O glioblastoma (GBM) é o tumor mais frequente do sistema nervoso central com um alto grau de malignidade e um prognóstico desfavorável. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e/ou quimioterapia, não há tratamento eficiente disponível para o GBM. Os prostanoide são derivados do ácido araquidônico e estão envolvidas com vários processos do desenvolvimento e progressão do câncer. O objetivo deste estudo foi analisar in vitro o perfil de diferentes prostanoides nas linhagens de GBM. Além de analisar o papel dos prostanoides e dos receptores na migração celular de GBM. Os resultados demostraram um perfil dos prostanoides da série 2 diferente entre as linhagens, além da expressão dos genes envolvidos na biossíntese de PGE2. Nos ensaios de migração os dados demostraram que os tratamentos realizados com os prostanoides exógenos aumentaram a migração celular e os tratamentos com os antagonistas de EP2 e EP4 diminuiram a migração. Em conjunto esses resultados, demonstram o papel importante dos prostanoides, especialmente PGE2, no processo de migração das células de GBM. / Glioblastoma (GBM) is the most common tumor of the central nervous system with a high degree of malignancy and poor prognosis. Despite advances in surgical techniques and radiation therapy and/or chemotherapy, there is no effective treatment available for GBM. The prostanoid are derived from arachidonic acid and are involved in many processes of development and progression of cancer. The aim of this study was to analyze in vitro profile of different prostanoids in the lines of GBM. In addition to analyzing the role of prostanoids and receptors on the cell migration of GBM. The results showed a profile of series 2 prostanoids the different between the cell lines, in addition to expression of genes involved in the biosynthesis of PGE2. In migration testing data showed that the treatments performed with exogenous prostanoids increased cell migration and treatment with antagonists of EP2 and EP4 decreased migration. Together these results demonstrate the important role of prostanoids, especially PGE2, in the migration process of the GBM cells.

Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.

Biotecnologias como a Ovum Pick-Up e a Produção In vitro de embriões (OPU-PIVE) tem sido uma importante ferramenta para alcançar o melhoramento genético rápido nos rebanhos, diminuindo o intervalo entre gerações. No Brasil, a PIVE está em fase de crescimento e representa 70,7% de toda a produção in vitro mundial. Contudo a OPU-PIVE é ainda ineficiente em vacas de leite, especialmente devido à sua reduzida população folicular. Muitos estudos têm mostrado um efeito positivo do FSH em gado de leite e corte. Recentemente, a pré-estimulação com FSH mostrou ser capaz de aumentar o diâmetro dos folículos aspirados e a porcentagem de embriões transferíveis. O FSH estimula o recrutamento dos folículos na fase antral, fazendo com que eles possam se desenvolver até o momento em que ocorre a divergência e um ou mais folículos se torne dominante. A hipótese do presente estudo é que o uso de FSH (200 mg), fracionado em 4 ou 6 aplicações, em vacas holandesas não lactantes, com emergência de onda folicular sincronizada, aumenta o número de folículos ovarianos, o número de oócitos viáveis e a quantidade de embriões na produção in vitro. Trinta e seis vacas Holandesas não lactantes foram utilizadas como doadoras de oócitos e distribuídas em três tratamentos: Controle (C), 4 aplicações de FSH (F4) e 6 aplicações de FSH (F6). Todas as vacas foram submetidas ao mesmo protocolo de sincronização de emergência de onda folicular, diferindo apenas pela administração e número de 4 ou 6 doses de FSH, conforme descrito acima. Em dia aleatório do ciclo estral (D0), todas as vacas receberam um dispositivo de P4 (Primer®, Tecnopec-Agener União, São Paulo, SP, Brasil) e 2 mg de benzoato de estradiol (Ric-BE®, Tecnopec-Agener União, São Paulo, SP, Brasil). Três dias após (D3), foi administrado 0,530 mg de Cloprostenol Sódico (Cioprostinn®, Innovare Biotecnologia e Saúde Animal Ltda, Monte Aprazível, SP, Brasil), induzindo a luteólise com a intenção de liberar espaço no estroma ovariano para o crescimento folicular e facilitar a visualização de folículos na OPU. Vacas do grupo C não receberam tratamentos adicionais. Vacas do grupo F4 receberam 200 mg de FSH (Folltropin - Bioniche Anim,al Health, Belleville, ON, Canadá) fracionados em 4 aplicações de equivalentes concentrações em intervalos de 12 h, iniciando no D4 pela manhã. Vacas do grupo F6 receberam 200 mg de FSH fracionados em 6 aplicações de equivalentes concentrações em intervalos aproximados de 12 h, tendo início no D3 pela manhã. No D7, o dispositivo foi removido e a OPU realizada concomitantemente à contagem dos folículos existentes nos ovários. Os oócitos considerados viáveis foram fertilizados in vitro com sêmen sexado de touros da raça Holandesa. Os dados foram analisados pelo PROC GLIMIX do SAS 9.3, utilizando contrastes ortogonais C1 (C x FSH) e C2 (F4 x F6). Não houve efeito de tratamento no número de folículos (C = 53,3 ± 4,9 vs FSH = 51,36 ± 3,1;P = 0,89), número total de oócitos (C = 19,46 ± 1,64 vs FSH = 18,47 ± 1,27; P = 0,55), número de oócitos viáveis (C = 12,57 ± 1,26 vs FSH = 12,70 ± 1,03; P= 0,606), taxa de recuperação de oócitos (C = 36,5% vs FSH = 36,0%; P = 0,48) e produção de embriões in vitro (C = 4,11 ± 0,52 vs FSH = 4,32 ± 0,46; P = 0,79). Apesar de não ter havido efeito no número de folículos, o tratamento com FSH alterou a distribuição dos mesmos, proporcionando o aumento no número de folículos médios (6 a 10 mm). No entanto, não houve efeito do tratamento com FSH no número de oócitos totais e viáveis recuperados, nem na produção de embriões. / Reproductive biotechnologies such as Ovum Pick-Up and in vitro Embryo production (OPU-IVEP) have been widely used as important tools to achieve faster genetic improvement in herds, diminishing the intervals between generations. In Brazil, in vitro Embryo Production (IVEP) is growing in popularity and accounts for 70.7% of all in vitro embryo production worldwide. However, the OPU-IVEP is still poorly efficient in high-producing dairy cattle, especially because of their reduced follicular population. Several studies have shown a positive effect of FSH on OPU-IVEP yeld. Recently, FSH pre-stimulation has shown to be able to increase the diameter of aspirated follicles and the percentage of transferable embryos. The hormone FSH stimulates follicle recruitment in the antral phase, in the way that they develop until the moment of divergence and one or more follicles becomes dominant. The hypothesis of this study is that the use of 200mg FSH split into 6 doses in non-lactating Holstein cows with a synchronized follicular wave emergence increases the number of follicles, the recovery rate and the number of embryos produced in vitro. Thirty six Holstein cows used as oocyte donors were homogenously allocated to one of three treatment groups in a 3x3 Latin square design: Control (C); 4 doses of FHS (FSH4); 6 doses of FSH (F6). All cows were synchronized using the same protocol for synchronization of follicular wave emergence, except for the administration and number of doses of FSH as previously described. At random days of the estrous cycle known as D0, all cows received an intravaginal P4 device (Primer®, Tecnopec-Agener União, São Paulo, Brazil) and 2mg estradiol benzoate (Ric-BE®, Tecnopec-Agener União). Three days after (D3), all cows received 0.530 mg D-Cloprostenol (Cioprostinn®, Innovare Biotecnologia e Saúde Animal Ltda, Monte Aprazível, SP, Brasil). Cows from the Control group received no additional treatment. Cows from group FSH4 were treated with 200 mg of FSH split in 4 doses of similar concentration given approximately 12 h apart, starting on D4 AM. Cows form group FSH6 were treated with 200 mg of FSH split in 6 doses of similar concentration given approximately 12 h apart, starting on D3 AM. On D7, the device was removed and OPU was performed concomitant with antral follicle count in each ovary. The oocytes considered as viable were sent to IVEP. Data was analyzed using the Glimmix of SAS 9.3, with orthogonal contrasts C1 (C x Treatment with FSH) and C2 (FSH4 x F6). There was no effect on the number of antral follicle (C = 53.3 ± 4.9 vs FSH = 51.36 ± 3.1;P = 0.89), number of total oocytes (C = 19.46 ± 1.64 vs FSH = 18.47 ± 1.27; P = 0.55), number of viable oocytes (C = 12.57 ± 1.26 vs FSH = 12.70 ± 1.03; P= 0.61), oocyte recovery rate (C = 36.5% vs FSH = 36.0%; P = 0.48) and number of embryos produced in vitro (C = 4.11 ± 0.52 vs FSH = 4.32 ± 0.46; P = 0.79). Although FSH treatment did not affect the number of follicles, it affected the distribution of them, increasing the number of follicles from 6 to 10 mm. However, FSH treatment did not alter the total number of oocytes and number of viable oocytes or embryo production.

Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

Low-load repetitive work (LLRW) and mental load are important risk factors for the development of workrelated muscle pain. The link between these risk factors and the development of pain is still not understood, but stimulation of chemo-sensitive receptors in the muscle probably plays an important role. It has been suggested that sensitising substances may accumulate in the muscle during LLRW, especially when combined with mental load. The overall purpose of this thesis was to try to shed some light on the effects of LLRW on the concentration of sensitising substances (glutamate, prostaglandin E2 (PGE2), norepinephrine (NE)) and on metabolism (lactate, pyruvate and oxygenation) in the trapezius muscle of healthy controls (CON) and subjects with trapezius myalgia (TM). A first step was to investigate whether females with TM exhibit higher absolute concentrations of glutamate and PGE2 in the affected muscle during rest. Using Microdialysis (MD) females with TM and asymptomatic controls were studied during four hours of rest. [Glutamate] and [PGE2] during rest did not differ between groups. A second step was to investigate, in a simulated occupational setting, the effects of LLRW on the concentration of sensitising substances and metabolism in the trapezius muscle of TM and CON, and whether increased work duration resulted in a progressive effect. Asymptomatic females were studied during baseline rest, 30 versus 60 min work and recovery, using MD and near infrared spectroscopy (NIRS). Subjects with TM were studied during baseline rest, 30 min work and recovery. [Glutamate] and [lactate] increased in response to work, but not progressively with increased work duration. [Glutamate] was at all time points significantly lower in TM. [PGE2]and oxygenation remained unchanged during work for CON, while for TM oxygenation decreased significantly during work. In TM [pyruvate] increased during both work and recovery, and a significant interaction between groups was found for [pyruvate] during recovery; while moderately increased in CON it increased progressively in TM. The effects of LLRW with and without superimposed mental load on intramuscular [NE], muscle activity and oxygen saturation in the trapezius were also investigated and compared. Using MD, electromyography and NIRS, healthy females were studied on two occasions; during 30 min LLRW and during 30 min LLRW with superimposed mental load. During work [NE], and muscle activity, were increased, while oxygenation decreased, but no differences between occasions. However, recovery of [NE] to baseline was slower after LLRW with superimposed mental load. The findings of the present thesis suggest: (i) no inflammation, or increased interstitial [glutamate] in TM; (ii) LLRW causes an increased anaerobic metabolism in both TM and CON; (iii) no effect of work duration was found; (iv) a significant difference in the effects of LLRW on the interstitial milieu of the trapezius muscle in TM as compared to CON; (v) LLRW causes a significant increase in [NE], but superimposed mental load does not cause a further increase; (vi) LLRW with a superimposed mental load may result in a slower recovery to baseline [NE] as compared with LLRW alone.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Die Mastzellen der Haut sind bedeutende Immuneffektorzellen in der Pathogenese der Caninen Atopischen Dermatitis (CAD; OLIVRY et al. 1997). Diese Zellen schütten in der Sofort- und in der Spätphase der Überempfindlichkeitsreaktion des Typs I Entzündungsmediatoren aus. Diätetisch verabreichte Fettsäuren werden in zelluläre Membranen eingebaut und sind somit in der Lage, die Produktion und Freisetzung dieser Entzündungsmediatoren zu beeinflussen. In der Praxis konnte gezeigt werden, dass eine diätetische Ergänzung von n6- und n3-Fettsäuren im Verhältnis von 5 zu 1 eine Linderung der klinischen Symptomatik bei 40% der an CAD leidenden Hunde herbeiführte (SCOTT et al. 1997). Das Ziel der vorliegenden Arbeit war es, zu überprüfen, welche Auswirkungen der Einbau supplementierter n6- und n3-Fettsäuren auf die Fettsäurenzusammensetzung und die Prostaglandinfreisetzung caniner Mastocytomzellen (C2) hat und ob diese Zellen in Bezug auf ihren Fettsäurenstoffwechsel als Modell für die CAD geeignet sind. Die Kultivierung der Zellen erfolgte in einem Grundmedium (DEH) oder in mit 14 µM Linol- (C18:2n6, DEH-LA), Gammalinolen- (C18:3n6, DEH-GLA), Arachidon- (C20:4n6, DEH-AA), a-Linolen- (C18:3n3, DEH-LnA), Eicosapentaen- (C20:5n3, DEH-EPA) oder Docosahexaensäure (C22:6n3, DEH-DHA) angereichertem Medium. Das Wachstum der C2 wurde in allen Kulturmedien über 11 Tage kontrolliert. Für die weiteren Untersuchungen wurden die Zellen am 4. bzw. 8. Tag geerntet, zweimal mit phosphatgepufferter Kochsalzlösung gewaschen und anschließend unter Stickstoff getrocknet. Die Ermittlung der Fettsäurenzusammensetzung der C2 erfolgte mittels Gaschromatographie nach Extraktion und Umesterung der Phospholipide. Dabei wurde L-a-Phosphatidylcholin-C17:0 als Interner Standard genutzt. Für die Bestimmung der Prostaglandine (PG) D2 und E2 wurden die Zellen mit dem Wespengift Mastoparan stimuliert. PGD2 wurde mittels eines PGD2-Methoxim-Enzym-Immunoassay (EIA) und PGE2 wurde mit Hilfe eines Radio-Immunassays (RIA) bestimmt. Die C2 zeigten in allen Kulturmedien eine Vermehrung lebender Zellen bis zum 8. Kultivierungstag, danach nahm die Zahl der abgestorbenen Zellen deutlich zu. Die Fettsäurensupplementierung beeinflusste das Zellwachstum nicht. Die erhöhte Zufuhr der Fettsäuren bewirkte eine Konzentrationserhöhung der entsprechenden Fettsäuren in den C2 (LA 4,9-fach, GLA 6,9-fach, AA 6-fach, LnA 9,3-fach, EPA 6,5-fach, DHA 8,4-fach). Weiterhin wurden signifikante Erhöhungen von Fettsäurenmetaboliten, die über die Elongasen und die D6-Desaturase aus den zugegebenen Fettsäuren gebildet werden, in den C2 gefunden. Produkte der D5-Desaturase waren dagegen nur in geringen Mengen nachweisbar. Ein zeitabhängiger Effekt des Einbaus der geprüften supplementierten Fettsäuren konnte nur für LA festgestellt werden, welche nach 8 Tagen in DEH-LA kultivierten C2 signifikant stärker eingebaut wurde als nach 4 Tagen. Die vorliegenden Ergebnisse lassen die Schlussfolgerung zu, dass in den C2 eine geringe Aktivität der D5-Desaturase vorliegt. Da eine niedrige Aktivität dieser Desaturase als möglicher Pathogenesemechanismus für das Auftreten der CAD verantwortlich gemacht wird, erscheinen die C2 als Modell für weitere Untersuchungen der CAD geeignet. Die durch Mastoparan stimulierte Freisetzung von PGE2 der C2 war bei der Kultivierung der Zellen im DEH-LnA und DEH-DHA signifikant erniedrigt und im DEH-AA und DEH-EPA signifikant erhöht. Die Ursache für die unterschiedlichen PGE2-Konzentrationen in C2 nach dem Zusatz der verschiedenen n3-Fettsäuren (LnA, EPA, DHA) ist bisher unklar. Verschiedene Möglichkeiten der Beeinflussung des Prostaglandinstoffwechsels durch diese Fettsäuren werden diskutiert. Auf Grund der erhaltenen Ergebnisse können die C2 als Modell genutzt werden, um die Mechanismen der Produktion von Prostaglandinen oder anderen Entzündungsmediatoren näher zu untersuchen und somit zur Erforschung der Pathogenesemechanismen der atopischen Dermatitis des Hundes sowie des Menschen beizutragen. / Cutaneous mast cells are considered as key immune effector cells in the pathogenesis of canine atopic dermatitis (CAD; OLIVRY et al. 1997). These cells release immediate-phase and late-phase mediators of inflammation. Dietary fatty acids are incorporated in cellular membranes and seem to influence mediator production and release. A dietary intervention with n6- and n3-fatty acids with a ratio from 5 to 1 alleviated clinical symptoms in 40% of atopic dogs (SCOTT et al. 1997). The purpose of this study was to examine the effects of n6- and n3-fatty acids on the fatty acid composition and the production of prostaglandins in canine mastocytoma cells (C2) as a possible model for CAD. Cells were cultured in a basic medium (DEH) or with additional 14 µM linoleic (C18:2n6, DEH-LA), gammalinolenic (C18:3n6, DEH-GLA), arachidonic (C20:4n6, DEH-AA), a-linolenic (C18:3n3, DEH-LnA), eicosapentaenoic (C20:5n3) or docosahexaenoic acid (C22:6n3, DEH-DHA). Cell growth was examined for 11 days in all media. The cells were harvested after 4 or 8 days, washed twice with phosphated buffered saline and dried under nitrogen for fatty acid analysis. The fatty acid composition was determined by gas chromatography after extraction and transesterification of the phospholipids using di-C17-phosphatidylcholin as internal standard. For measurment of prostaglandin (PG) D2 and E2 the C2 were stimulated with the wasp venom peptide mastoparan. PGD2 was measured by PGD2-methoxim-enzymimmunoassay (EIA) and PGE2 was determined by radioimmunoassay (RIA). Cell growth increased from day 1 to 8 and decreased thereafter in all media conditions. The supplied fatty acid did not influence the cell growth. Added fatty acids increased the concentration of these fatty acids in C2 (LA 4.9-fold, GLA 6.9-fold, AA 6-fold, LnA 9.3-fold, EPA 6.5-fold, DHA 8.4-fold). Futhermore elongated and D6-desaturated products of the corresponding fatty acids were significantly elevated, however D5-desaturated products were not measurable. An increased time dependent incorporation was only detectable for LA after culturing C2 in DEH-LA. The results let us assume that C2 has no activity of the D5-desaturase. If the assumed low activity of these desaturase is one of the mechanisms underlying the pathogenesis of CAD, C2 seems to be an adequate model for CAD. The production of PGE2 after stimulation with mastoparan was significantly reduced when C2 were cultured in DEH-LnA and DEH-DHA and was significantly increased when C2 were cultured in DEH-AA and DEH-EPA. The reason for the different PGE2-production in C2 after the treatment with the n3-fatty acids (LnA, EPA or DHA) being unsettled. The observed results suggest, that C2 could be used to investigate the mechanisms of production and release of prostaglandins or other mediators as a model to improve our understanding of the pathogenesis of canine or human atopic dermatitis.

Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.

No description available.