In recent decades, the acceptance has steadily increased that oxidative stress plays an important role in the development of chronic diseases, malignant neoplasia and the acceleration of the aging process. As one of the most common chronic diseases, hypertension is often associated with a misregulated renin-angiotensin-aldosterone system that causes chronic oxidative stress. Hypertension is a risk factor for neurological diseases such as vascular dementia (VaD) and many neurological disorders, including VaD, have an ROS-associated or inflammatory component in their etiology. Our group has already demonstrated AT-II-induced genotoxicity in kidney and myocardial cells and tissues. The aim of this dissertation was to investigate a possible association between AT-II and neurodegeneration that is triggered by neuronal genotoxicity of AT-II. First, we showed in two neuronal cell lines that AT-II causes dose-dependent genome damage. Subsequent experiments could attribute this toxicity to NOX-produced superoxide generated after AT-II binding to the AT1R. In addition, AT-II-induced depletion of the most important intracellular antioxidant - glutathione - was demonstrated. In vivo, we were able to show that AT1aR knockout mice after AT-II treatment showed significantly more genome damage in the subfornic organ (SFO) than wild-type mice. The SFO is one of the few structures in the brain with an interrupted blood-brain barrier, which makes it accessible and particularly sensitive to circulating AT-II. In the recent literature, these genome damages were also observed in kidney and heart tissues and prove an additional genotoxicity of AT-II independent of AT1aR and consequently independent of blood pressure. In summary, this work shows that increased AT-II levels in neuronal cells cause genome damage due to NOX-produced superoxide. It is hoped that these results will one day help to decipher the complete development of VaD. / In den letzten Jahrzehnten ist die Akzeptanz stetig größer geworden, dass oxidativer Stress eine bedeutende Rolle bei der Entstehung von chronischen Erkrankungen, malignen Neoplasien sowie der Beschleunigung des Alterungsprozesses spielt. Als eine der häufigsten chronischen Erkrankungen ist Hypertonie oft mit einem fehlregulierten Renin-Angiotensin-Aldosteron-System assoziiert, welches chronisch oxidativen Stress verursacht. Bluthochdruck ist ein Risikofaktor für neurologische Erkrankungen wie der vaskulären Demenz (VaD) und viele neurologischen Störungen, einschließlich der VaD, haben eine ROS-assoziierte beziehungsweise inflammatorische Komponente in ihrer Entstehung. Unsere Arbeitsgruppe konnte bereits eine AT-II-induzierte Genotoxizität in Nieren- und Myokardzellen bzw. -Gewebe nachweisen. Ziel dieser Dissertation war es, einen möglichen Zusammenhang zwischen AT-II und Neurodegeneration zu untersuchen, welche durch eine neuronale Genotoxizität von AT-II ausgelöst wird. Zunächst zeigten wir in zwei neuronalen Zelllinien, dass AT-II eine Dosis-abhängige Genomschädigung verursacht. Nachfolgende Experimente konnten diese Toxizität auf NOX-produziertes Superoxid zurückführen, das nach Bindung von AT-II an den AT1R generiert wird. Zudem konnte ein AT-II-induzierter Verbrauch des wichtigsten intrazellulären Antioxidans – Glutathion - nachgewiesen werden. In vivo konnten wir zeigen, dass AT1aR-Knockout-Mäuse nach AT-II-Behandlung signifikant mehr Genomschäden im Subfornikalorgan (SFO) aufwiesen als Wildtypmäuse. Das SFO hat als eine der wenigen Strukturen im Gehirn eine unterbrochene Blut-Hirn-Schranke, was es für zirkulierendes AT-II zugänglich und besonders empfindlich macht. Diese Genomschäden wurden in der neueren Literatur auch in Nieren- und Herzgewebe beschrieben und belegen eine zusätzliche, AT1aR- und damit Blutdruck-unabhängige Genotoxizität von AT-II. Zusammenfassend zeigt diese Arbeit, dass erhöhte AT-II-Konzentrationen in Nervenzellen Genomschäden durch NOX-produziertes Superoxid verursachen. Die Hoffnung ist, dass diese Ergebnisse dabei helfen, eines Tages die vollständige Entstehung der VaD zu entschlüsseln.

Memoria para optar al Título Profesional de Químico Farmacéutico / El fibroblasto cardíaco (FC) es la célula encargada de la homeostasis de la matriz extracelular (MEC), cumpliendo un rol estructural en el tejido cardíaco. Además, participan en el proceso inflamatorio aportando con citoquinas y quimioquinas; y también de esta forma participan en la homeostasis. El FC es capaz de expresar moléculas de adhesión celular como por ejemplo ICAM-1 y VCAM-1 lo que favorece la respuesta inflamatoria y el reclutamiento de leucocitos. Se conoce que Angiotensina II (ANG II) es un fuerte inductor de respuestas inflamatorias, pero se desconoce si en FC aumenta ICAM-1 y VCAM-1. La Resolvina E1 (RvE1) es un mediador lipídico que participa activamente en la resolución de la inflamación, y se desconoce si la RvE1 modula las respuestas inflamatorias inducidas por ANG II. El objetivo de este trabajo fue estudiar las vías transduccionales que son activadas por un estímulo proinflamatorio como la ANG II y un estímulo proresolutivo como la RvE1, determinando además su rol como moduladores de la expresión de proteínas de adhesión en el FC. Se demostró que tanto ANG II como RvE1 activan la vía transduccional ERK1/2 y que RvE1 modula la actividad de ésta frente al estímulo de ANG II. Igualmente se evidenció que ANG II aumentó la expresión de ICAM-1 y que el pretratamiento con RvE1 disminuye este efecto de ANG II sobre el FC, lo cual se tradujo en la disminución de la adhesión de monocitos derivados del bazo (SMC) a FC inducida por ANG II. Los resultados de este trabajo sugieren que ANG II induce la adhesión de SMC a FC a través de ICAM-1, lo cual fue prevenido por RvE1 mediante la modulación de la actividad enzimática de ERK1/2 / RESOLVINA-E1 PREVENTS THE INCREASE OF ICAM-1 INDUCED BY ANGIOTENSIN II IN CARDIAC FIBROBLASTS Cardiac fibroblast (FC) is the cell responsible for the homeostasis of the extracellular matrix (ECM), fulfilling a structural role in cardiac tissue. In addition, they participate in the inflammatory process contributing with cytokines and chemokines; and also in this way they participate in homeostasis. FC is capable of expressing cell adhesion molecules such as ICAM-1 and VCAM-1, which favors the inflammatory response and leukocyte recruitment. It is well known that Angiotensin II (ANG II) is a strong inducer of inflammatory responses, but it is unknown if in FC increases ICAM-1 and VCAM-1. Resolvin E1 (RvE1) is a lipid mediator that actively participates in the resolution of inflammation, and it is unknown whether RvE1 modulates the inflammatory responses induced by ANG II. The objective of this work was to study the transduction signalling pathways that are activated by a proinflammatory stimulus such as ANG II and by a proresolutive stimulus such as RvE1, also determining its role as modulators of the expression of cell adhesion protein in the FC. We show that both ANG II and RvE1 activate the transduction pathway ERK1/2 and that RvE1 modulates its activity against the stimulation of ANG II. We also shows that ANG II increased the expression of ICAM-1 and that pretreatment with RvE1 decreased this effect of ANG II on FC, which translated into a decrease in the adhesion of monocytes cells derived from the spleen (SMC) to FC induced by ANG II. The results of this work suggest that ANG II induces the adhesion of SMC to FC through ICAM-1, and RvE1 prevented this effect by modifying the enzymatic activity of ERK1/2

Orientador: Karin Adriane Henschel Pobbe Ramos / Resumo: Este trabalho tem o objetivo de investigar a prática do ensino da gramática no Ensino Fundamental II por professores de português que atuam em uma escola da Rede Pública estadual na cidade de Bauru, Estado de São Paulo, buscando um redimensionamento do papel do ensino da gramática e uma ressignificação da aplicabilidade dos conteúdos gramaticais. As investigações partiram da hipótese inicial de que o trabalho gramatical, mesmo com novas teorias que tiram o foco de tal abordagem linguística, ainda ocupa a maior parte da atividade docente em sala de aula. Trata-se de uma pesquisa qualitativa, cuja coleta dos dados foi feita por meio de um questionário aplicado a professores de Língua Portuguesa de uma Unidade Escolar da Rede Pública do Estado de São Paulo e por meio da observação das aulas de dois desses professores. O trabalho pode contribuir para um melhor entendimento a respeito do papel do ensino da gramática como especificidade das aulas de língua portuguesa, buscando esclarecer quanto do trabalho docente é dedicado a esses conteúdos, bem como as concepções e abordagens que sustentam essas atividades. Partiu-se da premissa de que o problema não é o ensino sistematizado e metalinguístico da gramática, mas sim a abordagem teórico-metodológica utilizada, que, na maioria das vezes, é opressora e não contribui para uma perspectiva dialógica e interacionista do trabalho com língua em sala de aula. A partir dessas reflexões, foi proposta uma discussão a respeito dos diferentes es... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Detta kandidatexamensarbete handlar om en konstruktion av ett säkerhetsystem för tåg med fokus på programmering av systemet. Programmeringen görs genom att använda realtidsoperativsystemet μC/OS-II som idag är en av världens mest kända och använda för mikrokontrollers. Programmet laddas sedan ner till en mikrokontroll för att testas. Användningen av μC/OS-II blir också syftet av detta arbete då jag vill visa hur användbar den är. Rapporten börjar med att beskriva de olika komponenter som ingår i tågsystemet och fortsätter genom att ta upp det programmeringskod som är det viktiga i detta arbete. Arbetet avslutas med ett test av säkerhetsystemet där resultaten blev lyckad. Arbetet kan sägas representera en tillämpning av realtidsoperativsystem. Rapporten börjar med att beskriva de olika komponenter som ingår i tågsystemet och fortsätter genom att ta upp det programmeringskod som är det viktiga i detta arbete. Arbetet avslutas med ett test av säkerhetsystemet där resultaten blev lyckad. Arbetet kan sägas representera en tillämpning av realtidsoperativsystem.

Leopoldo Lituma Agüero (Perú) - Expositor / Raúl H. Ascencio (Perú) - Expositor / Eduardo Torres Arancivia (Perú) - Expositor / Luis Cáceres (Perú) - Moderador / Cuarto conversatorio del proyecto ¿Imágenes de poder o el poder de las imágenes?

Indiana University-Purdue University Indianapolis (IUPUI) / Sensing, integrating, and relaying signals from the environment through proteins, metabolites, and lipids to the lung are critical for proper development. Moonlighting proteins, such as AIMP1, are a unique subset that serves at least two independent physiological functions. Encoded by gene AIMP1, AIMP1 has two known functions: (1) C-terminus EMAP II domain of full-length AIMP1 can be secreted out of the cell to chemoattract myeloid cells; (2) intracellular full-length protein interacts with tRNA synthetases in protein translation. However, despite the linkage of protein expression levels of with several lung pathologies such as bronchopulmonary dysplasia (BPD), effectively targeting the protein encoded by AIMP1 has been a challenge due to poorly understood mechanisms. This thesis explores physiological, signaling, and immunological moonlighting mechanisms of first, the extracellular EMAP II then the intracellular AIMP1. Experiments utilize both in vitro and in vivo models, including a murine model of BPD and Cre-mediated exon-deletion knockout. Experimental results provide evidence that in the BPD model, EMAP II levels are elevated and sustained – first in bronchial epithelial cells then in macrophages. Mice exposed to sustained and elevated EMAP II protein levels resemble the BPD phenotype while neutralization partially rescued the phenotype, implying EMAP II as a potential therapeutic target against BPD. Results from studies exploring EMAP II’s signaling mechanism identify transient stimulation of JAK-STAT3 phosphorylation, commonly found in inflammation-resolving macrophages. In contrast, it induces unique transcriptional changes that are reversible both by JAK-STAT inhibitor and siRNA-mediated knockdown of Stat3. Studies using AIMP1 knockout mouse reveal a novel function for the intracellular AIMP1. AIMP1 knockout mice exhibited neonatal lethality with a respiratory distress phenotype, decreased type I alveolar cell expression, and disorganized bronchial epithelium, suggesting a role in lung maturation. In vitro experiments suggest that a portion of AIMP1 residing in the cell’s membrane interacts with various phosphatidylinositols and contributes toward F-actin deposition and assembly. Data from these experimental studies provide insight into how the various functions of the promiscuous AIMP1 gene affect lung development. These studies exemplify not only characterize novel moonlighting mechanisms of AIMP1, but also highlight the importance of characterizing moonlighting proteins to promote therapeutic preventions. / 2020-02-21

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21014号 / 医博第4360号 / 新制||医||1028(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 武藤 学, 教授 木原 正博, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ausgangspunkt der Arbeit ist die klinische Beobachtung, dass Patienten mit arteriellem Hypertonus vermehrt Nierenerkrankungen entwickeln. Dabei zeigten sich in der Subgruppenanalyse vor allem erhöhte Inzidenzen der Niereninsuffizienz und der Nierenzellkarzinome. Als möglicher Pathomechanismus steht das Renin-Angiotensin-Aldosteron-System (RAAS-System) im Vordergrund. Dabei wird postuliert, dass erhöhte Angiotensin II-Spiegel zu einem Missverhältnis zwischen den Oxidations- und Reduktionspartnern in der Zelle führen, wodurch sich das oxidative Potential der Zelle ändert, und es vermehrt zur Bildung von Radikalen (ROS) kommt, die meist ungepaarte Elektronen in der Valenzschale oder instabile Verbindungen enthalten, wodurch sie besonders reaktionsfreudig mit Proteinen, Lipiden, Kohlenhydraten und auch der DNA interagieren. In der Folge kommt es zu DNA-Veränderungen in Form von Doppel- oder Einzelstrangbrüchen, DNA-Protein-Crosslinks, Basenmodifikationen und Basenverlusten, wodurch sich ein hohes mutagenes Potential ergibt. Dieser Ansatz zur Pathophysiologie bestätigte sich auch an den hier verwendeten porkinen Nierenzellmodell. Dabei zeigte sich nicht nur eine Veränderung der genomischen Stabilität nach Exposition gegenüber erhöhten Angiotensin II-Spiegeln, sondern auch eine Veränderung der DNA in Abhängigkeit von der Expositionsdauer der Zellen. Als nächster Schritt konnte die Modulation der Transkriptionsfaktoren Nrf 2 und NF-κB durch die Behandlung mit Angiotensin II und Sulforaphan nachgewiesen werden. Bei der Behandlung mit Sulforaphan ließ sich eine Nrf 2-Induktion nachweisen mit vermehrter Expression von antioxidativen und detoxifizierender Enzyme. Weiterhin zeigte sich im Rahmen der Behandlung erniedrigte NF-κB-Level. Bei der Modulation durch Angiotensin II stellte sich zunächst ein signifikant erniedrigtes Level an Nrf 2 in den Zellen dar, das im Verlauf von 24 Stunden anstieg und konsekutiv ließ sich eine maximale Proteinexpression zwischen 24 und 48 Stunden messen. Weiterhin wiesen die Zellen, die mit Angiotensin II behandelt wurden, erhöhte NF-κB Mengen/Zelle auf. Zudem zeigte sich der Einfluss erhöhter Glucosekonzentrationen auf eine progrediente genomischen Instabilität, die Veränderung der Transkriptionsfaktoren mit erhöhter Nrf 2-Induktion und mit Deregulation des Transkriptionsfaktors NF-κB wurde durch die Behandlung mit Sulforaphan nachgewiesen. Aufgrund dieser Rolle in der Tumorgenese sind mittlerweile einige Bestandteile des NF-κB- und des Nrf 2-Signalweges und auch Nrf 2-Aktivatoren wie Sulforaphan wichtige Zielstrukturen für die Entwicklung neuer Medikamente und Therapieoptionen. Besonders zeigt sich hierbei die Wichtigkeit bei Diabetes induzierten kardiovaskulären Folgeschäden mit frühzeitiger medikamentöser Behandlung. / The starting point of this work is the clinical observation that patients with arterial hypertension develop more renal diseases. The subgroup analysis showed an increased incidence of renal insufficiency and renal cell carcinoma. The renin-angiotensin-aldosterone system (RAAS system) has been implicated as a possible pathomechanism. It is postulated that increased angiotensin II levels lead to a mismatch between the oxidation and reduction partners in the cell, which alters the oxidative potential of the cell and results in increased formation of radicals (ROS), most of which contain unpaired electrons in the valence shell or unstable compounds, making them particularly reactive with proteins, lipids, carbohydrates, and DNA. As a result, DNA changes occur in the form of double or single strand breaks, DNA-protein crosslinks, base modifications, and base losses, resulting in a high mutagenic potential. This approach to pathophysiology was also confirmed in the porky kidney cell model. This showed not only a change in genomic stability after exposure to elevated angiotensin II levels, but also a change in DNA depending on the duration of exposure of the cells. Next, modulation of the Nrf 2 and NF-κB transcription factors by angiotensin II and sulforaphane treatment was demonstrated. Treatment with sulforaphane showed Nrf 2 induction with increased expression of antioxidant and detoxifying enzymes. Furthermore, treatment revealed decreased NF-κB levels. When modulated by angiotensin II, cells initially showed a significantly reduced level of Nrf 2, which increased over the course of 24 hours. In addition, cells treated with angiotensin II demonstrated increased NF-κB levels. Moreover, the influence of increased glucose concentrations on progressive genomic instability, the alteration of transcription factors with increased Nrf 2 induction and with deregulation of the transcription factor NF-κB was demonstrated by treatment with sulforaphane. Because of this role in tumorigenesis, some components of the NF-κB and Nrf 2 signaling pathways, as well as Nrf 2 activators such as sulforaphane, are now important targets for the development of new drugs and therapeutic options. The importance of this is particularly evident in diabetes-induced cardiovascular complications with early drug treatment.

Type II diabetes mellitus (TIIDM) causes multiple complications under chronic hyperglycemia. Long term persistent exposure to elevated glucose conditions is considered one of the major factors for diabetic complications. Pathologically, mechanical and biochemical stimuli will induce a signaling cascade in cardiac fibroblasts, which causes myocardial fibrosis and leading to ventricular stiffness. Non-enzymatically, high levels of glucose can react with long-lived proteins, such as collagen to form advanced glycation end-products (AGEs). AGEs have been shown to be associated with many of the diabetic cardiovascular complications due to their interaction with the receptor for AGE (RAGE). AGE/RAGE activation stimulates the secretion of growth factors, promotes increased collagen production that leads to tissue fibrosis, and increased RAGE expression. The purpose of this study is to identify the role for Rap1a in regulating fibrosis under TIIDM conditions, as well as to offer insight into the AGE-RAGE signaling cascade definition for cardiovascular extracellular matrix remodeling under TIIDM condition. To test our hypothesis, both loss-ofunction and gain-ofunction based experiments were performed to manipulate Rap1a protein expression in AGE-RAGE mediated fibrosis. Also, we down-regulated the activity of downstream molecules in the AGE-RAGE signaling cascade, such as protein kinase C-ζ (PKC-ζ) and ERK1/2 by specific inhibitor treatments, to test their positions in AGE-RAGE mediated fibrosis pathway. To perform our experiment in vivo, we used high fat diet to feed Rap1a heterozygous mice in order to build a Rap1a heterozygous diabetic animal model. Our results showed that Rap1a protein plays a key role in AGE-RAGE signaling pathway under TIIDM, and changes in Rap1a activity altered the signaling pathway. Also, we found that PKC-ζ is the upstream player relatively to ERK1/2, and Rap1a is the upstream player for both PKC-ζ and ERK1/2. By understanding the role Rap1a played in AGE-RAGE signaling cascade, a new molecular mechanism is found possibly to reduce the cardiac fibrosis in TIIDM patients.

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