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Made available in DSpace on 2015-07-22T20:49:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-25. Added 1 bitstream(s) on 2015-08-11T03:25:37Z : No. of bitstreams: 1 Publico-048.pdf: 2055730 bytes, checksum: 9a2ea073a704c8893b8b965d7f234ff5 (MD5) / Introdução: A pineal, moduladora da reprodução em mamíferos, influencia as gônadas e a ciclicidade estral por intermédio de alterações no eixo hipotálamo-ovário-adrenal. Objetivo: analisar a histomorfologia e a imunohistoquímica da adrenal de ratas pinealectomizadas e tratadas com melatonina. Material e métodos: foram utilizadas 40 ratas adultas, divididas em 4 grupos de 10 animais cada, a saber: GI – sem intervenção cirúrgica, com administração do veículo; GII – falsamente pinealectomizados com administração do veículo; GIII – pinealectomizados tratados com veículo; GIV- pinealectomizados com administração de melatonina (10μg/animal) durante a noite. Após 60 dias de experimento, todos os animais foram anestesiados, e a adrenal retirada e fixada em formol tamponado a 10%. Após processamento histológico para inclusão em parafina, os cortes foram corados pelo H.E e outros submetidos a métodos imunohistoquímicos para detecção da Caspase-3-clivada (apoptose) e fator de crescimento vascular (VEGF). Os dados obtidos foram submetidos à análise estatística (P<0,05). Resultados: morfologicamente, não houve modificações na zona glomerulosa em nenhum dos grupos estudados. No GIII, houve aumento da adrenal e da zona reticulada, com núcleos volumosos ricos em eurocromatina. No GIV houve a reversão deste quadro. Na imunohistoquímica houve queda de apoptose detectada pela Caspase-3 nos grupos GIII (zona fasciculada) e GIV (zonas fasciculada e reticulada). O VEGF teve sua menor reação na zona reticulada em todos os grupos, ocorrendo aumento da reação na zona fasciculada do GIII. Conclusão: nossos dados sugerem que a ministração exógena de melatonina reverte os efeitos da pinealectomia. / Introduction: The pineal gland is the modulator of mammal’s reproduction, it has some influence in gonads and in the estrous cycle thru hipotalamus-ovary-adrenal axis. Objectives: The objective of this study is to analyze the histomorphology and imunnohistochemical of the pinealectomized and treated with melatonin female rat’s adrenals. Material and methods: Forty adult female rats were divided in 4 groups of 10 animals each. GI – no surgery intervention, with vehicle administration; GII – falsely pinealectomized with vehicle administration; GIII – pinealectomized with vehicle administration; GIV – pinealectomized with melatonin administration (10μg/animal) during the night. After 60 days of experiment, all animals where anesthetized, and the adrenal removed and fixed in 10% formaldehyde. After histological processing to be embedded in paraffin, some slides were stained by H.E. and others submitted to imunnohistochemical to detection of Caspase-3 clivated (apoptosis) and vascular endothelial growth factor VEGF. The obtained data were submitted to statistics (P<0,05). Results: Morphologically, there weren’t modifications in the glomerulosa zone in any of the studied groups. In GIII there was a slightly increase of the adrenal size and the reticularis zone presented with volumous nucleus rich in eurocromatin. In GIV those findings from the previous group had been reversed. In immunohistochemical, there was a decrease in apoptosis detected by Caspase-3 in the GIII (fasciculate zone) and GIV (fasciculata and reticularis zone). VEGF had its smaller reaction in the reticularis zone in all groups, the only difference being the fasciculate zone in GIII, which had shown an increase of the reaction. Conclusion: our data suggests that exogenous melatonin can reverse the effects of pinealectomy. / TEDE / BV UNIFESP: Teses e dissertações

The timing of seasonal events in salmonids is thought to be controlled by endogenous circannual rhythm(s) which are entrained by the seasonally-changing daylength. This thesis investigates the role of the pineal gland in the perception of the photoperiodic zeitgeber and the subsequent transmission of this information to the brain through neural or hormonal pathways. Melatonin biosynthesis by isolated rainbow trout pineal glands was shown to exhibit a differential response to graded photic or thermal stimuli. In vitro experiments were carried out at 10±0.50 C as this provided optimum melatonin levels for radioimmunoassay analysis together with a pineal longevity of up to 14 days. By incorporating a variety of light intensities into the light/dark cycle, the salmonid pineal gland was shown to synthesise significantly different levels of melatonin even when light levels varied by only 0.5 lux. Early work on the salmonid pineal suggested it was unresponsive to red light, having a spectral sensitivity which peaks between 500 and 550 nm, this study has revealed that the pineal is also capable of responding to wavelengths between 660 to 800 nm, at which pineal reception was previously thought to be severely limited. No endogenous rhythm of melatonin secretion was observed within the isolated rainbow' trout pineal gland. Both Atlantic salmon and Atlantic halibut pineals exhibited elevated levels- of melatonin in response to the dark phase, however, they also appeared capable of maintaining this rhythm in the absence of external stimuli. This provides the first evidence that the daily rhythm of melatonin production in these species is controlled by an endogenous circadian oscillator located within the pineal II gland. The pinealectomy technique developed during the course of this thesis successfully abolished the diel rhythm of melatonin secretion and, together with an enucleation procedure, enabled the pineal to be identified as the predominant source of the dark phase melatonin in Atlantic salmon and rainbow trout. However, the lateral eyes did contribute significantly to plasma melatonin levels in both species. Long term experiments, involving pinealectomy and/or implantation of melatonin, were used to investigate the role of the pineal gland in the timing of rainbow trout maturation and smoltification in Atlantic salmon. Pineal removal at the summer or winter solstices did not significantly alter the timing of smoltification. However, significantly higher blood serum osmolarities following seawater challenge tests were observed in smolts implanted with melatonin. This, together with a significant growth increase shown by salmon parr within 1 month of implantation, indicates that melatonin may directly affect the development of salmonids through either a physiological response or by influencing the entrainment of endogenous rhythms. The increased growth observed in the implanted parr is also thought to be responsible for the unimodal population distribution and high percentage of S1 smolts within this group. Investigations into the role of the pineal gland in the timing of spawning in rainbow trout found that pineal removal at the summer solstice caused a 6 week delay in spawning time compared to intact fish. However, no clear effects on spawning time were observed when pineal removal, with or without melatonin implantation, was performed to coincide with the change from long to short daylengths which is known to advance spawning times. Although no significant effect in spawning times was observed between groups, the 4 month spawning period of the pinealectomised group compared to 1 month in the shampinealectomised fish also suggested that pineal removal may have caused a desynchronisation in spawning time. Pinealectomy and/or implantation did not alter egg size or fecundity, but plasma calcium levels were shown to be significantly lower in the pinealectomised trout over the spawning period. To summarise, the pineal gland and melatonin play a significant role in salmonid development. It is suggested that melatonin can influence biological systems through a direct physiological action while the pineal gland may synchronise circannual events through the photoneuroendocrine transduction of seasonal environmental information.

The physiological effects of the pineal gland on the prolactin cells of the adenohypophysis were examined in short photoperiod-exposed male hamsters, as well as in blinded male and female hamsters. Pituitary storage of prolactin was assessed by monitoring radioimmunoassayable prolactin levels in the pituitaries in vivo and the total amount of immunoreactive prolactin in vitro. The effects of the pineal on prolactin secretion were estimated by measuring immunoassayable prolactin titers in the serum. Prolactin synthesis was measured by the ability of anterior pituitaries to incorporate ³H-leucine into prolactin in vitro. Finally, the effects of blinding and the activated pineal on hypothalamic hypophysiotrophic activity was assessed by incubating pituitaries in the presence of neutralized, acidic extracts of the stalk-medium eminence (SME) region of the mediobasal hypothalamus. In the male hamster, the pineal gland inhibits PRL cell function which encompasses reductions in the synthesis, storage and release of prolactin. The depressions in prolactin release and in pituitary storage are evident as early as three weeks after males are deprived of light. However, the inhibitory influence of the pineal on prolactin synthesis may be only partially apparent by eight to nine weeks after male hamsters are deprived of light, and is not fully evident until 12 weeks of light restriction. In the blinded female hamster, the synthesis, storage and release of prolactin are also markedly suppressed. However, all aspects of prolactin cell inhibition in the female may not be pineal-mediated. Furthermore, it appears that there may be some direct hypothalamic mechanism by which orbital enucleation inhibits prolactin cell function that is independent of the pineal gland. Based on the data presented in this dissertation, it is concluded that the SME region of the female hamster contains inhibitory activity which may be specifically responsible for the inhibition of prolactin synthesis. Furthermore, blinding and the pineal gland may independently exert rather specific influences upon hypophysiotrophic activity within the SME region of the female hamster.

A glândula pineal regula diversos ritmos biológicos e respostas de defesa em indivíduos hígidos através da síntese noturna de melatonina. Por outro lado, é sabido que processos inflamatórios reduzem a produção deste hormônio na glândula pineal. Neste estudo utilizamos um arranjo de qPCR para investigar a expressão circadiana de 84 genes relacionados a sinalização via receptores do tipo toll e NF-&kappa;B em glândulas pineais de rato. Confirmamos ainda, a expressão de 14 proteínas em pinealócitos através de ensaios de imunocitoquímica. Nossos resultados indicam que 70 genes inflamatórios são expressos ritmicamente na glândula pineal de ratos, enquanto 7 não são expressos e outros 7 genes são expressos, mas sem ritmicidade. Grande parte dos genes examinados apresenta padrão de expressão circadiana com maior conteúdo transcricional na fase de claro, atingindo a máxima expressão no final desta fase. Após o apagar das luzes, a expressão destes genes é rapidamente reduzida. Um possível envolvimento do ritmo de glicocorticóides endógeno sobre o padrão dessa expressão gênica foi avaliado através do bloqueio de GR por mifepristona, o que induziu a regulação da expressão de 13 genes e redução do conteúdo plasmático de melatonina no ZT18. Ainda, avaliamos o efeito da ativação dos TLR1, TLR2 e TLR6 sobre a glândula pineal e observamos que zimosan e Pam3CSK4 ativam a via do fator de transcrição NF-&kappa;B e bloqueia a síntese de melatonina induzida por noradrenalina in vitro. Por fim, utilizamos o modelo de obseidade induzido por dieta hipercalórica para avaliar se o processo inflamatório de baixa intensidade regula a síntede se melatonina in vivo. Observamos que nestas condições, a dieta hipercalórica induz rápido aumento no peso corporal e redução da produção noturna de melatonina. O efeito protetor da melatonina sobre o ganho de peso induzido por dieta foi testado através da restauração da melatonina na água de beber noturna de animais expostos à dieta. Em conjunto, nossos resultados indicam que genes inflamatórios são expressos ritmicamente na glândula pineal de ratos e influenciam a produção circadiana de melatonina via reconhecimento de padrões moleculares associados à patógenos ou sinais de perigo / The pineal gland regulates several circadian rhythms as well as immune responses in healthy animals via rhythmic production of melatonin, the hormone of darkness. On the other hand, nocturnal melatonin levels are reduced in the course of inflammation. To date, it remains to be clear the mechanisms by which the immune system affects pineal melatonin synthesis. Here we used a qPCR array profiler to investigate circadian gene expression of 84 genes related to Toll-Like Receptors and Nuclear Factor kappa B signaling. We also examined the expression of 14 proteins in pinealocytes by immunocytochemistry. Our results indicate rhythmic expression of 70 inflammatory genes, while 7 genes were not expressed and 7 were expressed without rhythmicity. The overall majority of genes tested showed a pattern of expression with a cumulative diurnal increase that peaks at the light phase of ZT12 followed by a fast reduction in the expression as soon as the light is turned off. The possible involvement of endogenous glucocorticoid rhythm in the modulation of pineal\'s inflammatory gene expression were tested by blocking Glucocorticoid Receptor (GR) using mifepristone. This procedure modulated the expression of 13 genes. In addition, the blockade of GR reduced the circulating melatonin levels at ZT18. The activation of TLR1, TLR2 and TLR6 induces the nuclear translocation of NF-&kappa;B signaling and blocks noradrenaline-induced melatonin synthesis in vitro. In addition, high-fat diet feeding increases body weight and reduce the circulating melatonin levels at ZT18. The protective role of melatonin in diet-induced weight gain was also determined by giving these rats melatonin in their drinking water at night. Altogether, our results highlight that inflammatory genes are transiently expressed in the rat pineal gland and influences the daily fluctuation of melatonin synthesis

The genetic identification of molecular mechanisms responsible for circadian rhythm generation has advanced tremendously over the past 25 years. However the molecular identities of the avian clock remain largely unexplored. The present studies seek to determine candidate clock components in the avian species Gallus domesticus. Construction and examination of the transcriptional profiles of the pineal gland and retina using DNA microarray analysis provided a clear view into the avian clock mechanism. Investigation of the pineal and retina transcriptomes determined the mRNA profiles of several thousand genes over the course of one day in LD (daily) and one day in DD (circadian) conditions. Several avian orthologs of mammalian clock genes were identified and many exhibited oscillating patterns of mRNA abundance including several of the putative avian clock genes. Comparison of the pineal transcriptional profile to that of the retina revealed several intriguing candidate genes that may function as core clock components. Including the putative avian clock genes and several others implicated in phototransduction, metabolism, and immune response. A more detailed examination of several candidate photoisomerase/photopigment genes identified from our transcriptional profiling was conducted. These include peropsin (rrh), RGR-opsin (rgr), melanopsin (opn4) and cryptochrome 2 (cry2) genes. This analysis revealed several interesting patterns of mRNA distribution and regulation for these genes in the chick. First, the mRNA of all 4 genes is located within the Inner Nuclear Layer (INL) and Retinal Ganglion cell Layers (RGL) of the ocular retina, where circadian photoreception is present. Second, opn4 and cry2 mRNA is expressed in the photoreceptor layer of the chick retina where melatonin biosynthesis occurs. Lastly, the mRNA for all 4 candidate photopigment genes is regulated on a circadian basis in the pineal gland. As a whole these data yield significant insight into the mechanisms of the avian circadian system and present several candidate genes that may function to integrate photic information, and/or regulate circadian rhythm generation in birds.

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v, 175 leaves : photos., graphs, tables ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / David John Kennaway / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1979