O TNF atua diretamente sobre a pineal inibindo a via biossintética da melatonina. De forma semelhante o LPS, potente ativador da resposta imune inata, tem efeito inibitório sobre a produção de melatonina e seu precursor. A supressão da síntese noturna de melatonina é admitida de forma a permitir a montagem da resposta inflamatória, tanto durante o dia como à noite. Dados do grupo tem demonstrado que o NFKB é um fator constitutivamente expresso na glândula pineal e que o ritmo diário de translocação pode ser determinante para o início da síntese de melatonina. A inibição de sua ativação potencia a síntese de melatonina enquanto que fatores que classicamente promovem a sua ativação inibem esta produção. Nesta dissertação, verificamos as moléculas envolvidas no reconhecimento e na de sinalização desta citocina na pineal. Demonstramos que a pineal está apta a responder ao TNF, células gliais da pineal e as células secretoras, os pinealócitos, expressam constitutivamente o receptor TNF-R1. O TNF promove a translocação nuclear dos dímeros p50/p50 e p50/RelA do NFKB em glândulas pineais em cultura. Confirmamos a ativação desta via em pinealócitos pela análise da proteína inibitória IKB. Vimos que a ativação desse fator é essencial para expressão da iNOS e a produção de NO induzida por TNF em pinealócitos isolados. Além disso, mostramos que o TNF promove a redução da expressão do receptor TNF-R1 na membrana de pinealócitos. Em resumo, mostramos neste trabalho que a pineal está instrumentalizada a responder ao TNF, e que os pinealócitos são alvos diretos para o seu reconhecimento. Confirmamos a relevância do fator NFKB na resposta da pineal em situações de injúria, ampliando o conceito de que a pineal está apta a detectar moléculas do processo inflamatório. / TNF acts directly on the pineal gland by inhibiting the biosynthesis of melatonin. Similarly, LPS, a potent activator of the innate immune response has an inhibitory effect on the production of melatonin and its precursor. The suppression of nocturnal melatonin synthesis is admitted to allow the full mounting of the inflammatory response both during the day and night. Data from our laboratory have shown that the NFKB is constitutively expressed in the pineal gland and that the daily rate of translocation can be determinant for the onset of melatonin synthesis. Inhibition of their activation potentiates melatonin synthesis whereas factors that promote its activation classically inhibit this production. In this dissertation we find the molecules involved in recognition and signaling of this cytokine in the pineal. We demonstrated that the pineal is responsive to TNF, the pineal glial cells and secretory cells, the pinealocytes, constitutively express the TNF-R1. TNF promotes nuclear translocation of p50/RelA and p50/p50 NFKB dimers in the pineal glands in culture. We confirmed the activation of this pathway in pinealocytes by analyzing the inhibitory protein IKB. We have seen that the activation of this factor is essential for iNOS expression and NO production induced by TNF in isolated pinealocytes. Furthermore we show that TNF promotes the reduction of the expression of TNF-R1 receptor on the membrane of pinealocytes. In summary, we show here that the pineal is instrumented to respond to TNF and that the pinealocytes are direct targets for its recognition. We confirm the relevance of NFKB in the pineal response in situations of injury extending the concept that the pineal is able to detect molecules in the inflammatory process and respond accordingly.

O TNF atua diretamente sobre a pineal inibindo a via biossintética da melatonina. De forma semelhante o LPS, potente ativador da resposta imune inata, tem efeito inibitório sobre a produção de melatonina e seu precursor. A supressão da síntese noturna de melatonina é admitida de forma a permitir a montagem da resposta inflamatória, tanto durante o dia como à noite. Dados do grupo tem demonstrado que o NFKB é um fator constitutivamente expresso na glândula pineal e que o ritmo diário de translocação pode ser determinante para o início da síntese de melatonina. A inibição de sua ativação potencia a síntese de melatonina enquanto que fatores que classicamente promovem a sua ativação inibem esta produção. Nesta dissertação, verificamos as moléculas envolvidas no reconhecimento e na de sinalização desta citocina na pineal. Demonstramos que a pineal está apta a responder ao TNF, células gliais da pineal e as células secretoras, os pinealócitos, expressam constitutivamente o receptor TNF-R1. O TNF promove a translocação nuclear dos dímeros p50/p50 e p50/RelA do NFKB em glândulas pineais em cultura. Confirmamos a ativação desta via em pinealócitos pela análise da proteína inibitória IKB. Vimos que a ativação desse fator é essencial para expressão da iNOS e a produção de NO induzida por TNF em pinealócitos isolados. Além disso, mostramos que o TNF promove a redução da expressão do receptor TNF-R1 na membrana de pinealócitos. Em resumo, mostramos neste trabalho que a pineal está instrumentalizada a responder ao TNF, e que os pinealócitos são alvos diretos para o seu reconhecimento. Confirmamos a relevância do fator NFKB na resposta da pineal em situações de injúria, ampliando o conceito de que a pineal está apta a detectar moléculas do processo inflamatório. / TNF acts directly on the pineal gland by inhibiting the biosynthesis of melatonin. Similarly, LPS, a potent activator of the innate immune response has an inhibitory effect on the production of melatonin and its precursor. The suppression of nocturnal melatonin synthesis is admitted to allow the full mounting of the inflammatory response both during the day and night. Data from our laboratory have shown that the NFKB is constitutively expressed in the pineal gland and that the daily rate of translocation can be determinant for the onset of melatonin synthesis. Inhibition of their activation potentiates melatonin synthesis whereas factors that promote its activation classically inhibit this production. In this dissertation we find the molecules involved in recognition and signaling of this cytokine in the pineal. We demonstrated that the pineal is responsive to TNF, the pineal glial cells and secretory cells, the pinealocytes, constitutively express the TNF-R1. TNF promotes nuclear translocation of p50/RelA and p50/p50 NFKB dimers in the pineal glands in culture. We confirmed the activation of this pathway in pinealocytes by analyzing the inhibitory protein IKB. We have seen that the activation of this factor is essential for iNOS expression and NO production induced by TNF in isolated pinealocytes. Furthermore we show that TNF promotes the reduction of the expression of TNF-R1 receptor on the membrane of pinealocytes. In summary, we show here that the pineal is instrumented to respond to TNF and that the pinealocytes are direct targets for its recognition. We confirm the relevance of NFKB in the pineal response in situations of injury extending the concept that the pineal is able to detect molecules in the inflammatory process and respond accordingly.

Noch immer ist die Behandlung von Neuropathien mit den gängigen therapeutischen Mitteln für viele Patienten sehr unbefriedigend. Als erfolgsversprechender therapeutischer Ansatz werden zur Zeit Wege erforscht, welche direkt in die molekularen Entstehungsmechanismen pathologischer Veränderungen und regenerationsfördernder Mechanismen eingreifen, um dadurch eine Heilung von Nervenschäden zu ermöglichen. Bisher sind die Erkenntnisse über diese Mechanismen nicht vollständig genug, um daraus eine sichere Behandlungsmöglichkeit abzuleiten. Wegweisende Erkenntnisse deuten sich allerdings durch Studien von unterschiedlichen Vertretern des Zytokinnetzwerkes an - darunter auch TNF-alpha - welche als molekulare Ursache neuropathischer Veränderungen diskutiert werden. In dieser Studie wurde an Knockoutmäusen der Einfluss des jeweiligen TNF-alpha-Rezeptors auf morphologische Veränderungen nach CCI (Chronic constriction injury) und Crush-Verletzung des N. ischiadicus untersucht. Nach 3,7,15 und 36 Tagen (CCI) bzw. 3,7 und 28 Tagen (Crush) wurden in Methylenblau gefärbten Semidünnschnitten intakte und degenerierte Nervenfasern, Makrophagen, Angioproliferation, Ödembildung udn Veränderung des Anteils nicht neuronaler Zellen lichtmikroskopisch beurteilt. Zusätzlich wurden Mac-1+ Makrophagen immunzytochemisch erfasst. Die Ergebnisse zeigten in beiden Modellen und bei beiden Knockouttypen eine starke axonale Schädigung, die von einer großen endoneuroalen Makrophagenansammlung begleitet war. Bei TNF-R1-/- Mäusen war eine stärkere und verlängerte Degeneration mit entsprechend höheren Makrophagenzahlen sichtbar. In den Immunzytochemischen Färbungen wiesen die TNF-R1-/- Mäuse hingegen den geringsten Makropahgenanteil auf.Trotz der starken Schädigung war die anschließende Regeneration im Gegensatz zu WT und TNF-R2-/- Mäusen besser. Die Ödembildung war bei den TNF-R2-/- nach CCI besonders stark ausgeprägt und von einer schlechten Regeneration gefolgt. Während die gefundenen Daten auf eine Beteiligung beider Rezeptoren während degenerativer Prozesse hindeuten, scheint insbesondere TNF-R2 regenerationsfördernde Effekte zu vermitteln. / Current Treatment of neuropathic disorders is still dissatisfactory for many patients. A promising approach is the investigation of agents that directly interfere with molecular development of pathologic changes and regeneration. Up to now, consolidated findings of the underlying mechanisms are not yet sufficent to allow therapeutic intervention. Pathbreaking findings come from studies investigating different agents of the cytokine network - as e.g. TNF-alpha - that are discussed as molecular cause of neuropathic changes. This study investigated the influence of both TNF-alpha-receptors on morphologic changes after CCI (chronic constriction injury) and crush-injury of the sciatic nerve of TNF-R-knockoutmice. After 3,7,15 and 36 days (CCI), and 3,7 and 28 respectively (Crush),intact and degenerating nerve fibers, macrophages, angioproliferation, development of edema and changes in the amount of non-neuronal cells were acquired by light microscopy of toluidin-stained semithin sections. Additionally Mac-1+ macrophages were acquired via immuncytochemically stained sections. The results showed strong axonal damage in both knockout-types accompanied by large amounts of endoneurial macrophages. TNF-R1-/-mice showed a longer degeneration phase including respectively higher amounts of macrophages. In contrast the TNF-R1-/-mice revealed the fewest amount of macrophages in immunocytochemical sections. Despite the strong damage better nerve regeneration was observed compared to WT and TNF-R2-/-mice. Formation of edema was pronounced in TNF-R2-/- after CCI and followed by poorly regeneration. Whereas these findings point to a participation of both receptors in degeneration, TNF-R2 seems to support regeneration.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-25T23:07:30Z No. of bitstreams: 1 PatriciaIngridMacedoDeCastro_DISSERT.pdf: 960843 bytes, checksum: 3369f0056a2e2ab1f071631a2015066a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-04T20:48:06Z (GMT) No. of bitstreams: 1 PatriciaIngridMacedoDeCastro_DISSERT.pdf: 960843 bytes, checksum: 3369f0056a2e2ab1f071631a2015066a (MD5) / Made available in DSpace on 2016-08-04T20:48:06Z (GMT). No. of bitstreams: 1 PatriciaIngridMacedoDeCastro_DISSERT.pdf: 960843 bytes, checksum: 3369f0056a2e2ab1f071631a2015066a (MD5) Previous issue date: 2015-11-30 / A pr?-ecl?mpsia ? uma doen?a que afeta 3-8% das mulheres gr?vidas. Os fatores de risco para essa doen?a n?o s?o completamente compreendidos, mas incluem desregula??o da resposta imune oriundos de defeitos na placenta??o, fatores ambientais e gen?ticos. O presente estudo teve como objetivo investigar associa??o varia??o na quantidade de receptores de citocinas pr?-inflamat?rias (IL-1R, IL-6R e TNF-?R) estariam envolvidos com a pr?-ecl?mpsia. Receptores de citocinas (IL-1R2, TNF-?R1 e IL-6R) foram avaliados em c?lulas mononucleares das gr?vidas normotensas (controle n=11) e gr?vidas com pr?-ecl?mpsia (PE, n=24). Mulheres com pr?-eclampsia tinham peso mais elevado no in?cio da gravidez (p=0.0171). Foi observado uma diminui??o de mon?citos cl?ssicos, mas n?o de mon?citos intermedi?rios e n?o-cl?ssicos na pr?-ecl?mpsia. A frequ?ncia dos receptores de citocinas proinflamat?rias IL-1R2, TNF-?R IL-6R aderidos a membrana das subpopula??es de mon?citos (cl?ssicos, intermedi?rios e n?o cl?ssicos) e linf?citos (CD3+CD4+ e CD3+CD8+) estavam diminu?das em pacientes com pr?-ecl?mpsia, quando comparados com gr?vidas normais. A redu??o na quantidade de receptores de citocinas IL-1R2, TNF-?R1 e IL-6R em mon?ciots e linf?citos pode ser um fator mantenedor do estado inflamat?rio na pr?-eclampsia. / Preeclampsia is a disease specific of human pregnancy that affects 3-8% of pregnant women, and it is one of the three leading causes of maternal mortality and morbidity. The disease is characterized by hypertension and proteinuria after the 20th week of gestation. The risk factors for this disease are not completely understood but appear to include dysregulation of the immune response arising from defects in placentation, environmental and genetic factors. This study aimed to determine whether the variation in the amount of proinflammatory cytokine receptors IL-1R2, IL-6R and TNF-?R1 would be involved in preeclampsia. They were recruited women with preeclampsia (n=24) and women who evolved during pregnancy without changes in blood pressure (n=12) were recruited. Clinical and laboratory data were collected. The cytokine receptors (IL-1R2, TNF-?R1 and IL-6R) were assessed in mononuclear cells isolated from peripheral blood using flow cytometry (Control = 8; PE = 24). C-reactive protein (CRP) was determined by CRP ultrasensitive method (Control = 7; PE = 18) was performed using sera pregnant women. Women with preeclampsia had higher weight at the beginning of the pregnancy (p=0.0171) and lower gestational age at delivery (0.0008). Classical monocytes were decreased in preeclampsia but not intermediate or non-classical monocytes. The frequency of IL-1R2 pro inflammatory cytokine receptors is decreased in women with PE only in the subpopulation of non-classical monocytes (p = 0.0011). TNF-?R1 receptor and IL-6R, had a decreased frequency in the three subpopulations of monocyte (classic, intermediate and non-classical) when compared to women with normal pregnancy. An increase in IL-1R2 receptor in TCD4+ lymphocytes, but a decrease in TNF-receptor and IL-6R in women with preeclampsia were found. No differences in the frequency of those receptors in CD3+/CD8+ in preeclampsia. There was no difference in C-reactive protein in preeclampsia. The reduction in the amount of IL-1R2, TNF- ?R1 and IL-6R monocytes and lymphocytes can be involved in the regulation of inflammation observed in preeclampsia, contributing to disease.