As prostaglandinas (PGs) são derivadas do metabolismo do ácido aracdônico pela via das enzimas cicloxigenases (COX-1 e COX-2) e participam do controle do metabolismo ósseo. Os anti-inflamatórios não-esteroidais (AINEs), inibidores das COX, podem interferir negativamente com a formação óssea, atrasando o reparo de fratura de ossos longos, a fusão espinhal e a osseointegração de implantes. O presente trabalho teve por objetivo avaliar, quantitativamente, o efeito de diferentes tipos de AINEs (convencional, preferencial e seletivo para COX-2), assim como de um analgésico com efeito anti-inflamatório fraco, sobre o reparo ósseo alveolar, em ratos machos. Os animais foram divididos em 5 grupos experimentais: a) controle (administração de 1 mL água/dia), b) cetorolaco de trometamina (inibidor não-seletivo COX-1/COX-2; ingestão de 4 mg/kg/dia), c) nimesulida (inibidor preferencial de COX-2; ingestão de 5 mg/kg/dia), d) paracetamol (efeito anti-inflamatório fraco; ingestão de 80 mg/kg/dia), e) etoricoxibe (inibidor seletivo de COX-2; ingestão de 10 mg/kg/dia). As dosagens foram baseadas em trabalhos experimentais da literatura e na equivalência com a terapêutica humana. A administração foi realizada por gavage gástrica, iniciando logo após a extração do incisivo superior direito e seguindo por um periódo de 14 dias, após o que os ratos foram sacrificados, as hemi-maxilas contendo os alvéolos em reparação foram coletadas e processadas para inclusão em parafina, orientada de maneira a permitir cortes semi-seriados longitudinais de 6 μm de espessura (a intervalos de 60 μm), que foram corados pela hematoxilina e eosina. O percentual de tecido ósseo neoformado foi estimado por método de contagem diferencial de pontos, utilizando-se um microscópio óptico munido de câmera digital de vídeo para captura de imagens e um programa para histometria. Foram contados cerca de 1200 pontos (1182,9 ± 47,6 pontos; média ± EPM) no terço cervical alveolar de cada animal, em cerca de 8 secções histológicas intercaladas (8,6 ± 0,3 secções; média ± EPM). Após a aplicação de teste que comprovou a normalidade de todas as distribuições (teste de Kolmogorov-Smirnov, p > 0,10) aplicou-se a Análise de Variância (gl=4; F = 1,52; x2 = 0,13), que comprovou que o tratamento com os diferentes tipos de AINEs não interferiu com a formação óssea reparacional, neste modelo experimental. Os presentes resultados, obtidos em animais, não devem ser diretamente extrapolados para humanos, nem conduzir a uma inferência sobre o uso de AINEs na clínica odontológica. No entanto, as numerosas evidências experimentais e clínicas de que os diferentes tipos de AINEs podem ter efeitos indesejados na clínica ortopédica, somadas ao número reduzido de estudos voltados especificamente para a área odontológica, indicam a necessidade de mais investigações nessa área, com variação dos parâmetros experimentais e das ferramentas de avaliação, antes que se descarte a possibilidade de que os AINEs possam ter efeitos indesejados em procedimentos odontológicos que necessitam de formação óssea, principalmente no caso de utilização prolongada. / The cyclooxygenase enzymes COX-1 and COX-2 catalyze the conversion of arachidonic acid to prostaglandins (PGs), eicosanoids important for control of bone metabolism. The non-steroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors, may negatively interfere with bone formation and delay long bone fracture healing, spinal fusion and implant osseointegration. The aim of the present study was to investigate quantitatively whether different types of NSAIDs (conventional, preferential and COX-2-selective), as well as an analgesic drug with a weak anti-inflammatory action, can hinder alveolar bone healing, in male rats. The animals were divided in 5 experimental groups: a) control (oral administration of 1 mL water/day), b) cetorolac (non-selective COX-1/COX-2 inhibito - 4 mg/kg/day oral dose), c) nimesulide (preferential COX-2 inhibitor - 5 mg/kg/day oral dose), paracetamol (weak anti-inflammatory - 80 mg/kg/day oral dose), etoricoxib (selective COX-2 inhibitor - 10 mg/kg/day oral dose). The doses of NSAIDs were compatible to human therapy and in the range of investigations carried out in laboratory animals. The drugs were administered by gavage from the day of extraction of the upper right incisors until death 2 weeks later, when the hemi-maxillae containing the alveolar sockets were collected, decalcified and processed for paraffin embedding. Semi-serial longitudinal 6-μm-thick sections were cut at 60-μm intervals and stained with hematoxylin and eosin. The degree of new bone formation inside the alveolar socket was estimated by a differential point-counting method, using an optical microscopy with a digital camera for image capture and a public domain histometry software. A total of 1182,9 ± 47,6 points (mean ± SEM) were counted in the cervical alveolar third, in 8,6 ± 0,3 (mean ± SEM) histological sections per alveolus (final magnification 100x), the percentage of points lying on bone trabeculae being proportional to their volume density. After confirmation of a normal distribution (Kolmogorov-Smirnov normality test, p>0,10) and comparison among groups by Analysis of Variance (gl = 4; F = 1,52; x2 = 0,13), histometric data confirmed that none of the anti-inflammatory drugs have detrimental effects in the volume fraction of new bone trabeculae filling the tooth extraction socket. Although experimental results obtained in animals may not be directly extrapolated to human neither induce to inferences about the clinical use of NSAIDs, numerous evidences have confirmed the deleterious effects of NSAIDs in the orthopedic clinic. Considering the reduced amount of studies on this subject pertaining to the dental field, more investigations are needed varying the experimental parameters and techniques, before the possibility of deleterious effects of NSAIDs in dental procedures requiring new bone formation are discharged.

Dysmenorré är ett av de vanligaste gynekologiska tillstånden bland unga kvinnor med en prevalens kring 67%- 90% världen över. Tillståndet är uppdelat i primär- och sekundär dysmenorré och definieras som smärtsamma muskelkramper från nedre buken i kombination med bland annat illamående och diarré som uppkommer under menstruationen. Vid primär dysmenorré anses smärtan grunda sig i överproduktion av prostaglandiner i livmodern varpå det medför kontraktion i kärl och muskulatur. För behandling av dysmenorré avses smärtlindring genom inhibering av prostaglandinsyntesen det primära syftet vilket åstadkoms med icke-steroida antiinflammatoriska läkemedel (NSAID). Många kvinnor avstår från behandling med NSAID på grund av medförda biverkningar, kulturella anledningar eller utebliven effekt, som uppskattas till en fjärdedel av fallen. Detta har väckt intresse för icke syntetiska behandlingar som både är effektiva och har minimala biverkningar, såsom det växtbaserade läkemedlet ingefära. Syftet med arbetet är att värdera om ingefära är lika effektivt som NSAID avseende smärtlindring, hos kvinnor med primär dysmenorré. Denna litteraturstudie är baserad på fem randomiserade kontrollerade studier där två utav dem jämför NSAID med ingefära på primär dysmenorré, för bedömning om deras effektivitet i förhållande till varandra; två artiklar som jämför ingefära med placebo vid primär dysmenorré, för en säkerställning på ingefäras verkan; och en artikel som jämför NSAID med placebo på primär dysmenorré för styrkande av befintlig effekt. Studierna erhölls via databasen PubMed. Sammanställningen av resultaten påvisar att ingefära är effektivt som smärtlindring vid behandling av primär dysmenorré. I studierna som jämförde ingefära med placebo indikerade båda en signifikant skillnad avseende den smärtlindrande effekten (p<0.05). Däremot konstaterades ingen signifikant skillnad på smärtreduktionen mellan NSAID och ingefära (p>0.05). Det sammantagna resultatet av denna litteraturstudien är att ingefära är lika effektivt som NSAID vid primär dysmenorré avseende smärtlindring. Även fast ingefäras verkan har indikerats, krävs fler utförda högkvalificerade studier med höga deltagarantal för att säkerställa effekten av ingefära vid primär dysmenorré. Det kan även vara av intresse att analysera de specifika koncentrationerna av de verksamma komponenterna i ingefära, utvidga jämförelsen av ingefäras effekt med flera olika NSAID samt andra växtbaserade läkemedel och metoder som påvisat smärtlindring vid primär dysmenorré. / Dysmenorrhea is one of the most common gynecological condition reported by women with an estimated prevalence of 67% to 90% around the world. The condition is divided into primary and secondary dysmenorrhea and is defined as painful menstrual cramping in the abdominal region in combination with nausea, vomiting, insomnia, fatigue and diarrhea. The cause of the pain in primary dysmenorrhea is believed to result from an excessive increase in prostaglandin synthesis in the endometrium during the ovulatory cycle, which further causes vasoconstriction of the blood vessels in the uterine and increased uterine muscle contractions. The decrease of blood supply will result in ischemia and therefore induce pain. The treatments for dysmenorrhea include prostaglandin synthesis inhibitors such as Non -Steroidal Anti -Inflammatory Drugs (NSAID). NSAIDs are used as the first line therapy for pain relief of primary dysmenorrhea. In addition, one fourth of the patients don’t experience an effect of the drugs and some women don’t prefer the usage because of the lack of benefit, the adverse side effects or due to cultural reasons thus left with alternative treatments. Alternative non-synthetic treatments, such as herbal drugs, with effectiveness and low toxicity have been of high interest. Ginger, which is a herbal compound, have been reported to have beneficial effects on primary dysmenorrhea by inhibiting the prostaglandin synthesis and subsequent decrease of the pain. The aim of this study was to compare the effectiveness of ginger with NSAID on pain management in primary dysmenorrhea. This study is a literature review where five randomized controlled trials has been evaluated and analyzed. Two of them compared the pain management between NSAID and ginger in primary dysmenorrhea, two studies compared the pain management between placebo and ginger in primary dysmenorrhea and one study compared the pain management between NSAID and placebo in primary dysmenorrhea. All the studies where collected from the database PubMed. Overall the result showed that ginger influences pain relief in primary dysmenorrhea. In the two studies that evaluated gingers effect with placebo, both of them indicated a statistically significant difference in pain reduction (p<0.05). The treatment with ginger resulted in a pain reduction around 61% in the first study respectively around 34% in the second. In the studies which compared NSAID with ginger, the highest reduction NSAID could achieve was 56.5% whereas ginger achieved 60.1%. However, there were no statistically significant reduction in pain between them (p>0.05). The conclusion is that ginger as well as NSAID are effective in relieving pain in women with primary dysmenorrhea. Although the effect of ginger has been observed, further studies are necessary to establish gingers efficacy for the treatment of primary dysmenorrhea. Furthermore, it’s suggested that in the future extend the studies of ginger by comparing with multiple NSAID, other herbal compounds and methods used for primary dysmenorrhea with larger number of patients.

O veneno da serpente Crotalus durissus terrificus e seus componentes desencadeiam importantes efeitos biológicos que envolvem direta e/ou indiretamente, componentes do sistema circulatório. Contudo, não há estudos específicos na literatura sobre os efeitos do veneno crotálico ou de suas toxinas, em células endoteliais. As células endoteliais constituem a camada de revestimento interna dos vasos sanguíneos, denominada endotélio. Este tecido é metabolicamente ativo, com função protetora do sistema cardiovascular e desempenha papel central na regulação da função circulatória, através do controle da coagulação, permeabilidade e do tônus vascular. Neste contexto, este estudo teve como objetivo avaliar os efeitos do veneno de Crotalus durissus terrificus (VCdt), do seu componente majoritário, a crotoxina (CTX) e de suas subunidades, fosfolipase A2 (CB) e crotapotina (CA), sobre células endoteliais, em cultura, quanto à: i) viabilidade e proliferação celular; ii) integridade das monocamadas; iii) produção de óxido nítrico, de prostaciclina e mecanismos envolvidos neste efeito. Os resultados obtidos demonstram que o veneno de Crotalus durissus terrificus afetou a viabilidade e a integridade de células endoteliais em cultura, de modo tempo-dependente e apenas na maior concentração, sugerindo sua baixa toxicidade sobre as células endoteliais. A subunidade CB, mas não a CTX nem a crotapotina, reproduziu os efeitos causados pelo VCdt. Em concentrações não citotóxicas, tanto o veneno quanto as toxinas não alteraram a proliferação celular nem a produção basal de óxido nítrico pelas células endoteliais. Por outro lado, o veneno e a subunidade CB, mas não a CTX nem a CA causaram aumento significativo da produção de prostaciclina, via COX-1 e COX-2, sendo que a expressão protéica da isoforma COX-2 foi induzida por estes agentes. Além disso, foi demonstrado que a fosfolipase citosólica é relevante para o aumento da produção de prostaciclina, induzido pela CB. Adicionalmente, foi demonstrado que a atividade catalítica da subunidade CB é essencial para os efeitos descritos. Isto reforça a sugestão de que a subunidade fosfolipásica, isoladamente, possa contribuir para os efeitos do veneno total no endotélio. Nesse sentido, se houver alguma fração desta enzima na sua forma livre, no veneno total, sugere-se que ela contribua, de modo significativo, para os efeitos do veneno de Crotalus durissus terrificus no endotélio. / Crotalus durissus terrificus snake venom (CdtV) and their components induces systemic effects, which interfere with blood vessel system. Endothelial cells (EC) are central elements for haemostasis, regulating blood vessel-wall permeability, blood fluidity and adhesion properties of circulating leukocytes. However, there is no available data on the effects of this venom and its components on endothelial cells. In this study, the effects of CdtV, crotoxin (CTX) which is formed by two distinct subunits named crotapotin (CA) and phospholipase A2 (CB), on endothelial cells in vitro were investigated, analyzing EC viability and proliferation, EC monolayers integrity, release of both nitric oxide and prostacyclin (PGI2). CdtV, at the highest concentration, time-dependently decreased the viability of EC and the integrity of cell monolayers. The CB subunit, but not CTX nor CA, reproduced the effects caused by crude venom. In contrast, neither EC proliferation nor release of oxide nitric were affected by non-cytotoxic concentrations of CdtV or isolated toxins. However, at the same experimental condition, both CdtV and CB increased the prostacyclin release by endothelium through activation of COX-1 and -2 enzyme systems. Moreover, these toxins upregulated protein expression of COX-2 isoform, but did not alter constitutive expression of COX-1. On the other hand, neither CTX nor CA affected basal production of PGI2. Inhibition of cytosolic PLA2 (cPLA2) by AACOCF3 significantly reduced PGI2 increments caused by both CdtV and CB implying that cPLA2 cooperates for the synthesis of PGI2 induced by them. Inhibition of the catalytic activity of CB abrogated its ability to induce the release of PGI2, thus suggesting the importance of the phospholipase A2 enzyme activity for this effect. These findings provide evidence that CdtV and CB can directly activate EC and up-regulate cyclooxygenase pathways for production of prostacyclin, an important mediator of vasodilation and inflammation. Moreover, CB through its catalytic activity may significantly contribute for the stimulatory effect of CdtV in EC. Therefore, these findings indicate novel regulatory mechanisms for both CdtV and venom secretory PLA2 in endothelial cells.

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

<p>Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement <i>in vivo</i> has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation <i>in vivo</i> that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation.</p><p>The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples.</p><p>A methodology for measurement of the total amount (sum of free and esterified) of an F₂-isoprostane, 8-iso-prostaglandin F_2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F_2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride.</p><p>Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F_2α and a major prostaglandin F_2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation.</p><p>In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F_2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet.</p><p>In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation <i>in vivo</i> under basal conditions and in the pathology of diseases.</p>

Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement in vivo has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation in vivo that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation. The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples. A methodology for measurement of the total amount (sum of free and esterified) of an F2-isoprostane, 8-iso-prostaglandin F2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride. Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F2α and a major prostaglandin F2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation. In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet. In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation in vivo under basal conditions and in the pathology of diseases.

The kidney regulates the excretion of water and electrolytes, which maintains homeostasis and enables control of arterial blood pressure. Hyaluronan, a large negatively charged interstitial glucosaminoglycan, is heterogeneously distributed within the kidney, primarily found in the medulla. Medullary hyaluronan content changes depending on the state of body hydration and plays a part in fluid regulation through its water binding and viscoelastic properties. The aim of this thesis was to provide new insight into the regulation of intrarenal hyaluronan during different states of body hydration, during completion of kidney development, and during diabetes mellitus. Dehydration reduces medullary interstitial hyaluronan in parallel with reduced hyaluronan synthase 2 gene expression and increased urinary hyaluronidase activity. Acute hydration results in an increase in medullary hyaluronan, an increase that requires nitric oxide and prostaglandins. Urinary hyaluronidase activity decreases during hydration. The elevation of hyaluronan is important for reducing water permeability of the interstitium i.e. favoring diuresis. Changes in hyaluronan concentration constitute a morphoregulatory pathway that plays a key role in nephrogenesis. The reduction in neonatal hyaluronan depended on an angiotensin II mediated process that does not appear dependent on lymph vessel formation. If angiotensin II is blocked with an ACE inhibitor, hyaluronan accumulates, which results in structural and functional abnormalities in the kidney. Renomedullary hyaluronan is elevated during uncontrolled diabetes, which coincides with induction of hyaluronan synthase 2 mRNA, hyperglycemia, glucosuria, proteinuria and overt diuresis. The levels of hyaluronan are probably at a terminus ad quem as no further response was seen during hydration. The higher interstitial expression of hyaluronan during diabetes may be involved in the progression of diabetic nephropathy. This thesis in physiology provides new mechanistic insights into the regulation of renal hyaluronan during various aspects of fluid handling.

Le rejet chronique se manifeste dans le poumon par la bronchiolite oblitérante (BO), une pathologie inflammatoire et fibrotique menant à l’oblitération des bronchioles. L’étiologie exacte de cette maladie demeure inconnue. Certaines études suggèrent qu'un déséquilibre des leucotriènes (LT) sur les prostaglandines (PG) favorise la fibrose pulmonaire. Les taux des LT et des PG dans le poumon humain post-transplantation sont inconnus. Nous proposons qu'un déséquilibre de cystéinyl leucotriènes (CysLT) sur la PGE2 existe dans le poumon transplanté et pourrait être impliqué dans la pathogenèse de la BO. Aussi, les leucotriènes contribueraient à la fibrose par la transition épithélio-mésenchymateuse (TEM). Afin de vérifier ces hypothèses, nous avons déterminé les taux de CysLT et de PGE2 dans le liquide de lavage broncho-alvéolaire (LBA) provenant de poumons transplantés chez l'homme ainsi que leurs corrélations cliniques. Nous avons également déterminé la capacité des CysLT à induire l’expression des marqueurs de la TEM in vitro. Nous avons découvert des taux de CysLT et PGE2 supérieurs à la normale dans les LBA des greffés. Un pic prédominant de CysLT sur PGE2 est observée à 52 semaines postgreffe et deux facteurs de risque de la BO, les infections au CMV et à l’Aspergillus, sont associés au ratio CysLT/PGE2> 1. In vitro, les CysLT induisent une répression des marqueurs épithéliaux mais n’induisent pas l’expression de marqueurs mésenchymateux chez les cellules épithéliales bronchiolaires. / Chronic rejection occurs, in the lung, in the form of bronchiolitis obliterans (BO), an inflammatory and fibroproliferative disease that leads to the obliteration of the bronchioles. A concept of the pathogenesis of BO has been suggested and several risk factors are associated to it, however, the exact etiology of this disease remains unknown. Studies have suggested that an imbalance of leukotrienes (LT) over prostaglandins (PG) promotes pulmonary fibrosis. The levels of LT and PG in the human lung post-transplantation are unknown. We propose that an imbalance of cysteinyl leukotrienes (CysLT) on PGE2 exists in the transplanted lung and may be implicated in the pathogenesis of BO. We also suggest that leukotrienes contribute to fibrosis through epithelial-mesenchymal transition (EMT). In order to test these hypotheses, we have determined the levels of CysLTs and PGE2 in human transplanted lung bronchoalveolar lavage fluid (BALf) samples and their clinical correlations. We have also determined the capacity of CysLT to induce the expression of EMT markers in vitro. We found high average levels of CysLT and PGE2 in the BAL of transplant patients. A predominant peak of CysLT over PGE2 was observed at 52 weeks post-transplantation and two risk factors for BO, CMV infections and Aspergillus were associated with CysLT/PGE2 ratio> 1. According to our experimental parameters, CysLT can induce the repression of epithelial markers but do not induce the expression of mesenchymal markers in vitro in small airway epithelial cells.

Incidences of uterine infections in dairy cattle are high between parturition and Day 21 postpartum. Dairy cows with uterine infections are at risk for prolonged periods of days open and multiple services before becoming pregnant. Neutrophils are the first wave of immune system defense against uterine contamination. Neutrophil function seems to be mediated by reproductive hormones and good uterine health is related to properly functioning neutrophils. To elucidate the interaction between reproductive hormones, neutrophils and uterine health in dairy cows the objectives of this research were to evaluate: 1) changes in circulating white blood cell populations during the estrous cycle, 2) the effects of prostaglandin F₂[subscript α](PGF₂[subscript α]) on circulating white blood cell populations and 3) the effects of a two-injection PGF₂[subscript α] regimen on uterine neutrophil and bacterial populations and numbers of days open and services per conception. In the first experiment, the effect of stage of the estrous cycle on plasma neutrophil numbers was evaluated. Neutrophils were observed throughout the entire estrous cycle and numbers were greatest (P<0.05) on Day 14 (mid-cycle), when the corpus luteum was the dominant ovarian structure and plasma progesterone was at its acme. In the second experiment, plasma neutrophil numbers were examined in cows after injections of saline or the PGF₂[subscript α] pharmaceutical product, Lutalyse. Compared to saline, numbers of neutrophils were greater (P<0.05) 4 and 8 hr after Lutalyse injection. In the third experiment, neutrophil numbers were examined after injections of saline, Lutalyse or the PGF₂[subscript α] analog, Estrumate. Compared to saline, numbers of neutrophils did not differ (P>0.10) from cows injected with Lutalyse or Estrumate. In the fourth experiment, uterine bacterial populations and numbers of neutrophils were quantified in cows treated with Lutalyse or saline on Days 0 and 14 or 14 and 28 postpartum. Compared to saline, Lutalyse treatment decreased (P<0.05) total bacteria present in the uterus and increased (P<0.05) the number of uterine neutrophils. In experiment five, numbers of days open and services per conception were evaluated in cows treated with Lutalyse or saline on Days 0 and 14 or 14 and 28 postpartum. Compared to saline, Lutalyse decreased days open (154.7 ± 14.1 vs. 120.1 ± 7.9 days, respectively; P<0.05) and services per conception (3.0 ± 0.4 vs. 2.3 ± 0.2 services, respectively; P=0.09). These results suggest PGF₂[subscript α] treatment can increase neutrophil and depress bacterial cell populations in favor of the dairy cow's uterine health and may explain why fertility is improved when PGF₂α is administered early in the postpartum period. / Graduation date: 2012

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 5 uppsatser.