New roles of Rab GTPases on eye diseases: targeting VEGF secretion

RESUMO: A retina é composta, entre outras estruturas, pelo epitélio pigmentar da retina (EPR)e pela coróide. A região central da retina denomina-se mácula, e é a zona mais afetada na degenerescência macular relacionada com a idade, a forma mais comum de degenerescência da retina. Nesta doença, a secreção de fatores de crescimento pelo EPR é afetada, nomeadamente a do fator de crescimento vascular endotelial (VEGF), e pouco se sabe ainda sobre os mecanismos moleculares conducentes a esta condição. A família de proteínas Rab GTPases está envolvida nas vias intracelulares de sinalização e tráfego membranares, essenciais na transdução de sinais extracelulares em respostas biológicas. A sua crucial importância nestes mecanismos levou-nos a considerar o seu potencial envolvimento nas vias de secreção do VEGF, e a questionar-nos se teriam algum papel regulador sobre as mesmas. O principal objetivo deste trabalho é identificar Rab GTPases importantes para as vias de secreção e endocitose do VEGF no EPR. Essa identificação ajudará a esclarecer a patogénese da degenerescência macular da retina, e poderá servir para uma procura mais direcionada de novos agentes terapêuticos. A caracterização de dois modelos in vitro do EPR, células primárias isoladas de murganho e a linha celular B6-RPE07,levou-nos a concluir que são ambos semelhantes. Contudo, a linha celular foi escolhida como protótipo do EPR por permitir o acesso a um número ilimitado de células. No decurso deste trabalho, desenvolvemos e caracterizámos uma biblioteca de ferramentas moleculares que nos permitiram reduzir os níveis proteicos das proteínas Rab GTPases, com base na tecnologia de ácido ribonucleico (ARN) de interferência. O papel das proteínas Rab GTPases na secreção do VEGF no EPR foi estudado com base no silenciamento de apenas uma proteína, ou combinando várias, segundo a sua localização e funções intracelulares descritas. Este trabalho permitiu-nos concluir que as proteínas Rab GTPases são importantes intervenientes no processo de secreção de VEGF pelo EPR, e confirmar dados anteriores que relatam o envolvimento de algumas Rab GTPases endocíticas no processo. Propomos ainda um novo modelo para a interação destas proteínas no EPR, e sugerimos que a Rab10 e a Rab14 atuam negativamente sobre a Rab8, controlando o seu funcionamento. Os nossos resultados evidenciam a importância das proteínas Rab GTPases na secreção do VEGF pelas células do EPR, e servem de base a futuros estudos que melhor procurem compreender este mecanismo e de que modo a sua alteração se relaciona com a degenerescência da retina.--------ABSTRACT: Retinal pigment epithelium (RPE) and choroid are components of the mammalian retina, of which the central region is called macula. The most common form of retinaldegeneration, age-related macular degeneration (AMD), involves primarily deregulation of growth factors secretion by the RPE. Very little is known about the molecular mechanisms that lead to impairment of RPE’s homeostatic intracellular processes, namely the secretion of vascular endothelial growth factor (VEGF). Rab GTPases’ family regulates membrane targeting and traffic, being essential in the transduction of signal pathways. Given Rab proteins’ role in intracellular trafficking, we propose to identify key regulatory Rab proteins involved in either the secretory or the recycling pathways of VEGF in RPE. Understanding how Rab proteins’ function disruption could lead to retinal and choroidal pathology would ultimately contribute to find new therapeutic agents. Here, we characterized two mouse RPE in vitro cell models, primary cells and B6-RPE07 cell line, and concluded that both display important epithelial features as the RPE presents in vivo. Considering unlimited cell number and results reproducibility, we chose B6-RPE07 cells to further study Rab proteins’ function. To scrutinize the consequences of Rab proteins’ absence or diminished levels, we have developed novel molecular tools to achieve silencing of these key proteins using miRNA technology. We further addressed the effect of Rab proteins’ absence on VEGF secretion by performing an extensive screening where different Rab proteins were silenced, both individually and in multiple combinations considering their cellular/ compartment location. We conclude that Rab GTPases are important intervenients in VEGF secretion by RPE cells, confirming endocytic Rab proteins’ role in regulation of VEGF biology. We also propose a novel model for Rab proteins’ interaction in RPE. Our results suggest that Rab10 and Rab14 might influence Rab8 in a negative feedback mechanism, important for controlling VEGF secretion. Our achievements’ unravel Rab proteins’ role in VEGF secretion by RPE cells and are the basis for future studies to better understand RPE molecular secretory machinery.

The Evolution of Function in the Rab family of Small GTPases

Dissertation presented to obtain the PhD degree in Computational Biology.

Àrab en línia (2)

Des que es va concedir l’ajut (juliol 2006) fins a dia d’avui (desembre 2007) l’equip que porta endavant el projecte Àrab en línia (http://www.ub.edu/luga/AeL) ha estat desenvolupant el projecte Àrab en línia (2). A més a més, s’ha constituït en Grup Luga d’Innovació Docent de la UB (http://www.ub.edu/luga), ja que hi ha la ferma voluntat de seguir treballant en la mateixa direcció, des de la convicció que és una iniciativa que dóna resultats a mitjà i llarg termini. Els objectius que es varen proposar (elaborar paquets d’exercicis de llengua àrab adreçats al 2n nivell; produir materials compatibles amb els crèdits europeus i les assignatures semipresencials; i dissenyar unes pràctiques homogènies per al primer i segon nivell de llengua àrab) han estat plenament assolits. Seguint la dinàmica prevista, primer es va estudiar el material didàctic que s’havia adquirit i es varen dissenyar nous exercicis. D’altra banda, es va començar a fer una primera anàlisi dels exercicis que ja estaven penjats a la xarxa, tot i que aquest aspecte es va considerar millorable, ja que no es va aconseguir establir un sistema d’enquestes, sinó que els usuaris es van posar en contacte amb l’equip de manera espontània. Gràcies al servei analític de la pàgina web es va detectar que moltes de les visites procedien d’arreu del món, de manera que es va tornar a dissenyar la pàgina per tal d’adaptar-la als nous continguts, però de la mateixa manera es volia que la interfície estigués disponible, igualment, en altres idiomes. D’aquesta manera, ha passat a ser un autèntic portal d’aprenentatge de la llengua àrab, més si es té en compte que, a més a més del que s’havia previst, s’han inclòs materials que comencen des de la lecto-escriptura, així com també una part teòrica que l’estudiant pot d’utilitzar abans de fer les pràctiques.

La Primavera Àrab, primera gran crisi de l’acció exterior Post-Lisboa: la visibilitat, l’eficàcia i la coherència del nou marc institucional a prova

La reforma de Lisboa va establir un nou marc institucional en l'àmbit l'Acció Exterior de la Unió Europea. Un nou marc que ha de permetre a la UE consolidar-se com a actor global en el nou panorama internacional. S'espera que els canvis de Lisboa corregeixin algunes de les deficiències que la política exterior europea havia mostrat en crisis anteriors, mancances de visibilitat, eficàcia i coherència. Un any després de la seva entrada en vigor, les revoltes dels països àrabs durant la primavera de 2011 són la primera gran crisi que l'Acció Exterior post-Lisboa ha d'afrontar i l'oportunitat per avaluar si s'ha aconseguit corregir aquestes mancances del passat.

Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway.

GTPases of the Rab1 subclass are essential for membrane traffic between the endoplasmic reticulum (ER) and Golgi complex in animals, fungi and plants. Rab1-related proteins in higher plants are unusual because sequence comparisons divide them into two putative subclasses, Rab-D1 and Rab-D2, that are conserved in monocots and dicots. We tested the hypothesis that the Rab-D1 and Rab-D2 proteins of Arabidopsis represent functionally distinct groups. RAB-D1 and RAB-D2a each targeted fluorescent proteins to the same punctate structures associated with the Golgi stacks and trans-Golgi-network. Dominant-inhibitory N121I mutants of each protein inhibited traffic of diverse cargo proteins at the ER but they appeared to act via distinct biochemical pathways as biosynthetic traffic in cells expressing either of the N121I mutants could be restored by coexpressing the wild-type form of the same subclass but not the other subclass. The same interaction was observed in transgenic seedlings expressing RAB-D1 [N121I]. Insertional mutants confirmed that the three Arabidopsis Rab-D2 genes were extensively redundant and collectively performed an essential function that could not be provided by RAB-D1, which was non-essential. However, plants lacking RAB-D1, RAB-D2b and RAB-D2c were short and bushy with low fertility, indicating that the Rab-D1 and Rab-D2 subclasses have overlapping functions.

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis.

BACKGROUND: Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. RESULTS: Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. CONCLUSIONS: Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.

El Calila e Dimna al Llibre de les bèsties : Breu estudi comparatiu dels contes La serp que atemoreix, La pedra preciosa i La rata transformada en dona, en les versions àrab, catalana i castellana

Breu estudi comparatiu dels contes La serp que atemoreix, La pedra preciosa i La rata transformada en dona, de la versió àrab d'Ibn al-Muqaffa', la verió catalana de Ramon Llull a Llibre de meravelles i Llibre de les bèsties i la versió castellana de Calila e Dimna del 1252.

La Representació de l’Orient a la historieta gràfica : el món àrab i musulmà

El treball analitza la representació de l'Orient a la historieta gràfica, i d'una manera més especifica la imatge dels àrabs i els musulmans. La recerca demostra que els estereotips creats durant l'expansió europea dels segles XVIII i XIX, per l'orientalisme, han estat vigents i formant part de l'imaginari popular durant gairebé dos cents anys. La idisioncràsia popular de la historieta gràfica ha fet que aquests estereotips hagin influenciat la seva narrativa. El nou panorama social i polítc fruit, entre d’altres, del fenomen migratori de la última dècada del segle XX i l'aparició en escena del terrorisme integrista han comportat una transformació dels vells estereotips de l'orientalisme

Ya Adimou Chani ; El Hamed El Rab El Hanine / Si Mahmoud Hadjadji El Ouargli, chant [acc. voc. et instr.]

[Traditions. Afrique du Nord. Algérie]

Alguns aspectes formals de la traducció llatina de l'Alcorà de Robert de Ketton (ca. 1141-1143) i la seva relació amb el text original àrab

En el marc de la història de la transmissió escrita de l'Alcorà (al-Qur'an), l'article fa referència a l'evolució de la forma i l'estructura del text en relació amb l'oralitat del missatge original, a l'existència de diferents tradicions textuals en el si de la comunitat islàmica i al seu reflex en les traduccions. L'atenció se centra, sobretot, en la primera traducció sencera coneguda de l'Alcorà al llatí, realitzada per Robert de Ketton (ca. 1141-1143) a la Vall de l'Ebre, i se'n destaca el valor documental com a testimoni contemporani d'una tradició magribina i andalusina de tractament del text.

La conquesta àrab i la llengua catalana

Les relacions entre la llengua àrab i el català a la Catalunya Vella

Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.