Ingeniero Civil Electricista / El objetivo del presente trabajo de memoria consiste en implementar el middleware ROS en el robot de servicio Bender, así como implementar un sistema de navegación con soporte para SLAM online, e integrar dicho sistema junto con los módulos preexistentes del robot, al tiempo de actualizarlos a una nueva versión de URBI. Este proyecto es parte del trabajo de mejora continua realizado con el robot Bender, del Laboratorio de Robótica del Departamento de Ingeniería Eléctrica de la Facultad de Ciencias Físicas y Matemáticas de la Universidad de Chile y enmarcado dentro de la participación de este robot en el campeonato mundial de robótica Robocup, en su categoría Robocup@Home. Tras una presentación del contexto y antecedentes respecto a las características del middleware, se procedió a implementar un sistema de navegación en base al middleware ROS utilizando el algoritmo de SLAM Online GMapping. Para esto, se implementan diversos módulos ROS: control de la base robótica, publicación de odometría, manejo de marcos de referencia, generación de mapas de costos, módulo SLAM, lecturas del sensor láser, planificador de rutas local y global, teleoperación, servidor de mapas de entorno y localización. Implementados los módulos de navegación en ROS, se procedió a actualizar los módulos UObjects existentes a URBI 2.7.4 y finalmente a integrar el sistema ROS con el sistema URBI, ambos residentes en computadores diferentes y bajo distintos sistemas operativos. Finalizado el desarrollo del sistema y su integración, se realizaron diversas pruebas para verificar el módulo de navegación desarrollado en base a ROS, así como la integración de éste a los módulos preexistentes basados en URBI que fueron actualizados y la comunicación entre ambos componentes. En base a estas pruebas, se verifica que el trabajo realizado cumple a cabalidad los objetivos planteados, tanto al cumplir las necesidades de navegación (evasión de obstáculos, planificación dinámica de rutas, SLAM online), como al mantener las funcionalidades preexistentes e integrar los sistemas antiguos dentro de la nueva configuración del sistema.

Les leucémies aigues myéloïdes (LAM) sont caractérisées par une expansion clonale de cellule(s) souche(s) leucémique(s) bloquée à un stade précoce de maturation. Malgré les avancées thérapeutiques, leur pronostic reste sombre et des progrès thérapeutiques doivent encore être réalisés. Dans les LAM, les espèces réactives de l’oxygène (ROS) sont considérées comme, d’une part, participant à la leucémogenèse et, d’autre part, comme hautement impliquées dans la sensibilité aux chimiothérapies conventionnelles. Par ailleurs, l’équilibre redox qui participe aux dérégulations métaboliques associées au processus leucémique, dépend de nombreux régulateurs, dont la protéine S100A8, protéine connue pour son action stimulatrice sur la NADPH oxydase et sa valeur pronostique dans les LAM.Ce travail s’est donc intéressé à la caractérisation des désordres oxydatifs dans les LAM afin d’évaluer leur impact clinico-biologique, et d’autre part au rôle de la sécrétion de la S100A8 dans le microenvironnement médullaire. De plus, à partir de lignées leucémiques, nous avons étudié l’impact de la S100A8 exogène sur la production de ROS, la respiration mitochondriale et le métabolome des cellules blastiques.A partir d’une cohorte de 84 patients atteints de LAM de novo au diagnostic, nous avons mis en évidence des désordres de l’équilibre redox à la fois dans les cellules leucémiques, dans les cellules normales de l’environnement médullaire ainsi que sur les systèmes régulateurs antioxydants (SOD, GPX, glutathion…). De plus, nous avons montré que la production des ROS observée en réponse à des modulateurs de la mitochondrie, qui reflète indirectement la fonctionnalité mitochondriale, joue un rôle pronostique indépendant des facteurs pronostiques habituels. L’analyse de la S100A8 dans les plasmas médullaires montre une expression augmentée dans les LAM, d’origine monocytaire majoritairement et est associée à des anomalies moléculaires de bon pronostic (inv(16), NPM1) ou un sous-groupe de patients FLT3-ITD mutés présentant une meilleure survie. Enfin, l’étude de la S100A8 sur les lignées leucémiques a permis de mettre en évidence la diversité de ses effets sur la croissance cellulaire, l’apoptose, la production de ROS ainsi qu’une variation métabolique de la phosphocholine dont les mécanismes restent à explorer.En conclusion, mon travail apporte des éléments originaux sur les particularités de l’équilibre bio-énergétique dans les LAM. Il souligne, que l’impact de ses dérégulations sur le pronostic des patients résulte de la combinaison d’un ensemble de facteurs métaboliques, qui doivent être appréhender dans leur globalité pour une meilleure efficacité thérapeutique. / Acute myeloid leukemia (AML) is characterized by clonal expansion of leukemic(s) cell(s) blocked at an early stage of maturation. Despite therapeutic advances, their prognosis remains poor and therapeutic improvements are needed. In AML, reactive oxygen species (ROS) are considered to contribute to leukemogenesis and, on the opposite, standard chemotherapies exert cytotoxicity via ROS. In addition, the redox balance acts on metabolic dysregulation in AML and depends on many regulators, such as S100A8 protein, associated with worst prognostic in AML and known to stimulate NADPH oxidase.In this context, this work focuses on oxidative disorders, and S100A8 expression in bone marrow microenvironment according to clinical-biological characteristics and evaluate their prognostic impact in AML. In addition, we investigated the impact of exogenous S100A8 on ROS production, mitochondrial respiration, and metabolism in leukemia cell lines.In a cohort of 84 de novo AML at diagnosis, we demonstrate the existence of redox balance disorders on leukemic cells, on normal cells from bone marrow microenvironment, and on antioxidant systems (SOD, GPX, glutathione ...). In addition, ROS production observed in response to mitochondrial modulators indirectly reflects mitochondrial functionality plays a prognostic role independent of the current prognostic factors. The analysis of S100A8 in bone marrow plasmas shows a higher expression in AML than in healthy controls or other hematological neoplasms. This hyperexpression is predominantly of monocytic origin and is associated with molecular abnormalities of good prognosis such as (inv (16), NPM1) or with a subgroup of mutated FLT3-ITD patients with better survival. Finally, the study of S100A8 on leukemia cell lines highlights its heterogeneous effect on cell growth, apoptosis, ROS production and on NOX regulation. Furthermore, we observe a S100A8-phosphocholine change which remains to be explored.In conclusion, this work provides original information on bio-energetic balance in AML and their prognostic impacts, emphasizing that these metabolic alterations impact AML prognosis through complex interactions.

The mitochondria contain their own genome, which is organized in a dynamic high-order nucleoid structure consisting of several copies of mitochondrial DNA (mtDNA) molecules associated with proteins. The mitochondrial nucleoids are the units of mtDNA inheritance, and are the sites of mtDNA transcription, replication and maintenance. Therefore, the integrity of mitochondrial nucleoids is a key determinant of mitochondrial metabolism and the bioenergetic state of the cell. Deciphering the interaction of mtDNA with proteins in nucleoprotein complexes is fundamental to understand the mechanisms of mtDNA segregation leading to mitochondrial dysfunction and to develop therapies to treat diseases associated with mtDNA mutations. The work presented in this dissertation provides essential insights into the dynamics of mtDNA interaction with nucleoid proteins. In order to unveil the organization of the mitochondrial genome, we have mapped major regulatory regions of the mtDNA in vivo using mitochondrial-targeted DNA methyltransferases. In chapter 2, we have demonstrated that DNA methyltranferases are powerful tools in probing mtDNA-protein interactions in living cells. The DNA methyltransferases' accessibility to their cognate sites in the mtDNA is negatively correlated with the frequency and binding strength that protein factors occupy a specific site. Our results show that the transcription termination region (TERM) within the tRNALeu(UUR) gene is consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mTERF1 (mitochondrial transcription termination factor 1). DNA methyltransferases have also been shown to be effective in detecting changes in mitochondrial nucleoid architecture due to nucleoid remodeling. We were able to determine changes in the packaging state of mitochondrial nucleoids by monitoring changes in mtDNA accessibility. The impact of altered levels of major nucleoid proteins was assessed by monitoring changes in mtDNA methylation pattern. We observed a more condensed nucleoid state causing a decrease in mtDNA methylation when the levels of the mitochondrial transcription factor A (TFAM) were altered. Changes in mtDNA methylation pattern were also evident when cells were treated with ethidium bromide (EtBr) and hydrogen peroxide. The mtDNA nucleoids adopted a less compact state during rapid mtDNA replication after EtBr treatment. In contrast, we observed a more compact mtDNA, less accessible to DNA methyltransferase after hydrogen peroxide treatment. Our results indicate that mitochondrial nucleoids are not static, but are constantly been modulated in response to factors that affect the nucleoid environment. In chapter 3, we identified the in vivo DNA binding sites of major transcription regulatory proteins, TFAM and mTERF3 using a targeted gene methylation (TAGM) strategy. In this approach, the mtDNA binding protein is fused to a DNA methyltransferase as an attempt to selectively methylate the sites adjacent to the protein target DNA region. Knowledge on how proteins interact with the mtDNA in high-order structures, which function as a mitochondrial genetic unit, will help elucidate the segregation and accumulation of mutated mtDNA in diseased tissues.

For every robotics project, choosing a suitable framework and middleware for software and hardware is a challenging task which may influence the entire project. Robotics applications typically are resource constrained when it comes to computations and memory usage. They are built on different hardware platforms and applied in different domains. Therefore it is hard to introduce a common framework for all types of projects. However, in recent years several new attempts have been made and received attention from both researchers and industry. These frameworks are still under development and need to be extended. This paper discusses the different features that are needed for robotics frameworks and compares some of the available middleware and standards.

Hydrogen peroxide (H2O2), a membrane-permeable reactive oxygen species, is continuously produced by mitochondrial respiration, the membrane-associated NADPH oxidase complex, xathine oxidase catalyzed reaction. Although the cytotoxic effect of H2O2 is well documented, the role of H2O2 in synapse formation if still in its infancy. Here we test the role of H2O2 on the frequency of spontaneous synaptic currents (SSCs) at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of H2O2 dose-dependently enhances the frequency of spontaneous synaptic currents (SSC frequency). Treatment of the culture with membrane-permeable antioxidants N-acetylcysteine and sodium pyruvate significantly decreased SSC frequency, indicating endogenous reactive oxygen species play important roles in the regulation of spontaneous ACh release. Bath application of membrane non-permeable catalase, which breaks down H2O2 specifically, has no significant effect on SSC frequency, suggesting H2O2 is not an intercellular signaling molecule being produced and released from postsynaptic myocyte and affects the neurotransmitter release of presynaptic motoneuron. Much to our surprise is that the SSC frequency was significantly decreased while catalase was vii loaded into the myocyte through recording pipette. Furthermore, the SSC frequency facilitation induced by exogenously applied H2O2 was completely hampered while catalase was loaded into the myocyte. These results indicate although endogenous H2O2 in myocyte plays a crucial role on SSC frequency facilitation, this facilitation on the neurotransmitter release of presynaptic motoneuron is achieved through a retrograde factor other than H2O2 itself. Treatment of the culture with inhibitor of either NADPH oxidase does not have significant effect on SSC frequency. Bath application of mitochondria complex I, II and xanthine oxidase inhibitor significantly decreased SSC frequency, suggesting H2O2 derived from xanthine oxidase and mitochondria is responsible for the regulation of SSC frequency. Bath application of translation blocker anisomycin and cycloheximide could not attenuate the facilitation of H2O2. Addition of IGF-1 receptor inhibitor JB-1 to the culture significantly attenuated SSC frequency. Overall, our current results suggest that xanthine oxidase activity-derived H2O2 in myocyte induce the release of IGF-1 which retrogradely enhance the spontaneous neurotransmitter release from presynaptic motoneuron. Since synaptic activity is crucial in synaptogenesis and synapse maturation, results form viii our studies have shed some light on the molecular mechanism of the formation of developing neuromuscular synapse.

Heparan sulfate proteoglycans modulate many of the growth factor pathways that drive prostate cancer progression. Prior to being secreted into the extracellular matrix, the covalently attached HS chains are modified by sulfation which has been shown to increase the affinity of binding growth factors. The specific HSPG that I focus on in this dissertation is Perlecan (Pln). Previously, our group along with collaborators found that 54 percent of prostate cancer tumors had upregulated levels of Pln protein that correlated with increasing Gleason score [93]. The LNCaP-DU145-LN4 cell line series is introduced as a model for this subset of tumors because Pln levels increase 50-fold as the cells become more metastatic. It was found that three stress-induced transcription factors, HIF1α, NFkB, and ATF2, all stimulate Pln expression. ChIP analysis reveals that HIF1α and NFkB directly bind the Pln promoter while ATF2 does not. The ROS-generating NADPH Oxidase and the ROS-inducible p38 MAPK were also found to induce Pln expression. To address the subset of prostate cancer tumors that reach metastasis without upregulation of Pln, I focused on the 2-o-sulfotransferase enzyme and its effect on proliferation and invasion in the LNCaP-C4-2B cell model which does not show upregulation of Perlecan expression. 2OST RNAi resulted in a significant decrease in proliferation in each line of the series. 2OST RNAi in highly metastatic C4-2B cells caused a significant decrease in cell invasion. Cells with decreased levels of 2OST had increased accumulation of actin and E-cadherin suggesting the possible formation of adherens junctions. I also found that expression of 2OST increases four-fold as cells become more metastatic. I found HIF1α and ATF2 act in a direct manner while NFkB acts indirectly to stimulate 2OST expression. In summary, I have analyzed the effect of cellular stress on the expression of the Pln and 2OST genes and investigated the phenotype of 2OST knockdown in metastatic prostate cancer cells. These studies lead me to propose that the tumor stress response is necessary for prostate cancer progression due to the role of stress in the upregulation of extracellular HS that is required for growth factor signaling and metastatic behaviors.

Indiana University-Purdue University Indianapolis (IUPUI) / An autonomous car is a self-driving vehicle, that operates without human intervention and has the capability of sensing the environment around it. To achieve this, the autonomous vehicle mostly depends on multiple Sensors, Actuators, Machine learning, complex algorithms and processors for software execution. Developed Software, at that point, processes all the information obtained from sensors, plans the path, and the instructions are passed to the vehicle’s actuators, which are capable of controlling acceleration, steering, and brake systems. The rules that are hard-coded, algorithms for detection of object and obstacle avoidance, and predictive modelling control algorithms assist the software with observing traffic guidelines and navigate the vehicle accordingly. Free driving is anything but a simple assignment, and to make independent driving game plans is an extraordinarily critical capacity in the current programming planning field. Engineers and Researchers have been keeping huge endeavors to develop safe and precise algorithms to be incorporated in autonomous vehicles. ROS is a flexible and perfect middle ware tool for robotic applications. ROS offers the necessary tools to effortlessly get the sensors information, process that information, and produce a suitable response to actuators of the vehicle. This thesis work plans to exhibit how ROS could be utilized as a middle- ware tool to make the vehicle move autonomously by examining the surroundings and taking decision. The main focus of this thesis is to develop a one-tenth scale of an autonomous Racecar equipped with Jetson Nano as the on-board computer, ROS based software architecture, sensors, and a PWM driver and implement ADAS features such as Emergency Brake system, Lane Detection and Lane change on the autonomous Race car vehicle. At last, by following the strategies introduced in this thesis work, it is possible to build and develop an autonomousvehicle that uses ROS framework.

The retina is prone to oxidative stress from many factors which are also involved in the pathogenesis of degenerative diseases. In this study, we used the application of blue light as a physiological stress factor. The aim of this study was to identify the major source of intracellular ROS that mediates blue light-induced detrimental effects on cells which may lead to cytotoxicity. We hypothesized that outer segments are the major source of blue light induced ROS generation. In photoreceptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes and the recently found respiratory chain complexes may represent a major source for reactive oxygen species (ROS), beside mitochondria and chromophores. Therefore, we investigated this hypothesis and analysed the exact localization of the ROS source in photoreceptors in an organotypic culture system for mouse retinas. Whole eyeball cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Blue light impingement lead to an increase of ROS production (detected by H2DCFDA in live retinal explants), which was particularly strong in the photoreceptor outer segments. Nox-2 and Nox-4 proteins are sources of ROS in blue light irradiated photoreceptors; the Nox inhibitor apocynin decreased ROS stimulated by blue light. Concomitantly, enzyme SOD-1, a member of the antioxidant defense system, indicator molecules of protein oxidation (CML) and lipid oxidation (MDA and 4-HNE) were also increased in the outer segments. Interestingly, outer segments showed a mitochondrial-like membrane potential which was demonstrated using two dyes (JC-1 and TMRE) normally exclusively associated with mitochondria. As in mitochondria, these dyes indicated a decrease of the membrane potential in hypoxic states or cell stress situations. The present study demonstrates that ROS generation and oxidative stress occurs directly in the outer segments of photoreceptors after blue light irradiation.

Les dommages tissulaires associés à l'ischémie/reperfusion ont été largement étudiés. Plusieurs études ont montré que des dysfonctionnements mitochondriaux sont responsables de la survenue de ces dommages, et que la transition de perméabilité pourrait y être impliquée. Cette transition de perméabilité est médiée par l'ouverture du pore de transition de perméabilité (PTP). Cette ouverture du PTP pourrait survenir pendant la phase d'ischémie ou pendant la phase de reperfusion. L'objectif de ce travail était de visualiser l'ouverture du PTP dans des conditions d'ischémie/reperfusion sur cellules intactes (HMEC-1 et INS-1) et d'étudier son implication dans ce phénomène. Nous avons pu visualiser pour la première fois l'ouverture du PTP par le suivi des dommages qu'elle engendre (sortie du NADH et la chute du ΔΨ) induits par une ischémie/reperfusion. Nous avons constaté que l'activation du PTP a lieu pendant la phase d'ischémie tant dans les cellules HMEC-1 que dans les cellules INS-1. Cette induction a été prévenue dans les deux modèles cellulaires par la cyclosporine A. Nos résultats suggèrent également que le complexe I pourrait être impliqué dans la prévention de la chute du ΔΨ et de la sortie du NADH. Nous avons aussi montré que la capacité de rétention calcique des cellules perméabilisées diminue à l'ischémie et que cette diminution disparait après 60 minutes de reperfusion. Ainsi, la visualisation de l'ouverture du PTP dans un modèle d'ischémie/reperfusion constituerait une piste intéressante qui apporterait plus de certitude quant à l'implication du PTP dans ce phénomène. De plus, l'étude du phénomène d'ischémie in vitro, apporterait plus de réponses quant à l'implication des modifications du fonctionnement cellulaire dans les dommages tissulaires. / Several studies have shown that ischemia/reperfusion injury is strongly related to mitochondrial dysfunction. These studies have mostly focused on the involvement of the permeability transition in this phenomenon. The permeability transition is mediated by the opening of the mitochondrial permeability transition pore (mPTP). PTP activation may occur during ischemia or reperfusion. The aim of this work was to visualize mPTP opening during ischemia/reperfusion conditions on intact cells (HMEC-1 and INS-1) and to study its involvement in this phenomenon. For the first time, we observed the opening of the mPTP mediated by ischemia/reperfusion by monitoring the damages caused by its activation (NADH efflux and ΔΨ decrease). This mPTP activation occurred during ischemia in HMEC-1 and INS-1 cells. However, mPTP induction was prevented by cyclosporine A in both cell models. Furthermore, our results showed the involvement of complex I in the prevention of NADH efflux and ΔΨ decrease. A decrease in the mitochondrial calcium retention capacity was also shown in permeabilised cells during ischemia, which disappear after 60 minutes of reperfusion. Thus, the visualisation of the mPTP opening in a model of ischemia / reperfusion is an interesting observation indicating the involvement of PTP in this phenomenon. Furthermore, the study of ischemia in vitro would provide answers regarding the involvement of cellular function changes in tissue damage.

L’objectif de cette thèse est de définir l’effet de l’environnement maternel sur la qualité des graines de A.thaliana, H.annuus, B.oleracea et H.vulgare, caractériser deux mutants d’A.thaliana affectés au niveau d’enzymes de la membrane plasmique et caractériser l’activité de ces deux protéines. Des dosages de O₂•⁻ et de radicaux ont été faits sur des graines issues de plantes ayant subi des stress thermique et/ou hydrique et n’ont pas montré d’effet des stress. Des traitements de vieillissement accéléré ont eu des effets négatifs sur la vigueur et la viabilité des graines. Des radicaux ont été identifiés par RPE haut champ au niveau de l’enveloppe et du péricarpe. Etant donné la localisation de ces radicaux, ils ne sont pas de bons marqueurs de la qualité des graines. Des mutants KO d’A.thaliana de quinones réductases (QR) et de AIR12 ont été caractérisés. Les QR sont des enzymes cytosoliques ayant une affinité pour la membrane. Elles catalysent la réduction de quinones en dihydroquinones et jouent un rôle protecteur contre le stress oxydatif. A pH alcalin par contre, les dihydroquinones se déprotonent et s’autooxydent entrainant la formation de semiquinones et de O₂•⁻. AIR12 est un cytochrome b 561 ancré à la membrane du côté de l’apoplaste réductible par l’ascorbate et le O₂•⁻. Ces protéines pourraient être impliquées dans un transfert d’électrons à travers la membrane via la vitamine K1. Les protéines recombinantes NQR et AIR12 ont été produites. A pH alcalin, AIR12 est réduit par les semiquinones et pourrait donc récupérer les électrons du produit de NQR. En présence de membrane plasmique, la production de O₂•⁻ est augmentée avec NQR et diminuée avec NQR et AIR12. Les QR semblent avoir un rôle anti- et pro-oxydant selon les cas et AIR12 un rôle anti-oxydant. Le transfert d’électrons entre les deux protéines pourrait se faire via les semiquinones à pH alcalin et via O₂•⁻. / The aim of this work is to define the effects of adverse environmental conditions on seed quality of A.thaliana, H.annuus, B.oleracea et H.vulgare), characterize two A.thaliana mutants affected in plasma membrane proteins and characterize the activity of the two proteins. Seeds were harvested from plants subjected to drought and/or thermal stress. Then O₂•⁻ and organic radicals in seeds were measured showing no impact of the stress. Accelerated ageing had a negative effect on seed vigor and viability. Radicals have been identified by high field EPR: epi-catechin and Mn(II) in the testa and melanin in the pericarp. There was no correlation between these radicals and the seed quality, therefore these radicals were not found to be good markers of seed quality. A.thaliana KO mutants of quinone reductase (QR) and AIR12 have been characterized. QR are cytosolic enzymes that have an affinity for the membrane. QR catalyze the reduction of quinone to dihydroquinone. Thus they are known to be protective enzymes against oxidative stress. However, at alkaline pH, dihydroquinone deprotonize and form semiquinones and O₂•⁻. AIR12 is a b561 cytochrome anchored to the apoplastic side of the membrane. These proteins could be implied in a transmembrane electron transport via vitamin K1. Recombinant proteins and NQR and AIR12 were produced. At alkaline pH, AIR 12 was reduced by the semiquinone, AIR12 could form a redox couple with vitamin K1 as electron shuttle across the membrane. In the presence of plasma membrane, the production O₂•⁻ was increased with NQR and reduced with NQR and AIR12. QR appear to have an anti- and pro-oxidant according to the conditions and AIR12 an anti-oxidant role. Electron transfer between the two proteins could be done via the semiquinone at alkaline pH and via O₂•⁻.