Androgen has been shown to stimulate proliferation of osteoblast-like MC3T3-E1 cells. However, the molecular mechanism responsible for this effect remains to be elucidated. In the present study we demonstrate herein the non-genomic effect of androgen on osteoblast-like MC3T3-E1 cells involving activation of a PI(3)K/Akt signaling pathway and stimulating proliferation. In studies of steroids signaling, 5a-dihydrotestosterone (DHT), testosterone and 17b-estradiol but not dexamethasone or progesterone induced a rapid and transient phosphorylation of Akt in MC3T3-E1 cells. The androgen-induced Akt activation reached to the climax after 15 min and gradually diminished to baseline after 60 min. This induction of androgen was unaffected by actinomycin D and was specifically blocked by androgen receptor (AR) antagonist hydroxyflutamide (HF) or transfection of siRNA-AR. Treatment of MC3T3-E1 cells with PI(3)K inhibitor LY294002 or transfection with kinase-deficient Akt blocked androgen-induced cells proliferation. Moreover, androgen-induced activation of Akt was abolished by inhibitors of Src kinase, Gi-protein and phospholipase C showing the involvement of these effectors in androgen signaling pathway. Further, androgen-induced activation of Akt was dependent on intracellular calcium as shown by the effect of EGTA and intracellular calcium chelator BAPTA/AM. Fluorescence microscopy showed translocation of phospho-Akt from cytosol into nucleus after androgen treatment but no change in the subcellular distribution of phospho-Akt when HF or LY294002 pretreatment was administered to the cells. These results strongly suggest that phosphorylation of Akt in osteoblast cells is mediated by androgen receptor and the androgen-induced translocation of Akt is an important step in the androgen/AR signaling pathway that mediates osteoblast cells proliferation.

Cette thèse vise à démontrer l’importance de la PI 3-kinase dans la régulation hormonale de la réabsorption rénale du sodium. Ce contrôle extrêmement précis, notamment par l’aldostérone, s’effectue au niveau du néphron distal. Nous avons utilisé comme modèle l’épithélium de cellules A6, dérivées du tubule distal de Xenopus Laevis. Le transport unidirectionnel de sodium s’effectue en deux étapes: depuis, l’entrée à partir du milieu luminal par des canaux sodiques épithéliaux (ENaCs) insérés dans la membrane apicale, jusqu’à la sortie vers le liquide extracellulaire par des pompes Na+/K+-ATPases, situées dans la membrane basolatérale. L’insuline augmente ce transport de sodium et la PI 3-kinase semble assurer un rôle-clef. Nous avons étudié l’importance de chacun des 3-phosphoinositides produits par la PI 3-kinase, sur le transport du sodium en ajoutant au milieu cellulaire des formes «perméantes» de ces phospholipides. Parmi ceux-ci, le PIP3 et dans une moindre intensité le PI(3,4)P2 augmentent ce transport. En revanche, le PI3P, le PI(3,5)P2, ainsi que le PI(4,5)P2 n’ont pas d’effet sur lui. Nous avons démontré par la technique du Western blot que la 3-phosphatase PTEN est exprimée dans les cellules A6. Cette phosphatase déphosphoryle le PIP3 en PI(4,5)P2. Nous avons surexprimé PTEN dans les cellules A6. Ceci réduit l’augmentation du transport du sodium induite par l’insuline, ainsi que celle induite par addition de la forme «perméante» de PIP3. Nous avons ensuite vérifié si d’autres agents qui activent la PI 3-kinase, stimulent également le transport de sodium à travers cet épithélium. A cette fin, nous avons d’abord vérifié que l’EGF et le peroxyde d’hydrogène, connus pour stimuler la PI 3-kinase dans d’autres systèmes, activent également cette enzyme dans les cellules A6. Tous deux augmentent ce transport. L’importance de l’augmentation induite par H2O2 est comparable à celle de l’insuline, tandis que l’effet de l’EGF est plus transitoire. Un dosage d’activité de la PI 3-kinase, nous a permis de démontrer que l’intensité de l’activation de la PI 3-kinase est corrélée avec l’amplitude de l’augmentation du transport du sodium. Par comparaison avec l’effet de l’insuline et de l’H2O2, l’EGF augmente faiblement l’activité de la PI 3-kinase et induit une faible augmentation du transport. Nous avons également examiné si la voie des MAPK influence la stimulation du transport du sodium par ces différents agents. Cette voie ne semble pas impliquée dans l’effet de l’insuline ou du peroxyde d’hydrogène. Par contre, elle diminue la stimulation du transport de sodium par l’EGF. L’effet de l’EGF sur le transport semble résulter d’un compromis entre l’activation de la voie de la PI 3-kinase qui l’augmente et l’activation de la voie des MAPK qui le diminue. En conclusion, une augmentation de PIP3, soit par addition de PIP3 exogène, soit par augmentation endogène sous l’effet de l’insuline ou d’autres agents stimulant la PI 3-kinase, augmente le transport du sodium tandis qu’une diminution de PIP3 endogène (par surexpression de PTEN) le diminue. L’importance de l’activation de la PI 3-kinase est quantitativement corrélée avec l’importance de l’augmentation du transport du sodium. La PI 3-kinase est donc un médiateur-clef de la régulation rénale de ce transport.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / It is known that 33% of traffic accidents worldwide are caused by drunk driving or drowsiness [1] [2], so a drowsiness level detection system that integrates image processing was developed with the use of Raspberry Pi3 with the OpenCV library; and sensors such as MQ-3 that measures the percentage of alcohol and the S9 sensor that measures the heart rate. In addition, it has an alert system and as an interface for the visualization of the data measured by the sensors a touch screen. With the image processing technique, facial expressions are analyzed, while physiological behaviors such as heart rate and alcohol percentage are measured with the sensors. In image test training you get an accuracy of x in a response time of x seconds. On the other hand, the evaluation of the operation of the sensors in 90% effective. So the method developed is effective and feasible. / Revisión por pares

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

O Brasil é o 18ª maior produtor de carne ovina e, apesar de ser em grande parte informal, é uma cultura crescente no país. Dentre as enfermidades infecciosas que acomete a ovinocultura a broncopneumonia é uma das mais recorrentes, no entanto, não há muitos estudos sobre esta enfermidade em pequenos ruminantes no Brasil. Por isso, esta pesquisa teve como objetivo verificar a ocorrência de doenças respiratórias no estado de São Paulo e Rio de Janeiro causadas por bactérias e vírus. Para a realização desse projeto foi utilizado a técnica de lavado traqueobrônquico transtraqueal e coleta de sangue total para obtenção do soro em 99 ovinos. Essas amostras foram submetidas a teste de virusneutralização para identificação de anticorpos contra vírus da Parainfluenza Tipo 3 (PI-3), Herpesvírus Bovino Tipo 1 (BoHV- 1), Vírus Sincicial Respiratório Bovino (BRSV) e Vírus da Diarreia Viral Bovina (VDVB). Utilização do teste de imunodifusão em gel de agarose e Eradikt® para detectar a presença de anticorpos contra Lentivírus de Pequenos Ruminantes (LVPR). Isolamento e identificação bioquímica para M. haemolytica e Pasteurella multocida. Cultivo e isolamento, identificação bioquímica e PCR foram testes utilizados para identificação de micoplasmas (Mycoplasma bovis, M. agalactiae, M. mycoides subsp. capri). Das 99 amostras coletadas, 33 foram de ovinos doentes e 66 de ovinos sadios. Não houve identificação de M. haemolytica e P. multocida, nem presença de anticorpos contra BoHV-1 e VDVB. No entanto, observou-se a prevalência de 52,52%, 48,48% e 21,87% de PI-3, BRSV e LVPR respectivamente. Em relação as bactérias aeróbias, houve maior frequência de isolamento de Bacillus sp. e Gram-negativas não fermentadoras. Apesar de identificar bactérias da classe Mollicutes em colônias isoladas em 23,28% das amostras, houve apenas uma identificação com os oligonucleotídeos utilizados, o M. mycoides subsp. capri, primeiro isolamento em ovinos no estado de São Paulo e Rio de Janeiro. Houve diferença estatística significativa (p<0,05) entre animais com broncopneumonia e manifestações clínicas como taquipneia, hipertermia, secreção nasal, tosse, dispneia, crepitação fina e ronco e entre animais com broncopneumonia e não realizam de quarentena e não separam animais doentes. A broncopneumonia envolve vários fatores, incluindo o manejo, agente infeccioso e a imunidade do animal. Por isso, é necessário conhecer todos esses aspectos e associá-los para uma melhor prevenção e tratamento. / Brazil is the 18th largest producer of sheep meat and, despite being largely informal, is a growing crop in the country. Among the infectious diseases that affect sheep production, bronchopneumonia is one of the most recurrent, however, there are not many studies on this disease in small ruminants in Brazil. Therefore, this research aimed to verify the occurrence of respiratory diseases in the state of São Paulo and Rio de Janeiro caused by bacteria and viruses. For the accomplishment of this project was used tracheobronchial lavage technique transtracheal and collection of whole blood to obtain serum in 99 sheep. These samples were submitted to virus neutralization test to identify antibodies against Parainfluenza Type 3 (PI-3), Bovine Herpesvirus Type 1 (BoHV-1), Bovine Respiratory Syncytial Virus (BRSV) and Bovine Viral Diarrhea Virus (BVDV). Use of the Eradikit ® and agarose gel immunodiffusion test to detect the presence of antibodies against Small Ruminant Lentiviruses (LVPR). Isolation and biochemical identification for M. haemolytica and Pasteurella multocida. Cultivation and isolation, biochemical identification and PCR were used to identify mycoplasmas (Mycoplasma bovis, M. agalactiae, M. mycoides subsp. Capri). Of the 99 samples collected, 33 were from diseased sheep and 66 from healthy sheep. There was no identification of M. haemolytica and P. multocida, nor presence of antibodies against BoHV-1 and BVDV. However, the prevalence of 52.52%, 48.48% and 21.87% of PI-3, BRSV and LVPR, respectively, was observed. In relation to aerobic bacteria, there was a higher frequency of isolation of Bacillus sp. and Gram-negative non-fermenters. Despite identifying Mollicute class bacteria in isolated colonies in 23.28% of the samples, there was only one identification with the oligonucleotides used, M. mycoides subsp. capri, first isolation in sheep in the state of São Paulo and Rio de Janeiro. There was a significant statistical difference (p <0.05) between animals with bronchopneumonia and clinical manifestations such as tachypnea, hyperthermia, nasal secretion, cough, dyspnea, fine crackling and snoring, and between animals with bronchopneumonia and quarantine and separation of diseased animals. Bronchopneumonia involves several factors, including management, infectious agent and the immunity of the animal. Therefore, it is necessary to know all these aspects and to associate them for a better prevention and treatment.

Diplomová práca sa zaoberá návrhom embedded dohľadového systému, ktoré je implementované na Raspberry Pi 3 B zariadenie. Uvedený systém obsahuje algoritmy pre detekcie pohybu a detekcie objektov, ktoré sú realizované pomocou OpenCV funkcie. Vyhodnocené informácie sú prístupné na webový server.

One of the main challenges for advanced driver assistance systems (ADAS)is the environment perception problem. One factor that makes ADAS hardto implement is the large amount of different conditions that have to betaken care of. The main sources for condition diversity are lane and roadappearance, image clarity issues and poor visibility conditions. A review ofcurrent lane detection algorithms has been carried out and based on that alane detection algorithm has been developed and implemented on a mixedcriticality platform. The thesis is part of a larger group project consisting offive master thesis students creating a demonstrator for autonomous platoondriving. The final lane detection algorithms consists of preprocessing stepswhere the image is converted to gray scale and everything except the regionof interest (ROI) is cut away. OpenCV, a library for image processing hasbeen utilized for edge detection and hough transform. An algorithm for errorcalculations is developed which compares the center and direction of the lanewith the actual vehicle position and direction during real experiments. Thelane detection system is implemented on a Raspberry Pi which communicateswith a mixed criticality platform through UART. The demonstrator vehiclecan achieve a measured speed of 3.5 m/s with reliable lane keeping using thedeveloped algorithm. It seems that the bottleneck is the lateral control ofthe vehicle rather than lane detection, further work should focus on controlof the vehicle and possibly extending the ROI to detect curves in an earlierstage. / En stor utmaning för avancerade förarstödsystem (ADAS) är problemet med uppfattning av miljön runt omkring. En faktor som gör ADAS svårt att implementera är den stora mängd olika förhållanden som måste tas hand om. De största källorna till olikheter är utseendet på körfältet och vägen, dåliga siktförhållanden samt otydliga bilder. En granskning av nuvarande algoritmer för körfältsdetektering har utförts och baserat på den har en körfältsdetekteringsalgoritm utvecklats och implementerats på ett blandkritiskt system. Avhandlingen är en del av ett större grupprojekt bestående av fem mastersstudenter som ska skapa en demonstrator för autonom konvojkörning. Den slutgiltiga körfältsdetekteringsalgoritmen består av förbehandlingssteg, där bilden konverteras till gråskala och allt utom intresseområdet är bortklippt. OpenCV, ett bibliotek för bildbehandling har använts för kantdetektering och houghtransformation. En algoritm som jämför körfältets mittpunkt och riktning med fordonets faktiska position och riktning har utvecklats och används i experiment för kontroll av fordonet. Körfältsdetekteringsalgoritmen har implementeras på en Raspberry Pi som kommunicerar med en blandkritisk plattform genom UART. Demo-fordonet kan uppnå en uppmätt hastighet på 3,5 m/s med pålitlig väghållning med den utvecklade algoritmen. Det verkar som att flaskhalsen är kontroll av fordonet i sidled och inte körfältsdetektering, ytterligare arbete bör fokusera på kontroll av fordonet och eventuellt utöka synfältet för att detektera kurvor i ett tidigare skede.

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L’endocytose est le processus qui permet l’entrée spécifique et active dans la cellule du milieu extracellulaire et des substances qu’il contient. Ces dernières années, plusieurs mécanismes d’endocytose ont été identifiés, mais seule la voie impliquant la clathrine a été bien caractérisée. Il a été montré que certains récepteurs de cytokines comme celui de l’interleukine-2 (IL-2) empruntent une voie d’endocytose indépendante de la clathrine. Précédemment, mon laboratoire a identifié huit facteurs impliqués dans cette voie d’entrée dont l’actine et la dynamine. Nous savons notamment que l’activation de la RhoGTPase Rac1 permet l’induction des kinases Pak1 et Pak2 régulant ainsi la cortactine, le recrutement de N-WASP et la polymérisation d’actine nécessaire à l’internalisation de l’IL-2R. Cependant, les facteurs en amont de cette cascade Rac-Pak étaient inconnus et le rôle du cytosquelette d’actine restait mal défini. Les travaux que j’ai effectué durant mon doctorat ont mis en évidence que la PI3K en interagissant avec l’IL-2R, permettait la production locale de PI(3,4,5)P3 qui induisait le recrutement de Vav2; un facteur conduisant à l’activation de Rac1. De plus, j’ai montré que Rac1 activé était ensuite recruté par la PI3K, permettant ainsi l’activation locale de la cascade Rac/Pak lors de l’entrée de l’IL-2R. Ces données, qui ont fait l’objet d’une publication, ont montré que la PI3K n’était pas seulement impliquée dans la signalisation de l’IL-2, mais également dans son endocytose. Par la suite, la réalisation d’un crible par ARNi portant sur l’étude du rôle de 324 protéines humaines lors de l’entrée de l’IL-2R, a permis l’identification de 65 nouveaux facteurs impliqués dans ce mécanisme. Parmi ces protéines, j’ai étudié plus particulièrement le rôle du complexe WAVE connu pour être induit à la membrane plasmique par Rac1 et la PI3K. J’ai confirmé l’implication de ce complexe lors de l’entrée de l’IL-2R et mis en évidence qu’il interagissait avec le récepteur. De plus, cette étude a montré que le complexe WAVE était impliqué précocement dans ce mécanisme, probablement en permettant le recrutement du récepteur aux pieds de protrusions membranaires que le complexe pourrait induire. Cette localisation spécifique du récepteur suggère ainsi un nouveau mécanisme de formation des vésicules d’endocytose dont le puits s’initierait à partir des invaginations existantes à la base des protrusions membranaires. / Endocytosis is a basic and essential process used by eukaryotic cells to internalize, actively and specifically, a wide range of molecules. To date, several endocytic routes have been characterized, but the only well studied pathway is clathrin-dependent. This study focuses on a poorly characterized mechanism, the clathrin-independent endocytosis, used by several cytokine receptors such as the interleukin 2 receptors (IL-2R). Previously, my lab identified 8 factors in this process, most of them are related to actin polymerization and dynamin. We now know that the activation of the RhoGTPase Rac1 is required to induce Paks that regulate cortactin, N-WASP recruitment and actin polymerization essential for IL-2R uptake. However, upstream factors involved in the Rac-Pak cascade were unknown and the actin cytoskeleton function poorly characterised. During my PhD, I showed that IL-2R and the phosphatidylinositol 3-kinase (PI3K) interact, leading to the local production of phosphatidylinositol (3,4,5)-triphosphate (PIP3) and the recruitment by this lipid of the RacGEF Vav2, an activator of Rac1. In addition, I showed that activated Rac1 was able to interact with PI3K allowing the local activation of the Rac-Pak cascade during IL-2R entry. These published data highlight the dual role of PI3K as a regulator of both IL-2R endocytosis and IL-2 signaling. In an attempt to identify new actors involved in the endocytosis of IL-2R, I then performed a small interfering RNA (siRNA) screening, targeting 324 proteins involved mainly in membrane deformation. From this screening we identified 65 proteins and among them we found the WAVE complex as a new factor implicated in IL-2R uptake. Interestingly, to be recruited and activated at the plasma membrane, the WAVE complex required Rac1 and PI3K, two proteins essentials during IL-2R entry. First, I confirmed the results obtained from the screening and found that IL-2R can interact with the WAVE complex. In addition, I observed an early involvement of this complex during IL-2R uptake, which could be needed for the localization of the receptor at the basis of plasma membrane protrusions. These results reveal a new model for the formation of endocytic vesicles: IL-2R is recruited at the basis of WAVE-induced membrane protrusions initiating the pit and vesicle.