Submitted by Chaylane Marques (chaylane.marques@ufpe.br) on 2015-03-10T19:47:28Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Definitiva completa corrigida Rebeca.pdf: 9019274 bytes, checksum: 67e0a3de6025215bb0dc15d5ec51bb9e (MD5) / Made available in DSpace on 2015-03-10T19:47:28Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Definitiva completa corrigida Rebeca.pdf: 9019274 bytes, checksum: 67e0a3de6025215bb0dc15d5ec51bb9e (MD5) Previous issue date: 2012 / CNPQ / Moringa oleifera é uma arbórea, tolerante à seca e a solos pobres, no semiárido nordestino, compreendendo uma alternativa para os sertanejos que vivem a nível de subsistência. É principalmente utilizada na limpeza de água por apresentarem propriedades coagulantes e floculantes. As sementes apresentam destacado valor comercial contendo alto teor de óleo com qualidades ímpares para produção de biocombustível. Estudos recentes indicam que quando a planta sofre uma exposição prévia a um estresse, ela tem a capacidade de responder mais rápido e com mais vigor a um evento de estresse recorrente, esse fenômeno é conhecido como endurecimento (hardening). Isso implica que as plantas tem a capacidade de memória (stress imprint). O objetivo do trabalho foi avaliar se plantas jovens de Moringa oleifera são capazes de carregar na memória estresses hídricos recorrentes ocorridos desde a germinação das sementes até o porte de plantas jovens e com isso apresentar maior tolerância à seca. As sementes foram germinadas em baixos potenciais osmóticos 0,0; - 0,1; - 0,2; - 0,3; - 0,4 e - 0,5 MPa. Foram utilizadas plantas jovens com 50 dias após a emergência, originadas da germinação de três potenciais osmóticos 0,0; - 0,3 e - 0,4 MPa. Para cada tratamento de germinação houve dois regimes hídricos: as irrigadas (100% da capacidade de pote) e as estressadas (10% da capacidade de pote), totalizando seis tratamentos. Foram realizados dois ciclos de déficit hídrico intercalados por 10 dias de reidratação. Foram analisados os parâmetros de germinação, trocas gasosas, bioquímica, enzimática e biometria. As sementes de M. oleifera não germinaram em potenciais osmóticos menores que - 0,4 MPa. As plantas jovens de M. oleifera foram tolerantes aos ciclos recorrentes de seca em decorrência do alto o conteúdo hídrico relativo (CHR), da alta eficiência do uso de água (EUA), da manutenção no conteúdo de clorofila, da síntese de novos aminoácidos, além do aumento da atividade de enzimas antioxidantes e de carotenóides. Após o primeiro ciclo a taxa fotossintética foi recuperada rapidamente, indicando a preservação do metabolismo mesofílico. As plantas estressadas provenientes do tratamento - 0,3 e - 0,4 MPa apresentaram maior tolerância ao déficit hídrico do que as plantas estressadas provenientes do tratamento 0,0 MPa. Sugerindo que as sementes submetidas a um estresse prévio, no período germinativo, tem a capacidade de adquirir memória à seca, possibilitando à M. oleifera uma maior tolerância a ciclos recorrentes.

A domesticação dos equinos e a sua utilização para esportes fez com que a nutrição destes animais passasse por mudanças significativas. O alto requerimento energético pode ser suprido com forragem conservada e grandes quantidades de amido. Este manejo, porém, predispõe os equinos a problemas digestivos e metabólicos, podendo levar a cólica, endotoxemia e laminite. Outra forma de atingir o requerimento energético é a adição de gordura na dieta, já que óleos vegetais e animais podem conter até três vezes mais energia que grãos em igual volume. Sabe-se que óleos de diferentes fontes diferem quanto a propriedades relacionadas aos seus componentes ativos; óleos podem conter componentes como ω-3, ω-6, γ-orizanol em diferentes quantidades e estes compostos podem influenciar o metabolismo. O presente estudo teve por objetivo avaliar o efeito de óleos de diferentes origens e composição sobre o metabolismo bioquímico, oxidativo e função de células polimorfonucleares de equinos. Durante 15 semanas foram utilizados cinco equinos machos, mestiços, que receberam ou óleo mineral, ou óleo de soja, ou óleo de arroz, ou óleo de linhaça, ou óleo de peixe, em delineamento experimental quadrado latino 5x5. Cada período experimental foi composto por três semanas. Foram avaliados perfil bioquímico, metabolismo oxidativo e \"burst\"-respiratório de granulócitos. Houve efeito do tratamento sobre os teores de triglicérides, fração HDL de colesterol e hemoglobina, contagem de eritrócitos, hematócrito e na intensidade de produção de EROs por granulócitos estimulados com Sa-PI. Das variáveis do perfil bioquímico, pode-se observar que a adição dos óleos de soja, arroz, linhaça e peixe na dieta reduziu os triglicérides plasmáticos, a inclusão de óleos em até 5% da MS nas dietas não representou grande desafio oxidativo, apenas as concentrações de hemoglobina foram menores no tratamento com óleo de soja. O óleo de linhaça aumentou o teor de ácido linolênico plasmático e teve efeito sobre a produção de EROs in vitro por granulócitos dos equinos. / Sports use made significant changes in horses nutrition necessary. The high energy requirement is supplied with conserved forage, and large amounts of starch. This management, however, predisposes horses to digestive and metabolic disorders, leading to colic endotoxemia and laminitis. Another way to achieve the energy requirement is adding fat to the diet, as vegetable and animal oils may contain up to three times more energy than grains in equal volume. It is known that oils from different sources differ in their active components related properties, thay may contain ω-3, ω-6, γ-oryzanol, in different quantities and these compounds can influence metabolism. The aim of this study was to evaluate the effect of different oils from different origins and composition on the maintenance of the horses biochemical and oxidative metabolism and on polymorphonuclear cells function. A Latin square design was made with five crossbreed horses fed during three weeks with the five treatments: mineral (control), soy, rice, linseed and fish oil. Biochemical profiles, oxidative metabolism respiratory-burst of granulocytes were evaluated. Treatment effect was found on the triglycerides, HDL cholesterol and hemoglobin levels, erythrocyte count, hematocrit and intensity of ROS production by granulocytes stimulated with Sa-PI. Conclusions: The addition of oil to the diet reduced plasma triglycerides, fat diets didnt offer major oxidative challenge and hemoglobin showed to be a more sensitive oxidative marker. Linseed oil increases the plasma levels of linolenic acid and had effects on the production of in vitro ROS by granulocytes of horses.

A cisplatina ainda é um dos agentes quimioterápicos mais efetivos. No entanto, em elevadas doses pode ocorrer hepatotoxicidade. Alguns antioxidantes têm sido mostrado amenizar a hepatotoxicidade induzida pela cisplatina mas o mecanismo molecular envolvido não está bem esclarecido.No presente estudo nós investigamos moleculares subjacente ao efeito protetor da dimetiltiuouréia (DMTU), um conhecido eqüestrador de radical hidroxil, contra a lesão oxidativamitocondrial hepática induzida pela cisplatina em ratos. Ratos Wistar machos adultos ( 200 a 220g) foram divididos entre 4 grupos de 8 animais cada. O grupo controle foi tratado apenas com uma injeção intraperitoneal (i.p.) de solução salina (1 ml/ 100g de peso). Ao grupo DMTU foi administrado apenas DMTU (500 mg/kg de peso, i.p., seguido de 125 mg/kg, i.p., duas vezes ao dia até o sacrifício). Ao grupo cisplatina foi administrado uma injeção única de cisplatina (10 mg/kg de peso, i.p.). Ao grupo DMTU + cisplatina foi administrado DMTU (500mg/kg de peso, i.p.), pouco antes da injeção da cisplatina (10 mg/kg de peso, i.p.), seguido por injeções de DMTU (125 mg/kg de peso, i.p.) duas vezes ao dia até o sacrifício ( 72 horas após o tratamento). A hepatotoxicidade foi evidenciada no grupo cisplatina pelo aumento dos níveis séricos de alanina (ALT) e aspartato (AST)aminotransferases. O mecanismo de hepatotoxicidade induzido pela cisplatina mostrou-se envolvido na rigidez de membrana; na redução da razão glutationa reduzida em relação a glutationa oxidada (GSH/GSSG); na redução dos níveis de ATP, GSH e NADPH; na lipoperoxidação; na lesão oxidativa da cardiolipina e de proteínas com grupos fidrílicos. Mais ainda, a morte celular por apoptose foi também demonstrada e os achados fortemente sugerem a participação do xi mecanismo sinalizador mitocondrial neste processo; o DMTU não apresentou nenhum efeito direto sobre a mitocôndria e inibiu substancialmente a lesão mitocondrial induzida pela cisplatina, prevenindo a hepatotoxicidade. Todos os seguintes efeitos induzidos pela cisplatina foram previnidos pelo DMTU: (a) elevação dos níveis séricos de AST e ALT; (b) redução dos níveis de ATP hepático;(c)peroxidação lipídica;(d)oxidação da cardiolipina; (e)oxidação de proteínas sulfidrílicas; (f) rigidez da membrana mitocondrial; (g) oxidação de GSH; (h)oxidação de NADPH e (i) morte celular por apoptose. Os resultados mostraram o papel principal da mitocôndria e dos radicais hidroxilas na proteção do fígado saudável contra a lesão hepática induzida pela cisplatina, delineando um número de etapas que podem ser consideradas no desenvolvimento de futuros agentes citoprotetores / Cisplatin is still one of the most effective chemotherapeutic agents. However, at higher doses hepatotoxicity may occur. Some antioxidants have been shown to ameliorate cisplatin-induced hepatotoxicity but the involved molecular mechanism has not been clarified. In the present study we investigated the molecular mechanism underlying the protective effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, against liver mitochondrial oxidative damage induced by cisplatin in rats.Adult male Wistar rats (200 to 220g) were divided into 4 groups of 8 animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1ml/100g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until sacrifice). The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 hours after the treatment). epatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatininduced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process; DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial injury, therefore preventing the hepatotoxicity. All the following cisplatin-induced xiv effects were prevented by DMTU: (a) elevation of AST and ALT serum levels; (b) decreased hepatic ATP levels; (c) lipid peroxidation; (d)cardiolipin oxidation; (e) sulfhydryl protein oxidation; (f) mitochondrial membrane rigidification; (g) GSH oxidation; (h) NADPH oxidation and (h) apoptotic cell death. Results show the central role of mitochondria and hydroxyl radicals in the protection of healthy liver against cisplatin-induced injury, highlighting a number of steps that might be considered in the development of novel cytoprotective agents.

Salamanders have an extraordinary ability to regenerate appendages after loss or amputation, irrespective of age. My dissertation research explored the possibility that regenerative ability is associated with the evolution of novel, salamander-specific genes. I utilized transcriptional and genomic databases for the axolotl to discover previously unidentified genes, to the exclusion of other vertebrate taxa. Among the genes identified were multiple mmps (Matrix metalloproteases) and a jnk1/mapk8 (c-jun-N-terminal kinase) paralog. MMPs function in extracellular matrix remodeling (ECM) and tissue histolysis, processes that are essential for successful regeneration. Jjnk1/mapk8 plays a pivotal role in regulating transcription in response to cellular stress stimuli, including ROS (reactive oxygen species). Discovery of these novel genes motivated further bioinformatic studies of mmps and wet-lab experiments to characterize JNK and ROS signaling. The paralogy of the newly discovered mmps and orthology of 15 additional mmps was established by analyses of predicted, protein secondary structures and gene phylogeny. A microarray-analysis identified target genes downstream of JNK signaling that are predicted to function in cell proliferation, cellular stress response, and ROS production. These inferences were validated by additional experiments that showed a requirement for NOX (NADPH oxidase) activity, and thus presumably ROS production for successful tail regeneration. In summary, my dissertation identified novel, salamander-specific genes. The functions of these genes suggest that regenerative ability is associated with a diverse extracellular matrix remodeling and/or tissue histolysis response, and also stress-associated signaling pathways. The bioinformatic findings and functional assays that were developed to quantify ROS, cell proliferation, and mitosis will greatly empower the axolotl embryo model for tail regeneration research.

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...