Dans la cellule, les ARN pré messagers contenant des introns sont normalement retenus au noyau par leur interaction avec des facteurs d’épissage. Cependant, les ARN partiellement et non épissés des rétrovirus doivent entrer dans le cytoplasme pour servir de matrice pour la synthèse de certaines protéines telles que Env, Gag et Gag-Pol ainsi que d’ARN génomique qui sera empaqueté dans les nouveaux virions. Un mécanisme post-transcriptionnel utilisé par les lentivirus pour éviter la séquestration nucléaire de ces ARNm dépend d’une protéine virale appelée Rev. Pour assurer sa fonction d’exportation, Rev doit transiter entre le noyau et le cytoplasme et doit aussi pouvoir former des multimères. Par conséquent, Rev est dotée de domaines fonctionnels lui procurant ces habiletés. On retrouve le domaine riche en arginines qui contient le domaine de liaison à l’ARN et le signal de localisation nucléaire (NLS), un second domaine, riche en leucines, porte le signal d’exportation nucléaire (NES) et finalement le domaine de multimérisation. Bien que les protéines Rev du virus de l’immunodéficience humaine de type 1 (VIH-1) et bovine (VIB) aient été caractérisées, aucune étude n’a été réalisée pour la protéine Rev du virus de la maladie de Jembrana (JDV) et très peu sur le virus de l’immunodéficience féline (VIF). Comme les domaines fonctionnels et la voie d’importation des protéines Rev déjà caractérisées sont différents, nous supposons que chaque protéine Rev possède une organisation qui lui est propre et que les mécanismes de transport nucléo-cytoplasmique diffèrent entre les virus. Ce projet a pour objectif de caractériser ces domaines pour la protéine Rev du JDV et ceux du VIF ainsi que les mécanismes permettant leur transport nucléaire. L’utilisation de mutants de la protéine Rev de ces virus couplés à la protéine de fluorescente verte (EGFP) exprimés dans des cellules appropriées et observés par microscopie a permis d’identifier des séquences NLS et NES différentes de celles déjà caractérisées. Le NLS de la protéine Rev du JDV a été identifié et est composé des résidus arginines de la séquence 76-RRPARRPPIRR-87 avec un NoLS composé des mêmes résidus en plus des arginines R74, R103 et R104. Son NES est composé des résidus hydrophobes de la séquence 116-MAELEERFEDLAL-128 et est du type de l’inhibiteur de la protéine kinase (PKI pour « protéine kinase inhibitor »). Pour la protéine Rev du VIF, son NLS est composé des résidus basiques de la séquence 84-KKKRQRRRRKKKAFKK-99. Le NoLS est composé des mêmes acides aminés en plus du résidu K82. De plus, les essais d’importation nucléaires et d’interaction semblent indiquer que les voies d’importation utilisées diffèrent entre les virus et que plusieurs voies peuvent être utilisées. Ces travaux pourront éventuellement servir de base pour identifier de nouvelles cibles thérapeutiques contre les lentivirus. / In the cell, pre-messenger RNAs containing introns are normally retained in the nucleus by their interaction with splicing factors. However, the partially and unspliced ​​RNAs of retroviruses must enter the cytoplasm to serve as a template for the synthesis of certain proteins such as Env, Gag and Gag-Pol as well as genomic RNA to be packaged in the new virions. A post-transcriptional mechanism used by lentiviruses to prevent nuclear sequestration of these mRNAs depends on a trans-activator, the viral protein Rev. To ensure its export function, Rev must be able to shuttle between the nucleus and the cytoplasm and to form multimers. As a result, Rev has functional domains that provide these abilities: the arginine-rich domain, which contains the RNA binding domain and the nuclear localization signal (NLS), a second domain, rich in leucine, corresponding to the nuclear export signal (NES) and finally the multimerization domain. Although the Rev proteins of the human and bovine immunodeficiency virus (HIV-1 and BIV respectively) have been characterized, no studies have been performed for the Jembrana disease virus (JDV) Rev protein and very little on the feline immunodeficiency virus (FIV). Since the functional domains and import pathway of the already characterized Rev proteins are different, we assume that each Rev protein has its own organization and that the nucleo-cytoplasmic transport mechanisms differ between viruses. The goal of this project is to characterize these domains for the JDV and FIV Rev proteins as well as to elucidate mechanisms for their nuclear transport. The use of Rev mutants fused to the EGFP expressed in appropriate cells and observed by microscopy has identified NLS and NES sequences that differ from those already characterized. JDV Rev NLS is composed of arginine residues in the 76-RRPARRPPIRR-87 sequence with a NoLS composed of the same residues with the addition of arginine R74, R103 and R104. JDV Rev NES is composed of hydrophobic residues in the 116-MAELEERFEDLAL-128 sequence and is of the protein kinase inhibitor type (PKI). For the FIV Rev protein, its NLS is composed of basic residues in the 84-KKKRQRRRRKKKAFKK-99 sequence. FIV Rev NoLS is composed of the same residues with the addition of the lysine at position 82. In addition, the nuclear import and interaction tests suggest that the import routes used by Rev differ between the different viruses studied and that more than one import pathway may be used. This work could serve as a basis for identifying new therapeutic targets against lentiviruses.

The present final thesis discusses the transmission of a narrowly directional parametric sound beam through an amplitude-modulated ultrasonic wave, utilizing the effect of auto-demodulation in a nonlinear medium and ensuring the subsequent processing of the input signal for the parametric sound transmitter. Emphasis is placed on the mathematical tools that relate to parametric sound transmission in a nonlinear medium. The basic part of the thesis describes a parametric speaker and the associated amplitude modulation techniques, which constitute a major prerequisite for the processing of the transmitted signal. In the following section, the author then analyzes the computational intensity of these techniques, considering applicable hardware approaches. Finally, the fabrication and practical use of the proposed solution are discussed, including the measurement of typical parameters such as the spatial radiation characteristics, total harmonic distortion, and transmission channel bandwidth.

Made available in DSpace on 2016-04-29T13:32:24Z (GMT). No. of bitstreams: 1 Janaina Cristina Barea.pdf: 21166264 bytes, checksum: 6570a6aa5ae55c56d7f44b749b5371c1 (MD5) Previous issue date: 2009-06-10 / The 17 apparatus that were under the care of the Nucleus of Studies in History of the Psychology of the PUC-SP (NEHPSI) were donated, in 2004, by priest Salesiano João Modesti, of Araras SP. His find turns from the inquiry of doctorate of Iolanda Brandão on The Salesianos in the Brazilian Psychology , defended in the Program of Postgraduate Studies in Social Psychology of this University. Brought of Italy in the beginning of the 1950 years, them apparatus were used then for teaching, inquiry and installment of psychotechnic services. After a first exhibition carried out in the PUC-SP, the NEHPSI he has been a guest to repeat it in institutions of teaching and professionals, having carried out exhibitions in the UNICAMP, in the University São Marcos, in the Federal University of São João del Rei, in the PUC/Minas, Campus Poços of Caldas, in the Regional Council of 3rd Psychology Region Bahia and Sergipe and in the Institute of Psychology of the University of São Paulo. Registers of observation not organized are analysed here only to illustrate the effective participation of the visitors while getting in touch with the apparatus. The objective of this inquiry is to check if the exhibition of the apparatus, together with other documents of the period, can be a resource for the teaching of history of the psychology of Brazil. A special cutting out (seven apparatus for measure of intelligence) was done for that, with two Exhibitions (pupils of the degree course in psychology of the PUC-SP and of pedagogy of the Faculty Oswaldo Cruz) with questionnaires specially prepared for the visitors. The results show that the Exhibition builds knowledge and besides interesting, it can be a resource for the teaching of History of the Psychology / Os 17 aparelhos que se encontraram sob o cuidado do Núcleo de Estudos em História da Psicologia da PUC-SP (NEHPSI) foram doados, em 2004, por Pe. Salesiano João Modesti, de Araras SP. Seu achado resulta da pesquisa de doutorado de Iolanda Brandão sobre Os Salesianos na Psicologia Brasileira , defendida no Programa de Estudos Pós-Graduados em Psicologia Social dessa Universidade. Trazidos da Itália no início dos anos 1950, os aparelhos eram então utilizados para ensino, pesquisa e prestação de serviços psicotécnicos. Depois de uma primeira exposição realizada na PUC-SP, o NEHPSI tem sido convidado a repeti-la em instituições de ensino e profissionais, tendo realizado exposições na UNICAMP, na Universidade São Marcos, na Universidade Federal de São João del Rei, na PUC/Minas, Campus Poços de Caldas, no Conselho Regional de Psicologia 3ª Região Bahia e Sergipe e no Instituto de Psicologia da Universidade de São Paulo. Registros de observação assistemática são aqui analisados apenas para ilustrar a participação efetiva dos visitantes ao entrarem em contato com os aparelhos. O objetivo desta pesquisa é verificar se a exposição dos aparelhos, juntamente com outros documentos do período, pode ser um recurso para o ensino de história da psicologia do Brasil. Um recorte especial (sete aparelhos para medida de inteligência) foi feito para isso, com duas Exposições (alunos do curso de graduação em psicologia da PUC-SP e de pedagogia da Faculdade Oswaldo Cruz) com questionários especialmente elaborados para os visitantes. Os resultados mostram que a Exposição constrói conhecimento e além de interessante, pode ser um recurso para o ensino de História da Psicologia

Los cuerpos nucleares de Cajal (CBs) son estructuras nucleares implicadas en la biogénesis de ribonucleoproteínas nucleares y nucleolares de pequeño tamaño (snRNPs y snoRNPs) requeridas para el procesamiento nuclear de pre-mRNAs y pre-rRNAs, respectivamente. El CB concentra la proteína coilina, un marcador molecular de esta estructura, snRNPs, el factor de supervivencia de las neuronas motoras (SNM) y las proteínas que comparte con el nucleolo Nopp140 y fibrilarina. Los CB son estructuras dependientes de transcripción, pero los mecanismos de ensamblaje molecular de estos cuerpos nucleares son poco conocidos.En este estudio se utilizan métodos de inmunofluorescencia, expresión ectópica de proteínas del CB y métodos bioquímicos para analizar la importancia de dos modificaciones postraduccionales, la metilación de la coilina y la conjugación con SUMO1 del factor SMN para el ensamblaje molecular de los CBs. Se ha utilizado la línea celular MCF7 como un modelo de hipometilación endógena debido al déficit del gen MTAP. La hipometilación de la coilina conduce al desensamblaje de los CBs y a la relocalización nucleolar de la coilina no metilada. Este efecto revierte en células transfectadas que expresan el gen MTAPwt, indicando que el grado de metilación de la coilina marca su destino nuclear.Respecto a la importancia de la sumoilación en el ensamblaje de los CBs, hemos demostrado la existencia de un subtipo de CBs que concentran SUMO1 y la conjugasa de SUMO Ubc9. En neuronas, hemos detectado la presencia de SUMO durante la fase de reformación de CBs, en la respuesta al estrés. Los experimentos de inmunoprecipitación confirman la interacción de SUMO-1 con el factor SMN y demuestran que la lisina K119, portadora de una secuencia consenso de sumoilación, es esencial para la regulación del número de CBs. / Cajal bodies (CBs) are nuclear structures involved in the biogenesis of small nuclear and nucleolar ribonucleoproteins (snRNPs and snoRNPs) required for nuclear processing of pre-mRNAs and pre-rRNAs, respectively. CBs concentrate the protein coilin, a molecular marker of this structure, snRNPs, the survival of motor neurons factor (SMN) and proteins shared with the nucleolus Nopp140 and fibrillarin. CBs are transcription-dependent structures, but the mechanisms of molecular assembly of these structures are poorly understood.In this study we used inmunofluorescence, ectopic expresion of CB proteins and biochemical methods to analyze the importance of two posttranslational modifications, methylation of coilin and conjugation of SMN with SUMO1, for the molecular assembly of CBs. The cell line MCF7 has been used as a model of endogenous hypomethylation due to the lack of MTAP gene. Coilin hypomethylation leads to the disassembly of CBs and nucleolar relocation of unmethylated coilin. This effect reverses in transfected cells expressing the gene MTAPwt, indicating that the degree of methylation of coilin directs its nuclear destination.On the importance of sumoylation in the assembly of CBs, we have demonstrated the existence of a subset of CBs which concentrate SUMO1 and the SUMO1 conjugase Ubc9. In neurons, we detected the presence of SUMO1 during the reformation of CBs in response to stress. Immunoprecipitation experiments confirm the molecular interaction of SUMO1 with SMN and demonstrate that lysine 119, carrying the SMN sumoylation consensus sequence, is essential for regulating the number of CBs.

The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. <p>Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.<p>The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.<p>In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses. <p>In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand.<p>/<p>Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.<p>Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée. <p>Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11. <p>Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.