Cette thèse est une grammaire descriptive du tat de l’Apshéron, une langue iranienne de la branche sud-ouest parlée en Azerbaïdjan. Il s’agit de la première description d’un dialecte tat musulman dans une langue occidentale. Après une introduction détaillée présentant le contexte sociolinguistique et la phonologie, le présent travail aborde les différentes parties du discours, le marquage des fonctions grammaticales, la morphologie verbale (dérivation, classes morphologiques du verbe, locutions verbales, emplois et valeurs des formes conjuguées). Les principaux faits de syntaxe de la phrase complexe sont ensuite décrits : subordonnées relatives, complétives, et adverbiales, coordination de prédicats.La description, effectuée dans une perspective typologique, s’appuie sur l’analyse de textes spontanés récoltés sur le terrain, de traductions de l’azéri vers le tat, et sur les connaissances personnelles de l’auteur, locutrice native. Elle est suivie en annexe de textes extraits du corpus, partiellement traduits, ainsi que d’un lexique recensant les lexèmes utilisés dans l’étude et dans les textes. / This thesis is a descriptive grammar of Tat (an Iranian language of the South-Western branch) as spoken on the Absheron Peninsula, east of Baku in the Republic of Azerbaijan. It is the first description of a Muslim variety of Tat in a Western European language.After a detailed introduction outlining the sociolinguistic context and the phonology, the present study discusses the parts of speech, the marking of grammatical relations and verbal morphology of Absheron Tat (verbal derivation, verb classes, complex predicates, formation and use of inflected verb forms). This is followed by a survey of complex sentences, viz. relative clauses, complement clauses, adverbial subordinates as well as coordination.The present work adopts a typological point of view and is based on the analysis of texts originating from the author’s fieldwork and tales translated from Azeri into Tat, in addition to the author’s competence as a native speaker. The appendix presents samples of the text corpus (some of them also translated) and a glossary listing items that feature in the grammatical description and the texts.

The concept of decentering originated with Piaget, who defined decentering as a feature of operational thought, the ability to conceptualize multiple perspectives simultaneously. Feffer applied Piaget’s concept of decentering to the cognitive maturity of social content. This study used Feffer’s Interpersonal Decentering scoring system for stories told about TAT pictures to investigate the developmental hierarchy of decentering for children and adolescents. The participants originated from the Berkeley Guidance Study, a longitudinal sample of more than 200 individuals followed for more than 60 years by the Institute of Human Development at the University of California, Berkeley. The hypotheses tested were: (1) chronological age will be positively related to Decentering as reflected in Feffer’s Interpersonal Decentering scores obtained annually between ages 10 and 13 and at 18; (2) children born into higher class homes would have higher Age 12 Decentering scores; (3) children born later in birth order will have higher Age 12 Decentering scores; (4) children whose parents were observed to have closer bonds with their children at age 21 months will have higher Age 12 Decentering scores; (5) adolescents with higher scores from the Decentering Q-sort Scale (derived from adolescent Q-sorts) will have higher Age 12 Decentering scores; and (6) participants who have higher Age 12 Decentering scores will self-report higher CPI Empathy scale scores at Age 30. A repeated measures ANOVA tested Hypothesis 1. Pearson product-moment correlation coefficients tested Hypotheses 2-6. Age and Decentering scores were unrelated, as was birth order; social class findings were mixed. Parents’ bonds with child and Age 12 Decentering were negatively correlated (closer bonds predicted higher Decentering), as were Age 12 Decentering and Age 30 Empathy (higher early Decentering predicted lower adulthood Empathy). Girls (age 12) tended to decenter more consistently and had higher Decentering scores than boys.

acase@tulane.edu / Peptides and peptide nucleic acids (PNAs) have long been recognized as promising tools and potential therapeutics. Yet the cell membrane remains a significant barrier to their intracellular targets. Conjugation to cell penetrating peptides like pTat48-60 (tat) and pAntp43-68 (penetratin) facilitates delivery, however delivery efficiencies remain low. Improving the performance of known cell penetrating peptides by rational design is hindered by the lack of explicit design principles. Instead, here we use synthetic molecular evolution to generate and screen a cell penetrating peptide library containing 8,192 tat/penetratin hybrid peptides to identify sequences with improved ability to deliver a splice correcting PNA sequence. The parent sequences poorly deliver PNA to cells; however, at 5M peptide-PNA, the top performing PNA conjugated Delivery Peptide (PDEP) daughter sequence showed an 80-fold increase over PNA only treated cells in properly spliced luciferase mRNA and 33-fold higher standardized luminescence values than the top performing parent sequence penetratin. The PDEPs identified in this study are effective in multiple cell types, and also deliver a peptide cargo to cells. The capabilities of the PDEPs make them a valuable research tool for delivery of membrane impermeable PNA or peptide sequences. This dramatic improvement in performance following a single iteration of synthetic molecular evolution is an indication of the power of this approach to peptide sequence optimization. / 1 / William Berkeley Kauffman

Cell-penetrating peptides (CPPs) mediate the delivery of macromolecules across the plasma membrane of live cells. These peptides are therefore important due to the potential of making the delivery of protein probes or therapeutics a routine procedure. However, CPP-mediated delivery is currently an inefficient process. CPP-protein conjugates are internalized into cells by endocytosis and the macromolecules remain trapped inside endosomes instead of reaching the target cellular localization. To solve this problem, we report a delivery methodology which relies on the use of a chimera of the TAT and of the Influenza HA2. TAT is a prototypical CPP that can promote macropinocytosis in live cells and HA2 is a pH-sensitive peptide that destabilizes lipid membranes upon acidification. I demonstrate that HA2-TAT can deliver a variety of macromolecular cargos into live mammalian cells by a simple co-incubation protocol. A model is described where TAT causes the endocytic uptake of cargos present in the media and that HA2 disrupts the endosomal membrane upon endosomal acidification. In addition, using red blood cells as a model system, HA2-TAT binds to membranes in a pH-dependent manner and causes the formation of pores through which macromolecules can diffuse. Additionally, the pro-apoptotic domain (PAD) peptide is also successfully delivered by HA2-TAT and shows significant apoptosis in cells through macropinocytosis.

Bien que le traitement antirétroviral (ART) supprime efficacement la multiplication du VIH-1 chez les patients infectés, l’ART ne guérit pas l'infection. En effet, si l'ART est arrêté, nous observons un rebond viral. Celui–ci est principalement dû à l'activation stochastique de cellules latentes qui contiennent le génome viral intégré mais ne produisent pas de virus et ne sont donc pas ciblées par l'ART ou le système immunitaire. Ces cellules latentes sont peu nombreuses (1-10 par million de cellules T-CD4+ quiescentes) mais elles apparaissent rapidement après la primo infection et constituent donc un obstacle majeur à l'éradication virale. La stratégie la plus prometteuse pour supprimer ces cellules, dite "Shock and Kill", est de les réactiver pour qu'elles soient ensuite ciblées par l'ART et/ou lysées par les cellules T cytotoxiques. Un certain nombre d’agents de sortie de latence (LRAs) ont été mis au point pour réactiver ces cellules. Ils ciblent les protéines cellulaires telles que les Histone-désacétylases (HDAC) ou la protéine kinase C. La plupart d'entre eux présentent donc des effets non spécifiques et parfois une toxicité. Tat est la protéine du VIH-1 qui permet la transcription virale et favorise la traduction des gènes viraux. Tat est la protéine clé pour la levée de latence et l'initiation de la production des protéines virales par la cellule latente. Sur la base des structures RMN de Tat disponibles, nous avons identifié par dynamique moléculaire les conformations les plus stables de Tat. Cela nous a permis d'identifier par criblage in silico des ligands potentiels de Tat. Dix molécules ont été sélectionnées. Une molécule appelée D10 se fixe spécifiquement à la protéine Tat et augmente son activité de transactivation d'environ 4 fois. De plus, D10 présente une activité LRA sur les lignées cellulaires latentes JLat-9.2 et OM-10.1. L’activité LRA de D10 sur ces lignées représente 50 à 70% de celle du SAHA (vorinostat), un inhibiteur des HDAC candidat LRA en cours d’essais cliniques (Phase 2). Sur les cellules latentes de patients VIH traités, D10 à 50 nM a une activité LRA très efficace, 80% supérieure à celle de la bryostatine-1 qui agit sur la PKC et est considéré comme le LRA le plus prometteur actuellement. Le mécanisme d’action de D10, à semble être la stabilisation du complexe de transcription Tat-TAR. Cet effet est observé à 30 nM D10. En utilisant une approche chémoinformatique nous avons sélectionné 11 analogues de D10, dit N1-N11. Certains de ces analogues (N5, N8) montrent un effet plus fort que D10 sur l’augmentation de la transactivation de Tat ainsi que pour l’effet LRA sur les lignées cellulaires latentes. Ce résultat nous a permis d'ébaucher une relation structure chimique / activité LRA de ces molécules. Nous avons donc identifié de nouveaux agents de sortie de latence du VIH-1 ciblant Tat, plus spécifiques que les LRAs ciblant les protéines cellulaires. Ce sont les premiers activateurs de Tat identifiés. / Despite its efficiency to prevent viral multiplication, antiretroviral therapy (ART) is unable to cure patients with HIV-1. Indeed if ART is stopped, a viral rebound is observed. This increase in blood viral load is due to the activation of HIV-1 reservoirs, among which latently-infected memory CD4+ T cells. These cells are rare (1 per million of quiescent T cells) and appear very quickly following infection. To purge this long-lived reservoir the "Shock and Kill" approach was developed. This strategy relies on the use of latency reversing agents (LRAs) to induce reservoir activation. All LRAs developed until now target cellular proteins such as Histone deacetylases or protein kinase C. These LRAs are not specific for viral transcription and displayed modest effects ex vivo. Here we present a new LRA family that binds to and activates HIV-1 Tat which is the key regulator for viral transcription and latency reversal. These compounds are not cytotoxic and specifically activate Tat transcriptional activity. They were less efficient than available LRAs on HIV-1 latent cell lines. Nevertheless, when tested on latent T-cells from HIV-1 patients, the lead compound D10 was ~ 80% more efficient than bryostatin-1, one of the best LRA available to date. This effect was observed at 50 nM, which corresponds to the D10 concentration required for this compound to stabilize the Tat-TAR transcription complex. These molecules are the first Tat activators available.

La protéine Tat du VIH-1 est principalement connue pour son rôle majeur dans l’élongation de la transcription des gènes viraux. Cependant, plusieurs études ont montré que Tat pouvait être impliquée dans la rétrotranscription, étape précoce du cycle de réplication du VIH-1 et antérieure à l’intégration du génome viral dans la cellule hôte. Si Tat est impliquée dans la rétrotranscription, sa présence dans la particule virale pourrait procurer au virus un avantage cinétique. Pourtant, malgré une étude de protéomique l’ayant mis en évidence dans des virions purifiés, Tat n’est toujours pas considérée comme étant incorporée aux particules virales. Récemment, nous avons montré une interaction entre Tat et la cyclophiline A (CypA), une prolyl isomérase cellulaire. Cette protéine est connue pour être incorporée dans les particules virales via son interaction avec le domaine capside (CA) de la protéine Gag virale.Les données sur l’interaction de Tat avec CypA et l’implication de Tat dans l’étape précoce de rétrotranscription, nous ont conduits à formuler l’hypothèse suivante : L’interaction de Tat avec CypA permettrait son incorporation dans les particules virales et Tat serait donc présente dans les toutes premières étapes de l’infection des cellules hôtes par le VIH-1.Dans les travaux présentés, nous avons confirmés par GST pulls down et co-immunoprécipitation l’existence d’un complexe CA-CypA-Tat. Le complexe a pu être purifié par gel filtration. Puis, nous avons montré la présence de Tat dans les particules virales par deux approches complémentaires : une approche par western blot sur des particules virales purifiées, l’autre par microscopie à force atomique couplée à la microscopie à fluorescence. Grâce à un test fonctionnel, nous avons montré que Tat, associée au VIH-1, est fonctionnelle et délivrée dans le cytoplasme des cellules nouvellement infectées. Enfin, les interactions entre CA, CypA et Tat ont été caractérisées par différentes approches biochimiques faisant appel à des protéines purifiées. Les données de thermophorèse indiquent que l’affinité de Tat pour le complexe CA-CypA est plus forte que pour la CypA seule. Donc, dans une cellule infectée, Tat se fixerait préférentiellement sur la CypA déjà complexée à CA. Le complexe CA-CypA étant majoritairement retrouvé au niveau du site d’assemblage du virus, cela pourrait favoriser l’incorporation de Tat dans les virus.La mise en évidence de Tat dans les particules virales permettra sans doute d’apporter un angle de vue nouveau sur la réplication du VIH-1, en particulier sur les étapes précoces du cycle viral. Ces résultats pourraient initier le développement d’inhibiteurs de l’incorporation de Tat pour une application thérapeutique. / HIV-1 protein Tat is essentially known for its key role in the transcription elongation of the viral genes. However, several studies showed that Tat could favor reverse transcription. This early and essential step of HIV-1 replication cycle takes place before the integration of viral genome into cellular DNA and therefore prior to the production of the viral proteins. If Tat is involved in reverse transcription, its presence in viral particles might give a kinetic advantage to the virus. Yet, Tat is still not considered as incorporated into the virus, even though a proteomic study identified Tat in purified viral particles. Our team recently demonstrated an interaction between Tat and cyclophilin A (CypA), a cellular prolylisomerase. The latter is known for being incorporated into HIV-1 virions via its binding to the capsid (CA) domain of HIV-1 Gag protein.The evidences for Tat positive effect on reverse transcription and Tat-CypA interaction led us to formulate the following hypothesis: the Tat-CypA interaction enables HIV-1 Tat protein incorporation into viral particles.In the present work, we confirmed, using both co-immunoprecipitation and GST-pull down assays, the existence of a CA-CypA-Tat complex. In addition, this complex could be purified by gel filtration. We accordingly demonstrated the presence of Tat inside viral particles by two complementary methods: the western blot analysis of purified virions and atomic force microscopy coupled with fluorescence microscopy. Through a functional test, we found that Tat, incorporated by HIV-1, is functional and delivered to the cell cytoplasm of newly infected cells. Additionally, CA, CypA and Tat interactions were characterized by several biochemical techniques using purified proteins. Thermophoresis results indicated that Tat affinity is stronger for the complex CA-CypA than for CypA alone. Hence, in infected cells, Tat will preferably bind a CypA protein already complexed with CA compared to cytosolic CypA, thereby favoring Tat encapsidationThe demonstration that Tat is present into viral particles may bring a new point of view on HIV-1 replication cycle, especially for viral reverse-transcription and transcription steps. Our results could initiate the development of Tat encapsidation inhibitors that might have future therapeutic application.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.