Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

C. glabrata est une levure commensale de l’Homme à l’origine d’infections opportunistes chez les patients immunodéprimés. Les neutrophiles sont les premières cellules recrutées sur le site de l’infection. La production de formes réactives de l’oxygène par ces cellules, initiée dans le phagosome par la NADPH oxydase 2 est un évènement majeur de la maturation du phagosome et fait l’objet de l’étude réalisée. L’objectif de ce travail était de développer des outils expérimentaux permettant l’évaluation du stress oxydant subi par C. glabrata lors de son internalisation par les phagocytes. La levure non pathogène S. cerevisiae, phylogénétiquement plus proche de C. glabrata, a été choisie comme organisme contrôle permettant une étude comparative entre les deux levures. C. glabrata est efficacement internalisée en absence d’opsonisation, par la lignée PLB-985-neutrophile contrairement à S. cerevisiae. L’utilisation de sondes organiques pour la mesure des FRO dans le phagosome est limitée puisque que le marquage des levures par ces sondes n’est pas spécifiquement localisé. Le développement de biosenseurs de FRO à partir de protéines fluorescentes, dont la localisation est maitrisée grâce à un système de marquage, est présenté. Les études réalisées suggèrent cependant que les protéines fluorescentes subissent des modifications dans le phagosome indépendantes de la production de FRO. Des tests de viabilité effectués sur les levures après phagocytose montrent que l’élimination des levures dépend fortement de facteurs indépendants de la production de FRO. Plusieurs méthodes indiquent néanmoins que les phagosomes avec C. glabrata contiennent moins de FRO que les phagosomes avec S. cerevisiae. C. glabrata semble éliminer les FRO plus efficacement que S. cerevisiae. / C. glabrata is a human commensal yeast responsible for opportunistic infections in immunocompromised patients. Neutrophils are the first cells to be recruited to the infection site. Production of reactive oxygen species (ROS) in the phagosome by the phagocyte NADPH oxidase 2 is a major event of the phagosome maturation and is the subject of the study. The aim of this work was to develop experimental tools allowing the evaluation of the oxidative stress endured by C. glabrata during its internalization by phagocytes. The non-pathogenic yeast S. cerevisiae, phylogenetically close to C. glabrata, was chosen as a control organism allowing a comparative study between the two yeasts. Unlike S. cerevisiae, non-opsonized C. glabrata is efficiently internalized by the PLB-985 cell line. The utilization of organic dyes for the detection of ROS in the phagosome lacks precision since the staining of the yeasts by those dyes is not specifically localized. The development of ROS biosensors based on fluorescent proteins, whose localization can be controlled due to a new staining procedure, is presented here. However the results suggest that fluorescent proteins undergo modifications in the phagosome independently from the ROS production. Viability tests performed on the yeasts after phagocytosis showed that yeast removal depends mainly on factors independent from ROS production. Several methods indicate nevertheless that the phagosomes of C. glabrata contain less ROS than the phagosome of S. cerevisiae. C. glabrata appears to suppress ROS more efficiently than S. cerevisiae.

Att klippa sly är en uppgift som behöver uträttas på plats där det växer, vilket oftast är ute i skogen. Skogen är för en maskin sällan en väldefinierad miljö och förhållanden som arbetet ska uträttas i förändras konstant. Att automatisera arbete som ska uträttas i svårkontrollerad miljö innebär många utmaningar, moment som för en människa är triviala är ofta inte möjliga för en maskin som arbetar under föränderliga omständigheter. Det här arbetet undersöker vilka typer av verktyg som kranarm och mjukvara som kan användas för att lösa den typen av problem samt ett förslag till en praktisk lösning på problemet med slyklippning med hjälp av dessa verktyg. Projektet avslutades då lösningsförslaget fortfarande är i prototypstadium och är inte ämnat för industri utan behöver vidare förfining innan lösningen kan tillämpas i praktiken. / Cutting brush wood is a task that needs to be performed where it grows, which is usually in a forest. The forest is an environment that is difficult to map for a machine and the environment the task is to be performed in is constantly changing. To automate a task that is to be performed in such environment comes with a range of challenges, tasks that are trivial for a human being may not be possible for a machine that is working inside a shifting environment. This thesis is looking at what type of tools such as jib and software may be used to solve these types of problems and will also present a proposed practical solution for brush wood cutting using these tools. The projects cut-off was at a prototype state and is not meant for industrial application but will need some further refinement before it is applied practically.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23086号 / 医博第4713号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 藤田 恭之, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This master's thesis describes design and development of an application for estimation of vehicle speed from both recorded video file and from camera stream.  It explains the procecess of a camera calibration, vehicle detection and tracking and describes the robot operating system as a target platform. The application uses library OpenCV for most of tasks, to access video application uses a FFmpeg library. Results can be printed to the terminal window, they can also be logged to a file or published in ROS. Application is written in C++ language, some parts in Python.

The role of lipids and ROS in cardioprotective mechanism of chronic hypoxia Cardiovascular diseases, mainly ischemic heart disease is one of the most frequently cause of morbidity and mortality in developed countries. Therefore effective protection of the heart against ischemia and reperfusion injury is the crucial aim of experimental and clinical cardiology. One of the main streams of cardiovascular research is looking for possibilities of natural heart resistance augmentation. Adaptation to chronic hypoxia is one possibility how to protect the heart against ischemia-reperfusion injury. Chronic hypoxia increases resistance of the myocardium to acute deficiency of oxygen leading to vetricular arrythmias, postischemic contractile dysfunction and necrotic changes in the tissue. Recently, it has been shown that reactive oxygen species (ROS) play an important role in the cardioprotective mechanism of chronic hypoxia. It is known that oxidative stress has a harmful effect in acute ischemia-reperfusion however ROS generated during the adaptation to hypobaric intermittent chronic hypoxia play a role in the induction of cardioprotection. In this study, we demonstrated that adaptation of adult rats to chronic hypoxia increased the activity and protein abundance of manganese superoxide dismutase (MnSOD) in the...

The complexity of mobile robots increases as more parts are added to the system. Introducing microcontrollers into a mobile robot abstracts and modularises the system architecture, creating a demand for seamless microcontroller integration. The Robot Operating System (ROS) used by ABB’s new mobile robot, the mobile YuMi prototype (mYuMi), allows standardised robot software libraries and packages to simplify robotic creations. As ABB is porting over from ROS1 to ROS2, the ROS2 compatible Microcontroller Robot Operating System (micro-ROS) will be incorporated into the system to smoothly integrate microcontrollers into mYuMi. In order to display the validity of micro-ROS, this project used tracing and latency measurements with external applications to test the remote communication between mYuMi using ROS2 and microcontrollers using micro-ROS, with three different microcontrollers tested. The communication was evaluated in different scenarios with a test bench, using ping pong communication to get the round-trip time. A reinforcement of the test results was presented by demonstrating the use of micro-ROS live in a prototype developed, where mYuMi controlled a 1D rangefinder and an RC servo motor by utilising two microcontrollers. The results concluded that the micro-ROS delay could be analysed in theory with external applications, equivalent micro-ROS functionality should apply to most microcontrollers, and the test results and prototype displayed the potential of micro-ROS matching ROS2 in terms of delay and stability.

No description available.

Palmitate (PA), one of the most prevalent saturated fatty acids, causes myocardial dysfunction. However, the mechanisms by which PA induces cell apoptosis and autophagy remain to be elucidated. We showed that autophagy was induced in an mTORC1-dependent way and played a protective role against PA-induced apoptosis, which was verified by pretreatment with 3-methyladenine (3MA) and rapamycin. However, p62 began to accumulate after 18 h treatment with PA, suggesting prolonged exposure to PA lead to an impairment of autophagic flux. PA enhanced ROS production as well as activated p38-mitogen-activated protein kinase (p38 MAPK) and c-jun NH2 terminal kinases (JNKs). The antioxidant N-Acety-L-Cysteine (NAC) was found to attenuate the JNK and p38 MAPK activation with a concomitant reduction of PA-induced autophagy and apoptosis. Furthermore, both JNK and p38 MAPK inhibitors were shown to directly abrogate caspase 7 cleavage as well as the conversion of LC3BI to LC3BII. Thus, we demonstrate that PA stimulates autophagy and apoptosis via ROS-dependent JNK and p38 MAPK pathways.

According to the WHO, 38 million individuals were living with human immunodeficiency virus (HIV), 25.4 million of which were using antiretroviral therapy (ART) at the end of 2019. Despite ART-mediated suppression of viral replication, ART is not a cure and is associated with viral persistence, residual inflammation, and metabolic disturbances. Indeed, due to the presence of viral reservoirs, lifelong ART therapy is required to control viremia and prevent disease progression into acquired immune deficiency syndrome (AIDS). Successful ART treatment allows people living with HIV (PLHIV) to achieve a similar life expectancy to uninfected individuals. However, recent studies have illustrated the presence of increased comorbidities, such as accelerated, premature immune aging, in ART-controlled PLHIV compared to uninfected individuals. Studies suggest that both HIV-infection and ART-treatment lead to mitochondrial dysfunction, ultimately resulting in cellular exhaustion, senescence, and apoptosis. Since mitochondria are essential cellular organelles for energy homeostasis and cellular metabolism, their compromise leads to decreased oxidative phosphorylation (OXPHOS), ATP synthesis, gluconeogenesis, and beta-oxidation, abnormal cell homeostasis, increased oxidative stress, depolarization of the mitochondrial membrane potential, and upregulation of mitochondrial DNA mutations and cellular apoptosis. The progressive mitochondrial damage induced by HIV-infection and ART-treatment likely contributes to accelerated aging, senescence, and cellular dysfunction in PLHIV. This review discusses the connections between mitochondrial compromise and cellular dysfunction associated with HIV-and ART-induced toxicities, providing new insights into how HIV and current ART directly impact mitochondrial functions and contribute to cellular senescence and aging in PLHIV. Identifying this nexus and potential mechanisms may be beneficial in developing improved therapeutics for treating PLHIV.