Дисертації з теми "Adhesive plaque"

Lors de l'assemblage de wafers par adhésion moléculaire, un mince film d'air est piégé entre les deux wafers, créant ainsi un système fluide/structure couplé. La qualité finale de l'assemblage dépend fortement de la dynamique de ce système. L'initiation et la propagation du collage ont été étudiées, en régime transitoire, en utilisant un modèle de plaques minces couplée avec l'équation de Reynolds. La résolution numérique de l'équation, ainsi que la mesure optique du déplacement vertical de la plaquette durant le collage, nous a permis de valider le modèle et de mieux comprendre la dynamique du collage. Dans la continuité de cette étude, nous avons proposé une expression analytique de la courbure finale de l'assemblage en fonction des forces en jeu pendant le collage, ceci en utilisant à nouveau la théorie des plaques minces et en considérant l'existence d'un saut de déformation transverse le long de l'interface collée. Ce modèle a été validé par une expérience, impliquant le collage de wafers d'épaisseur différentes et en prenant soin de contrôler l'ensemble des forces agissant sur ces wafers. Nous observons une influence importante du film d'air sur la forme finale des wafers. En complément, un modèle du travail d'adhésion a été développé prenant en compte, à la fois, la rugosité d'interface et la quantité d'eau adsorbée. La différence de répartition de l'eau à l'interface de collage, nous permet d'expliquer les résultats expérimentaux montrant des valeurs d'énergie de séparation supérieure à celle de l'adhésion. Enfin, nous proposons une nouvelle méthode de mesure du travail d'adhésion pour la géométrie entière des wafers, utilisant la mesure de la taille d'une bulle cylindrique intentionnellement créée, par un petit objet, à l'interface de collage.

Les oligodendrocytes sont les cellules myélinisantes du système nerveux central. La gaine de myéline ainsi que l’oligodendrocyte qui la synthétise, sont les cibles du processus pathologique dans la Sclérose En Plaques (SEP), une maladie neurologique autoimmune et démyélinisante du système nerveux central. Les progrès thérapeutiques des 10-15 dernières années ont permis une meilleure prise en charge de la composante autoimmune de la maladie. En revanche nous sommes toujours aussi démuni pour corriger la composante démyélinisante, qui pourtant est la cause du handicap sensori-moteur permanent. Dans le but de mettre en place des thérapies de réparation myélinique chez les patients atteints de SEP, des efforts sont développés pour identifier des cibles thérapeutiques qui permettraient de contrôler la production et la dispersion des cellules précurseurs d’oligodendrocytes (OPCs) dans la moelle épinière et le cerveau. Le développement embryonnaire et néonatal constitue une période privilégiée pour examiner ces phénomènes. Dans ce contexte, mon travail de thèse a porté sur l’identification de nouvelles molécules contrôlant la spécification régionale et la migration des OPCs ainsi que leur interaction avec les axones dans le système nerveux central en développement chez la souris. Une première contribution, portant sur l’étude de la molécule d’adhésion TAG-1, m’a permis de montrer que TAG-1 régule la structure de la myéline et des axones dans le nerf optique de souris. Dans un deuxième temps, j’ai exploré l’origine des oligodendrocytes dans le tronc cérébral de souris, et mon travail m’a permis de montrer que le facteur de transcription Hoxa2 réprime l’oligodendrogenèse.

Thesis (M. S.)--Civil and Environmental Engineering, Georgia Institute of Technology, 2007.
Committee Chair: Mulalo Doyoyo; Committee Co-Chair: Reginald Desroches; Committee Member: Laurence J. Jacobs.

Plaquetas e células tumorais interagem em uma reação cruzada com proteínas do plasma, via integrina αIIbβ3 e αvβ3, respectivamente. A integrina αvβ3 também encontra-se presente na angiogênese tumoral. O objetivo desse trabalho foi avaliar a GST-INS, uma disintegrina recombinante do veneno de Bothrops insularis em eventos da progressão tumoral. Em condições estáticas, GST-INS foi capaz de inibir totalmente a adesão de células HUVECs e SK-MEL-28 às plaquetas em comparação ao controle e ao Aggrastat® (inibidor seletivo da integrina αIIbβ3). Além de inibir a TCIPA (agregação plaquetária induzida por células tumorais) a GST-INS também inibiu a invasão de SK-MEL-28 em substrato de matrigel. Células t.End.1 ou SK-MEL-28 pré-incubadas com GST-INS não formaram túbulos no substrato de matrigel. Análise por microscopia confocal mostrou que GST-INS liga-se a integrina αv presente nas células SK-MEL-28. Nossos resultados sugerem que essa disintegrina pode ser utilizada como potencial ferramenta no estudo e desenvolvimento de antiangiogênicos e antimetastáticos.
Platelets and tumor cells interact in a cross-react with plasma proteins via integrin αIIbβ3 and αvβ3 , respectively.The integrin αvβ3 is also strongly stimulated in tumor angiogenesis. The aim of this study was to evaluate the ability of GST-INS, a recombinant disintegrin from Bothrops insularis venom on events of tumor progression. Under static conditions, GST-INS was able to completely inhibit the adhesion of endothelial cells (HUVECs) and melanoma cells (SK-MEL-28) to platelets compared to control and Aggrastat® (selective inhibitor of integrin αIIbβ3). In addition, GST-INS inhibit TCIPA (platelet aggregation induced by tumor cells) GST-INS also inhibited SK-MEL-28 on matrigel invasion substrate. t.End.1 cells or SK-MEL-28 pre-incubated with GST-INS were not able to form tubules in matrigel substrate. Analysis by confocal microscopy showed that GST-INS binds to integrin αv present in SK-MEL-28 cells. The results suggest that disintegrin can be used as a potential tool in the study and development of antiangiogenic and antimetastatic.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-07T17:05:57Z No. of bitstreams: 1 2015 - Gilmar Correia Silva.pdf: 1482826 bytes, checksum: 7a35ec4d7a85ca255722822c502c454d (MD5)
Made available in DSpace on 2017-04-07T17:05:57Z (GMT). No. of bitstreams: 1 2015 - Gilmar Correia Silva.pdf: 1482826 bytes, checksum: 7a35ec4d7a85ca255722822c502c454d (MD5) Previous issue date: 2015-05-22
CAPES
This study aimed to evaluate the quality of panels Pinus caribaea var caribaea produced from lignosulfonato based adhesive (LS), urea formaldehyde (UF) and mixtures thereof, variations in time and pressing temperature. For that, were determined the physical and chemical properties of wood and adhesives, the chemical composition of the LS and its chemical bonds by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR), and pure in composition with different catalysts , the substitution effect of different percentages of UF by LS in the production of panels on the technological properties. They were also produced panels with modified LS acid. The predetermined nominal density of the panels was 0,70g/cm?. LS used in solid form was diluted to 45% in distilled water. The production of the panels was performed in three steps by varying the press temperature (140, 160 and 180?C). In the first step was applied at 140?C for composite panels 100% UF and thereafter was to be replaced by LS in the proportions of 20, 40, 60, 80 and 100%. In a second step, the first step three treatments with results from inferior mechanical properties were tested at temperatures of 160 to 180?C. In the third stage they were produced composite panels only with LS adhesive and acid in the previous three temperatures. The results of basic and apparent wood density were 0,54 and 0,60g/cm?, respectively. The extractive content of the wood was 1,80%, the macromolecular components present in the cell wall of the wood (cellulose, hemicellulose and lignin) followed the standard for the species, and pH and buffering capacity of the timber. NMR analysis showed the same pattern for lignin derivatives in their chemical bonds. Regarding the physical properties of the boards produced at 140?C, smaller percentages of thickness swelling and water absorption were checked in particleboards produced with 100% UF. For mechanical properties, no significant difference occurred between the panels made with 100% UF panels and modified with up to 40% LS. The panels produced with temperatures of 160 and 180?C generate similar results and in most higher in temperature at 180?C. Since the panels produced with LS and acid had generally improved in all properties, especially those with higher temperature
O presente trabalho teve como objetivo geral avaliar a qualidade de pain?is aglomerados de Pinus caribaea var. caribaea produzidos a partir de adesivo ? base de lignosulfonato (LS), ureia-formalde?do (UF) e suas misturas, sob varia??es de tempo e temperatura de prensagem. Para tanto, foram determinadas as propriedades f?sicas e qu?micas da madeira e dos adesivos, a composi??o qu?mica elementar do LS e suas liga??es qu?micas por meio da espectroscopia de infravermelho (IV) e resson?ncia magn?tica nuclear (RMN), puro e em composi??o com diferentes catalisadores, o efeito da substitui??o de diferentes porcentagens de UF por LS na produ??o dos pain?is sobre as propriedades tecnol?gicas. Tamb?m foram produzidos pain?is com LS modificados com ?cido. A densidade nominal preestabelecida dos pain?is foi de 0.70 g/cm?. O LS utilizado na forma s?lida foi dilu?do a 45% em ?gua destilada. A produ??o dos pain?is foi realizada em tr?s etapas variando a temperatura de prensagem (140, 160 e 180?C). Na primeira etapa foi aplicada a temperatura de 140?C para pain?is compostos com 100% de UF e a partir da? houve a sua substitui??o por LS nas propor??es de 20, 40, 60, 80 e 100%. Numa segunda etapa, tr?s tratamentos da primeira etapa com resultados de propriedades mec?nicas inferiores foram testados nas temperaturas de 160 e 180?C. Na terceira etapa foram produzidos pain?is compostos apenas com o adesivo LS e ?cido nas tr?s temperaturas anteriores. Os resultados da densidade b?sica e aparente da madeira foram de 0,54 e 0,60g/cm?, respectivamente. O teor de extrativos da madeira foi de 1,80%, os componentes macromoleculares presentes na parede celular da madeira (celulose, hemicelulose e lignina) seguiram o padr?o para a esp?cie, assim como o pH e a capacidade tamp?o da madeira. A an?lise de RMN mostrou o mesmo padr?o para derivados de lignina em suas liga??es qu?micas. Em rela??o ?s propriedades f?sicas dos pain?is produzidos a 140?C foram verificadas porcentagens menores de inchamento em espessura e absor??o de ?gua nos pain?is produzidos com 100% de UF. Para as propriedades mec?nicas, os tratamentos que tiveram composi??o modificada com at? 40% de LS, n?o apresentaram diferen?a significativa com o tratamento produzido com 100% de UF. Os pain?is produzidos com temperaturas de 160 e 180?C geraram resultados similares e na maioria superiores na temperatura de 180?C. J? os pain?is produzidos com LS e ?cido apresentaram em geral, melhoria em todas as propriedades, com destaque para aqueles com maior temperatura.

In this thesis, a number of mathematical models of haptotactic cell migration are developed. The modelling of haptotaxis is presented in two distinct parts - the first comprises an investigation of haptotaxis in pre-necrotic avascular tumours, while the second consists of the modelling of adhesion-mediated haptotactic cell migration within tissue, with particular attention paid to the biological appropriateness of the description of cell-extracellular matrix adhesion. A model is developed that describes the effects of passive and haptotactic migration on the cellular dynamics and growth of pre-necrotic avascular tumours. The model includes a description of the extracellular matrix and its effect on cell migration. Questions are posed as to which cell types act as a source of the extracellular matrix, and the model is used to simulate the possible effects of different matrix sources. Simulations in one-dimensional and spherically symmetric geometry are presented, displaying familiar results such as three-phase tumour growth and tumours comprising a rim of proliferating cells surrounding a non-proliferating region. Novel effects are also described such as cell population splitting and tumour shrinkage due to haptotaxis and appropriate extracellular matrix construction. The avascular tumour model is then extended to describe the internalisation of labelled cells and inert microspheres within multicell tumour spheroids. A novel model of adhesion-receptor mediated haptotactic cell migration is presented and specific applications of the model to tumour invasion processes are discussed. This model includes a more biologically realistic description of cell adhesion than has been considered in previous models of cell population haptotaxis. Through assumptions of fast kinetics, the model is simplified with the identification of relationships between the simplified model and previous models of haptotaxis. Further simpli.cations to the model are made and travelling wave solutions of the original model are then investigated. It is noted that the generic numerical solution routine NAG D03PCF is not always appropriate for the solution of the model, and can produce oscillatory and inaccurate solutions. For this reason, a control volume numerical solver with .ux limiting is developed to provide a better method of solving the cell migration models.

Since their accidental introduction into the Great Lakes in mid- to late-1980s, the freshwater zebra mussels, Dreissena polymorpha, have colonized most lakes and waterways across eastern North America. Their rapid spread is partly attributed to their ability to tenaciously attach to hard substrates via an adhesive apparatus called the byssus, resulting in serious environmental and economic impacts. A detailed ultrastructural study of the bysuss revealed a 10 nm adhesive layer at the attachment interface. Distributions of the main adhesive amino acid, 3,4-dihydroxyphenylalanine (DOPA), and its oxidizing (cross-linking) enzyme, catechol oxidase, were determined histochemically. It was found that, upon aging, DOPA levels remained high in the portion of the byssus closest to the interface, consistent with an adhesive role. In contrast, reduced levels of DOPA corresponded well with high levels of catechol oxidase in the load-bearing component of the byssus, presumably forming cross-links and increasing the cohesive strength.

Grande parte do tempo clínico do Médico Dentista é, atualmente, gasto a substituir restaurações dentárias realizadas no passado devido a fraturas ou fenómenos de cárie secundária. Pesquisou-se, assim, para a realização desta monografia em diferentes bases de pesquisa acerca dos fenómenos de cárie e cárie secundária. Um dos objetivos desta revisão bibliográfica estudar a microbiologia associada á cárie secundária, a histopatologia da lesão, o diagnóstico e os problemas associados ao diagnóstico. pretende-se, também, nesta monografia devido á sua pertinência investigar o potencial bacteriostático e bactericida dos diversos materiais disponíveis e em que situações esse efeito é potenciado, de modo a auxiliar o clinico na sua decisão no momento de restauração dentária, procurando prevenir ou adiar a necessidade de efetuar uma nova restauração. Para a realização desta monografia foram realizadas pesquisas nos motores de busca PubMed, B-on, Science Direct e Sci-elo. Depois de selecionados os artigos com interesse e com a utilização de seguintes palavras-chave e combinações de palavras sendo que no final foram selecionados 58 Artigos, dentro dos últimos 10 anos na língua portuguesa e na língua inglesa. Apesar da microbiologia e dos fenómenos histopatológicos de desenvolvimento da lesão estarem bem documentados, não existe consenso no que toca ao diagnóstico e tratamento das lesões. Existem diversos métodos de diagnóstico ao alcance da prescrição do Médico Dentista, sendo que, no entanto, nem todos estão indicados para o diagnóstico de lesões de cárie. Face á grande variedade de materiais restauradores e sistemas adesivos existentes no mercado torna-se imperativo que o Médico Dentista tenha os conhecimentos necessários sobre o potencial antibacteriano desses mesmos materiais. Sendo, assim, capaz de concluir mediante a situação apresentada: as vantagens, desvantagens e potencialidades dos diversos materiais á sua disposição na prevenção de lesões de cárie inicial e cárie secundária, aumentando a sobrevivência das peças dentárias. Conclui-se que a cárie secundária não é um fenómeno exclusivo a nenhum tipo de material forrador, ou restaurador não existindo também nenhum material capaz de efetuar com elevadas taxas de sucesso a sua prevenção. Contudo, atualmente, vem-se procurando a utilização de materiais que proporcionem um maior selamento da estrutura dentária, diminuindo a microinfiltração e consequentemente diminuindo os fenómenos de cárie secundária.
Nowadays Dentists spend a lot of their clinical time renewing previously made restorations due to fractures or the occurrence of secondary caries. One of the objectives of this work lies on studying the microbiology associated with secondary caries its histopathology, diagnostic and the problems associated with that diagnostic. It’s also na objective due to its pertinence to study the antibacterial potential of many dental materials in a way that helps the Dentist to take the best course of action in his clinical practice. In the search it was used the follow search engines PubMed, B-on, Science Direct e Sci-elo with the use and combination of different search terms it was select 58 articles with interest in the last 10 years within the Portuguese and English languages. Although the fact the microbiology and histopathology of the secondary carious lesion is well documented there is no scientific agreement when we speak about the diagnostic or treatment of this lesions. Due to the many options of restorative materials and adhesive systems available it becomes imperative that the Dentist has the knowledge about the antibacterial potential of those materials. With this knowledge the Dentist will be able to know the vantages, disadvantages and the potential of the different restorative materials in their role on the prevention of carious lesions improving the survival of the teeth It can be concluded that the dental carie it is not a phenomon exclusive to any type of restorative or lining material at the same time that it is not available in the market capable of preventing it with high success rates. However nowadays it is being pursued the use of materials that offer a better sealing of the dental structure lowering the microleakage consequently dropping the incidence of secondary caries.

Objectifs: Chez les patients atteints de sclérose en plaques (SEP), des lymphocytes pro-inflammatoires utilisent des molécules d’adhérence afin de parvenir à traverser la barrière hémo-encéphalique (BHE) et former des lésions multifocales dans le système nerveux central (SNC). Dans le contexte de la SEP, les lymphocytes CD4 auto-agressifs polarisés en TH17 (sécrétant de l’IL-17) sont reconnus comme contribuant à la formation des lésions. Le rôle des lymphocytes CD8 TC17 est quant à lui encore mal défini. L’identification de marqueurs de surface spécifiquement exprimés par les lymphocytes TH17 et TC17 faciliterait la caractérisation de ces sous-populations pathogéniques et fournirait de nouvelles cibles thérapeutiques pour traiter la SEP. Méthodologie: Nous avons identifié MCAM lors d’analyses protéomiques de cellules endothéliales de la BHE humaine et de lymphocytes T humains. Nous avons caractérisé le phénotype et la fonction de ces cellules exprimant MCAM ex vivo, in vitro, in situ et in vivo, à partir de matériel obtenu de témoins (contrôles), de patients atteints de SEP et d’animaux atteints d’encéphalomyélite auto-immune expérimentale (EAE). Résultats: MCAM est exprimé à la fois par les cellules endothéliales de la BHE humaine et par une sous-population de lymphocytes T effecteurs mémoire CD161+ et CCR6+. Les lymphocytes CD4 et CD8 MCAM+ expriment plus d’IL-17, IL-22, GM-CSF et granzyme B (Gz B) que les lymphocytes MCAMneg. De plus, l’expression de MCAM est fortement augmentée à la surface des lymphocytes T CD4+ et CD8+ lors des poussées de SEP, alors que les traitements immunomodulateurs en diminuent l’expression. In situ, l’expression de MCAM par les cellules endothéliales de la BHE est plus marquée au site des lésions de SEP et d’EAE, et on retrouve des lymphocytes CD4 et CD8 MCAM+ au sein de ces infiltrats périvasculaires du SNC. In vitro, les lymphocytes CD8 MCAM+ causent plus de mort oligodendrocytaire et bloquer MCAM diminue la transmigration des CD8 TC17 et des CD4 TH17 à travers les cellules endothéliales de la BHE humaine. In vivo, dépléter les lymphocytes CD4 ou CD8 MCAM+ améliore les signes cliniques de l’EAE par transfert. Par ailleurs, l’expression de MCAM est régulée à la hausse à la surface des lymphocytes CD4 et CD8 de la souris transgénique TCR1640, un modèle animal d’EAE spontanée. Finalement, bloquer MCAM atténue les déficits neurologiques chroniques aussi bien du modèle d’EAE induite avec le MOG35-55 que du modèle d’EAE spontanée. Conclusion: Nos données démontrent que les lymphocytes encéphalitogéniques produisant de l’IL-17 et présentant une capacité effectrice et migratoire marquée expriment MCAM. MCAM pourrait servir de biomarqueur en SEP et constituer une cible thérapeutique valable pour traiter les conditions neuroinflammatoires.
Objective: In multiple sclerosis (MS), pro-inflammatory lymphocytes use adhesion molecules to cross the blood-brain barrier (BBB) and accumulate in central nervous system (CNS) lesions. CD4 T lymphocytes polarized into auto-aggressive encephalitogenic TH17 (IL-17 secreting) are known to partake in MS lesion formation. Much less is known about the role of CD8 TC17. Identification of specific surface markers and adhesion molecules expressed by TH17 and TC17 lymphocytes would allow further characterization of these pathogenic subsets and would provide new therapeutic targets in MS. Methodology: We identified MCAM in a proteomic screen of human BBB endothelial cells (ECs) and on a subset of T lymphocytes. We characterized the phenotype and function of MCAM-expressing cells ex vivo, in vitro and in situ using human and mouse material obtained from controls, MS subjects and Experimental Autoimmune Encephalomyelitis (EAE) animals. Results: MCAM is expressed by human BBB-ECs and by human effector memory CD161+ and CCR6+ T lymphocytes. Both CD4 and CD8 MCAM+ lymphocytes express more IL-17, IL-22, GM-CSF and Gz B than MCAMneg lymphocytes. Moreover, MCAM is strikingly up-regulated in human on CD4+ and CD8+ T lymphocytes during MS relapses, while treatment decreases MCAM expression. In situ, MCAM+ CD8 and CD4 T lymphocytes are present in perivascular infiltrates of MS and EAE CNS specimens, while MCAM expression is up-regulated on BBB-ECs within lesions. In vitro, MCAM+ CD8 T lymphocytes display higher killing capacity of oligodendrocytes, and MCAM blockade reduces CD8 TC17 and CD4 TH17 transmigration across human BBB-ECs. In vivo, depletion of MCAM+ cells from reactivated CD4 T lymphocytes and from CD8 T lymphocytes decreases clinical symptoms in adoptive transfer EAE. Furthermore, expression of MCAM is up-regulated on CD4 and CD8 T lymphocytes in the TCR1640 transgenic mice, a model of spontaneous EAE. Finally, blocking MCAM in both MOG35-55-induced and spontaneous primary progressive EAE attenuates chronic neurological deficits. Conclusions: Our data demonstrate that encephalitogenic IL-17-producing lymphocytes with high effector and migratory capacity express MCAM, and that MCAM could serve as a biomarker for MS and a valuable target for the treatment neuroinflammatory conditions.

But : La perte de l’intégrité de la barrière hémo-encéphalique (BHE) est l’une des caractéristiques principales de la sclérose en plaques. Cette augmentation de la perméabilité est associée à une désorganisation des molécules de jonction serrée et à une augmentation de l’expression de molécules d’adhérence essentielles à l’extravasation des cellules immunitaires. Identifier de nouvelles molécules impliquées dans ce processus est donc crucial pour le développement de nouvelles thérapies contre la sclérose en plaques visant à promouvoir l’intégrité de la BHE et à diminuer la migration des leucocytes dans le système nerveux central (SNC) au cours du processus neuro-inflammatoire. Dans cette étude, le rôle spécifique de la molécule d’adhérence ALCAM, qui est exprimé à la surface des cellules endothéliales de la BHE (CE-BHE) et de certains sous-types de leucocytes, a été évalué. Méthodologie : À l’aide d’une analyse protéomique exhaustive, notre laboratoire a identifié ALCAM comme étant une molécule d’adhérence surexprimée par les CE-BHE mises en culture dans un milieu pro-inflammatoire. Dans le but d’étudier le rôle spécifique d’ALCAM durant la diapédèse leucocytaire, nous avons induit chez des souris de type sauvages et des souris ALCAM déficientes l’encéphalite auto-immune expérimentale (EAE), le modèle animal de la sclérose en plaques. Le rôle d’ALCAM a aussi été étudié à l’aide d’un système d’adhérence sous flux laminaire. Cet appareil, qui imite un capillaire cérébral, permet de suivre en temps réel le mouvement des leucocytes, soumis à une pression physiologique, dans un tube couvert à sa base par des CE-BHE. Résultats : En utilisant ce système d’adhérence, j’ai pu démontrer que des anticorps dirigés contre ALCAM réduisent de façon significative le roulement et l’adhérence de monocytes CD14+ humains à la surface de CE-BHE. Par ailleurs, ces anticorps préviennent de façon marquée la diminution de la vitesse moyenne des cellules au cours de l’expérience. Par le fait même, j’ai aussi observé une réduction significative de l’extravasation des monocytes traités avec de l’anti-ALCAM au travers de CE-BHE dans un modèle statique de migration. Subséquemment, j’ai démontré que ces monocytes migrent plus rapidement et en plus grand nombre au travers d’une barrière constituée de cellules endothéliales méningées à comparer à des CE-BHE. Bien que des observations similaires ont été effectuées en utilisant des lymphocytes T CD4+ humains ex vivo, j’ai été incapable de reproduire ces résultats à l’aide de cellules Th1 et Th17 réactivées in vitro. Par opposition à nos données in vitro, j’ai découvert que les souris déficientes en ALCAM développent une EAE active plus sévère que celle observée chez des souris de type sauvages. Cette EAE est par ailleurs associée à une infiltration périvasculaire de lymphocytes T pro-inflammatoires et de monocytes/macrophages de type M1 plus marqué chez les souris ALCAM déficientes. L’induction d’une EAE par transfert adoptif, dans laquelle des cellules immunitaires de type sauvage réactivées par du MOG sont injectées à des souris déficientes en ALCAM, suggère que la pathophysiologie observée durant l’EAE active serait liée à l’absence d’ALCAM au niveau de la BHE. Une caractérisation de la barrière des souris ALCAM déficientes non immunisées a par la suite révélé une réduction de l’expression de certaines molécules de jonction serrée. Une analyse plus poussée a par ailleurs démontré qu’ALCAM est lié indirectement à des molécules de jonction serrée des CE-BHE, ce qui expliquerait l’augmentation de la perméabilité de celle-ci chez les souris déficientes en ALCAM. Une analyse de la perméabilité intercellulaire de la BHE effectuée in vitro a d’autre part corrélé ces résultats. Conclusion : Collectivement, nos données prouvent qu’ALCAM joue un rôle prépondérant dans la diapédèse des monocytes, mais pas des lymphocytes Th1 et Th17 au travers de la BHE. Par ailleurs, nos résultats suggèrent qu’ALCAM remplit une fonction biologique cruciale favorisant le maintien de l’intégrité de la BHE en agissant comme molécule adaptatrice intermédiaire entre les molécules de jonction serrées et le cytosquelette. De cette façon, l’absence d’ALCAM au niveau des CE-BHE promeut indirectement le recrutement de leucocytes pro-inflammatoires dans le SNC des souris atteintes de l’EAE en augmentant la perméabilité des vaisseaux sanguins de la BHE.
Aim: The loss of blood-brain barrier (BBB) integrity is a hallmark of multiple sclerosis. It is associated with a disorganization of junctional molecules and an upregulation of cell adhesion molecules essential for immune cell transmigration. Identifying novel key players involved in this process is thus crucial for the development of MS therapies aimed at promoting BBB integrity and decreasing leukocytes trafficking into the central nervous system (CNS) during neuroinflammation. In this study, the specific role of the adhesion molecule ALCAM, found on BBB endothelial cells (BBB-ECs) and subsets of leukocytes, was assessed. Methods: We first identified ALCAM as an important molecule upregulated during inflammation in a proteomic screen of in vitro cultured primary human BBB-ECs. In order to study the effects of ALCAM on leukocyte transmigration, both active and passive experimental autoimmune encephalomyelitis (EAE) was induced in ALCAM KO and WT animals. The specific role of ALCAM during leukocyte transmigration was also assessed using a modified adhesion assay under sheer-stress, in which leukocytes flow across a capillary-like channel lined with a monolayer of BBB-ECs under physiological pressure. Results: Using the modified adhesion assay, we demonstrated that anti-ALCAM blocking antibodies significantly reduce the rolling and the adhesion of human CD14+ monocytes interacting with primary human BBB-ECs, as well as prevent their overall decrease in velocity. Concurrently, we also observed a significant reduction in the migration of ex vivo CD14+ monocytes, across a monolayer of human BBB-ECs. These monocytes also migrated more rapidly and in higher number across meningeal endothelial cells, as compared to BBB-ECs. While similar observations were made using ex vivo CD4+ T lymphocytes, we failed to reproduce these results using in vitro activated Th1 and Th17 cells. In opposition to our in vitro data, ALCAM KO mice developed a more severe active EAE associated with a significant increase in perivascular infiltration of pro-inflammatory lymphocytes (Th1/Th17) and M1 monocytes/macrophages, as compared to WT controls. In addition, EAE transfer experiments, in which ALCAM KO mice received WT MOG-reactivated splenocytes, suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. Phenotypic characterization of un-immunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further analysis showed that ALCAM is indirectly associated with tight junction molecules of the BBB-ECs, which explains the increased CNS parenchymal blood vessel in vivo permeability in ALCAM KO animals. Correlating with these data, primary culture of mouse brain BBB-ECs was shown to possess a lower TEER and an increased permeability coefficient. Conclusion: Collectively, our data provide evidence of the implication of ALCAM in monocyte transmigration, but not Th1 or Th17 lymphocyte diapedesis across CNS endothelium. Our results also point to a biologically crucial function of ALCAM in maintaining BBB integrity by acting as an adaptor molecule between tight junctions and the cytoskeleton. As such, the absence of ALCAM at the level of BBB-ECs indirectly promotes the recruitment of pro-inflammatory leukocytes in the CNS of EAE animals by increasing the BBB vessels permeability.

La sclérose en plaques est une maladie neuroinflammatoire idiopathique caractérisée par la formation de lésions focales de démyélinisation, qui apparaissent suite à l’infiltration périvasculaire de cellules immunitaires et à l’augmentation de la perméabilité de la barrière hémato-encéphalique. L’encéphalomyélite auto-immune expérimentale (EAE) est le modèle animal de cette maladie. Cependant, ce modèle présente des différences importantes avec la sclérose en plaques. L’objectif de ce projet de maîtrise était d’approfondir la caractérisation d’un nouveau modèle transgénique d’encéphalomyélite auto-immune expérimentale spontanée, le modèle TCR1640, afin de valider celui-ci pour l’étude des phénomènes physiopathologiques qui surviennent à différents stades de la sclérose en plaques, ainsi que pour le développement de nouveaux traitements de la maladie. La souris TCR1640 porte un récepteur des cellules T (TCR) transgénique autoréactif, qui reconnaît un peptide de la myéline et déclenche une réaction auto-immune contre la myéline endogène au sein du système nerveux central (SNC). Des observations faites in situ et in vitro ont permis d’identifier des changements qui surviennent de façon très précoce dans l’unité neurovasculaire chez les animaux TCR1640 présymptomatiques, et qui sont liés à la présence d’un profil immunitaire périphérique proinflammatoire. Lors des phases actives de l’EAE spontanée, les animaux TCR1640 au stade chronique présentent une inflammation accrue du système nerveux central associée à une infiltration leucocytaire massive, par rapport aux animaux au stade aigu de la maladie. Une étude in vivo a également permis de moduler la maladie développée par des animaux ayant subi une immunisation passive avec des cellules T auxiliaires en provenance de souris TCR1640. Enfin, l’implication de nouvelles molécules d’adhésion cellulaire dans le développement et le maintien de l’EAE spontanée a été suggérée par des observations in vitro. L’ensemble de ces résultats suggère que le modèle TCR1640 présente plusieurs avantages pour l’étude de la physiopathologie de maladies neuroinflammatoires telles que la sclérose en plaques, et servira d’outil afin de valider de nouvelles stratégies thérapeutiques.
Multiple sclerosis is an idiopathic inflammatory disease of the central nervous system. It is characterized by the formation of focal perivascular lesions and demyelination of the surrounding area, which appear concomitantly to a massive immune cell infiltration and disruption of the blood brain barrier. Experimental autoimmune encephalomyelitis is the animal model most extensively used for the study of multiple sclerosis. Unfortunately, this model does not mimic many aspects of the human disease. The goal of this project is to further the characterization of a new transgenic model of spontaneous experimental autoimmune encephalomyelitis, the TCR1640 model, and to validate it as a relevant tool for the study of multiple sclerosis physiopathology and treatment. The TCR1640 mouse possesses a transgenic T cell receptor which recognizes a myelin peptide and triggers an autoimmune response against endogenous myelin in the central nervous system. In situ and in vitro observations have led to the identification of early changes which appear at the neurovascular unit in presymptomatic TCR1640 animals. This early disruption of blood brain barrier homeostasis is linked to the establishment of a proinflammatory immune profile in the periphery. Animals at the chronic stage show sustained inflammation of the central nervous system parenchyma and massive leukocyte infiltration, compared to animals in acute phase of disease. An in vivo experiment has allowed modulating the disease by treatment with a multiple sclerosis-approved therapy, in wild type mice which had received reactivated CD4+ T cells from TCR1640 animals. Finally, the implication of new cell adhesion molecules in the development and maintenance of spontaneous experimental autoimmune encephalomyelitis has been suggested by in vitro study of melanoma cell adhesion molecule (CD146) and activated leucocyte cell adhesion molecule (CD166). The results obtained in this study suggest that the TCR1640 model is a valuable asset in the study of neuroimmune diseases such as multiple sclerosis. It could also be used to validate new therapeutic strategies for the treatment of this disease.

Indiana University-Purdue University Indianapolis (IUPUI)
Alzheimer's disease (AD) is a national priority, with nearly six million Americans affected at an annual cost of $200 billion and no available cure. A better understanding of the mechanisms underlying AD is crucial to combat its high and rising incidence and burdens. Most cases of AD are thought to have a complex etiology with numerous genetic and environmental factors influencing susceptibility. Recent genome-wide association studies (GWAS) have confirmed roles for several hypothesized genes and have discovered novel loci associated with disease risk. However, most GWAS-implicated genetic variants have displayed modest individual effects on disease risk and together leave substantial heritability and pathophysiology unexplained. As a result, new paradigms focusing on biological pathways have emerged, drawing on the hypothesis that complex diseases may be influenced by collective effects of multiple variants – of a variety of effect sizes, directions, and frequencies – within key biological pathways. A variety of tools have been developed for pathway-based statistical analysis of GWAS data, but consensus approaches have not been systematically determined. We critically review strategies for genetic pathway analysis, synthesizing extant concepts and methodologies to guide application and future development. We then apply pathway-based approaches to complement GWAS of key AD-related endophenotypes, focusing on two early, hallmark features of disease, episodic memory impairment and brain deposition of amyloid-β. Using GWAS and pathway analysis, we confirmed the association of APOE (apolipoprotein E) and discovered additional genetic modulators of memory functioning and amyloid-β deposition in AD, including pathways related to long-term potentiation, cell adhesion, inflammation, and NOTCH signaling. We also identified genetic associations to amyloid-β deposition that have classically been understood to mediate learning and memory, including the BCHE gene and signaling through the epidermal growth factor receptor. These findings validate the use of pathway analysis in complex diseases and illuminate novel genetic mechanisms of AD, including several pathways at the intersection of disease-related pathology and cognitive decline which represent targets for future studies. The complexity of the AD genetic architecture also suggests that biomarker and treatment strategies may require simultaneous targeting of multiple pathways to effectively combat disease onset and progression.

La sclérose en plaques (SEP) est caractérisée par des infiltrations périvasculaires de cellules immunitaires et par de la démyélinisation au sein du système nerveux central (SNC). Ces deux paramètres de la maladie sont associés à la fragilisation de la barrière hémato-encéphalique (BHE). En ce sens, le recrutement des cellules présentatrices d’antigène (CPA) myéloïdes, telles que les monocytes, les macrophages et les cellules dendritiques, dans le SNC à travers la BHE, est une étape cruciale dans l’initiation et la persistance de l’inflammation cérébrale. Nerve injury-induced protein (Ninjurin)-1 est une nouvelle molécule d’adhérence qui médie une interaction de type homophilique et dont l’expression sur l’endothélium vasculaire de la BHE humaine fut identifiée grâce à une analyse protéomique des protéines associées à la BHE. Les résultats présentés dans ce mémoire montrent que l’expression de Ninjurin-1 augmente dans un contexte inflammatoire dans les cultures primaires de cellules endothéliales de la BHE (CE-BHE) et sur les CPA myéloïdes humaines ex vivo et générées in vitro. De plus, les CPA infiltrantes retrouvées dans les lésions cérébrales de patients atteints de SEP et dans le SNC des souris atteintes d’encéphalomyélite autoimmune expérimentale (EAE), le modèle murin de la SEP, expriment de hauts niveaux de Ninjurin-1. À l’aide du modèle in vitro de la BHE, la neutralisation de Ninjurin-1 restreint spécifiquement la migration des monocytes à travers les CE-BHE sans affecter le recrutement des lymphocytes, ni la perméabilité des CE-BHE. Enfin, les souris atteintes d’EAE et traitées avec un peptide bloquant dirigé contre Ninjurin-1 présentent une maladie moins sévère ainsi qu’une diminution des CPA infiltrant le SNC et ce comparé au groupe contrôle. Ces résultats suggèrent que Ninjurin-1 est une molécule d’adhérence de la BHE impliquée dans le recrutement de CPA myéloïdes au sein du SNC et qu’elle peut être considérée comme une cible thérapeutique potentielle en SEP.
Multiple Sclerosis (MS) is characterized by perivascular infiltrations of immune cells and by demyelination in the central nervous system (CNS). These two hallmarks of the disease are associated with the disruption of the blood-brain barrier (BBB). The recruitment of monocytes, macrophages and dendritic cells, the so-called myeloid antigen-presenting cells (APCs), in the CNS through the BBB is thought to play a crucial role in the initiation and the persistence of the disease. Therefore the identification of the molecular mechanisms involved in the migration of myeloid APCs into the CNS is considered a valid therapeutic option in MS. Nerve injury-induced protein (Ninjurin)-1, a novel adhesion molecule that mediates homophilic binding, was found to be expressed in the vascular endothelium of the BBB following a proteomic screen of human BBB-associated proteins. Ninjurin-1’s expression increases during an inflammatory context in primary cultures of endothelial cells of the BBB (BBB-ECs) and on ex vivo and in vitro generated myeloid APCs. In addition, infiltrating APCs in human MS lesions and in the CNS of the murine model of MS, the mice affected with experimental autoimmune encephalomyelitis (EAE), express high levels of Ninjurin-1. Using an experimental model of the BBB, the neutralization of Ninjurin-1 specifically restricts the migration of monocytes across the BBB-ECs without affecting the recruitment of lymphocytes or the permeability of the BBB-ECs. Finally, EAE mice treated with a Ninjurin-1 blocking peptide have reduced disease severity and a reduced infiltration of myeloid APCs in the CNS, as compared to the control group. Our results show that Ninjurin-1 is an adhesion molecule of the BBB involved in the recruitment of myeloid APCs to the CNS and is also a potential therapeutic target to dampen CNS inflammatory processes, as occurs in MS.