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<p>Trajectory formation is an important quality of human movement wherein the analysis of its kinematic structure often permits inferences regarding the underlying sensorimotor organization. The studies reported herein aimed to reveal how task-specific encoding of saccades contribute to two movement models. We first examined saccades for changes in the evidence for online spatial corrections in response to different instances of visual tracking. Results from our first study indicated the sensorimotor operations associated with making timed series responses permitted unhindered operation of online corrections in predictive saccades. These saccades imposed a demand on spatial working memory without suffering diminished corrections typical of single delayed memory-guided saccades. Next, we queried the role of ventral stream visuomotor processing, as well as the contribution of basic stimulus features to explaining known modulations of online saccadic corrections. Our results are consistent with the explanation that ventral stream contribution to target metrics influence trajectory kinematics, but did not induce diminished online corrections if vision of that illusory target was available at saccade onset. Using another movement task, we examined how different saccadic tracking behaviours dissociated the contribution of various sensorimotor operations involved in oculomotor preparation and execution to eye-hand coupling. By observing changes in manual trajectory in an oculo-manual task, we contrasted the effects of saccadic tracking in predictive and reactive modes, overt and covert, horizontal and vertical, spatially congruent or perpendicular axes of primary motion relative to eye-hand coupling. From these data we concluded that saccadic encoding induced execution-dependent coupling, and optionally motor planning-dependent coupling when task constraints specified temporal synchrony between eye and hand movements. Moreover, eventual motor execution was a prerequisite for the emergence of oculomotor planning-dependent coupling effects. This implied no apparent contribution to the coupling dynamic as an exclusive function of shifts in the spatial allocation of attention without oculomotor output.</p> / Doctor of Philosophy (PhD)

Das COP9 Signalosom (CSN) ist ein hoch konservierter Proteinkomplex, der an der Regulation des Ubiquitin (Ub)-26S Proteasomsystems (UPS) beteiligt ist. Das UPS ist die wichtigste Proteolysemaschinerie in eukaryotischen Zellen, bei der Proteine über eine dreistufige Kaskade der Enzyme E1-E3 mit einer Ub-Kette markiert werden, die als Erkennungssignal für den Abbau durch das 26S Proteasom dient. Das CSN gilt als Paralog zum Lid, einem Subkomplex des 26S Proteasoms, und interagiert mit einer Vielzahl von Proteinen, unter anderem mit E3-Ligasen und Kinasen. In dieser Arbeit konnte die direkte Bindung des CSN an das 26S Proteasom gezeigt werden, was zu einem Einfluss auf die Peptidaseaktivität des 26S Proteasoms in vitro führt. In Flag-Pulldown-Experimenten aus B8 Mausfibroblasten, die stabil mit Flag-CSN2 transfiziert waren, wurde ein vollständiger Flag-CSN-Komplex nachgewiesen, der mit dem 26S Proteasom assoziiert vorliegt. Co-Immunpräzipitationen beider Komplexe in vitro wiesen auf eine konzentrationsabhängige Verdrängung des Lid-Subkomplexes durch das CSN hin. Diese Interaktion führte zur Reduktion der proteolytischen Aktivität des 26S Proteasoms. Darüber hinaus wurde eine assoziierte deubiquitinierende Aktivität am CSN entdeckt und als USP15 identifiziert. Die Charakterisierung von USP15 zeigte, dass es durch die am CSN assoziierte Kinase CK2 phosphoryliert und stabilisiert wird. Erstmalig konnte durch Inhibitorstudien mit ortho-Phenanthrolin eine Metallabhängigkeit der Aktivität von USP15 nachgewiesen werden, die zur Identifizierung eines bisher unbekannten Zn-Fingers führte. Mutationsanalysen des Zn-Fingers zeigen, dass dieser für die Bindung und Spaltung von Ub-Ketten, nicht aber von linearen Ub-Konstrukten, notwendig ist. In Zellexperimenten konnte nachgewiesen werden, dass USP15 die E3 Ligase Rbx1 stabilisiert, was vermutlich auf eine Umkehr der Autoubiquitinierung zurückzuführen ist. Das CSN scheint somit sowohl das 26S Proteasom als auch die E3-Ligasen direkt zu beeinflussen. Die Ergebnisse dieser Arbeit stellen eine Vertiefung der Erkenntnisse über das CSN als Regulator des UPS dar. / The COP9 signalosome (CSN) is a conserved protein complex that is involved in the regulation of the ubiquitin (Ub)/26S proteasome system (UPS). The UPS is the most important degradation machinery in eukaryotic cells. By the concerted action of three enzymes, E1-E3, proteins are labelled with a Ub-chain that serves as a recognition signal for the degradation by the 26S proteasome. The CSN is homologous to the lid, a subcomplex of the 26S proteasome, and interacts with numerous proteins, including E3 Ub ligases and kinases. In this study a direct interaction of the CSN with the 26S proteasome could be shown which has consequences for the peptidase activity of the 26S proteasome in vitro. In Flag-pull-down experiments from mouse B8 fibroblasts, that permanently expressed Flag-CSN2, an intact Flag-CSN complex was detected that is associated with the 26S proteasome. Co-immunoprecipitation of both complexes in vitro indicated a concentration-dependent replacement of the lid subcomplex by the CSN. This interaction led to a decrease of the proteolytic activity of the 26S proteasome. Moreover, a deubiquitinating activity associated with the CSN was discovered and identified as USP15. The USP15 was phosphorylated by the CSN-associated kinase CK2 that stabilised the enzyme. For the first time inhibitor studies with ortho-phenanthroline demonstrated a metal-dependency for the activity of USP15 that could be attributed to a formerly unidentified Zn-finger. Mutational analysis of the Zn-finger showed that it is necessary for the binding and cleavage of poly-Ub-chains but not for linear Ub-constructs. Cell culture experiments demonstrated a stabilisation of the E3 ligase Rbx1 by USP15 most likely by reversing its autoubiquitination. Therefore the CSN seems to directly influence the 26S proteasome as well as E3 ligases in their functions. These results expand the present knowledge on the CSN as a regulator of the UPS.

Une régression des principales modalités sensorielles (vision, audition, goût et odorat) est bien rapportée à l'âge. La perception tactile est influencée par différents paramètres. Un grand nombre de ces paramètres ont été bien étudiés dans la littérature tels que le système nerveux central, la densité des mécanorécepteurs dans la peau et les seuils de détection vibro-tactile. Ces paramètres ont été étudiés pour comprendre l'affaiblissement de la perception tactile au fil du temps et les différences naturelles entre les hommes et les femmes. Le doigt humain est toujours utilisé pour qualifier la qualité et les propriétés d'une surface. Par conséquent, un panel de différent âge et sexe est toujours utilisé par différents secteurs industriels afin de comprendre les besoins des consommateurs. Cependant, cette méthode est très coûteuse, longue et subjective. L’objectif de ce travail était de développer une méthodologie qui permette d'avoir une objectivation tactile semblable au panel, cela signifie qu'on doit prendre en compte les effets de l'âge et du sexe. Cette thèse a fourni des mesures in vivo pour 40 doigts humains. Nos développements proposent un doigt artificiel capable de mimer le doigt humain, en tenant l’effet de l'âge et du sexe. Pour atteindre notre objectif, nous avons développé plusieurs approches qui combinent à la fois, la topographie multi-échelle du doigt humain, ces propriétés mécaniques anisotropes, les propriétés tribologiques et l’effet de l’aire réelle de contact et la direction du toucher sur la force de frottement et les vibrations générées. Le développement d’un algorithme de traitement du signal, a permis d’identifier des coefficients représentatifs de la qualité de la surface touchée. L’ingénierie de l'émotion a été un autre axe de recherche dans de ces travaux. L’instrumentation du doigt humain avec un dispositif laser-doppler, a permis d’évaluer la qualité tactile des échantillons en fonction de l'émotion générée pendant le processus du toucher. Cette émotion est étudiée par la variation fréquentielle du débit sanguin. Le mise au point d’un doigt artificiel bio-inspiré qui possède des propriétés biophysiques proches du doigt humain, a permis de réaliser un démonstrateur de toucher qui peut intégrer l’âge et le sexe d’un panel. Ce dispositif répond au cahier des charges défini dans le projet ANR «plasticTouchDevice», et permet aux industriels de la plasturgie de mener des expertises de leurs innovations en ayant recours à un dispositif qui permet d’intégrer l’effet de l’âge et du sexe dans la métrologie de la qualité du toucher des matériaux plastiques. / A decline in the main sensory modalities (vision, hearing, taste, and smell) is well reported to occur with advancing age, it is expected a similar change to occur with touch sensation and perception. The tactile perception is influenced by different parameters. Many of those parameters have been well studied in the literature such as central nervous system, the density of mechanoreceptors in the skin and the vibro-tactile detection thresholds. These parameters have been studied in order to understand the weakens of tactile perception over time and the natural differences between men and women. The human finger is always used to qualify the quality and the properties of a surface. Therefore, a panel of different age and gender always used by companies in order to understand the customers’ needs. However, this method is very expensive, time-consuming and subjective. The objective of this work is to give a solution that avoid the limits of the actual method, but in the same time it should keep their advantages (age and gender). This thesis provided in vivo measurements for 40 human fingers. Our developments propose an artificial finger capable of mimicking the human finger, taking into account the effects of age and gender. To achieve our goal, we have developed several approaches that combine the multi-scale topography of the human finger, the anisotropic mechanical properties, the tribological properties, the effect of the real contact area, the direction of touch on the friction force and the vibrations generated by the human finger. The development of a new signal processing algorithm has made it possible to identify coefficients representative of the quality of the affected surface. The engineering of emotion has been another area of research in this work. Instrumentation of the human finger with a laser Doppler device, allowed to evaluate the tactile quality of the samples according to the emotion generated during the process of the touch. This emotion is studied by the frequency variation of the blood flow. The development of a bio-inspired artificial finger that has biophysical properties close to the human finger, has made it possible to realize a haptic touch demonstrator that can integrate the age and gender of a panel. This device meets the specifications defined in the ANR project "PlasticTouchDevice", and allows plastics industry to conduct expertise of their innovations by using a device that allows the integration of the effects of age and of gender in the metrology of the quality of the touch of plastic materials.

Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.