Orientador: Cláudia Vianna Maurer-Morelli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T13:52:07Z (GMT). No. of bitstreams: 1 Barbalho_PatriciaGoncalves_M.pdf: 8177547 bytes, checksum: 9a3923a19cb47c5609f933774ee68213 (MD5) Previous issue date: 2013 / Resumo: O Danio rerio é um peixe teleósteo popularmente conhecido como zebrafish que têm se destacado como modelo animal favorável para investigações genéticas devido à facilidade de manipulação in vivo, por sua transparência nas fases embrionária e larval e por seu desenvolvimento externo. Recentemente, foi demonstrado que o zebrafish é capaz de exibir padrão comportamental e alteração da atividade eletrográfica durante crise epiléptica como visto em roedores, tornando-o um modelo promissor para as investigações moleculares das epilepsias. Os estudos sobre os diferentes eventos já conhecidos das epilepsias no zebrafish estão apenas começando e, portanto, há ainda muito que ser investigado para uma melhor caracterização deste modelo para estudos em epilepsia. Estudos clínicos e em modelos animais mostraram que a crise epiléptica eleva os níveis da interleucina-1 beta e induz morte neuronal. Nesse sentido, o presente trabalho se propôs a investigar (i) o perfil temporal de expressão do transcrito do gene da interleucina-1 beta (il1b) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol por transcriptase reversa-PCR quantitativa e também, sua relação da expressão com a idade em que é realizada a indução da crise no cérebro imaturo e (ii) a morte neuronal pela histoquímica do Fluoro-Jade B (FJB) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol. Neste trabalho conseguimos estabelecer com sucesso as condições ideais para o acasalamento desta espécie e obtenção de embriões e, para a criação de larvas. Além disso, padronizamos o processamento histológico para a criosecção do cérebro do zebrafish. A análise temporal do transcrito do gene il1b revelou um padrão de expressão similar ao observado em roedores. A marcação por FJB não identificou inequivocadamente a presença de morte neuronal após crise epiléptica / Abstract: Danio rerio is a teleost fish popular named as zebrafish that has emerged as a suitable animal model for genetic investigations due to its features such as: facility for in vivo manipulation, transparency during embryonic and larval stages and external development. Recently, It was demonstrated that larvae and adult zebrafish (Danio rerio) seizure model mimics the behavior, eletrographic and c-fos features that are well established in rodent models of epilepsy, making the zebrafish as a promising model for studying the molecular mechanisms underlying epilepsies. However, the potential of the zebrafish model for epilepsy studies has been partly described since the molecular changes and neuronal loss after seizure has not yet been investigated. Proinflammatory cytokines, such as interleukin-1beta (il1b), has shown to be upregulated in surgical specimens of pharmacoresistent patients and in experimental rodent models of seizure, however the profile of this cytokine in zebrafish model for epilepsy is unknown. For this reason, and in view of the importance of inflammatory response in the pathophysiology of epilepsy, we sought to investigate the temporal transcript profile of interleukin-1b (il1b), a proinflammatory cytokine, in adult and immature (larvae) zebrafish brain after Pentylenetetrazole-evoked seizure, as well its aged-related expression in the developing brain by reverse transcriptase-quantitative PCR and neuronal death by Fluoro-Jade B (FJB) staining in adult zebrafish brain. Zebrafish breeding was successfully established during this work as well as the protocol for histological procedures using the zebrafish brain. Our results showed a short up-regulation of il1b mRNA levels after seizure in immature and adult zebrafish brain similar as with those patterns observed in rodent models. FJB did not show a reliable neuronal death staining in the zebrafish brain after seizure / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Acute Lung injury (ALI) and the more severe acute respiratory distress syndrome (ARDS) are respiratory maladies that present immense clinical challenges. ALI affects 200,000 individuals annually and features a 40% mortality rate. ALI can be initiated by both pathogenic and sterile insults originating locally in the lungs or systemically. While immense research has been poured into this disease in an effort to find a therapeutic strategy, the heterogeneously diffuse nature of the disease has not yielded a cure for the disease. Death from this disease is strongly attributed to reduced gas exchange from a severely compromised alveolar-capillary barrier. The only way currently to manage this disease is through enhanced ventilation and hyperoxic therapy. Hyperoxic therapy is a common treatment given to over 800,000 patients each year to treat respiratory maladies such as ALI. Prolonged exposure to oxygen at high concentrations results in the development of a condition known as hyperoxic acute lung injury (HALI). In this disease, the formation of reactive oxygen species damages healthy tissue and impairs gas exchange. Hyperoxia is also a well-documented murine sterile lung injury model that replicates the symptoms of ALI in lung injury patients. The ability of non-lethal dosages of hyperoxia to resolve without lung fibrosis also enables the study of molecules associated with ALI resolution and repair, a process not clearly understood. Inflammation in ALI is associated with disease progression, however pharmaceutical interventions aimed at targeting the inflammatory cascade have failed in clinical trials for ALI. Recent reports point to an aberrant injury resolution mechanisms that may be more strongly correlated with morbidity and mortality. There seems to be a homeostatic imbalance between endogenous inflammation progression and resolution initiation. This is especially the case with HALI, as significant ROS generation results in depletion of redox regulating antioxidants. Resolution mechanisms associated with ALI in the oxygen toxicity setting is poorly understood. Polyunsaturated fatty acids such eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential fatty acids that show immense antioxidant and anti-inflammatory action in cases of acute injury. The lung mucosa is rich in DHA and following inflammatory insult DHA is readily converted to resolution phase interaction products (resolvins), which have shown immense proresolutionary potential in recent reports of acute injury. In the presence of aspirin, more potent and longer-acting aspirin-triggered resolvins are formed. The effects of resolvins and their aspirin triggered epimers have not been studied in an oxygen toxicity setting and are the focus of this dissertation. For the first time, we show that one of these resolvin molecules, aspirin triggered resolvin d1 (AT-RvD1), can enhance resolution of hyperoxic acute lung injury. In vitro results reveals that AT-RvD1 treatment resulted in reduced interaction of two key players in the HALI inflammatory cascade, the macrophage and alveolar epithelium. AT-RvD1 was able to blunt macrophage cytokine secretion as well as inhibit epithelial cell cytokine secretion and adhesion molecule expression. More importantly, AT-RvD1 blunted cytokine mediated leukocyte-epithelial cell interaction in vitro. In a sublethal hyperoxic injury model, mice given AT-RvD1 following hyperoxia exposure displayed reduced HALI pathological severity. ATRvD1 treatment resulted in reduced alveolar-capillary permeability, tissue inflammation, proinflammatory mediator secretion, epithelial cell death, and leukocyte influx. Taken together these novel results demonstrate the therapeutic potential of resolvins in the oxygen toxicity setting. These results also arouse the idea that resolvins could be used to lessen the comorbidities associated with oxygen therapy and improve recovery times of ALI patients.

The P2X7R is a functionally distinct member of the P2X non-selective cation channels and has been implicated in the initiation of immune responses. One of the most extensively characterised immune responses of the receptor is to signal the rapid aggregation of the inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived macrophages [BM-MΦ]). Expression of the P2X7R has been identified in all three populations both at the transcriptional (P2X7A variant) and protein levels. Activation with lipopolysaccharide (LPS) (2h) induced a rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL- 1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly lower levels. Furthermore, like with IL-1β, LPS did not induce IL-1α release in BM-DC. Interestingly, subsequent challenge with ATP evoked IL-1α release in BM-DC alone, with little or no detectable levels observed in activated BM-MΦ. This rapid IL-1β release (but not IL-6) was potently inhibited in both macrophages and DC with a P2X7R-specific inhibitor (A-740003) providing evidence that is predominantly a P2X7R-driven process. Treatment with A-740003 also potently inhibited IL-1α release from BM-DC suggesting that the ATP-P2X7R and caspase-1 activation might have a role in the release of the cytokine. Expression of gain-of-function P2X7K and loss-of- function P2X7J splice variants has been identified in both BM-DC and BM-MΦ, at the level of transcription. The possibility that a differential baseline or LPS-induced expression (at the transcriptional level) of P2X7J and P2X7K variants accounts for the diverse cytokine responses observed in BM-DC and BM-MΦ was also explored. However, the levels of expression for the various splice variants of interest (P2X7K and P2X7J) were found to be similar between the two cell types. The results of these investigations identify some subtle but intriguing differences in the mechanism of P2X7R activation and IL-1 release between DCs and macrophages. Purinergic signalling is increasing being implicated in the regulation of immune responses both in potentiating or suppressing inflammation. However, further work is required to decipher how the dynamic interplay between different purines can influence the immune activation of different cell types and indeed different cell subsets.

P2X7-Rezeptoren stellen Schlüsselmoleküle bei der Entstehung und Aufrechterhaltung proinflammatorischer Zustände, chronischer Schmerzen sowie der neuroglialen Kommunikation dar. Ihre Aktivität wird durch eine Vielzahl zellbiologischer Mechanismen beeinflusst. Dazu gehört die allosterische Modulation durch extrazelluläre niedermolekulare Stoffe. Die Entwicklung selektiver und potenter P2X7-Modulatoren ist darum Gegenstand intensiver Forschung. Bisher sind jedoch keine Pharmaka für die klinische Anwendung verfügbar. Die Untersuchung zugelassener pharmakologischer Substanzen in einem akademischen Screening erbrachte eine hohe Trefferrate für P2X7-Rezeptoren. In dieser Arbeit wird die P2X7-Wirkung einiger der potentesten allosterischen Modulatoren genauer charakterisiert. Das Antihistaminikum Clemastin stellt dabei einen positiven allosterischen Modulator dar, der den Rezeptor gegenüber niedrigeren ATP-Konzentrationen sensibilisiert. Ivermectin, ein häufig angewendetes Anthelminthikum, konnte als potenzierender Modulator des humanen P2X7-Rezeptors charakterisiert werden. Mit den Phenothiazinen Prochlorperazin und Trifluoperazin zeigen sich schließlich ZNS-gängige Inhibitoren der ATP-induzierten P2X7-Aktivität, die für weiterführende in vivo-Untersuchungen hilfreiche pharmakologische Werkzeuge darstellen können.:1. Bibliographische Beschreibung 4 2. Abkürzungsverzeichnis 5 3. Einleitung 6 Überblick über die purinerge Signalübertragung 6 Pathophysiologische Bedeutung des P2X7-Rezeptors 8 Speziesspezifische Eigenschaften des P2X7-Rezeptors 10 P2X7-Rezeptoren in der Pharmakotherapie 10 Rationale der Studie 11 Clemastin 12 Ivermectin 12 Die Neuroleptika Prochlorperazin und Trifluoperazin 13 4. Publikationen 15 5. Zusammenfassung der Arbeit 64 6. Literaturverzeichnis 68 7. Anlagen 74 Erklärung über die eigenständige Abfassung der Arbeit 74 Lebenslauf 75 Danksagung 76

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.

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Mehrere Studien haben gezeigt, daß die Cyclooxygenase-2 (COX-2) eine bedeutende Rolle sowohl bei Entstehung als auch Progression maligner Tumoren spielt. COX-2-Inhibitoren werden bereits in klinischen Studien zur Krebstherapie getestet. COX-2 ist die induzierbare Isoform der Cyclooxygenase - dem Schlüsselenzym der Synthese von Prostaglandinen und anderen Eicosanoiden. Im Tier- und Zellkulturmodell konnten COX-Hemmer anti-Tumor-Effekte hervorrufen. Es ist jedoch unklar, ob diese Effekte durch Hemmung des COX-Enzyms oder durch COX-unabhängige Mechanismen vermittelt werden. Wir untersuchten daher die Auswirkung der COX-Inhibition zum einen durch den selektiven COX-2-Hemmer NS-398 sowie zum anderen durch COX-Isoform-spezifische RNA-Interferenz (RNAi) in zwei humanen Ovarialkarzinomzellinien (OVCAR-3 und SKOV-3). OVCAR-3 zeigte eine konstitutive COX-1-Expression und eine durch IL-1beta induzierbare COX-2-Expression. SKOV-3 war COX-1- und COX-2-negativ. IL-1beta führte bei OVCAR-3 zu einer vermehrten Produktion von Prostaglandin E2 (PGE2), die durch eine gegen die COX-2 gerichtete siRNA gehemmt werden konnte, wohingegen COX-1-siRNA keinen Effekt hatte. Das deutet darauf hin, daß die COX-2 die Hauptquelle von PGE2 in OVCAR-3 ist. 1mikroM NS-398 waren ausreichend, um die PGE2-Produktion und somit auch die COX-2 in OVCAR-3 zu inhibieren. Höhere Konzentrationen NS-398 (>10mikroM) hatten einen antiproliferativen Effekt. Auch in der COX-2-negativen Zellinie SKOV-3 trat diese Wachstumshemmung auf; sie war nicht durch exogene Zufuhr von PGE2 (10mikroM) reversibel. Durchflußzytometrische Zellzyklusanalyse ergab, daß der Wachstumshemmung in beiden Zellinien ein G0/G1-Zellzyklusarrest zugrunde liegt. Dagegen führten weder COX-1- noch COX-2-Ausschaltung durch RNAi zu ähnlichen Auswirkungen auf Proliferation bzw. Zellzyklus. Diese Ergebnisse zeigen, dass ein COX-2-unabhängiger Mechanismus für den durch NS-398 induzierten G0/G1-Arrest verantwortlich ist. / Several studies have provided evidence that the enzyme Cyclooxygenase-2 (COX-2) plays an important role in tumor development and progression. COX-2-inhibitors are already evaluated in clinical trials as cancer therapeutics. COX-2 is the inducible isoform of cyclooxygenase - the rate-limiting enzyme in the synthesis of prostaglandins and other eicosanoids. COX-inhibitors cause antitumor effects in animal models and in cell culture experiments. However, it is not clear, whether these effects are due to inhibition of the COX-enzyme or mediated via a COX-independent mechanism. We therefore investigated the effects of COX inhibition by the selective COX-2-inhibitor NS-398, as well as by COX-isoform specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1, and an inducible COX-2 expression. COX-2 was induced through stimulation with Interleukin-1beta, leading to production of high levels of Prostaglandin E2 (PGE2). SKOV-3 cells were negative for both COX isoforms. Selective COX-2-suppression by RNAi reduced PGE2 production in OVCAR-3, whereas COX-1-siRNA had no effect on PGE2 synthesis. Thus, COX-2 is the main source of PGE2 in OVCAR-3 cells. In these cells, 1microM NS-398 was sufficient to completely inhibit PGE2-synthesis - and thus the activity of the COX-2 enzyme. Increasing amounts of NS-398 (>10microM) had an antiproliferative effect. This growth inhibition was also observed in the COX-negative cell line SKOV-3, it could not be reverted by exogenous addition of PGE2 (10microM). Flowcytometric analysis of the cell cycle revealed that this growth inhibition was based on a G0/G1-cell-cycle-arrest. In contrast, suppression of COX-1 or COX-2 by RNAi had no effect on proliferation or cell cycle progression. These results suggest that a COX-independent mechanism is responsible for the G0/G1-arrest induced by NS-398.