic model of CRC, and decreased the number and size of metastatic nodules in an experimental model of lung metastasis. Collectively, these results identify MAG1 as a COXIB-induced inhibitor of the Wnt/β-catenin signaling pathway, with tumor-suppressive and anti-metastatic activity in experimental colon cancer.

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

Characterization of the role of AKAP-Lbc/p38 complex in the hypertrophic response of the heart

In response to chronic stress the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy, increased cellular apoptosis and fibrosis, which ultimately causes cardiac dysfunction and heart failure. Increasing evidence suggest the role of scaffolding and anchoring proteins in coordinating different signaling pathways that mediate the hypertrophic response of the heart. In this context, the family of Α-kinase anchoring proteins (AKAPs) emerged as important regulators of the cardiac function. During my thesis work I have conducted two independent projects, both of them aiming at elucidating the role of AKAPs in the heart. It has been shown that AKAP-Lbc, an anchoring protein that possesses an intrinsic Rho- specific exchange factor activity, organizes a signaling complex that links AKAP-Lbc- dependent activation of RhoA with the mitogen activated protein kinase (MAPK) p38. The first aim of my thesis was to study the role of this novel transduction pathway in the context of cardiac hypertrophy. Here we show that transgenic mice overexpressing in cardiomyocytes a competitor fragment of AKAP-Lbc, which specifically disrupts endogenous AKAP-Lbc / p38 complexes, developed early dilated cardiomyopathy in response to two weeks of transverse aortic constriction (TAC) as compared to controls. Interestingly, inhibition of the AKAP-Lbc / p38 transduction pathway significantly reduced the hypertrophic growth of single cardiomyocytes induced by pressure overload. Therefore, it appears that the AKAP- Lbc / p38 complex is crucially involved in the regulation of stress-induced cardiomyocyte hypertrophy and that disruption of this signaling pathway is detrimental for the heart under conditions of sustained hemodynamic stress. Secondly, in order to identify new AKAPs involved in the regulation of cardiac function, we followed a proteomic approach which allowed us to characterize AKAP2 as a major AKAP in the heart. Importantly, here we show that AKAP2 interacts with several proteins known to be involved in the control of gene transcription, such as the nuclear receptor coactivator 3 (NCoA3) or the ATP-dependent SWI/SNF chromatin remodeling complex. Thus, we propose AKAP2 as a novel mediator of cardiac gene expression through its interaction with these transcriptional regulators.

Role of myosin V in actin cable organization in fission yeast

Functional specialization is tightly linked to the ability of eukaryotic cells to acquire a particular shape. Cell morphogenesis, in turn, relies on the capacity to establish and maintain cell "polarity", which is achieved by orienting the trafficking of signaling molecules and organelles towards specific cellular locations and/or membrane domains. The "oriented" transport is based upon cytoskeletal polymers, microtubules and actin filaments, which serve as tracks for molecular motors. These latter generate motion that is translated either into pulling forces or directed transport. Fission yeast, a rod-like unicellular eukaryote, shapes itself by restricting growth at cell tips through the concerted activity of microtubules and actin cables. Microtubules, which assemble into 2-6 bundles and run parallel to the long axis of the cell, serve to orient growth to the tips. Growth is supported by the actin cytoskeleton, which provides tracks, the cables, for motor-based transport of secretory vesicles. The molecular motors, which bind cargos and deliver them to the tips along cables, are also known as type V myosins (hereafter indicated as myosin V). How the bundles of parallel actin filaments, i.e. the cables, extend from the tips through the cell and whether they serve any other purpose, besides providing tracks, is poorly understood. It is also unclear how the crosstalk between the two cytoskeletal systems is achieved. These are the basic questions I addressed during my PhD. The first part of the thesis work (Chapter two) suggests that the sole function of actin cables in polarized growth is to serve as tracks for motors. The data indicate that cells may have evolved two cytoskeletal systems to provide robustness to the polarization process but in principle a unique cytoskeleton might have been able to direct and support polarized growth. How actin cables are organized within the cell to optimize cargo transport is addressed later on (Chapter three). The major finding, based on the actin cable defect of cells lacking myosin Vs, is that actin filaments self-organize through the activity of the transport motors. In fact, by delivering cargos to cell tips and exerting physical pulling forces on actin filaments, Myosin Vs contribute not only to polarize cargo transport but also actin tracks. Among the cargos transported by Myosin V, which may be relevant to its function in organizing cables, there is likely the endoplasmic reticulum (ER). Actin cables, which run parallel to cortical ER, may serve as tracks for Myosin V. Myosin V-driven displacement, in turn, may account for the dynamic expansion and organization of ER during polarized growth as suggested in Chapter four. The last part of the work (Chapter five) highlights the existence of a crosstalk between actin and microtubules. In absence of myosin V, indeed, microtubules contribute to actin cable organization, likely playing a scaffolding/tethering function. Whether or not the kinesin 1, Klp3, plays any role in such process has to be demonstrated. In conclusion the work proposes a novel role for myosin Vs in actin organization, besides its transport function, and provides molecular tools to further dissect the role of this type of myosin in fission yeast. - La sp��cialisation fonctionnelle est ��troitement connect��e �� la capacit�� des cellules eucaryotes d'acqu��rir une forme particuli��re. La morphogen��se cellulaire �� son tour, est bas��e sur la capacit�� d'��tablir et de maintenir la polarit�� cellulaire, polarit�� r��alis��e en orientant le trafic des mol��cules signales et des organelles vers des zones cellulaires sp��cifiques. Ce transport directionnel d��pend des polym��res du cytosquelette, microtubules et microfilaments, qui servent comme des voies pour les moteurs mol��culaires. Ces derniers engendrent du mouvement, traduit soit en force de traction soit en transport directionnel. La levure fissipare, un eucaryote unicellulaire en forme de b��tonnet, acqui��re sa forme en limitant sa croissance aux extr��mit��s par l'action concert��e des microtubules et de l'actine. Les microtubules, qui s'assemblent de fa��on antiparall��le et parcourent la cellule parall��lement �� l'axe longitudinal, servent �� orienter la croissance aux extr��mit��s. Cette croissance est permise par le cytosquelette d'actine, fournissant des voies, les c��bles, pour le transport actif des v��sicules de s��cr��tion. Les moteurs mol��culaires, responsables de ce transport actif sont aussi appel��s myosines de type V (par la suite appel��s myosines V). La mani��re dont ces c��bles s'��tendent depuis l'extr��mit�� jusqu'�� l'int��rieur de la cellule est peu connue. De plus, on ignore ��galement si ces c��bles pr��sentent une fonction autre que le transport. L'interaction entre les deux cytosquelettes est ��galement obscure. Ce sont ces questions de base auxquelles j'ai tent�� de r��pondre lors de ma th��se. La premi��re partie de cette th��se (chapitre II) sugg��re que les c��bles d'actine, pendant la croissance polaris��e, fonctionnent uniquement comme des voies pour les moteurs mol��culaires. Les donn��es indiqueraient que les cellules ont fait ��voluer deux syst��mes de cytosquelette pour assurer plus de robustesse au processus de polarisation, bien que, comme nous le verrons, un syst��me unique est suffisant. Au chapitre III, nous verrons comment les c��bles d'actine sont organis��s �� l'int��rieur de la cellule afin d'optimiser le transport des cargo. La d��couverte majeure, r��alis��e en observant des cellules dont la myosine V fait d��faut, est que ces filaments d'actine s'auto organisent gr��ce au passage des moteurs mol��culaires le long de ces voies. En r��alit��, en d��livrant les cargos aux extr��mit��s de la cellule et en exer��ant des forces de traction sur les c��bles, les myosines V contribuent non seulement �� polariser le transport mais ��galement �� polariser les voies elles m��mes. Nous verrons ��galement au chapitre IV, que parmi les cargos importants pour l'organisation des c��bles, il y aurait le r��ticulum endoplasmique (RE). En effet, les c��bles d'actine, qui s'��talent parall��lement au RE cortical, pourraient servir comme voie pour la myosine V. Cette derni��re en retour pourrait ��tre responsable de l'expansion dynamique et de l'organisation du RE pendant la croissance polaris��e.

Infantile spasms is associated with deletion of the MAGI2 gene on chromosome 7q11.23-q21.11.

Infantile spasms (IS) is the most severe and common form of epilepsy occurring in the first year of life. At least half of IS cases are idiopathic in origin, with others presumed to arise because of brain insult or malformation. Here, we identify a locus for IS by high-resolution mapping of 7q11.23-q21.1 interstitial deletions in patients. The breakpoints delineate a 500 kb interval within the MAGI2 gene (1.4 Mb in size) that is hemizygously disrupted in 15 of 16 participants with IS or childhood epilepsy, but remains intact in 11 of 12 participants with no seizure history. MAGI2 encodes the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 that interacts with Stargazin, a protein also associated with epilepsy in the stargazer mouse.