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.

Dans la recherche de thérapie antitumorale de plus en plus innovante, nous avons évalué un traitement local basé sur l'utilisation de plasma froid. Le plasma froid (dans ce cas, <40°C) est un gaz ionisé par un apport d'énergie. Il contient des charges (électrons, ions), des radicaux libres et des molécules excitées. Il peut être généré à l'extrémité de cathéter permettant un traitement locorégional comme le traitement de dysplasie ou encore de tumeurs non résécables. Une activité antitumorale importante du plasma a été mise en évidence in vitro sur diverses lignées tumorales (colorectale, pulmonaire, pancréatique et cérébrale). Par ailleurs les cellules tumorales sont plus sensibles au plasma que les cellules normales. Les ROS générés sont à l'origine des principaux mécanismes d'action du plasma. Ils induisent de nombreux dommages à l'ADN, suivi d'un arrêt du cycle cellulaire conduisant à l'apoptose des cellules. Les études de tolérance ont mis en évidence l'innocuité de faibles doses de plasma sur le tissu traité permettant de définir les doses de plasmas utilisable dans le cadre de traitements antitumoraux. En utilisant des tumeurs xénogreffées en sous cutané et l'imagerie de bioluminescence, une activité antitumorale du plasma froid a été mise en évidence pour la première fois in vivo avec une augmentation de la survie des souris traitées d'environ 60%. Le traitement induit un arrêt de la prolifération tumoral avec une induction d'apoptose dans l'ensemble de la tumeur sans augmenter la surface de nécrose. L'effet antitumoral a également été démontré en utilisant le plasma gun sur un modèle de tumeurs colique et pancréatique en situation orthotopique chez la souris avec une augmentation de la survie (115%) accompagné d'une diminution de la métastasie. Ces résultats obtenues dans une démarche de recherche translationnelle montrent l'intérêt potentiel du plasma comme nouvel agent antitumoral.

Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

In diabetes hyperglycemia and lack of insulin signaling are key factors in the induction of diabetic sensory neuropathy. The combination of these factors in diabetes may enhance oxidative stress and trigger distal nerve damage in the peripheral nervous system. The link between elevated reactive oxygen species (ROS) levels and nerve degeneration is not clear. We tested the hypothesis that elevation of 4-hydroxy-2-nonenal (4-HNE) induced by oxidative stress in diabetes impairs mitochondrial activity and axonal regeneration in dorsal root ganglion (DRG) neurons. Also, we investigated the association between mitochondrial dysfunction and altered mitochondrial proteome in the axons of streptozotocin–induced diabetic rats. Research design and methods. Cultured adult rat DRG sensory neurons were treated exogenously with 4-HNE, and cell survival, axonal morphology, and level of axon outgrowth assessed. Western blot and fluorescence imaging were used to determine changes in the levels of adducts of 4-HNE and abnormalities in the mitochondria. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in the mitochondria. Results. 4-HNE impaired axonal regeneration, mitochondrial activity and induced aberrant axonal structures along the axons, which mimicked axon pathology observed in nerve isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic neuropathy. Proteins associated with mitochondrial dysfunction, oxidative phosphorylation and biosynthesis were down regulated in diabetic samples. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria. CNTF and resveratrol reversed abnormalities in the mitochondrial membrane potential induced by diabetes and treatment of neurons with 4-HNE. CONCLUSIONS. Elevation of 4-HNE levels in diabetes was associated with impaired mitochondrial function and might be an important link between increased ROS levels and nerve degeneration in diabetic neuropathy. Abnormal mitochondrial function correlated with a down-regulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons.

The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised. Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation. mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase.  Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction. We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway. Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst. A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype. Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.

Pre-gestational diabetes increases the risk of congenital malformation in the offspring and both morbidity and mortality in the diabetic mother and her offspring. During pregnancy, high glucose levels act as a teratogen through several cellular and biochemical pathways and increased production of reactive oxygen species (ROS) has a central role in diabetic embryopathy. The aim of this work was to investigate the importance of genetic predisposition for congenital malformations and to study the genes involved in the teratogenic process of diabetic pregnancy. The crossbreeding of two rat strains, with both low and high incidence of diabetes-induced malformations, indicated that strain-specific maternal factors, such as disturbed serum levels of amino acids, triglycerides, and β-hydroxybutyrate, were associated with malformation. In addition, disturbed fetal expression of genes involved in ROS defense and development (Shh, Bmp4, Ret and Gdnf) in mandible and heart, and decreased activity of Gapdh and Aldose Reductase were associated with the teratogenic process, and the trans-generational heredity of the mother determined the type of malformations induced by maternal diabetes. In rat embryos, a diabetic environment in utero changed the expression of genes involved in ROS defense (Nrf2, Gpx1 and Cat), development of mandible and heart (Msx2, Shh, Bmp4, Ret and Gdnf), and neural tube closure and apoptosis (Pax3 and p53). The changes were divergent with tissue-specific alterations of gene expression in developing mandible, heart anlage, and whole embryo. Disruption of the Receptor for Advanced Glycation End products (RAGE) had a protective effect against diabetic embryopathy in mice, and the blockage of RAGE diminished ROS production in the offspring: this supported oxidative stress being a necessary etiological component in diabetic embryopathy. Maternal metabolic state and genetic susceptibility influence fetal outcome in experimental diabetic pregnancy. Disturbed protection against oxidative stress and tissue-specific derangements in the expression of developmental genes play pivotal roles in the teratogenic mechanism, and enhanced levels of Advanced Glycation End products (AGE) and RAGE-induced oxidative stress are involved in diabetic dysmorphogenesis.

Η κυτταροφαγία βακτηρίων από τα πολυμορφοπύρηνα κύτταρα του αίματος του ανθρώπου επιτυγχάνεται με την ενεργοποίηση διαφόρων σηματοδοτικών μονοπατιών. Η σηματοδότηση ξεκινάει με την προσκόλληση των βακτηρίων στην πλασματική μεμβράνη των κυττάρων και καταλήγει στην αναδιοργάνωση του κυτταροσκελετού, στο σημείο επαφής, για τη δημιουργία του φαγοσώματος. Διάφορες μορφές δραστικού οξυγόνου, όπως το Η2Ο2, παράγονται παρουσία βακτηρίων, και δρουν ως σηματοδοτικά μόρια. Στην εργασία αυτή μελετήθηκε η παραγωγή και η συμμετοχή του Η2Ο2 στην κυτταροφαγία E.coli από τα ανθρώπινα πολυμορφοπύρηνα κύτταρα. Για το σκοπό αυτό, χρησιμοποιήθηκε το εξειδικευμένο φθορίζον μόριο, διυδροροδαμίνη (DHR) - που οξειδώνεται από το Η2Ο2 - καθώς και φθορίζοντα βακτήρια E. coli. Πειράματα κυτταρομετρίας ροής έδειξαν ότι τα πολυμορφοπύρηνα παράγουν Η2Ο2 παρουσία E. coli, ενώ παράλληλα δείχτηκε με έμμεσο τρόπο, με ανοσοαποτύπωση, η μετατόπιση της κυτταροπλασματικής υπομονάδας p47 για την συγκρότηση μεμβρανικής NADPH οξειδάσης. Το ένζυμο αυτό παράγει υπεροξεικά ιόντα τα οποία μετατρέπονται, στη συνέχεια, σε Η2Ο2 από τη δεσμουτάση SOD. Ο ενεργός ρόλος του Η2Ο2 στη ρύθμιση της κυτταροφαγίας, επιβεβαιώθηκε με τη χρήση εξειδικευμένων αναστολέων για τα ένζυμα της σύνθεσής του, N-εθυλμαλειμίδιο (N-ethylmaleimide - nem) για τη NADPH οξειδάση και διεθυλδιθειοκαρβαμικό (diethyldithiocarbamate – ddc) για την δεσμουτάση του υπεροξεικού ανιόντος. Οι αναστολείς αυτοί μείωσαν και την παραγωγή του Η2Ο2 και την κυτταροφαγία βακτηρίων σε καλλιέργεια λευκών αιμοσφαιρίων. Ανοσοαποτυπώματα για τις ΜΑΡ κινάσες, p38 και ΕΡΚ1/2, από εκχύλισμα λευκών αιμοσφαιρίων, μετά από καλλιέργεια παρουσία των παραπάνω αναστολέων, έδειξαν ότι κατά την κυτταροφαγία E. coli, μόνο η φωσφορυλίωση της ERK1/2 μειώνεται όταν μειώνεται η παραγωγή Η2Ο2. Είναι γνωστό ότι το Η2Ο2 απενεργοποιεί φωσφατάσες πρωτεϊνικών τυροσινών (PTPs). Από τα αποτελέσματα μπορεί να υποτεθεί ότι το Η2Ο2 που παράγεται κατά την κυτταροφαγία, απενεργοποιεί τις PTPs, ενισχύοντας τη δράση κινασών τη φωσφορυλίωση της ERK1/2 και να ολοκληρωθεί η διαδικασία της κυτταροφαγίας. Αναστολή της παραγωγής του Η2Ο2 αυξάνει τη δράση των PTPs, την αποφωσφορυλίωση της pERK1/2 και τελικά να μειώνει την κυταροφαγία. / Phagocytosis by human polymorphonuclear blood cells, is achieved by the activation of several signaling pathways. Signaling is initiated with the attachment of the bacteria on the plasma membrane of the cells and is completed with the remodeling of actin on that sight leading to the formation of the phagosome. Certain reactive oxygen species, such as Η2Ο2, which are produced in the presence of bacteria, have been referred to act as signaling molecules. In the present work, we investigated the production and participation of Η2Ο2 in the phagocytosis of E. coli by the human polymorphonuclear cells. For this purpose specific fluorescent probe dihydrorhodamine (DHR) - which is oxidised by H2O2 - and fluorescent E. coli were used. Flow cytometry analysis revealed the production of Η2Ο2 by polymorphonuclears, during phagocytosis of E. coli, while in parallel, it was indirectly shown with immunoblotting, the translocation of cytoplasmic p47 subunit for the assembly of the membrane NADPH oxidase. This enzyme produces superoxide ions which are then converted in H2O2 by superoxide dismutase (SOD). The active role of Η2Ο2, in the regulation of phagocytosis, was confirmed with the use of specific enzyme inhibitors of its synthesis, N-ethylmaleimide (nem) for the NADPH oxidase and diethyldithiocarbamate (ddc) for superoxide dismutase. These inhibitors decreased E. coli phagocytosis in polymorphonuclear cells cultures along with the decrease of Η2Ο2 production. Immunoblot analysis of ΜΑΡ kinases p38 and ERK1/2, from crude extracts of cultured white blood cells, in the presence of the above inhibitors, showed that the phosphorylation of ERK1/2 was reduced when H2O2 was blocked. On the other hand, p38 phosphorylation was not influenced at all. It is known that the target molecules of Η2Ο2 are a family of protein tyrosin phosphatases (PTPs) which are getting inactivate. From the above data, it may be assumed that Η2Ο2, which is produced by the polymorphonuclear blood cells, during phagocytosis, inactivates these enzymes, in order to promote the activity of kinases enhancing the phosphorylation of ERK1/2, among others, so that phagocytosis to be completed. Thus, when the H2O2 production is inhibited, the activity of the PTPs increases, leading to the dephosphorylation of pERK1/2 and the observed decrease of bacterial uptake.

Orientador: Suely Cunha Amaro Mantovani / Resumo: A criação de robôs que podem operar autonomamente em ambientes controlados e não controlados tem sido, um dos principais objetivos da robótica móvel. Para que um robô possa navegar em um ambiente interno desconhecido, ele deve se localizar e ao mesmo tempo construir um mapa do ambiente que o rodeia, a este problema dá-se o nome de Localização e Mapeamento Simultâneos- SLAM. Tem-se como proposta neste trabalho para solucionar o problema do SLAM, o uso de um sensor RGB-D, com 6 graus de liberdade para perceber o ambiente, o qual é embarcado em um robô. O problema do SLAM pode ser solucionado estimando a pose - posição e orientação, e a trajetória do sensor no ambiente, de forma precisa, justificando a construção de um mapa em três dimensões (3D). Esta estimação envolve a captura consecutiva de frames do ambiente fornecidos pelo sensor RGB-D, onde são determinados os pontos mais acentuados das imagens através do uso de características visuais dadas pelo algoritmo ORB. Em seguida, a comparação entre frames consecutivos e o cálculo das transformações geométricas são realizadas, mediante o algoritmo de eliminação de correspondências atípicas, bPROSAC. Por fim, uma correção de inconsistências é efetuada para a reconstrução do mapa 3D e a estimação mais precisa da trajetória do robô, utilizando técnicas de otimização não lineares. Experimentos são realizados para mostrar a construção do mapa e o desempenho da proposta. / Doutor

Este trabalho apresenta a modelagem matemática para um manipulador móvel composto por uma base móvel (o robô móvel Twil) e um manipulador (o manipulador WAM da Barrett). Os modelos cinemático e dinâmico para a base móvel, manipulador e manipulador móvel são apresentados. Como o manipulador móvel é um sistema não linear, uma estratégia de controle baseada em linearização por realimentação da dinâmica da plataforma seguida por uma transformação não suave para tratar a não holonomicidade do modelo cinemático é proposta. Então o método de backstepping é utilizado para obter as entradas do modelo dinâmico. Um controlador de torque calculado é proposto para o manipulador, Estas técnicas de controle são utilizadas simultaneamente para controlar o manipulador móvel. A implementação dos controladores propostos, na forma de plugins para o gerenciador de controladores é feita no ROS, assim os controladores são executados em tempo real. A maioria dos controladores existentes no ROS são do tipo SISO baseados em controle PID e independentes para cada junta, sendo que neste trabalho controladores MIMO não lineares são implementados. / This work presents a mathematical modelling for a mobile manipulator composed by a mobile base (the Twil mobile robot) and a manipulator (the Barrett WAM manipulator). The kinematic and dynamic models for the mobile base, the manipulator and the mobile manipulator are presented. As the the mobilie manipulator is a non-linear system, a control strategy based on feedback linearization of the platform dynamics followed by a non-smooth transform to handle the non-holonomicity of its kinematic model is proposed. Then, the backstepping method is used to obtain the inputs for the dynamic model. A computed torque controller is proposed for the manipulador. These control techniques are used simultaneously to control the mobile manipulator. The implementation of the proposed controllers is done in ROS as plugins for the controller manager so that the controllers run in real-time. Most controllers existing in ROS are independent joint SISO controllers based on the PID control law while in this work MIMO non-linear controllers are implemented.