Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Orientador: José Luiz Laus / Banca: Carlos Augusto Araújo Valadão / Banca: Paula Diniz Galera / Resumo: Estudou-se a eficácia do meloxicam e do carprofeno, aplicados por diferentes vias, em uveítes experimentais em cães. Realizou paracentese de câmara anterior em dois momentos (M0 e M1), com intervalo de cinco horas entre si. Em M0 e M1, colheram-se 0,2 ml de humor aquoso e determinou-se a concentração de proteína total e de prostaglandina E2 (PGE2). Em um primeiro período, constituíram-se quatro grupos (n = 5), que receberam meloxicam ao final de M0 pelas vias subcutânea (GIm), subconjuntival (GIIm) e tópica (GIIIm). Um quarto grupo não recebeu tratamento (Controle). Decorridos sete dias, os animais foram submetidos aos mesmos procedimentos adotados previamente e receberam carprofeno. Avaliação clínica foi também realizada, assim como histopatologia da conjuntiva dos animais dos grupos GIIm e GIIc. Os resultados foram avaliados estatisticamente (p LÜ 0,05). Em todos os grupos, encontrou-se aumento significativo dos níveis protéicos e de PGE2 em M1 (p < 0,001). Não se observou diferença significativa entre os grupos para os valores de proteína total e de PGE2 em M1 (p > 0,05). Observou-se correlação positiva entre proteína total e PGE2 (p < 0,05) apenas no GIm, GIc, GIIIm, GIIIc e GIIm. Exsudado inflamatório de caráter agudo e hemorragia discreta foram vistos à histopatologia após a aplicação de ambos os fármacos (p > 0,05). O meloxicam e o carprofeno foram ineficazes em inibir a síntese de PGE2 e o influxo de proteínas para a câmara anterior, por qualquer uma das vias testadas. A redução nos níveis de 44% proteínas, quando o carprofeno foi utilizado pela via tópica, sugere que por esta via, ele pode ser utilizado como adjuvante no controle da uveíte em cães. / Abstract: Efficacy of meloxican and carprofen, administered by different routes, in experimental uveitis in dogs were studied. Anterior chamber paracenteses was accomplished at two different moments (M0 and M1), with a five hour interval among them. At M0 and M1, 0,2 ml of aqueous humor were collected and total protein and prostaglandin E2 (PGE2) concentration was determined. Four groups were formed in a first period (n = 5), which received meloxican at the end of M0, by the following routes: subcutaneous (GIm), subconjunctival (GIIm), and topical (GIIIm). A fourth group that received no treatment was instituted (Control). Seven days after, animals underwent the same procedures described previously and received carprofen. Clinical evaluation was also performed, as well as conjunctival histopathology of the conjunctiva of the animals of GIIm and GIIc. Results were evaluated statistically (p LÜ 0,05). In all groups, protein and PGE2 values enhanced significantly in M1 (p < 0,001). Protein and PGE2 values, did not change significantly between groups at M1 (p > 0,05). Positive correlation among total protein and PGE2 (p < 0,05) was only noted in GIm, GIc, GIIIm, GIIIc and GIIm. Inflammatory exudate of acute character and mild hemorrhage were seen at histopathology, after both agents were administered. Meloxican and carpofen were unable to inhibit PGE2 synthesis and the protein influx to the anterior chamber by any of the tested routes. The lowering of 44% in protein levels, when carprofen was used by the topical route, suggests that by this route, it can be used as an adjuvant to control uveitis in dogs. / Mestre

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.

<p>Prostaglandin (PG) E₂ is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE₂ has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE₂ is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE₂ synthesising enzymes are not completely established.</p><p>PGE₂ was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA₂a (cPLA₂a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE₂. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH₂. The conversion of PGH₂ to the final product PGE₂ was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA₂α was markedly advantageous for the formation of PGE₂.</p><p>Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE₂ in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA₂a.</p>

<p>Aim: The aim of this essay is to study the communication strategy that the company SATS developed before taking over the company e2. The following questions are to be answered: - what was the planned communication strategy before taking over the company e2? - did SATS follow their original plan when implementing this strategy? – Which strengths and weaknesses of the communication strategy can be identified based on opinions of the employees?</p><p>Material/Method: Together with literature and document studies, a qualitative method has been used and seven personal interviews have been conducted. One informant was selected due to professional position; the other six respondents were former employees of the affected company e2.</p><p>Main results: The main results from the study show that the company SATS did in fact follow their plan very accurately with only some minor changes such as an earlier started education program and exclusion of an initially planned evaluation activity. Communication about the takeover of e2 to employees was quick and clear. This can be seen as strength of the strategy. The employees were positive to all of the activities that involved personal contact and face to face meetings. However the employees were negative to the limited possibilities for contribution of opinions and also to the lack of a thorough evaluation of the communication activities.</p>

Aim: The aim of this essay is to study the communication strategy that the company SATS developed before taking over the company e2. The following questions are to be answered: - what was the planned communication strategy before taking over the company e2? - did SATS follow their original plan when implementing this strategy? – Which strengths and weaknesses of the communication strategy can be identified based on opinions of the employees? Material/Method: Together with literature and document studies, a qualitative method has been used and seven personal interviews have been conducted. One informant was selected due to professional position; the other six respondents were former employees of the affected company e2. Main results: The main results from the study show that the company SATS did in fact follow their plan very accurately with only some minor changes such as an earlier started education program and exclusion of an initially planned evaluation activity. Communication about the takeover of e2 to employees was quick and clear. This can be seen as strength of the strategy. The employees were positive to all of the activities that involved personal contact and face to face meetings. However the employees were negative to the limited possibilities for contribution of opinions and also to the lack of a thorough evaluation of the communication activities.

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 members of the ubiquitously expressed E-protein subfamily of basic Helix-Loop-Helix (bHLH) transcription factors, E12/E47, SEF2-1 and HEB, have important roles as regulators of gene expression in various differentiation processes, including lymphocyte development and myogenesis. In myogenesis, E-proteins are proposed to function as obligate heterodimer partners for members of the MyoD family of muscle-specific bHLH transcription factors. The calcium ion (Ca2+) is a universal cellular messenger involved in regulation of a variety of cellular functions, including transcription. The Ca2+-bound form of the Ca2+-binding protein calmodulin (Ca2+/CaM) has been shown to inhibit DNA binding of E-proteins, but not tissue specific bHLH transcription factors, through direct physical interaction with the DNA binding basic sequence. The main focus of this thesis is on the role of Ca2+-binding proteins in regulation of bHLH transcription factors. Solution structure analysis of CaM in complex with the CaM-binding basic sequence of an E-protein revealed a novel type of protein-protein interaction with alternative binding modes in a complex of a CaM dimer surrounding the dimer of the E-protein sequence. This model for the interaction was further supported by mutational analysis, since every amino-acid substitution in the CaM binding basic sequence of E12 only partially affected the interaction with CaM. The mechanism of Ca2+/CaM regulation of transcriptional activation by E-proteins was studied using a cell culture system. CaM overexpression inhibited transcriptional activation by E12, E47 and SEF2-1 but not by MyoD. Ca2+/CaM inhibition of DNA binding in vitro directly correlated with the inhibitory effects of Ca2+ stimulation and CaM overexpression on transcription in vivo in a series of E12 basic sequence mutants. Furthermore, in vivo DNA binding of E12, but not a CaM resistant mutant of E12, was inhibited by overexpression of CaM. The data indicate that Ca2+/CaM can inhibit transcriptional activation by E-proteins through formation of a CaM-E-protein complex that can not bind DNA. An in vitro myogenesis system was used to investigate the potential role of the CaM-E-protein interaction in regulation of differentiation. CaM resistant mutants of E12 were inhibitory in MyoD initiated myogenic conversion of transfected fibroblasts, and inducers of intracellular Ca2+ activated, and Ca2+-channel blockers inhibited, transcriptional activation by E12, but not by a CaM resistant mutant of E12, with MyoD. The data support a model that Ca2+/CaM plays a role in initiation of myogenic differentiation through inhibition of E-protein dimers that can function as competitors to the CaM resistant MyoD/E-protein heterodimers required for myogenesis. The potential involvement of the Ca2+-binding calretinin proteins in regulation of bHLH transcription factors was also studied. Calretinin and the alternative splice variant calretinin-22k have been proposed to function as Ca2+-buffer proteins. Calretinin expression is restricted primarily to neuronal tissues. Calretinin and calretinin-22k are also found expressed in colon cancers, but not in normal colon tissue, and a role for calretinins in tumorigenesis has been proposed. We show that calretinins can inhibit DNA binding and transcriptional activation by E12 through basic sequence interaction. Endogenous E12/E47 and calretinin co-localize in a subset of cells in a proliferating colon cancer cell line and can be co-immunoprecipitated from the cell extract. A model is proposed in which calretinin overexpression can contribute to tumorigenesis through inhibition of the anti-proliferative function of E-proteins. The role of the E-protein E2-2 in lymphocyte development was studied using genetically altered mice with mosaic deletion of the E2-2 gene. The proportion of cells with a functional E2-2 allele was increased in the B- and T-lymphocyte populations, indicating a role for E2-2 not only in B-cell development, as reported before, but also in T-cell development.

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.