The World Health Organization classifies glioblastoma (GBM) as a type IV astrocytoma, making it one of the most fatal tumors that exists. Despite the advances in chemotherapy, surgery, and radiation treatments that improve a patients length of survival, the overall trajectory of the disease remains unchanged. It has been shown that GBM cells produce significant levels of prostaglandins, including prostaglandin D2 (PGD2). PGD2 possesses pro- and anti-tumorigenic properties. Hence, a more complete understanding of PGD2 activity in GBM could yield more effective treatments against GBM. Through techniques like RT-PCR, immunohistochemistry, and HPLC tandem mass spectrometry, we were able to confirm the presence of the PGD2 synthesis in GBM cell lines. We treated GBM cell lines with various concentrations of exogenous PGD2 over 72 hours and observed its effects on cell count, apoptosis, mitosis and viability. Our results suggest that PGD2 possesses contradictory functions in GBM depending on concentration (mM PGD2 vs. nM PGD2) and receptor activation. / A Organização Mundial de Saúde classifica glioblastoma (GBM) como um astrocitoma tipo IV, fazendo uns dos tumores mais fatais que existe. A pesar dos avanços em quimioterapia, cirurgia e radioterapia que melhoram a longevidade de sobrevivência, a trajetória geral da doença permanece imutável. Tem sido demonstrado que células de GBM produzem níveis significativos de prostaglandinas, incluindo prostaglandina D2 (PGD2). PGD2 possui propriedades pro- e anti-tumorigenicos. Então, um entendimento mais completo da atividade de PGD2 em GBM pode gerar tratamentos mais efetivos. Através de técnicas como RT-PCR, imunohistoquimicas e HPLC espectrometria de massa em tandem, conseguimos confirmar a presença da síntese de PGD2 em linhagens de GBM. Tratamos linhagens de GBM com concentrações variáveis de PGD2 exógeno durante 72 horas e observamos seus efeitos na contagem de células, apoptose, mitose e viabilidade. Nossos resultados sugerem que PGD2 possui funções opostas em GBM dependendo em concentração (mM PGD2 vs. nM PGD2) e ativação de receptores.

The World Health Organization classifies glioblastoma (GBM) as a type IV astrocytoma, making it one of the most fatal tumors that exists. Despite the advances in chemotherapy, surgery, and radiation treatments that improve a patients length of survival, the overall trajectory of the disease remains unchanged. It has been shown that GBM cells produce significant levels of prostaglandins, including prostaglandin D2 (PGD2). PGD2 possesses pro- and anti-tumorigenic properties. Hence, a more complete understanding of PGD2 activity in GBM could yield more effective treatments against GBM. Through techniques like RT-PCR, immunohistochemistry, and HPLC tandem mass spectrometry, we were able to confirm the presence of the PGD2 synthesis in GBM cell lines. We treated GBM cell lines with various concentrations of exogenous PGD2 over 72 hours and observed its effects on cell count, apoptosis, mitosis and viability. Our results suggest that PGD2 possesses contradictory functions in GBM depending on concentration (mM PGD2 vs. nM PGD2) and receptor activation. / A Organização Mundial de Saúde classifica glioblastoma (GBM) como um astrocitoma tipo IV, fazendo uns dos tumores mais fatais que existe. A pesar dos avanços em quimioterapia, cirurgia e radioterapia que melhoram a longevidade de sobrevivência, a trajetória geral da doença permanece imutável. Tem sido demonstrado que células de GBM produzem níveis significativos de prostaglandinas, incluindo prostaglandina D2 (PGD2). PGD2 possui propriedades pro- e anti-tumorigenicos. Então, um entendimento mais completo da atividade de PGD2 em GBM pode gerar tratamentos mais efetivos. Através de técnicas como RT-PCR, imunohistoquimicas e HPLC espectrometria de massa em tandem, conseguimos confirmar a presença da síntese de PGD2 em linhagens de GBM. Tratamos linhagens de GBM com concentrações variáveis de PGD2 exógeno durante 72 horas e observamos seus efeitos na contagem de células, apoptose, mitose e viabilidade. Nossos resultados sugerem que PGD2 possui funções opostas em GBM dependendo em concentração (mM PGD2 vs. nM PGD2) e ativação de receptores.

Osteocytes are believed to be the mechanosensory cells that detect and respond to mechanical loading. Physiological loading by oscillatory fluid flow (OFF) activates osteocytes to increase intracellular calcium concentration and release prostaglandin E2 (PGE2). Osteocytes are also sensitive to chemical stimulations such as serotonin, which can also increase PGE2 release. However, it is unclear whether mechanical stimulation can influence osteocyte sensitivity towards serotonin. In this thesis, MLO-Y4 osteocyte-like cells were subjected to serotonin with or without precondition by OFF, the responses of intracellular calcium and PGE2 release were measured. Serotonin increased intracellular calcium and PGE2 release in osteocytes. The effects were significantly reduced by OFF precondition, suggesting mechanical precondition by OFF can reduce osteocyte sensitivity towards serotonin. In terms of mechanisms, OFF- and serotonin-induced calcium responses depended on intra- and extracellular calcium stores. ATP was found to partially mediate OFF modulation of serotonin-induced PGE2 release but not calcium.

Osteocytes are believed to be the mechanosensory cells that detect and respond to mechanical loading. Physiological loading by oscillatory fluid flow (OFF) activates osteocytes to increase intracellular calcium concentration and release prostaglandin E2 (PGE2). Osteocytes are also sensitive to chemical stimulations such as serotonin, which can also increase PGE2 release. However, it is unclear whether mechanical stimulation can influence osteocyte sensitivity towards serotonin. In this thesis, MLO-Y4 osteocyte-like cells were subjected to serotonin with or without precondition by OFF, the responses of intracellular calcium and PGE2 release were measured. Serotonin increased intracellular calcium and PGE2 release in osteocytes. The effects were significantly reduced by OFF precondition, suggesting mechanical precondition by OFF can reduce osteocyte sensitivity towards serotonin. In terms of mechanisms, OFF- and serotonin-induced calcium responses depended on intra- and extracellular calcium stores. ATP was found to partially mediate OFF modulation of serotonin-induced PGE2 release but not calcium.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for blood disorders and autoimmune diseases. Following HSCT, these cells must successfully migrate to the marrow niche and replenish the blood system of the recipient. This process requires both non-cell and cell-autonomous regulation of hematopoietic stem and progenitor cells (HSPCs). A transgenic reporter line in zebrafish allowed the investigation of factors that regulate HSPC migration and function. To directly observe cells in their endogenous microenvironment, confocal live imaging was used to track runx1:GFP+ HSPCs as they arrive and lodge in the niche. A novel cellular interaction was observed that involves triggered remodeling of perivascular endothelial cells during niche formation. A chemical screen identified the TGF-beta pathway as a regulator of HSPC and niche interactions. Chemical manipulation of HSPCs was used to improve engraftment and repopulation capability following transplantation. Runx1:GFP fish treated with prostaglandin E2 (PGE2) during embryogenesis exhibit increased runx1+ cells in the AGM and CHT, consistent with previous in situ data. This increase in HSPCs is maintained into adulthood, even in the absence of prolonged PGE2 exposure. Kidney marrow from these treated fish can outcompete control marrow in transplantation assays. The ability of PGE2 to confer a long-term advantage on sorted mouse marrow populations in competitive transplantation assays was tested. I found that PGE2-treated short-term (ST)-HSCs, but not long-term (LT)-HSCs show enhanced transplantability in recipients compared to control animals. My studies demonstrate that the effects of PGE2 on HSC function persist over substantial time despite transient exposure. A population of short-term HSCs can engraft and give rise to long-term multilineage reconstitution following PGE2 treatment. Collectively, our studies have led to novel insights regarding the pathways involved in HSC migration, homing, and repopulation.

The neuropeptides dynorphin and cholecystokinin (CCK), and their associated pronociceptive effects were investigated in the RVM. Utilizing a nerve-injury model (SNL), RT-PCR analysis revealed increases (p<0.05) of prodynorphin mRNA, and bradyinkin, B1- and B2-receptor mRNA, post-SNL, 14-days, 2-days, and 14-days, respectively. Administration of dynorphin into the RVM produced both acute and long-lasting (>30-days) tactile hypersensitivity. Administration of the B1-antagonist, DALBK and the B2-antagonist, Hoe-140, into the RVM significantly attenuated dynorphin-induced tactile hypersensitivity. Nerve-injury induced tactile hypersensitivity was significantly reversed by RVM administration of dynorphin antiserum or the B2-antagonist, Hoe-140. These data suggest that dynorphin is up-regulated in the RVM in nerve-injury, and via the activation of bradykinin receptors in the RVM, produces abnormal pain. Like dynorphin, CCK is up-regulated in the RVM in nerve-injury, with studies suggesting that elevated levels of CCK in the RVM mediate pronociceptive activity through CCK2 receptor activation, resulting in enhanced spinal nociceptive transmission. At present, it is unknown what key neurotransmitters are mediating this RVM CCK-driven effect at the level of the spinal cord. Here, spinal cerebrospinal fluid (CSF) levels of serotonin (5-HT) and prostaglandin E2 (PGE2) were measured in the lumbar spinal cord in naïve rats following CCK administration into the RVM. Following RVM CCK microinjection, an approximate 5-fold increase in spinal (CSF) PGE2 levels was observed, as compared to baseline controls. PGE2 levels showed a progressive increase with peak levels observed at the 80-minute post-CCK injection timepoint, whereas 5-HT levels in the spinal CSF remained unchanged following CCK administration into the RVM. This release of PGE2 coincided with the timecourse for CCK-induced mechanical hypersensitivity. Administration of the CCK2-antagonist YM022 prior to CCK into the RVM, significantly attenuated (>50%) the release of PGE2 in the spinal cord. The non-selective COX-inhibitor naproxen and the 5-HT3 antagonist ondansetron, both administered intrathecally, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. In summary, these data suggest a bradykinin- or CCK2-receptor antagonist could be used alone or in conjunction with current therapies in the treatment of chronic pain.

No description available.

Japanese quail were used as animal models in four experiments to evaluate the effects of supplementing diets with different lipids on bone chemical, mechanical, and histological properties. In Exp. 1, laying hens were fed a basal diet containing either 5% soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO). The addition of SBO in the maternal diet increased the levels of total n-6 fatty acids and arachidonic acid (AA, 20:4n-6) in yolk and tibial bones of newly hatched progeny (P<0.01), whereas the maternal FO diet elevated the concentrations of total n-3 fatty acids, eicosapentaenoic acid (EPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and total saturated acid, but greatly decreased the amount of AA in both egg and progeny tibiae (P<0.01). The maternal HSBO diet resulted in the accumulation of trans-18:1 fatty acid in egg yolks and tibiae at hatch. The addition of FO or HSBO to the maternal diet significantly lowered the ex vivo PGE2 production of tibiae in newly hatched quail compared to those from hens given the SBO or CF diets (P<0.01). In Exp. 2, the addition of different lipids in the maternal diets did not affect growth, tibial length, diameter or collagen content of the progeny. However, supplementing the maternal diet with 5 % FO or HSBO increased the percent bone ash , increased bone pyridinium crosslinks of collagen, enlarged the cartilaginous proliferative and hypertrophied zones, increased diaphyseal cortical thickness of the tibiae in embryos (P<0.05), and subsequently increased tibial shear force, stiffness (P<0.05) and improved cortical thickness, density and trabecular density in early growth and development of progeny compared to those from hens consuming the SBO or CF diets (P<0.05). In Exp. 3, male quail at one month of age were fed a basal diet containing either 5% SBO, HSBO, CF or FO for seven months. Long-term supplementation in the diets of different lipids did not affect body weight, food intake, tibial length or diameter, but the FO group had the highest tibial percent ash, and both FO and HSBO increased tibial mineral content in aged quail compared to those fed the SBO or CF diets (P<0.05). At 8 months of age, quail fed FO had the highest concentrations of (n-3) fatty acids (20:5n-3, 22:5n-3, 22:6n-3) but the lowest amounts of 20:4n-6 in lipids from tibial cortical bone, whereas the SBO and CF diets greatly elevated (n-6) fatty acids and 20:4n-6 levels. The HSBO diet which contains t18:1 fatty acid resulted in t18:1 accumulation in bone. Long-term supplementation with FO or HSBO increased tibial shear force, stiffness and shear stress, as well as improved cortical thickness and density compared with the SBO or CF diets ( P<.05). In Exp. 4, the addition of SBO or CF to the diet for seven months decreased tibial mineral content compared to the FO diet (P<0.05). Quail fed SBO increased collagen concentration in the tibiae (P<0.05), but the level of collagen crosslinks was higher in quail fed FO or HSBO compared to those given the SBO or CF diets (P<0.05). The PGE2 production in bone organ culture and marrow was greatly increased in quail maintained on the SBO or CF diets (P<0.05). PGE2 production in the bone microenvironment was negatively correlated with the tibial percent ash and collagen crosslinks but had a positive correlation with tibial collagen concentration. The results of these studies demonstrate that either supplementing the maternal diets with or long-term exposure to different lipids alters the chemical composition and metabolism of skeletal tissue in both embryos and aged quail. Maternal dietary SBO or CF had an adverse effect on bone growth and development in embryos. Likewise, long-term exposure to SBO or CF diet impaired bone metabolism and remodeling. In contrast, the FO or HSBO diet had beneficial effects on bone modeling in embryos and remodeling in adult quail. / Ph. D.

Le métabolisme lipidique affecte la maturation et la différenciation des cellules osseuses. L'objectif de ma thèse est d'approfondir deux aspects du métabolisme lipidique mal connus, soit les actions de la phospholipase D (PLD) et celles des récepteurs de prostaglandine PGE2 pendant la différenciation des cellules. Une lignée humaine, les Saos-2 et les ostéoblastes primaires issus de calvaria de souriceaux ont servi de modèles cellulaires de la minéralisation physiologique. La culture d'aorte ex vivo sous des conditions d'hyperphosphatémie a été utilisée pour reproduire la calcification de l'aorte qui est un modèle ex vivo de calcification cardiovasculaire (CCV). Nous avons montré que l'expression et l'activité de la PLD augmentent dans les Saos-2 et les ostéoblastes primaires au bout du 5ème jour de la différenciation tandis qu'elles s'accroissent au bout du 6ème jour de traitement de l'aorte dans un milieu d'hyperphosphatémie. Les inhibiteurs de PLD diminuent l'activité de phosphatase alcaline (TNAP) dans les ostéoblastes et dans l'aorte calcifiée tandis que la surexpression de la PLD1 dans les Saos-2 l'augmente. Dans une deuxième partie de ce travail, nous avons suivi la variation d'expression des récepteurs de PGE2 au cours de la maturation des Saos-2. L'expression du gène EP3 augmente au stade tardif de la minéralisation tandis que celle d'EP4 diminue. Pour conclure, ces résultats indiquent que l'activité de la PLD en affectant l'activité de la TNAP pourrait moduler finement la minéralisation physiologique et la CCV et que la minéralisation s'accompagne d'un changement d'expression des récepteurs de PGE2, dans les Saos-2 / Lipid metabolism affects the maturation and the differentiation of bone cells. The aim of my PhD thesis is to explore two unknown sides of lipid metabolism which are the actions of phospholipase D (PLD) and those of prostaglandin PGE2 receptors during cell differentiation. Human lineage, Saos-2 cells and primary osteoblasts from calvaria of mice were used as cellular models of physiological mineralization. The ex vivo aorta culture under hyperphosphatemia conditions has been used to reproduce the calcification of the aorta, which is an ex vivo model of cardiovascular calcification (CVC). We showed that the expression and the activity of PLD increased in Saos-2 and primary osteoblasts after the fifth day of differentiation while in the aorta under hyperphosphatemia condition, PLD activity increased at the end of the sixth day. PLD inhibitors decreased the activity of alkaline phosphatase (TNAP) in osteoblasts and in calcified aorta while the overexpression of PLD1 in the Saos-2 increased it. In the second part of this work, we monitored the variation of the expression of PGE2 receptors during the maturation of Saos-2 cells. The EP3 gene expression increased in the late stage of the mineralization while that of EP4 decreased. In conclusion, these results indicated that the PLD activity by affecting the activity of TNAP could modulate the physiological mineralization and CVC. We showed that the mineralization is dependent of the change of the expression of PGE2 receptors in Saos-2 cells

Hormonal-mediated uterine quiescence involves the maintenance of a decreased inflammatory responsiveness. However, no study has investigated whether labor induction with prostanoids is associated with changes in maternal serum hormones. The objective of this study was to determine whether changes in circulating levels of progesterone, estradiol and estriol from admission to delivery are associated with successful labor induction with dinoprostone. A cohort of 81 pregnant women at term was followed from admission to birth until delivery, during the period of 2010-2011. The study was performed at the Hospital of the Federal University of Santa Maria, a tertiary care hospital. Unselected subjects were recruited and blood samples were obtained at admission and immediately before delivery. Sixteen patients had vaginal delivery after spontaneous labor, 12 required emergency cesarean after spontaneous labor and 16 underwent elective cesarean. Thirty-seven patients had labor induction with dinoprostone. Eligible patients received a vaginal insert of dinoprostone (10 mg), and patients were followed up until delivery. Progesterone (P4), estradiol (E2) and estriol (E3) plasma level and P4/E2, P4/E3 and E3/E2 ratio changes were observed from admission to immediately before birth, and the association of these measures with the resulting clinical classification outcome (route of delivery and induction responsiveness) were assessed. Progesterone plasma level decreased from admission to delivery in patients who underwent successful labor induction with dinoprostone [vaginal and cesarean delivery after induced labor: 23% (P<0.001) and 18% (P<0.025) decrease, respectively], but not in those whose induction failed (6.4% decrease, P>0.05). Estriol and estradiol levels did not differ between groups. Successful dinoprostone-induced labor was associated with maternal progesterone level decrease along time. While a causal relationship between progesterone decrease and effective dinoprostone-induced labor can not be established, it is tempting to propose that it may contribute for progesterone withdrawal and favor labor induction in humans. / A quiescência uterina mediada por hormônios envolve a manutenção de uma responsividade inflamatória reduzida. Contudo, nenhum estudo investigou se a indução do parto com prostanóides está associada com alterações em hormônios séricos maternos. Os objetivos deste estudo foram determinar se as alterações nos níveis circulantes de progesterona, estradiol e estriol desde a admissão até o parto estão associados à indução bem sucedida do parto com dinoprostona. Uma coorte de 81 mulheres grávidas a termo foi acompanhada desde a admissão até o parto, durante o período de 2010-2011. O estudo foi realizado no Hospital da Universidade Federal de Santa Maria, um hospital de cuidados terciários. Indivíduos não selecionados foram recrutados e amostras de sangue foram obtidas na admissão e imediatamente antes do nascimento. Dezesseis pacientes tiveram parto vaginal após trabalho de parto espontâneo, 12 necessitaram a realização de cesariana de emergência após trabalho de parto espontâneo e 16 foram submetidas à cesárea eletiva. Trinta e sete pacientes tiveram indução de trabalho de parto com dinoprostona. As pacientes elegíveis receberam um pessário de inserção vaginal de dinoprostona (10 mg), e foram acompanhadas até o parto. Os níveis plasmáticos de progesterona (P4), estradiol (E2) e estriol (E3) e as relações P4/E2, P4/E3 e E3/E2 foram observadas da admissão até imediatamente antes do nascimento, e a associação destas medidas com a classificação clínica resultante foi avaliada (via de parto e resposta à indução). Os níveis plasmáticos de progesterona diminuíram desde a admissão até o nascimento em pacientes que responderam à indução com dinoprostona [parto vaginal e cesáreo após trabalho de parto induzido: redução de 23% (P<0.001) e 18% (P<0.025), respectivamente], mas não nos quais a indução falhou (redução de 6.4%, P>0.05). Os níveis de estriol e estradiol, e as relações P4/E2, P4/E3 e E3/E2 não foram diferentes entre os grupos. O sucesso da resposta à indução de parto com dinoprostona esteve associado com a redução no nível de progesterona materna ao longo do tempo. Enquanto uma relação causal entre a redução na progesterona e o trabalho de parto efetivo induzido pela dinoprostona não pode ser estabelecida, é tentador propor que possa contribuir para a retirada da progesterona e favorecer a indução do parto em humanos.