Made available in DSpace on 2016-08-17T18:39:37Z (GMT). No. of bitstreams: 1 3649.pdf: 6160139 bytes, checksum: e2fc962eefef9b03fa83e0311a505531 (MD5) Previous issue date: 2010-11-23 / Financiadora de Estudos e Projetos / In this research molecular docking was used to study enzime-ligand complexes of Cyclooxygenase 1 (COX-1) and Cyclooxygenase 2 (COX-2) with pyrimidine derivatives, aiming at understanding the possible mechanisms of action of these compounds and, thus, suggest modifications that could increase their specificity. The chosen ligands were a series of 25 substituted pyrimidines with known activity. The three-dimensional structures of these compounds were obtained by molecular modeling and that of the enzymes from the PDB and PDBSum under the codes 2OYE and 1CX2 for COX-1 and COX-2 respectively. The binding sites chosen for the docking studies were 20 Å around the crystallographic ligands IM8-700 (2OYE) and SC-558 (1CX2). In the COX-1 formed complexes the SO2Me moiety is positioned in such a way as to form hydrogen bonds with Ile517 and Phe518. The benzo[b]thiophen-2-ylmethyl-[2-(4-methanesulfonylphenyl)-6-trifluoromethylpyrimidin-4- yl]amine formed the most favorable complex with COX-1. In COX-2, the enzyme-ligand interaction pattern shows the SO2Me group in the side pocket, forming hydrogen bonds with His90 and Arg513 and the different substituent groups of the pyrimidine ring form hydrogen bonds with Arg120 and Tyr355. The presence of a small lipophilic pocket in COX-2 and the docking results suggest that the ligands 2, 15, 17, 22 and 23 may have their activity enhanced by the addition of a hydrophobic group on the phenyl or thiophenyl rings so this group can interact within this pocket. In both cases the mechanism of inhibition is probably competitive. As the search for new anti-inflammatory drugs must deal with a subtle balance of COX-2 and COX-1 inhibitions, the ligands 2 and 22 that showed better results for COX-2 rather than for COX-1 would be the most promising ones and therefore, those that should be tested in vivo. / Neste trabalho, o docking molecular foi utilizado para o estudo da formação de complexos alvoligante das enzimas Ciclooxigenase 1 (COX-1) e Ciclooxigenase 2 (COX-2) com derivados pirimidínicos, com a finalidade de entender os possíveis mecanismos de ação e, assim, propor modificações nos compostos visando diminuir prováveis efeitos colaterais. Os ligantes escolhidos foram uma série de 25 pirimidinas substituídas com atividade conhecida. As estruturas tridimensionais destas moléculas foram obtidas por modelagem molecular e as das enzimas dos bancos de dados PDB e PDBSum sob o código 2OYE e 1CX2 para COX-1 e COX- 2 respectivamente. Os sítios de ligação escolhidos para os cálculos de docking foram de 20 Å ao redor dos ligantes cristalográficos IM8-700 (2OYE) e SC-558 (1CX2). Das pirimidinas analisadas as que formaram complexo com a COX-1 se orientaram com a porção SO2Me formando ligações de hidrogênio com a Ile517 e Phe518. Sendo o benzo[b]tiofen-2-ilmetil-2-(4- metanosulfonilfenil)-6-trifluorometilpirimidina-4-il]amina o mais favorável para a formação de complexo com a COX-1. Para a COX-2, os compostos que se ligaram mostram um padrão que inclui ligações de hidrogênio entre a porção SO2Me e a His90 e Arg513 do bolso lateral e entre a Arg120 e Tyr355 com os grupos substituintes do anel pirimidínico. A presença de um pequeno bolso lipofílico na COX-2 e os resultados de docking permitem sugerir que os ligantes 2, 15, 17, 22 e 23 poderiam mostrar melhor atividade mediante a adição de um grupo hidrofóbico no anel fenila ou tiofenila para que este grupo se posicione dentro desse bolso. Em ambos os casos o tipo de inibição provável é o competitivo. Como a busca por novos fármacos antiinflamatórios deve lidar com um equilíbrio entre a inibição de COX-2 e COX-1, os ligantes 2 e 22 que apresentaram resultados favoráveis para a COX-2 e não tanto para a COX-1 seriam os mais promissores e, portanto, aqueles que poderiam ser testados in vivo.

L'inflammation est considérée comme un promoteur de la cancérogenèse. La cyclooxygénase-2 (COX-2), la forme inductible de la famille des cyclooxygénases est un médiateur important de l'inflammation. Cette enzyme est constitutivement exprimée dans un grand nombre de cancers tels que les cancers du sein, du colon ou de la prostate. De nombreuses études mettent en évidence que COX-2 est surexprimée lors des étapes pré-néoplasiques. La COX-2 représente de ce fait une cible thérapeutique potentielle en chimioprévention et également pour le traitement des cancers. L'utilisation d'inhibiteurs synthétiques de COX-2 qui ciblent l'activité enzymatique est le seul traitement clinique actuellement disponible pour réduire l'activité de COX-2. Cependant, ces agents présentent des effets secondaires sévères, ce qui limite leur prise chronique chimiopréventives ou au cours des traitements anti-cancéreux. Une stratégie alternative pour cibler la fonction de COX-2 est d'inhiber son expression. Un grand nombre d'études montrent que certains produits naturels (la curcumine, le resveratrol ou l'apigénine par exemple) inhibent, préférentiellement l'expression de COX-2, sans être toxique. Notre projet analyse l'effet de l'acide cinnamique, un produit naturel extrait de la plante Cinnamonium cassia, sur l'expression de COX-2 au cours de la cancérogenèse dans le but d'évaluer son intérêt en chimioprévention. Nous avons utilisé comme modèle les cellules mammaires non carcinogènes, MCF10A stimulées avec un ester de phorbol, le 12-phorbol myristate 13-acetate (PMA), qui induit l'expression de COX-2. Nous avons observé une diminution de l'expression de COX-2 au niveau de l'ARNm et de la protéine après le traitement avec différentes concentrations d'acide cinnamique (1 et 10μM). L'analyse des mécanismes impliqués dans la diminution de l'expression de COX-2 a mis en évidence que l'acide cinnamique régule l'expression de COX-2 de façon post-transcriptionnelle en réduisant la stabilité de son ARNm. Cet effet est associé à une prévention de la diminution de l'expression de microARN (miR)-16 et une inhibition de l'expression de p38 induite par l'acide cinnamique en réponse au traitement avec le PMA. / Inflammation is considered a cancer-promoting factor. Cyclooxygenase-2 (COX-2), the inducible form of the family of cyclooxygenases is an important mediator of inflammation, which has been found constitutively expressed in many forms of cancer including breast, colon or prostate. A number of studies show that COX-2 is stably expressed since the early pre-neoplastic stages. This encourages us to consider COX-2 as a potential target in chemoprevention as well as in the treatment of cancer. Synthetic inhibitors of COX-2, which target enzymatic activity, are the only clinical strategy to counteract COX-2. However, these compounds present severe side effects, a fact that limits their prolonged intake, like requested in chemoprevention or during anti-cancer treatment.An alternative strategy to target COX-2 is at the level of its expression. A number of studies show that several natural compounds including curcumin, resveratrol or apigenin preferentially target COX-2 expression without showing toxicity.Our study analyses the effect of cinnamic acid, a natural compound derived from Cinnamonium cassia on COX-2 expression during carcinogenesis, with the final aim to evaluate its potential in chemoprevention/therapy.For our chemopreventive purposes, we used the non-carcinogenic breast cell line MCF10A, stimulated by the phorbol ester 12-phorbol myristate 13-acetate (PMA), which typically induces COX-2. We show a reduction of induced COX-2 expression after treatment with different concentrations of cinnamic acid (1 and 10μM). The analysis of the mechanisms involved in COX-2 protein expression decrease shows that cinnamic acid regulated COX-2 expression at the post-transcriptional level by reducing COX-2 mRNA stability. This effect is associated with the ability of cinnamic acid to prevent downregulation of miR-16 expression and p38 activation in response to PMA treatment.

Les statines sont des molécules hypocholestérolémiantes, inhibiteurs compétitifs de l'hydroxyméthylglutaryl-CoA réductase, possédant des propriétés anti-inflammatoires et anti-oxydantes dépendantes et indépendantes de leur capacité à réduire le cholestérol. L'hème-oxygénase-1, une enzyme responsable du catabolisme de l'hème participe à la résolution de l'inflammation, notamment via ses propriétés anti-oxydantes. Le système enzymatique cyclooxygénase-2/prostaglandine synthase-1 microsomale catalyse la transformation de l'acide arachidonique en prostaglandine E2, médiateur biologique important dans la régulation de l'hémostase des vaisseaux, la croissance cellulaire, l'inflammation et la douleur. La cyclooxygenase-2 et l'heme-oxygenase-1 étant des cibles des statines, et jouant un rôle majeur dans l'inflammation et la fibrose, le but de ce travail a été d'élucider les mécanismes moléculaires impliqués dans la régulation de l'expression de ces enzymes par les statines dans les macrophages et les fibroblastes. Dans les fibroblastes, nous avons démontré que l'induction de l'hème-oxygénase-1 par deux statines différentes, la simvastatine et la fluvastatine, est dépendante de la voie du mévalonate et de la géranygéranylation des protéines. Nous avons pu également démontrer le rôle des facteurs de transcription CCAAT/enhancer-binding protein beta et gamma et upstream stimulatory factor 1 ou 2 dans cette induction, en utilisant des ARN interférants. Dans les macrophages, nous avons mis en évidence que les statines induisent l'hème-oxygénase-1 par un mécanisme dépendant du monoxyde d'azote. La petite protéine G Rho A/C semble être impliquée dans cette régulation ainsi que le facteur de transcription CCAAT/enhancer-binding protein beta. Finalement, nous avons analysé le rôle des statines dans la régulation des cyclooxygénase-2 et la prostaglandine synthase-1 microsomale dans des myofibroblastes hépatiques humains. Nous avons mis en évidence que les statines induisent l'expression de ces deux enzymes, par un mécanisme impliquant la voie de la Rho A/C. La conséquence de cette activation est une libération de la prostaglandine E2 qui inhibe la prolifération des myofibroblastes hépatiques. Au niveau transcriptionnel, l'élément de réponse nuclear factor-kappa B et cAMP response element/E box régions ainsi que GATA les régions riches en GC participent à la régulation des promoteurs de la cyclooxygénase-2 et de la prostaglandine synthase-1 microsomale, respectivement. En resumé, nos travaux confirment que les statines jouent un rôle protecteur dans les macrophages et les fibroblastes en induisant hème-oxygénase-1, la cyclooxygénase-2 et la prostaglandine synthase-1 microsomale, des enzymes qui jouent un rôle majeur dans le contrôle de l'inflammation et la fibrose. / Statins are selective competitive inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase administered for the treatment of hypercholesterolemia. These molecules have multiple pleiotropic effects in addition to lowering cholesterol such as anti-inflammatory and anti-oxidant properties. Heme-oxygenase-1 is responsible for the catabolism of heme and has important anti-oxidant and anti-inflammatory effects. Cylooxygenase-2, along with microsomal prostaglandin E synthase-1, metabolizes arachidonic acid into prostaglandin E2, a biological mediator with important effects on vascular tone, cell growth, inflammation and pain. Because cyclooxygenase-2 and heme-oxygenase-1 are targets for statins and play a key role in inflammation and fibrosis, we aimed to investigate the molecular mechanisms underlying the regulation of these enzymes by statins in macrophages and fibroblasts.In fibroblasts, simvastatin and fluvastatin induced HO-1 expression in a mevalonate and geranylgeranylated-dependent manner. We further demonstrated a role of the transcription factors CCAAT/enhancer-binding protein beta and gamma and upstream stimulatory factor 1 or 2 in statin-dependent induction of heme-oxygenase-1 using small interfering RNA and dominant-negative constructs.In macrophages, we showed that statins i- increase the level of expression of heme-oxygenase-1 and ii- nitric oxide can play a role in statin-dependent induction of heme-oxygenase-1 , iii- RhoA/C is one of the target of statins, iv- the transcription factor CCAAT/enhancer-binding protein beta is involved in the regulation of heme-oxygenase-1 by statins.Finally, since cyclooxygenase-2 and heme-oxygenase-1 play a role in fibrosis and inflammation, we analyzed the effect of statins in human hepatic myofibroblasts, the fibrogenic cells of the liver. Statins significantly upregulated cyclooxygenase-2 and microsomal prostaglandin E synthase-1 and inhibited cell proliferation in a PGE2-dependent manner via inhibition of RhoA/C activity. Further analysis of the transcription factors involved showed a role for nuclear factor kappa B and cAMP response element/Ebox regions of cyclooxygenase-2 promoter and GATA and GC rich box regions for microsomal prostaglandin E synthase-1.Overall, our thesis results highlight the molecular mechanisms of statin-dependent regulation of two important enzymes in inflammation and fibrosis, in macrophages and fibroblasts. They confirm that some of the protective effects of statins go through the upregulation of heme-oxygenase-1, cyclooxygenase-2 and microsomal prostaglandin E synthase-1.

L'insuffisance rénale chronique (IRC) se caractérise par la diminution progressive et irréversible des fonctions renales. Elle s'accompagne d'une accumulation d'un ensemble de toxines responsable du syndrome urémique. Le syndrome urémique touche tous les organes, et de façon préoccupante le système cardiovasculaire. Il est associé à une dysfonction endothéliale, à la production d'un stress oxydant et d'une inflammation. L'objectif de cette thèse était d'identifier les mécanismes moléculaires responsables des complications du syndrome urémique. La première partie a tenté d'identifier des épissages alternatifs associés à l'IRC. Deux épissages alternatifs ont été identifiés. Cependant, le petit nombre d'épissages alternatifs trouvé au vue du nombre de gènes étudiés, nous permet de conclure que si l'IRC peut être responsable de l'apparition d'épissages alternatifs, ce phénomène n'est pas déterminant dans la régulation de l'expression des gènes responsable des complications de l'IRC. Dans la deuxième partie, nous avons montré par une étude clinique que le taux d'une toxine urémique, l'acide indole acétique (IAA), était associé à la mortalité et à la survenue d'événements cardio-vasculaires. In vitro, l'IAA induit un stress oxydant et un signal inflammatoire par l'induction de la cyclooxygénase 2 (COX-2). Une voie inflammatoire non génomique impliquant aryl hydrocarbon receptor (AhR), p38 MAPK et NF-κB est responsable de l'induction de la COX-2 endothéliale par l'IAA. Nos travaux ont identifié de nouvelles cibles thérapeutiques dont la modulation pourrait avoir un impact sur la mortalité cardiovasculaire des patients présentant une maladie rénale chronique. / Chronic kidney disease (CKD) is characterized by an irreversible decrease in kidney functions. Accumulation of uremic toxins is implicated in the uremic syndrome. Uremic syndrome affects all organs and particularly the cardiovascular system. The aim of this thesis was to identify and understand the molecular mechanisms implicated in the uremic syndrome.The first part attempted to ascertain the existence of alternative splice events associated with CKD. Two alternative splicing were identified. The small number of alternative splice events highlighted allows us to conclude that this phenomenon does not seem to be a key event in the modulation of gene expression during CKD.In the second part of this work, we demonstrated that the plasmatic concentration of an uremic toxin, Indole-3-acetic acid (IAA), is associated with all-cause mortality and major cardiovascular events. In vitro, we demonstrated that IAA induced endothelial cyclooxygenase-2 expression and endothelial oxidative stress production. IAA activated an endothelial Aryl hydrocarbon receptor/P38MAPK/NF-κB pathway. The activation of this inflammatory AHR dependant pathway could play a critical role in the increase of cardiovascular morbidity and mortality observed during CKD.Our work provides new therapeutic targets. The modulation of their activation could reduce cardiovascular mortality in patients with chronic kidney disease.

Seit Anfang der 90-iger Jahre ist bekannt, dass nichtsteroidale Antirheumatika die Inzidenz des kolorektalen Karzinoms vermindern können. Diese antitumoröse Aktivität wird wahrscheinlich über die Cyclooxygenase-2 (COX-2) vermittelt, welche die Biosynthese von Prostaglandin H2, einer Vorstufe der Prostanoide, katalysiert. Eine Überexpression der COX-2 wurde für verschiedene Karzinome beschreiben. In dieser Studie wurde die Expression und tumorbiologische Relevanz der COX-2 im malignen Melanom, als Vertreter nichtepithelialer Tumoren, untersucht. Im Western blot wurde COX-2 Protein in den sechs untersuchten Melanomzelllinien nachgewiesen. Mittels eines spezifischen ELISAs wurde PGE2 im Überstand der Zelllinien nachgewiesen. Die PGE2-Biosynthese wurde durch den COX-2-spezifischen Inhibitor NS 398 konzentrationsabhängig inhibiert. Die IC50 der COX-2 für NS 398 wurde mit etwa 6 microM bestimmt. NS 398 inhibierte die Matrigelinvasion aller sechs Melanomzelllinien, ohne Einfluss auf die Proliferation zu haben. Die Invasionshemmung war PGE2-unabhängig, weil i) exogenes PGE2 die Invasionshemmung nicht wieder aufhob, ii) die erforderliche Konzentration von NS 398 zur Invasionshemmung im 8-fachen Bereich der IC50 der COX-2 lag. Die COX-2 ist Melanomzellen konstitutiv exprimiert und synthetisiert PGE2. NS 398 hemmt die Invasion von Melanomzellen in PGE2-unabhängiger Weise und könnte somit über ein sog. Non-COX-target wirken. / Accumulating evidence indicates that nonsteroidal anti-inflammatory drugs can reduce the incidence of colorectal cancers in humans. This antineoplastic activity is largely related to the inhibition of the inducible cyclooxygenase-2 (COX-2), which catalizes the biosynthesis of prostaglandin H2 the precursor of prostanoids. Elevated expression of COX-2 has been described in several types of epithelial tumors. In this study we evaluate the expression and function of COX-2 in malignant melanoma as a model of a non-epithelial tumor. With Western blot COX-2 protein was detected in all of six malignant melanoma cell lines. These cell lines produced prostaglandinE2 (PGE2) which was measured by a specific ELISA. PGE2 biosynthesis was blocked in a concentration dependent manner by the COX-2-specific inhibitor NS 398. The COX-2 IC50 for NS 398 was determined with 6 microM. In all six cell lines treatment with NS 398 reduced the invasion through a matrigel coated membrane while cell proliferation was not influenced. The inhibition of invasion was not mediated by PGE2, because i) exogenous PGE2 did not restore invasion, ii) the concentration needed for inhibitory effects on invasion was 8-fold higher than the IC50 of the COX-2. COX-2 is constitively expressed in malignant melanoma cells and is capable to produce PGE2. NS 398 reduces melanoma cell invsaion in a PGE2-independent manner, thus it likely further acts via a non-COX-target.

Zur Abschätzung der Prognose von Tumorerkrankungen und zur Therapieplanung können neben konventionellen klinischen Parametern auch molekulare Prognosemarker im Tumorgewebe bestimmt werden. In der vorliegenden Studie haben wir vier verschiedene potentielle molekulare Prognosefaktoren im Ovarialkarzinom und teilweise auch im Mammakarzinom untersucht: die Cyclooxygenase-2 (COX-2), das humane ELAV-ähnliche Protein HuR, das Oberflächenantigen CD24 und die Mitogen-aktivierte Protein Kinase Phosphatase-1 (MKP-1). Dabei lag der Schwerpunkt auf der Untersuchung der Cyclooxygenase-2 (COX-2), die sowohl in der Entzündungsreaktion als auch bei der Entstehung und Progression maligner Tumoren eine wichtige Rolle spielt. Wir konnten zeigen, dass eine erhöhte Expression der COX-2 beim Ovarialkarzinom und beim Mammakarzinom signifikant mit einer schlechteren Prognose assoziiert ist. In Zellkulturmodellen haben wir verschiedene Strategien zur Inhibition der COX-2 angewendet, nämlich die pharmakologische Inhibition durch NS-398 sowie die spezifische Inhibition durch RNA Interferenz. Dabei ergab sich, dass COX-2 Inhibitoren neben der Wirkung auf die COX-2 auch über anderen Zielproteine die Proliferation von Ovarialkarzinomzellen hemmen und zu einem Zellzyklusarrest führen. Bei weiteren Untersuchungen zur Regulation der COX-2 konnten wir zeigen, dass das RNA-stabilisierende Protein HuR mit der COX-2 Expression korreliert und ebenfalls ein Prognosefaktor für das Ovarialkarziom ist. Unsere Ergebnisse bilden eine Grundlage für klinische Studien zur Untersuchung des möglichen Effektes von COX-Inhibitoren in der Therapie maligner Tumoren. / Molecular prognostic markers can be determined in tumor tissue and can be used - in addition to conventional clinicopathological parameters - to estimate patient prognosis and to plan the therapy of malignant tumors. In this study we have investigated the expression of four different molecular prognostic factors in ovarian carcinoma and partially in breast carcinoma: cyclooxygenase-2 (COX-2), the human ELAV-like protein HuR, the surface antigen CD24, as well as the mitogen-activated protein kinase phosphatase-1 (MKP-1). For further evaluation, we have focused on COX-2, which plays an important role in tumor biology and inflammation. Increased expression of COX-2 in tumor tissue was associated with poor prognosis in ovarian carcinoma and breast carcinoma. In cell culture models, we have used two different strategies for inhibition of COX-2: pharmacological inhibition and RNA interference. We found that COX-2 inhibitors act on other cellular targets in addition to COX-2 and inhibit proliferation of ovarian carcinoma cells by induction of cell cycle arrest. In further studies we could show that the RNA-stabilizing protein HuR is associated with increased COX-2 expression and is an prognostic factor in ovarian carcinoma, as well. These results provide a basis for further evaluation of COX-inhibitors in tumor therapy.

環氧合酶-2(COX-2)是在花生四烯酸(AA)轉化為前列腺素H₂(PGH₂)的過程中最重要的限速酶，PGH2再進一步被合成為各種前列腺素，包括前列腺素E₂(PGE₂), 因此，COX-2在前列腺素的合成中起著舉足輕重的作用。COX-2在受到例如感染和炎症等刺激的情況下被誘導，迅速大量地產生。越來越多的證據證明瞭COX-2在許多細胞反應和病理生理過程中起重要作用, 其中, 對COX-2在炎症中的作用研究最深入。 / 囊性纖維化病(CF)是一種由於編碼囊性纖維化跨膜轉導調節器(CFTR)基因的突變所引起的常染色體隱性遺傳疾病。CFTR是在上皮細胞中廣泛表達的環磷酸腺苷(cAMP)依賴的陰離子通道。愈來愈多的證據顯示, CF的呼吸道上皮處於過量炎症因子和前列腺素的微環境中, 最終導致了在CF肺部病變中觀察到的超炎症反應. 但其中的機制仍未闡明. 本研究觀察到, 相對於野生型人類支氣管上皮細胞系(16HBE14o-), CF的人類支氣管上皮細胞系(CFBE41o-)中NFκB的活化, COX-2的表達和PGE₂的產量增加. 此外, CFTR基因敲除小鼠顯示出升高的NFκB活性和COX-2表達水準, 提示CFTR基因的缺失介導了超炎症反應的信號. 我們還驗證了一條PKA和CREB參與介導的PGE₂產生的正回饋通路. 更重要的是, 在CFBE41o-細胞中過表達CFTR顯著地抑制了COX-2的表達. 用LPS或者PGE₂處理16HBE14o-細胞導致了野生型CFTR表達的顯著升高. 這些實驗結果提示了CFTR可能參與對COX-2/PGE₂的負調節. 因此, CFTR負調節PGE₂介導的炎症反應. 這個調節機制的缺陷可能導致在CF炎症反應的組織中觀察到的過量的NFκB活化和過量PGE₂產生. / 我們證實了睾丸中也存在這條CFTR負調節COX-2/PGE₂的通路. 由於隱睾處於比陰囊溫度高的腹腔中, 在隱睾中, 我們觀察到了高溫導致的CFTR下調,伴隨著COX-2的上調以及緊密連接蛋白(ZO-1, occludin)的下調. 這種CFTR和COX-2的負相關在小鼠睾丸高熱動物模型以及CFTR基因敲除小鼠模型中也被證實. 為了模擬隱睾的病理狀況, 我們提高原代睾丸支援細胞的培養溫度至37°C. 與在32°C培養條件下的對照細胞相比, 37C培養的支持細胞中CFTR表達顯著下調, 而COX-2表達顯著上調. 用CFTR的抑制劑CFTRinh-172處理支持細胞48小時後, COX-2的表達也上升了. 抑制或者敲除支持細胞中的CFTR都引起了ZO-1和occludin表達水準的下降, 從而損傷了支持細胞間的緊密連接. NFκB或者PGE₂的抑制劑都能逆轉ZO-1和occludin表達水準的下降. PGE₂同樣導致了支援細胞間緊密連接的損傷. 以上結果提示CFTR對緊密連接的調節作用是通過NFκB/COX-2/PGE₂通路實現的. 本研究闡明了在支持細胞中, CFTR通過負調節NFκB/COX-2/PGE₂通路調節緊密連接, 從而參與了隱睾導致的生精障礙的病理過程. / 總之, 本研究論證了CFTR/COX-2/PGE₂通路在CF呼吸道的超炎症反應以及隱睾導致的生精障礙兩個病理過程中的作用, 說明了CFTR在呼吸系統和男性生殖系統中維持細胞因子穩態的重要作用. CF肺中CFTR的缺失或者隱睾病中CFTR表達水準的下降可能導致了呼吸道中過剩炎症反應和生精障礙. / Cyclooxygenase-2 (COX-2) is a pivotal rate-limiting enzyme responsible for the production of prostaglandins by converting arachidonic acid (AA) to prostaglandin H₂ (PGH₂), which is further metabolized to various prostaglandins, including PGE₂. COX-2 is inducible and increases dramatically upon stimulation, such as infection and inflammation. Accumulating evidences have demonstrated the important role of COX-2 in many cellular responses and pathophysiological processes, especially inflammation. / Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-dependent anion channel expressed in many epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased COX-2 expression and over-production of prostaglandin E₂ (PGE₂) in human CF bronchial epithelial cell line (CFBE41o-) with elevated NFκB activity compared to a wild-type bronchial epithelial cell line (16HBE14o-). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NFκB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE₂ involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o- cells were challenged with LPS as well as PGE₂, indicating possible involvement of CFTR in the negative regulation of COX-2/PGE₂. These results suggest that CFTR is a negative regulator of PGE₂-mediated inflammatory response, defect of which may result in excessive activation of NFκB, leading to over production of PGE2 as seen in inflammatory CF tissues. / This negative regulation of COX-2/PGE₂ pathway by CFTR was also identified in the testis in the present study. Downregulation of CFTR accompanied by upregulation of COX-2/PGE₂ and downregulation of tight junction proteins, including ZO-1 and occludin, were observed in a cryptorchidism mouse model with elevated testis in the abdomen, at which the temperature is several degrees higher than that in the scrotum. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and in CF mice. Culturing primary Sertoli cells at a temperature of 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression compared to the physiological condition of 32°C. Increase of COX-2 expression was also detected 48 hours after administrating CFTRinh-172 to the cells. Inhibition or knockdown of CFTR led to decreased ZO-1 and occludin expression and impaired tight junction in Sertoli cells, which could be mimicked by PGE₂, but reversed by NFκB and COX-2 inhibitors, suggesting that regulation of tight junction by CFTR is mediated by NFκB /COX-2/PGE₂ pathway. This study illustrates that CFTR may be involved in regulating testicular tight junctions through its negative regulation of NFκB/COX-2/PGE₂ pathway in Sertoli cells, defect of which may result in spermatogenesis defect in cryptorchidism. / Taken together, the present study has demonstrated the role of CFTR/ NFκB /COX-2/PGE₂ pathway in two pathological processes, exaggerated inflammation in CF airway and defective spermatogenesis in cryptorchidism, indicating that CFTR is critical for maintaining cytokine homeostasis in respiratory system and male reproductive system. Defect of CFTR in CF lung and downregulation of CFTR in cryptorchidism may contribute to the excessive lung inflammation and impaired spermatogenesis respectively. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 109-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.vii / ABBREVIATIONS --- p.xii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- Chpter 1: Overview --- p.1 / Chapter 1.1 --- CFTR and Cystic Fibrosis --- p.1 / Chapter 1.1.1 --- Cystic Fibrosis --- p.1 / Chapter 1.1.2 --- Structure of CFTR --- p.2 / Chapter 1.1.3 --- Mutations of CFTR --- p.2 / Chapter 1.1.4 --- Channel and signal transduction function of CFTR --- p.3 / Chapter 1.1.5 --- Interaction of CFTR with other proteins --- p.4 / Chapter 1.1.6 --- Regulation of CFTR --- p.5 / Chapter 1.2 --- COX-2 and PGE₂ --- p.6 / Chapter 1.2.1 --- Biosynthesis of PGE₂ --- p.6 / Chapter 1.2.2 --- Pathophysiologic roles of COX-2 and PGE₂ --- p.7 / Chapter 1.2.3 --- Role of COX-2/PGE₂ in inflammation --- p.7 / Chapter 1.2.4 --- Regulation of COX-2 --- p.8 / Chapter 1.2.4.1 --- Regulation of COX-2 by NF-κB --- p.9 / Chapter 1.2.4.2 --- Regulation of COX-2 by CREB --- p.10 / Chapter 1.3 --- Link between CFTR and NF-κB --- p.11 / Chapter 1.4 --- General hypothesis and aims of study --- p.12 / Chapter 2 --- Chapter 2: CFTR negatively regulates COX-2/PGE₂ positive loop in feedback loop in inflammation --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Airway inflammation in Cystic Fibrosis --- p.13 / Chapter 2.1.2 --- Current theories on the causes of pulmonary inflammation in CF --- p.13 / Chapter 2.1.2.1 --- Theory one --- p.14 / Chapter 2.1.2.2 --- Theory two --- p.16 / Chapter 2.1.3 --- Role of airway epithelia in CF airway inflammation --- p.16 / Chapter 2.1.4 --- Link between CFTR and NF-κB in pulmonary inflammation --- p.17 / Chapter 2.1.5 --- Link between CFTR and COX-2/PGE₂ in pulmonary inflammation --- p.18 / Chapter 2.1.6 --- Hypothesis and aims of study --- p.18 / Chapter 2.2 --- Materials and methods --- p.20 / Chapter 2.2.1 --- Cell culture materials --- p.20 / Chapter 2.2.2 --- Animals --- p.20 / Chapter 2.2.3 --- Chemicals, drugs and assay kits --- p.20 / Chapter 2.2.4 --- Antibodies --- p.22 / Chapter 2.2.5 --- Cell culture. --- p.22 / Chapter 2.2.6 --- Animal models and procedures --- p.23 / Chapter 2.2.7 --- Manipulation of RNA and QRT-PCR --- p.23 / Chapter 2.2.8 --- Manipulation of protein and Western blot --- p.25 / Chapter 2.2.9 --- Histological and morphological --- p.27 / Chapter 2.2.9.1 --- Tissue section. --- p.28 / Chapter 2.2.9.2 --- Hematoxylin and eosin staining --- p.28 / Chapter 2.2.9.3 --- Immunohistochemistry --- p.28 / Chapter 2.2.10 --- PGE₂ EIA --- p.29 / Chapter 2.2.11 --- Statistical analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Increased expression of NF-κB and COX-2 in the lung of CF mice --- p.31 / Chapter 2.3.2 --- Defect of CFTR leads to increased COX-2 expression in CF cell line --- p.31 / Chapter 2.3.3 --- Increased expression of COX-2 in CF cells is attributed to NF-κB activation --- p.33 / Chapter 2.3.4 --- A positive feedback loop from PGE₂ to COX-2 is mediated by PGE₂/cAMP/PKA/p-CREB pathway --- p.34 / Chapter 2.3.5 --- PGE₂ increase the expression of CFTR protein in 16HBE14o- but not in CFBE41o- cells --- p.35 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.5 --- Conclusion --- p.51 / Chapter 3 --- Chapter 3: Role of CFTR/COX-2/PGE₂ Pathway in the Regulation of Junctional Complex Proteins in Sertoli Cells and its Implication in Spermatogenesis Defect in Cryptorchidism --- p.53 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Spermatogenesis.p53 / Chapter 3.1.1.1 --- Structure of the seminiferous tubules --- p.53 / Chapter 3.1.1.2 --- Role of Sertoli cells in spermatogenesis --- p.55 / Chapter 3.1.1.3 --- Role of junctional complexes in spermatogenesis --- p.55 / Chapter 3.1.2 --- Junctional complexes in the testis --- p.59 / Chapter 3.1.2.1 --- Tight Junction --- p.59 / Chapter 3.1.2.2 --- Anchoring Junction. --- p.60 / Chapter 3.1.2.3 --- Cross talk between TJs and AJs --- p.60 / Chapter 3.1.3 --- Cryptorchidism --- p.61 / Chapter 3.1.3.1 --- Causes and consequences of Cryptorchidism --- p.61 / Chapter 3.1.3.2 --- Elevated temperature caused by cryptorchidism greatly contributes to defective spermatogenesis --- p.62 / Chapter 3.1.3.3 --- Changes of Sertoli cells in cryptorchidim contributing to defective spermatogenesis. --- p.62 / Chapter 3.1.3.4 --- Disruption of junctional complexes in heat shock and cryptorchidism. --- p.65 / Chapter 3.1.4 --- CFTR and spermatogenesis --- p.66 / Chapter 3.1.4.1 --- Expression of CFTR in Sertoli cells in testis --- p.66 / Chapter 3.1.4.2 --- Temperature sensitive processing of CFTR protein --- p.66 / Chapter 3.1.4.3 --- CFTR and junctional complex --- p.67 / Chapter 3.1.4.4 --- CFTR and male reproduction --- p.68 / Chapter 3.1.4.5 --- Role of CFTR in spermatogenesis --- p.68 / Chapter 3.1.5 --- Prostaglandins and male fertility --- p.69 / Chapter 3.1.5.1 --- Expression of COX-2 in testis. --- p.69 / Chapter 3.1.5.2 --- Role of prostaglandins in spermatogenesis --- p.70 / Chapter 3.1.5.3 --- Regulation of junctional complexes by PGE₂ --- p.70 / Chapter 3.1.5.4 --- Prostaglandins in cryptorchidism --- p.72 / Chapter 3.1.6 --- Hypothesis and aims of study --- p.73 / Chapter 3.2 --- Materials and Methods --- p.74 / Chapter 3.2.1 --- Cell culture materials --- p.74 / Chapter 3.2.2 --- Drugs and Reagents --- p.74 / Chapter 3.2.3 --- Antibodies --- p.74 / Chapter 3.2.4 --- Animals --- p.75 / Chapter 3.2.4.1 --- Mice artificial cryptorchidism model --- p.75 / Chapter 3.2.4.2 --- Mice testes hyperthermia model --- p.75 / Chapter 3.2.5 --- Sertoli cell primary culture --- p.76 / Chapter 3.2.6 --- siRNA against CFTR and transfection --- p.76 / Chapter 3.2.7 --- Examination of assembly and destruction of assembly of inter-Sertoli TJs --- p.77 / Chapter 3.2.8 --- Manipulation of RNA and Real-Time Quantitative RT-PCR (QRT-PCR) --- p.77 / Chapter 3.2.9 --- Manipulation of protein and western blot --- p.77 / Chapter 3.2.10 --- Histological and morphological studies --- p.78 / Chapter 3.2.10.1 --- Immunofluorescence of ZO-1 Staining in Sertoli cells --- p.78 / Chapter 3.2.10.2 --- Immunofluorescent staining of ZO-1, Occludin and β-Catenin in testes --- p.78 / Chapter 3.2.11 --- PGE₂ EIA --- p.79 / Chapter 3.2.12 --- Statistical Analysis --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.3.1 --- Downregulation of CFTR is associated with upregulation of COX-2 in mice cryptorchidism model, mice testes hyperthermia model, and CF mice testes --- p.79 / Chapter 3.3.2 --- Negative regulation of COX-2 by CFTR is mediated by NF-κB --- p.81 / Chapter 3.3.3 --- Decreased tight junction proteins expression and increased anchoring junction proteins expression in cryptorchid testes. --- p.81 / Chapter 3.3.4 --- Elevation of culture temperature results in downregulation of CFTR and upregulation of COX-2 in primary cultured rat sertoli cells --- p.82 / Chapter 3.3.5 --- Defect of functional CFTR leads to increased COX-2 expression. --- p.83 / Chapter 3.3.6 --- CFTR regulates TJ protein expression and TJ formation through NF-κB/COX-2/PGE₂. --- p.83 / Chapter 3.4 --- Discussion --- p.100 / Chapter 3.5 --- Conclusion --- p.104 / Chapter 4 --- Chapter 4: General Discussion --- p.105 / Chapter 4.1 --- The immunosuppressive function of PGE₂ in CF lung disease and cryptorchidism-induced infertility. --- p.105 / Chapter 4.2 --- Importance of CFTR/ NF-κB /COX-2/PGE₂ pathway in inflammation-based diseases. --- p.106 / Chapter 4.3 --- Possible implications of CFTR/NF-κB /COX-2/PGE₂ pathway in cancer --- p.107 / Chapter 4.4 --- Concluding remarks --- p.108

Adverse effects of cyclooxygenase (COX) inhibitors on bone healing have previously been demonstrated in diaphyseal fracture models in animals. In spite of that, they are widely used as postoperative analgesics in orthopaedic surgery. After joint replacement, a bone repair process starts at the interface between bone and cement. If this process is disturbed, the prosthesis may never become rigidly fixed to the bone, leading to migration and with time loosening. This thesis investigates the effects of a selective COX-2 inhibitor (parecoxib or celecoxib) on bone healing in metaphyseal bone in a rat model and on knee prosthesis migration after total knee replacement, as measured with radiostereometric analysis. Blood loss, postoperative recovery, and the 2-year subjective outcome, were also measured. In addition, a hemoglobin dilution method for blood loss estimation, used in this thesis, was evaluated. In the first study, pull-out force of a screw inserted in metaphyseal bone of the tibia in rats was only marginally decreased by parecoxib after 7 days but not after 14 days. In the second and third study, celecoxib treatment resulted in less pain postoperatively in conjunction with total knee replacement (TKR), but no effects were seen on blood loss, range of motion, subjective outcome, or prosthesis migration after 2 years. Comparing the true blood loss of blood donors with the blood loss estimated by the hemoglobin dilution method, this method was found to underestimate the true blood loss. It is therefore not suitable for calculation of the absolute blood loss volume, but may be used for a rough estimate. In summary, celecoxib and presumably other cyclooxygenase inhibitors seems not likely to increase the risk of prosthesis loosening.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 4 uppsatser.

Brucella est une bactérie intracellulaire facultative à Gram négatif responsable d’une zoonose, la brucellose. Pour persister dans l’organisme, Brucella agit comme un pathogène furtif en modulant la réponse immunitaire de l’hôte. La cyclooxygénase 2 (COX-2) est l’enzyme responsable de la synthèse des prostanoïdes, des médiateurs lipidiques dérivés de l’acide arachidonique (AA) présentant des propriétés immunorégulatrices. Cette thèse est centrée sur l’étude de cette voie métabolique au cours de l’infection par Brucella in vitro dans des cellules dendritiques (DC) humaines et murines ainsi qu’in vivo chez la souris en comparant différentes routes d’infection. Nous avons mis en évidence la capacité de l’infection à stimuler in vitro la production d’AA ainsi que l’expression de Ptgs2. In vivo, la comparaison des différentes routes d’inoculation a montré que l’infection intradermale induit une signature génique inflammatoire caractérisée par l’expression de Ptgs2 et d’Ifng. L’utilisation de NS-398, un inhibiteur spécifique de COX-2 stimule la clairance bactérienne dans les ganglions cervicaux (CLN) drainant le site d’infection. Ces résultats ouvrent ainsi la voie à de nouvelles stratégies thérapeutiques dans le traitement de la brucellose. La seconde partie de la thèse traite de l’implication des infections bactériennes dans l’initiation des processus oncogéniques. Nous y présentons une revue répertoriant l’ensemble des mécanismes pouvant contribuer à l’initiation oncogénique ainsi qu’un projet que nous développons au laboratoire portant sur l’initiation d’un lymphome folliculaire à la suite d’une stimulation antigénique chronique suite à l’infection par B. abortus. / Brucella is a facultative intracellular Gram-negative bacterium, responsible for a zoonosis called brucellosis. To persist into the host, Brucella acts as a stealthy pathogen by modulating the host immune response. The cyclooxygenase 2 (COX-2) is the enzyme responsible for the synthesis of prostanoids, a family of lipid mediators derived from the arachidonic acid (AA) and presenting immunomodulatory properties. Here, we have studied the impact of this pathway during Brucella infection in vitro in human and murine dendritic cells (DCs) as well as in vivo by comparing different infection routes. We have highlighted the ability of the infection to stimulate the AA synthesis and Ptgs2 expression. In vivo, by comparing different inoculation routes we showed that intradermal infection induces a specific inflammatory gene signature characterized by an important expression of Ptgs2 and Ifng. The use of NS-398, a specific inhibitor of COX-2 stimulates the bacterial clearance in the cervical lymph nodes (CLN) draining the site of infection. These results might open the way to new therapeutic strategies in the treatment of brucellosis. In a second part of the thesis, we discuss the involvement of bacterial infections in initiating oncogenic processes. Here, we present a review listing all the mechanisms that contribute to the oncogenic initiation and a project that we are developing in the laboratory dealing with the initiation of follicular lymphoma following chronic antigenic stimulation during B. abortus infection.