Normal tissue structure and function are maintained by a dynamic interaction between epithelial cells and the stroma consisting of fibroblasts, adipose, vasculature and resident immune cells, and a multitude of cytokines and growth factors. Stroma was usually studied in the background of the malignant lesion, only in recent years researchers have started considering its role before carcinogenic lesions appear. Recent studies have shown that stromal cells and their products can cause the transformation of adjacent cells through transient signaling during phenomena like adipogenesis and inflammation by secreting various cytokines and chemokines into the matrix which can lead to apoptosis resistance, proliferation, mutations etc. Research in the last few years has demonstrated a functional role for stroma in the initiation and progression of breast, colon and prostate carcinomas. In this study effect of adipogenesis and/or inflammation on prostaglandin biosynthesis is investigated and the effects that these prostaglandins can have on epithelial cells is highlighted. This work demonstrates that normal colonic fibroblasts CCD18Co can produce anti-tumorigenic and pro-tumorigenic prostaglandins during adipogenesis and that this signaling is mediated via COX-2 activation. Although deoxycholic acid (DCA), a secondary bile acid that is responsible for inflammation in the gastro-intestinal tract, induces COX-2 signaling in the fibroblasts the downstream signaling of prostaglandin synthases is suppressed. Adipogenesis also leads to an increased polyamine catabolism. Effects of the prostaglandins were studied on various epithelial colon cancer cell lines. It was seen that 15d-PGJ2 causes growth inhibition and apoptosis in all cell lines tested and it was demonstrated that an activated K-RAS suppressed this phenomena. It was also seen that 15d-PGJ2 treatment could induce MAPK signaling and that an activated K-RAS suppressed JNK activation via AKT and MKK4. In conclusion this work reports that colonic fibroblasts can produce anti-tumorigenic factors like 15d-PGJ2 which may then induce apoptosis in epithelial cancer cells. This would be suppressed by an activated K-RAS and at the same time 15d-PGJ2 mediated MAPK signaling could confer a growth advantage for these cells and thus aid in tumor progression.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A asma é um distúrbio crônico pulmonar caracterizado por inflamação, obstrução e remodelamento brônquico, levando a sintomas como sibilo, tosse e falta de ar. A terapia antiasmática consiste em corticosteroides inalados e agonistas &#946;2 de curta ou longa duração. O tratamento é limitado por efeitos colaterais e refratariedade de alguns pacientes, justificando a necessidade de novas terapias. Estudos demonstram que a 15-deoxy-delta- 12,14-prostaglandina J2 (15d-PGJ2), um ligante endógeno de receptores ativados por proliferadores de peroxissomos do tipo gama (PPAR-&#947;), é capaz de reduzir a expressão de citocinas pró-inflamatórias, o que pode resultar em benefícios no tratamento de doenças com esse perfil. O objetivo deste estudo foi avaliar o potencial anti-inflamatório e antiasmático da 15d-PGJ2 em modelos experimentais de asma. Camundongos A/J machos foram sensibilizados nos dias 0 e 7 através de injeção subcutânea (s.c.), contendo ovoalbumina (OVA) e Al(OH)3, e desafiados com 4 instilações intranasais (i.n.) de OVA em intervalos semanais. O tratamento com 15d-PGJ2 (30 e 100 &#61549;g/Kg, s.c.) foi realizado 30 min antes dos desafios a partir da terceira provocação antigênica. Em outro modelo, camundongos A/J foram desafiados intranasalmente com extrato de ácaro 3 vezes por semana durante 3 semanas. As administrações de 15d-PGJ2 (30, 70 e 100 g/Kg, s.c. e 0,65; 1,5 e 2,3 g/animal, i.n.) foram realizadas a partir da 3 semana, 30 min antes dos desafios. As análises ocorreram 24 h após o último desafio. Nossos resultados mostraram que, em camundongos previamente sensibilizados e desafiados com OVA, a administração de 15d-PGJ2 limitou significativamente o influxo peribrônquico de eosinófilos e neutrófilos, bem como a produção de muco por células caliciformes e fibrose sub-epitelial, além da hiperreatividade das vias aéreas e produção de IL-5. A redução do epitélio brônquico e das citocinas IL-13 e TNF-&#945; foram observadas somente na maior dose administrada. No modelo HDM a inflamação e o remodelamento foram atenuados em todas as doses administradas do composto, enquanto que a hiperresponssividade brônquica foi inibida apenas nas doses de 70 e 100 &#956;g/Kg (via sistêmica) e na dose intermediária dada topicamente (1,5 &#956;g/animal, i.n.). Os níveis de citocinas foram atenuados pelo tratamento subcutâneo, porém somente os níveis de IL-17, eotaxina-1 e TNF-&#945; foram inibidos com a dose intranasal de 0,65 g/animal. O aumento da expressão de NF-&#954;B, induzido por provocação com HDM também foi reduzido significativamente pela administração de 15d-PGJ2. Em conjunto, nossos dados indicam que o tratamento com 15d-PGJ2 inibe alterações cruciais associadas à patogênese da asma, em modelos experimentais distintos da doença, demonstrando possuir grande potencial para controlar e reverter inflamação, hiperreatividade e remodelamento pulmonar desencadeados por provocação alérgica. / Asthma is a chronic pulmonary disorder characterized by inflammation, obstruction and airway remodeling, leading to symptoms such as wheezing, coughing and breathlessness. Asthma therapy is based on inhaled corticosteroids and short or long term-&#946;2 agonists. The treatment is limited by side effects and some refractory patients, justifying the study for new therapies. Studies have demonstrated that 15-deoxy-delta-12 ,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPAR-&#947;) can reduce pro-inflammatory cytokines expression, providing a protective effect in diseases with this profile. The aim of this study was to evaluate the anti-inflammatory and antiasthmatic properties of 15d-PGJ2 in murine models of experimental asthma. A/J mice rats were sensitized on days 0 and 7 by subcutaneous (s.c.) injection , containing ovalbumin (OVA) and Al(OH)3, and challenged with 4 intranasal OVA instillations at weekly intervals. 15d-PGJ2 treatment (30 and 100 &#956;g/Kg) was performed 30 min before the challenges from the third antigen challenge. In another model, A/J mice were intranasally (i.n.) challenged with mite extract 3 times per week for 3 weeks. The administration of 15d-PGJ2 (30, 70 and 100 &#956;g /Kg, s.c. and 0.65, 1.5 and 2.3 &#956;g / animal, i.n.) were performed from the 3rd week, 30 min before the challenges. The analyzes were 24 h after the last challenge. Our results showed that in previously OVA-sensitized and challenged mice, administration of 15d-PGJ2 limited significantly (p <0.05), eosinophilic and neutrophilic inflammation and mucus production by goblet cells and sub-epithelial fibrosis, as well as airways hyperreactivity and IL-5 production. The reduction of bronchial epithelium and IL-13 and TNF-&#945; were observed only at the highest dose administered. In HDM model, inflammatory and remodeling parameters were attenuated in all administered doses of compound, whereas bronchial hyperresponsiveness was inhibited only at doses of 70 and 100 &#956;g/kg (s.c.) and 1.5 &#956;g/animal (i.n.). Serum cytokine levels were attenuated by subcutaneous treatment, but only IL-17, Eotaxin-1 and TNF-&#945; was inhibited by intranasal dose of 0.65 &#956;g/ animal. The increased expression of NF-kB induced by HDM challenge was also significantly reduced by the administration of 15d-PGJ2. Together, our data indicate that treatment with 15d-PGJ2 inhibits critical changes associated with the pathogenesis of asthma in different experimental models of the disease, demonstrating great potential to control and reverse pulmonary inflammation, hyperresponsiveness and remodeling triggered by allergen challenge.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A asma é um distúrbio crônico pulmonar caracterizado por inflamação, obstrução e remodelamento brônquico, levando a sintomas como sibilo, tosse e falta de ar. A terapia antiasmática consiste em corticosteroides inalados e agonistas &#946;2 de curta ou longa duração. O tratamento é limitado por efeitos colaterais e refratariedade de alguns pacientes, justificando a necessidade de novas terapias. Estudos demonstram que a 15-deoxy-delta- 12,14-prostaglandina J2 (15d-PGJ2), um ligante endógeno de receptores ativados por proliferadores de peroxissomos do tipo gama (PPAR-&#947;), é capaz de reduzir a expressão de citocinas pró-inflamatórias, o que pode resultar em benefícios no tratamento de doenças com esse perfil. O objetivo deste estudo foi avaliar o potencial anti-inflamatório e antiasmático da 15d-PGJ2 em modelos experimentais de asma. Camundongos A/J machos foram sensibilizados nos dias 0 e 7 através de injeção subcutânea (s.c.), contendo ovoalbumina (OVA) e Al(OH)3, e desafiados com 4 instilações intranasais (i.n.) de OVA em intervalos semanais. O tratamento com 15d-PGJ2 (30 e 100 &#61549;g/Kg, s.c.) foi realizado 30 min antes dos desafios a partir da terceira provocação antigênica. Em outro modelo, camundongos A/J foram desafiados intranasalmente com extrato de ácaro 3 vezes por semana durante 3 semanas. As administrações de 15d-PGJ2 (30, 70 e 100 g/Kg, s.c. e 0,65; 1,5 e 2,3 g/animal, i.n.) foram realizadas a partir da 3 semana, 30 min antes dos desafios. As análises ocorreram 24 h após o último desafio. Nossos resultados mostraram que, em camundongos previamente sensibilizados e desafiados com OVA, a administração de 15d-PGJ2 limitou significativamente o influxo peribrônquico de eosinófilos e neutrófilos, bem como a produção de muco por células caliciformes e fibrose sub-epitelial, além da hiperreatividade das vias aéreas e produção de IL-5. A redução do epitélio brônquico e das citocinas IL-13 e TNF-&#945; foram observadas somente na maior dose administrada. No modelo HDM a inflamação e o remodelamento foram atenuados em todas as doses administradas do composto, enquanto que a hiperresponssividade brônquica foi inibida apenas nas doses de 70 e 100 &#956;g/Kg (via sistêmica) e na dose intermediária dada topicamente (1,5 &#956;g/animal, i.n.). Os níveis de citocinas foram atenuados pelo tratamento subcutâneo, porém somente os níveis de IL-17, eotaxina-1 e TNF-&#945; foram inibidos com a dose intranasal de 0,65 g/animal. O aumento da expressão de NF-&#954;B, induzido por provocação com HDM também foi reduzido significativamente pela administração de 15d-PGJ2. Em conjunto, nossos dados indicam que o tratamento com 15d-PGJ2 inibe alterações cruciais associadas à patogênese da asma, em modelos experimentais distintos da doença, demonstrando possuir grande potencial para controlar e reverter inflamação, hiperreatividade e remodelamento pulmonar desencadeados por provocação alérgica. / Asthma is a chronic pulmonary disorder characterized by inflammation, obstruction and airway remodeling, leading to symptoms such as wheezing, coughing and breathlessness. Asthma therapy is based on inhaled corticosteroids and short or long term-&#946;2 agonists. The treatment is limited by side effects and some refractory patients, justifying the study for new therapies. Studies have demonstrated that 15-deoxy-delta-12 ,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPAR-&#947;) can reduce pro-inflammatory cytokines expression, providing a protective effect in diseases with this profile. The aim of this study was to evaluate the anti-inflammatory and antiasthmatic properties of 15d-PGJ2 in murine models of experimental asthma. A/J mice rats were sensitized on days 0 and 7 by subcutaneous (s.c.) injection , containing ovalbumin (OVA) and Al(OH)3, and challenged with 4 intranasal OVA instillations at weekly intervals. 15d-PGJ2 treatment (30 and 100 &#956;g/Kg) was performed 30 min before the challenges from the third antigen challenge. In another model, A/J mice were intranasally (i.n.) challenged with mite extract 3 times per week for 3 weeks. The administration of 15d-PGJ2 (30, 70 and 100 &#956;g /Kg, s.c. and 0.65, 1.5 and 2.3 &#956;g / animal, i.n.) were performed from the 3rd week, 30 min before the challenges. The analyzes were 24 h after the last challenge. Our results showed that in previously OVA-sensitized and challenged mice, administration of 15d-PGJ2 limited significantly (p <0.05), eosinophilic and neutrophilic inflammation and mucus production by goblet cells and sub-epithelial fibrosis, as well as airways hyperreactivity and IL-5 production. The reduction of bronchial epithelium and IL-13 and TNF-&#945; were observed only at the highest dose administered. In HDM model, inflammatory and remodeling parameters were attenuated in all administered doses of compound, whereas bronchial hyperresponsiveness was inhibited only at doses of 70 and 100 &#956;g/kg (s.c.) and 1.5 &#956;g/animal (i.n.). Serum cytokine levels were attenuated by subcutaneous treatment, but only IL-17, Eotaxin-1 and TNF-&#945; was inhibited by intranasal dose of 0.65 &#956;g/ animal. The increased expression of NF-kB induced by HDM challenge was also significantly reduced by the administration of 15d-PGJ2. Together, our data indicate that treatment with 15d-PGJ2 inhibits critical changes associated with the pathogenesis of asthma in different experimental models of the disease, demonstrating great potential to control and reverse pulmonary inflammation, hyperresponsiveness and remodeling triggered by allergen challenge.

This thesis aims to investigate fluoxetine, a widely prescribed SSRI antidepressant, for its role in the pathogenesis of NAFLD and uncover novel mechanisms by which it may contribute to drug-induced steatosis. We demonstrated that increased hepatic lipid accumulation was mediated, in part, via elevated serotonin production. The inhibition of hepatic serotonin synthesis prevented lipid accumulation in fluoxetine-treated hepatocytes demonstrating a causal role for serotonin in fluoxetine-induced hepatic steatosis. Interestingly, in several studies, serotonin signaling has been shown to impact prostaglandin biosynthesis. As prostaglandins have been implicated in the development of NAFLD, and fluoxetine has previously been shown to alter the production of prostaglandins I assessed the role of prostaglandins in fluoxetine-induced hepatic lipid accumulation. Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes, increased production of prostaglandin 15-deoxy-Δ12,14PGJ2 (PPARG agonist), and elevated PPARG targets involved in fatty acid uptake. Fluoxetine-induced lipid accumulation, 15-deoxy-Δ12,14PGJ2 production, and the expression of PPARG lipogenic genes were attenuated with a PTGS1 specific inhibitor. Taken together these findings suggested that fluoxetine-induced lipid accumulation was mediated via PTGS1 and its downstream product 15-deoxy-Δ12,14PGJ2. Given that Pparg was elevated following fluoxetine treatment, and PPARG regulates microRNA involved in hepatic lipid accumulation, my final project focused on PPARG’s role in altered miRNA expression. Indeed, fluoxetine treatment increased the miRNA expression of miR-122, an effect that was attenuated when fluoxetine treatment was combined with the PPARG antagonist GW9662, suggesting a fluoxetine-PPARG-miR122 axis contributing to hepatic steatosis. While these studies have only been performed in vitro, an understanding of the molecular changes associated with SSRI treatment may lead to the development of strategies to prevent the increased risk of adverse metabolic outcomes associated with the use of SSRI antidepressants. / Dissertation / Doctor of Philosophy (Medical Science) / In adults, major depressive disorder (depression) is one of the most common psychiatric illnesses. Recent data suggests that there are more than 4.1 million Canadians who currently suffer from depression. Depression is commonly treated using selective serotonin reuptake inhibitor (SSRI) antidepressants. While these antidepressants do help manage depressive symptoms, they can also cause unwanted side effects including a build-up of fat in the liver, leading to fatty liver disease. The goal of my research is to understand the link between SSRI use and the development of fatty liver disease. This thesis investigates the effects of fluoxetine (Prozac®), a commonly used SSRI antidepressant, on molecular pathways that can lead to the development of fatty liver disease. An understanding of the molecular changes with SSRI treatment may lead to the development of strategies to prevent the harmful effects of SSRI antidepressants on the liver.