O reconhecimento da flagelina é compartilhado pelo receptor transmembrânico TLR5 e citosólico NAIP5/NLRC4. Entretanto, pouco se sabe sobre os mecanismos efetores individuais induzidos a partir do reconhecimento extra e intracelular da flagelina. Aqui, nós demonstramos que macrófagos estimulados com a flagelina citosólica (FLA-BSDot) induziu a expressão de iNOS, enzima responsável pela produção do óxido nítrico (NO). A expressão de iNOS foi dependente do eixo NAIP5/NLRC4/caspase-1 e independente de IL-1β, IL-18 e MyD88, descartando a via de ativação dos TLRs. Ainda, esta via não requer a ativação do fator de transcrição IRF-1, mas envolve a ativação do NF-kB, assim como a clivagem da enzima PARP-1 (poly(ADP-ribose)polymerase-1). Por fim, avaliamos a relevância biológica desta via no controle das infecções por L. pneumophila e S. Typhimurium, dados que definem um mecanismo efetor adicional no controle de patógenos. / Recognition of flagellin is shared by transmembranic TLR5 and cytosolic NAIP5/NLRC4. However, little is known about the individual effector mechanisms induced by extra and intracellular flagellin. Here, we have demonstrated that cytosolic flagellin-stimulated macrophages (FLA-BSDot) induced iNOS expression, an enzyme responsible for the production of nitric oxide (NO). iNOS expression was dependent of the NAIP5/NLRC4/caspase-1 axis and independent of IL-1β, IL-18 and MyD88, discarding TLRs signaling pathway. Still, this pathway do not require the activation of IRF-1 transcriptional factor, but involves NF-kB activation as well as the cleavage of the enzyme, PARP-1 (poly(ADP-ribose)polymerase-1). Finally, we have evaluated the biological relevance of this pathway in the control of the infections by L. pneumophila e S. Typhimurium, which define an additional effector mechanism to the control of pathogens.

A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-<font face=\"Symbol\">kB. / Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-<font face=\"Symbol\">kB.

Clostridium difficile (CD) est l’entéropathogène le plus fréquemment responsable d'infections nosocomiales intestinales post-antibiotiques. L'apparition de cas graves liés à l'émergence de souches hypervirulentes ces dernières années a contribué à accroître la morbidité et la mortalité. Les toxines TcdA et TcdB contribuent directement aux lésions intestinales associées aux infections à CD (ICD), mais d'autres facteurs bactériens sont nécessaires à l’adhésion et la colonisation intestinale. Les flagelles de CD, qui confèrent la mobilité et la chimiotaxie, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l’hôte et aux lésions de la muqueuse. En effet, en activant le récepteur TLR5, les flagelles peuvent provoquer l'activation des cascades de signalisation cellulaire des MAPK et de NF-κB conduisant à la sécrétion de cytokines pro-inflammatoires. Notre objectif était d’étudier ce rôle potentiel des flagelles de CD in vitro et in vivo. Nous avons montré que l'interaction de la flagelline de CD avec le TLR5 active principalement la voie de NF-κB, et, dans une moindre mesure, la voie des MAPK, conduisant ainsi à la régulation de l'expression de gènes pro-inflammatoires et à la synthèse de médiateurs pro-inflammatoires. De plus, en utilisant un modèle murin d’ICD, nous avons démontré un effet synergique des flagelles et des toxines dans l’induction d’une réponse inflammatoire de la muqueuse caecale. Dans ce modèle, l'absence de flagelles diminue considérablement le degré d'inflammation de la muqueuse caecale et la seule présence de toxines, sans flagelles, ne suffit pas à provoquer d’importantes lésions épithéliales. Ces résultats mettent en évidence le rôle des flagelles de CD dans l’induction d’une réponse inflammatoire intestinale en synergie avec l’action des toxines bactériennes sur l'épithélium. / Clostridium difficile (CD) is the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. The appearance of severe cases related to the emergence of hypervirulent strains these last years has contributed to increased mortality and morbidity. The CD TcdA and TcdB toxins contribute directly to CD infection (CDI)-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the gut. The CD flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. Our objective was to study the potential role of CD flagella in vitro and in vivo. We reported that the interaction of CD flagellin-TLR5 predominantly activates the NF-κB, and, in a lesser degree, the MAPKs pathways, thus leading to up-regulation of pro-inflammatory gene expression and subsequent synthesis of pro-inflammatory mediators. Moreover, by using a mouse model of CDI, we demonstrated a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreased the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of CD flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.

L’activation de la signalisation NF-κB par de nombreux immunorécepteurs met en jeu un large signalosome. Afin de propager cette signalisation en réponse à différents stimuli, l’oligomérisation d’adaptateurs pourvus de domaines d’interaction protéine-protéine est nécessaire. Alors que certains adapteurs sont communs d’autres sont spécifiques à un immunorécepteur donné. Une des principales caractéristiques partagées par toutes ses protéines est leur capacité à être poly-ubiquitinylé de façon non-dégradative afin d’aboutir à une activation optimale de NF-κB. Ce projet avait pour objectif d’identifier de nouvelles déubiquitinylases impliquées dans la signalisation NF-κB. C’est ainsi que nous avons identifié USP34 comme étant un régulateur négatif de la signalisation NF-κB induite par le TCR dans des cellules Jurkats, une lignée de lymphocytes T immortalisés. Nos données suggèrent un modèle dans lequel USP34 permet d’éviter l’activation excessive de NF-κB, en agissant directement ou indirectement sur les dimères NF-κB/IκBα, en aval d’IKK, et en modulant l’affinité du facteur de transcription pour l’ADN. Parallèlement, l’étude du microenvironnement des membranes endocellulaires responsables du recrutement des signalosomes formés en réponse à l’activation du TCR, du TNFR et du CD40 a permis l’identification d’une protéine - clé de la signalisation NF-κB, la MTDH. Cette protéine du RE s’est révélée être un relais déterminant pour l’activation d’IKK et donc la propagation du signal NF-κB. / Large signalosome assembly is a prerequisite for NF-κB signaling upon engagement of various immunoreceptors. Adaptor proteins containing protein-protein interaction domains oligomerise in response to such stimuli in order to propagate signaling. Each immunoreceptor uses distinct adaptors, as well as common ones, to achieve that. The main characteristic shared by these proteins is their ability to undergo poly-ubiquitinylation in a non-degradative manner, leading to optimal NF-κB activation. In this work, we aimed to identify novel deubiquitinylating enzymes that control ubiquitinylation status. That is how USP34 came up to be a negative regulator of NF-κB signaling in TCR-activated Jurkat cells, a T lymphocyte cell line. Our data suggest a model whereby USP34 prevents excessive NF-κB activation by acting rather late, directly or indirectly on the NF-κB:IκBα dimers, downstream of IKK, altering transcription factor DNA binding affinity. In parallel, studies of the endocellular membrane microenvironment that hosts mature signalosomes in response to TCR-, TNFR- and CD40 ligation led to the identification of an ER-residing protein, Metadherin (MTDH), which seems to globally integrate signaling before forwarding it to downstream pathway components able to activate IKK.

Polychlorinated biphenyls (PCBs) are nongenotoxic hepatic tumor promoters. PCBs have been shown to cause oxidative stress, but the exact mechanism by which PCBs exert their tumor promoting activity is not clear. In our study, PCB-153, a non-coplanar congener, caused a transient increase in hepatic NF-B DNA binding activity and cell proliferation, while PCB-77, a coplanar congener, showed no effect. Our second study using a mouse model that was deficient in the p50 subunit of NF-kB (p50-/-) showed that NF-kB contributes to the changes in hepatocyte proliferation and apoptosis in response to PCB-153 treatment: a single dose of PCB-153 increased hepatic NF-B activity and cell proliferation in wild type mice, but not in the p50-/- mice; longer-term treatment with PCB-153 increased cell proliferation in p50-/- mice, but this increase was less than that in the wild type. In addition, p50-/- livers had more apoptosis than in the wild type, and PCB-153 inhibited apoptosis in the p50-/- livers. p50-/- livers had less cyclin D1 protein than the wild type, but that the mRNA levels were same. Bcl-xL protein was not changed by PCB-153, and wild type and p50-/- mice had the same level of Bcl-xL protein. In the third study, PCB-77 caused an increase in hepatic NF-kB DNA binding activity and cell proliferation during the promotion stage, and this increase was blocked by dietary supplementation of vitamin E, but the number and volume of placental glutathione S-transferase (PGST)-positive foci were slightly, though insignificantly, increased in the same animals. The apparent conflict could be due to different effect in different cells: high level vitamin E significantly inhibited PCB-77-induced cell proliferation in normal hepatocytes, while this inhibitory effect was much less in the PGST-positive hepatocytes. In conclusion, our studies show that a non-coplanar PCB can cause an increase in hepatic NF-kB DNA binding activity in rats and mice, and this increase contributes to the change in cell proliferation and apoptosis. Dietary vitamin E supplementation did not show protective effect on the formation of altered hepatic foci that were promoted by PCBs, although vitamin E supplementation decreased PCBs-induced hepatic NF-kB activation and cell proliferation.

ABSTRACT Metformin is the most common drug used against type 2 diabetes mellitus. However, it was only recently shown, in human and rat hepatocytes, that metformin-like 5-aminoimidazole-4-carboximide ribonucleoside (AICAR), acts via activation of the AMP-activated protein kinase (AMPK), an enzyme that plays a central role in lipid metabolism. Although it is well known that metformin is used in the treatment of type 2 diabetes and results in significant fat loss, no study has investigated the effects of this drug on adipocytes. In this report I studied the effects of metformin on the formation of fat deposits in mouse 3T3-L1 preadipocytes, as well as its effects on the activation of AMPK in these cells. Our results suggested that metformin significantly inhibits the transformation of pre-adipocytes into adipocytes. This is achieved via the inhibition of intracellular lipid accumulation during adipogenesis. In addition to its inhibition of intracellular lipid accumulation, metformin induced a significant increase in the phosphorylation of AMPK. It has been shown that AMPK activation with AICAR results in the inhibition of the nuclear factor-κB (NF-κB) induced gene expression. Since NF-κB is the key nuclear factor used by HIV-1 during the initiation of its gene transcription, I investigated the possibility of inhibiting HIV-1 replication in U1 cells with metformin and AICAR. I observed that AICAR and metformin inhibit HIV-1 replication in U1 cells. This inhibition wasparalleled by the accumulation of NF-κB in the cytoplasm of AICAR and metformin treated cells, and at the same time by a significant decrease in the concentration of this nuclear factor in the nucleus of these cells. However, I failed to observe any phosphorylation of AMPK by metformin and AICAR in U1 cells. In conclusion, metformin inhibits adipogenesis in mouse adipocytes and this inhibition is likely to take place via the activation of AMPK. AICAR and metformin have inhibitory properties against HIV-1 replication. However, this inhibition does not seem to be by the activation of AMPK.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-09-22T19:02:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-09-29T19:20:07Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Made available in DSpace on 2016-09-29T19:20:08Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) Previous issue date: 2016-09 / O câncer é apontado como a segunda maior causa de morte em todo o mundo, com previsão de em breve ser tornar a primeira. O câncer de próstata está entre os 5 tipos de câncer mais diagnosticados em homens, sendo que o câncer hepático está em segundo lugar em taxa de mortalidade entre homens e mulheres. Indícios apontam para uma ativação das vias da inflamação associados a uma inibição das vias de morte celular no processo de carcinogênese. A regulação destas vias torna-se alvo importante e complementar no controle do câncer, sendo estimulada a busca de biomoléculas com este potencial. As plantas são importante fonte de descoberta de novas biomoléculas com ampla utilização para o tratamento de diversas patologias. A família Myrtaceae possui diversas espécies que são apontadas como fortes candidatos em potencial nesta busca, incluindo as do gênero Myrciaria. A espécie Myrciaria plinioides não possui estudos referentes suas propriedades terapêuticas ou a atuação em vias de sinalização envolvidas na inflamação ou na carcinogênese. Neste contexto, este estudo teve por objetivo avaliar a atividade do extrato etanólico de M. plinioides em células de carcinoma hepatocelular (HepG2) e próstata (LNCaP) , através da análise de expressão dos marcadores p38-α, pp38-α, NF-κB e caspase-3, envolvidos na carcinogênese, e o efeito sobre a viabilidade celular através do método de MTT. A viabilidade das células foi alterada significativamente, em ambas as linhagens celulares quando tratadas com o extrato etanólico. A análise da expressão proteica demonstra significativa inibição da expressão de p38-α e caspase-3 nas células LNCaP, quando tratadas com extrato etanólico de M. plinioides seguido de LPS. Em células HepG2, somente houve alteração na expressão da caspase-3 na concentração de 200 μg/mL, com ou sem adição de LPS após tratamento com extrato. Os resultados deste estudo demonstraram redução da viabilidade celular nas duas linhagens tumorais, expressão diferenciada de proteínas envolvidas em apoptose, o que leva a indícios da ativação de mecanismos distintos pelo extrato em cada tipo celular. Estudos futuros para averiguar o mecanismo celular e a indução de morte em células tumorais de câncer de próstata e de fígado podem contribuir para a identificação e elucidação de novas biomoléculas com potencial antitumoral. / Cancer is touted as the second leading cause of death worldwide, forecast to soon be making the first. Prostate cancer is among the five most cancers diagnosed in men, and liver cancer is second in mortality between men and women. Evidence points to the activation of pathways of inflammation associated with an inhibition of cell death pathways in carcinogenesis. The regulation of these pathways becomes important and complementary target in cancer control, and stimulated the search for biomolecules with this potential. The plants are important source of discovery of new biomolecules with wide use for the treatment of various diseases. The Myrtaceae family has many species that are identified as potential candidates strong in this search, including the Myrciaria genre. The species Myrciaria plinioides not have studies on its therapeutic properties or performance in signaling pathways involved in inflammation or carcinogenesis. In this context, this study aimed to evaluate the activity of the ethanol extract of M. plinioides in hepatocellular carcinoma cells (HepG2) and prostate (LNCaP) by expression analysis of p38-α markers, PP38-α, NF-kB and caspase-3, involved in carcinogenesis, and the effect on cell viability by the MTT method. The viability of cells was significantly altered in both cell lines when treated with ethanolic extract. Protein expression analysis demonstrates significant inhibition of p38-α expression and caspase-3 in LNCaP cells, when treated with ethanolic extract of M. plinioides followed by LPS. In HepG2 cells there was only a change in the expression of caspase-3 at a concentration of 200 / ml, with or without addition of LPS after treatment with extract. The results showed reduction of cell viability in both tumor lines, differential expression of proteins involved in apoptosis, leading to evidence of activation by distinct mechanisms in each extract cell type. Further studies to investigate the cellular mechanism, and induction of death in tumor cells of prostate and liver cancer may contribute to the identification and elucidation of new biomolecules with antitumor potential.

The Royal Library (KB) of Sweden harbours great collections of different objects through a special law from 1661. This master degree paper is exploring a part of KB’s digitisation of those collections. It deals with the questions about what the KB had chosen for digitisation, why those choices were maid and who is responsible for making these decisions. The paper will also for example seek out different patterns and trends in the information. The questions were answered through examining10 of the KB’s earlier digitisation projects and tests with qualitative methods: personal interviews, interviews by phone and questionnaires. The interviews and questionnaires were performed with informants at KB. General information about KB’s digitisation and the results from the interviews and questionnaires are presented partly with each individual test and project and partly in two analyse tables. KB had chosen different kinds of objects from their collections, for example older books and posters. The main reasons for digitising objects and collections were preserving and giving access to the collections and objects. The copyright were important to notice and there were also marketing of the collections and the testing of technology. The people responsible for the decision-making were partly KB’s head council and partly the informants involved with the projects. / Uppsatsnivå: D

[Introductory paragraph] The present generation must confront a challenge. The challenge is to determine what it must do for the sake of future generations. This challenge is quite puzzling because the present generation, like its predecessors, will pass on to future generations a complex mix of goods, inventions, institutions and opportunities containing a range of benefits and burdens. In this thesis, I focus on one key intergenerational problem – anthropogenic climate change – considering some of the questions of intergenerational justice that it raises. While it has not always been the case, climate and climate change have recently taken on new significance as a process to which humans can, and in fact do, contribute. More specifically, while paleoclimatic data show substantial variation in the Earth’s climate (Masson- Delmotte, Schulz, Abe-Ouchi, Beer, Ganopolski, J.F. González Rouco, E. Jansen, et al., 2013: 385), an ever-growing mass of evidence shows that human activity – particularly the sustained emission of greenhouse gases (GHGs) – is beginning to change the global climate, with much greater changes still to come (IPCC, 2013b: 4, 19ff). This produces what is known as anthropogenic climate change, “a change in the state of the climate that can be identified (e.g., by using statistical tests) by changes in the mean and/or the variability of its properties, and that persists for an extended period, typically decades or longer”, and that results from human activities (IPCC, 2013a: 1448, 1450).