Transition of the cardiopulmonary circulation at birth requires functional closure of the ductus arteriosus (DA). The DA is an arterial shunt that is vital in the fetus for diverting the pulmonary circulation away from the uninflated lungs. Failure of the vessel to functionally close after birth is known as patent DA, which is the second most common congenital heart disease. Patent DA may seriously compromise neonatal health and current pharmacological treatments are often limited by serious complications or a significant failure rate, thereby increasing the necessity for surgical intervention. Recently, we were the first to show that genetic or pharmacological inactivation of cyclooxygenase (COX) -2 produces postnatal patent DA in mice. We also demonstrated that the DA expresses high levels of COX-2 during normal closure after birth, suggesting novel contractile actions of COX-2-dependent prostanoids in the DA. In humans, patent DA is more common in preterm infants than those born at full-term, however, mechanism(s) responsible for the reduced DA closure have not been identified. In the current studies, we examined COX-1 and COX-2 expression in the DA at multiple stages of gestation to determine whether alterations in the expression of these enzymes contribute to patent DA in preterm mice. Using real-time PCR, analysis of the time-course of COX-2 mRNA in the fetal mouse DA indicated that COX-2 expression significantly increased with advancing gestational age. The preterm (day 17.5) neonatal mouse DA showed attenuated COX-2 expression, as compared to the full-term (day 19.5) neonatal DA at 3 hours after birth. Furthermore, the DA of preterm neonatal mice showed incomplete closure after 3 hours of birth, a time-point when the DA of full-term neonates was completely remodeled. These data indicate a correlation between reduced DA closure and attenuated COX-2 expression. Additionally, COX-2 expression was significantly attenuated in the DA of mice deficient in the prostanoid receptor EP4, which also show a patent DA phenotype, suggesting the importance of this receptor for the induction of COX-2 required for DA closure. Overall, these studies suggest that attenuated expression of COX-2 may contribute to increased patent DA at preterm gestation.

Personalized medicine can be an approach to address the unsatisfactory treatment outcomes in inflammatory conditions such as cancer, arthritis, and cardiovascular diseases. A common feature of chronic diseases is the infiltration of pro-inflammatory macrophages at the disease loci. Infiltrating macrophages have been previously utilized for disease diagnosis. These features suggest that macrophages can be broadly applicable targets for simultaneous therapy and diagnosis. Cyclooxygenase-2 (COX-2), an enzyme involved in the biosynthesis of a lipid inflammatory mediator, prostaglandin E2 (PGE2), is over expressed in macrophages infiltrating the pathological site. Inhibition of PGE2 leads to reduced inflammation, pain and macrophage infiltration. To utilize macrophages for the purpose of simultaneous therapy and diagnosis, we proposed to integrate therapeutic and imaging capabilities on a single nanomedicine platform, referred as theranostics. A stable 19F MRI visible nanoemulsion platform was developed, incorporating celecoxib for COX-2 inhibition and near-infrared fluorescent dye(s) for fluorescence imaging. We hypothesized that inhibition of COX-2 in macrophages using a theranostic nanoemulsion will reduce the inflammation (and pain), and that this response can be visualized by monitoring changes in macrophage infiltration. In vitro characterization demonstrated that the theranostic displays excellent stability with no toxicity, and significant uptake in macrophages. Furthermore, it delivers celecoxib to macrophages and reduces PGE2 production from these cells. In vivo studies in a murine paw inflammation model showed nanoemulsion presence at the inflamed site, specifically in COX-2 expressing macrophages compared to neutrophils. Supporting our hypothesis, celecoxib delivered through a nanoemulsion demonstrated time-dependent reduction in fluorescence from the inflamed paw, indicative of reduced macrophage infiltration. In a neuropathic pain model, celecoxib delivered to macrophages led to reduced pain concomitant with reduced macrophage infiltration at the inflamed site compared to free drug control (cross reference: Kiran Vasudeva, Dissertation, 2015). In conclusion, inhibition of COX-2 in macrophages using theranostic nanoemulsions proves to be an effective and generalized strategy facilitating simultaneous therapy and diagnosis, which can be applied to many chronic diseases. The diagnostic information during therapy can be used to tailor the treatment and reduce patient variability leading to personalized medicine. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmaceutics / PhD; / Dissertation;

ABSTRACT Vascular permeability and angiogenesis are crucial events in the rodent and human uterus in early pregnancy and are regulated by vascular endothelial growth factor (VEGF) and prostaglandins liberated from arachidonic acid by cyclooxygenase 2 (COX2). These events coincide with the typical morphological features of the receptive uterus and are regulated by synchronized release of ovarian hormones (oestrogen and progesterone). However, administration of follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG), commonly used in assisted reproduction, affect the synchrony of the hormonal milieu, particularly by increasing oestrogen levels. This causes detrimental changes to the uterine morphology and affects vascular permeability at the site of implantation. In the present study, the expression of COX2 and VEGF was compared between control and hyperstimulated rat uteri during the peri-implantation period using immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes. immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes.

histoplasmose é uma doença granulomatosa crônica, cujo agente etiológico é o fungo dimórfico Histoplasma capsulatum. A infecção ocorre pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras que é responsável pela patogenia da doença. A imunidade celular do hospedeiro determina o grau das manifestações clínicas na histoplasmose, sendo a interação entre células T e macrófagos, fundamental para o controle da infecção e erradicação do H. capsulatum. Recentemente, nosso grupo de pesquisa demonstrou a participação de leucotrienos nos mecanismos de defesa do hospedeiro durante a histoplasmose. Neste trabalho descrevemos o papel das prostaglandinas, demonstramos que este mediador lipídico contribui para a patogênese da doença, pois sua inibição, com celecoxibe, resultou na sobrevivência de até 80% dos animais infectados com o inóculo letal de H. capsulatum, em contraste com 100% de mortalidade dos animais somente infectados. Além disso, a inibição das prostaglandinas resultou na diminuição (i) da síntese de citocinas pró-inflamatórias e da resposta imune celular e (ii) do recrutamento de neutrófilos e macrófagos para o espaço bronco-alveolar. Por outro lado, resultou no aumento (iii) de células TCD4+ no pulmão, (iv) na síntese de óxido nítrico por células do parênquima pulmonar, (v) na fagocitose de leveduras de H. capsulatum por macrófagos alveolares e (vi) da síntese de LTB4. Nossos resultados sugerem que prostaglandinas têm papel importante na patogênese na infecção por H. capsulatum, modulando a resposta imune do hospedeiro. / The Histoplasmosis is a chronic granulomatosas disease whose etiologic agent is pathogenic dimorphic fungus Histoplasma capsulatum. Infection occurs mainly by fungal inhalation that reaches the alveoli, where if transforms into leavenings that are responsible for pathogenic diseases. The cellular immunity of the host determines the degree of the clinical manifestations in histoplasmosis, being the interaction between cells T and macrophages, basic for the control of the infection and eradication of the H. capsulatum. Recently, our group of research demonstrated the participation of leukotrienes in the mechanisms of defense of the host during the Histoplasmosis. Beyond this important lipid mediator who participates in the immune reply against H. capsulatum. In this work, we describe another involved mediator, the prostaglandin. In the present work, we demonstrate that the prostaglandins contribute for pathogenic of the disease, being that during its inhibition with celecoxib it resulted in the survival of up to 80% of the infection-mice with inoculum lethal of H. capsulatum, in contrast with 100% of mortality infection-mice. Moreover, the inhibition of prostaglandins resulted in the reduction (i) of the synthesis of pro-inflammatory cytokines and the cellular immune response and (ii) in the migration of neutrophils and macrophages. For other hand, increased (iii) of cells TCD4+ in the lung, (iv) of the nitric oxide synthesis, (v) of phagocytosis of yeast of H. capsulatum for alveolar macrophages and (vi) of the synthesis of LTB4. Our results suggest that prostaglandins have important role in pathogenic in the infection for H. capsulatum, modulating the host immune response.

Made available in DSpace on 2017-07-24T19:22:17Z (GMT). No. of bitstreams: 1 Reila Mendes.pdf: 2640941 bytes, checksum: ae80dd043b80ca7c3792d907a25ab34f (MD5) Previous issue date: 2012-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Periodontal disease is an inflammatory chronic disorder caused by a small group of gram-negatives bacteria witch colonizes the subgengival area. This disease is characterized by the destruction of the periodontal tissues, including bone resorption and loss of clinical attachment level. Observational studies have shown an increase in the risk of cardiovascular diseases, specially atherosclerosis and hypertension, among the subjects affected by the periodontal disease. The literature also reports endothelial dysfunction among these patients. Data suggest that the systemic inflammation due to the biofilm present in periodontits and the reduction in the nitric oxide availability may be, at least in part, the cause of endothelial dysfunction, which leads to cardiovascular disorders. An increase in vascular COX-2 expression has been demonstrated among diseases related to endothelial dysfunction. COX-2 expression seems to have an important protector function through the production of arachidonic acid metabolites with vasodilator and antitrombotic properties. Therefore, COX-2 inhibition may represent negative cardiovascular implications. The purpose of this research was to evaluate the effect of COX-2 inhibition on the vascular reactivity and on the heart tissue of animals with periodontitis. At day 0, Wistar rats were subdivided into the following groups: ligature + etoricoxib (the animals received ligatures and were treated with etoricoxib 10 mg/kg p.o., for seven days, from the day 14), ligature + vehicle (the animals were treated with distilled water), sham + etoricoxib (the animals went through a sham procedure: the ligatures were positioned and immediately removed) and sham + vehicle. The rats were prepared for the data collection and sacrificed at day 21. Changes on the vasoconstrictor response were not observed. However, the group ligature + etoricoxib showed a tendency to have the vasodilator response reduced. All the groups showed histological cardiac alterations, especially the groups that received etoricoxib, when submitted to ischemia with isoprenaline. It is suggested, though, that COX-2 inhibition in an experimental model of periodontal disease may increase cardiovascular disorders. / A periodontite é uma doença inflamatória crônica iniciada e perpetuada por bactérias anaeróbicas gram-negativas que colonizam a área subgengival. Esta doença é caracterizada pela destruição do tecido periodontal de inserção, reabsorção óssea, infiltração de leucócitos e formação de bolsa periodontal. Estudos observacionais têm mostrado um significativo aumento do risco de doenças cardiovasculares, principalmente aterosclerose e hipertensão, entre pessoas com periodontite. A literatura mostra também a presença de disfunção endotelial nesses pacientes. Os dados sugerem que a inflamação sistêmica induzida pela microbiota presente na doença periodontal e a diminuição na biodisponibilidade do óxido nítrico podem ser, pelo menos em parte, a causa da disfunção endotelial, que por sua vez leva a doenças cardiovasculares. Um aumento na expressão vascular da ciclooxigenase-2 (COX-2) tem sido consistentemente demonstrado em patologias que apresentam disfunção endotelial. A expressão da COX-2 nesta condição parece ter um importante papel protetor através da produção de metabólitos do ácido araquidônico com propriedades vasodilatadoras e antitrombóticas. Dessa maneira, a inibição da COX-2 pode apresentar aspectos cardiovasculares negativos. Assim, a proposta deste trabalho foi avaliar o efeito da inibição COX-2 sobre a reatividade vascular e sobre o tecido cardíaco de animais com periodontite. No dia 0, ratos Wistar foram subdivididos nos seguintes grupos: ligadura + etoricoxibe (receberam ligaduras e foram medicados com etoricoxibe 10 mg/kg v.o. por sete dias, a partir do dia 14), ligadura + veículo (receberam água destilada), falso-operado + etoricoxibe (passaram pelo procedimento de falsa-cirurgia, as ligaduras foram colocadas e imediatamente removidas) e falso-operado + veículo. Os animais foram preparados para aferição dos dados e sacrificados no dia 21. Não foram observadas alterações na resposta vasoconstritora, porém o grupo ligadura + etoricoxibe mostrou uma tendência em ter a resposta vasodilatadora reduzida. Quando submetidos à isquemia com isoprenalina, todos os grupos apresentaram alterações histológicas cardíacas, especialmente os que receberam etoricoxibe. Sugere-se, pois, que a inibição da COX-2 em um modelo experimental de periodontite exacerbe alterações cardiovasculares.

Cheng Sze-Lok Alfred. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 175-211). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Celecoxib is a COX-2 selective non-steroidal anti-inflammatory drug which has been shown to inhibit growth and induce apoptosis in various cancer cell lines. Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and an apoptosis detection kit, we demonstrated that celecoxib was able to induce growth inhibition and apoptosis in a dose-dependent manner in 3 NPC cell lines: HK-1, Hone-1, and C666-1. Afterwards, a proteomic approach was used to study the underlying mechanisms involved in celecoxib-mediated effects on two COX-2 positive NPC cell lines (HK-1 and C666-1). Results showed that a total of 18 protein spots were differentially expressed in the HK-1 and C666-1 cells. On the other hand, we also compared the proteomic expression profile between an NPC cell line (C666-1) and a normal nasopharynx cell line (NP69) in order to study whether those differentially expressed proteins after celecoxib treatment were also involved in NPC carcinogenesis. Proteomics results with confirmation using Western blotting discovered that HSP27 phosphorylated of serine 82 (HSP27-pSer82) protein was up-regulated in C666-1 cells when compared with that in NP69 cells. After treatment with celecoxib, expression of HSP27-pSer82 protein was down-regulated in both HK-1 and C666-1 cells. These findings suggest that down-regulation of HSP27-pSer82 protein expression may have mediated the growth-inhibitory effects of celecoxib in HK-1 and C666-1 cells. Finally, other differential expressed proteins identified from proteomics with confirmation by immunocytochemical staining in the 2 NPC cell lines and 40 NPC patient specimens showed that down-regulation of annexin 2 and beta2-tubulin may be important in NPC formation. / COX-2 over-expression has been found in various cancers such as colorectal cancer, liver cancer and lung cancer. In vivo studies have shown that mice overexpressing COX-2 developed breast cancer whereas COX-2 knockout mice had reduced rates of cancer formation in the intestines and skin. In the present study, COX-2 expression in NPC patient biopsies was examined and correlated with the clinicopathological data of the patients. Immunocytochemical staining showed that COX-2 protein was over-expressed in 84.6% (66/78) of non-metastatic NPC patients and was associated with an advanced nodal stage (P<0.05). All these data support an important role for COX-2 in NPC pathogenesis. / In summary, this study is the first to identify HSP27-pSer82 protein as a potential target of celecoxib in NPC cells. Detailed investigations of the functional role of molecular targets identified in this study would improve our understanding of the chemotherapeutic effects of celecoxib and, in the long run, may lead to a more effective chemotherapeutic treatment to this common cancer. / Nasopharyngeal carcinoma (NPC) is prevalent in southern China. Although early stage patients have a high rate of cure with radiotherapy alone, the prognosis for those with stage III or IV disease remains poor due to subsequent development of distant metastases. Therefore there is an urgent need to develop novel biologic agents to improve treatment outcomes. / Chan, Ming Lok. / Adviser: Anthony T.C. Chan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3418. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 141-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

乳腺癌是女性最常見的腫瘤之一，多發生在女性絶經後，並具有雌激素依賴性。芳香化酶(CYP19)是雌激素生物合成過程中的關鍵酶，而芳香化酶抑製劑(AI)則被用於替代治療雌激素依賴性的乳腺癌。然而，AI在降低雌激素水平的同時能夠引起骨質酥鬆。此項研究的目的是找尋AI替代物。 / 黃酮類化合物是一種多酚化合物，廣泛分佈于植物中。我們先前的研究發現二氢黄酮陈皮素能夠抑制芳香化酶的生物活性，并且抑制芳香化酶高表達的乳腺癌生長。在本研究中，我們發現陳皮素在抑制腫瘤生長的同時能夠降低来曲唑引起的骨質流失。木犀草素是另外一種黄酮类化合物，它同樣能夠抑制芳香化酶的活性并減少骨流失。而與陳皮素不同的是，它能夠抑制芳香化酶的表達。在芳香化酶高表達的乳腺癌細胞(MCF-7 aro)中，木犀草素抑制芳香化酶活性的IC50是3 μM。在MCF-7 細胞中，5 μM的木犀草素能夠抑制CYP19 mRNA 的表達，螢光素酶報告實驗顯示木犀草素是通過作用于啟動子I.3和II來抑制CYP19的表達。蛋白印跡實驗表明木犀草素抑制CYP19表達的分子機制可能通過調節JNK信號通路進而減少AP-1的活性來實現。動物實驗結果顯示木犀草素能夠抑制MCF-7aro腫瘤的生長并改善來曲唑引起的骨流失。 / 環氧化酶(COX)是花生四烯酸轉化為前列腺素途徑中的一種關鍵酶。研究發現COX-2在乳腺癌組織中廣泛表達。本實驗研究了COX抑製劑在裸鼠動物模型中對乳腺癌腫瘤的作用機制。研究結果表明塞來昔布和阿司匹林在不影響血液中雌激素水平的情況下抑制乳腺癌腫瘤的生長。蛋白印迹實驗顯示這兩種藥物能夠降低腫瘤中COX-2，Cyclin A和Bcl-xL的表達。miR-98, miR-222和miR-145也能夠被塞來昔布和阿司匹林影響。 / 本研究表明陳皮素，木犀草素及COX抑制劑有潛力成為替代AI的化學治療藥物或共同治療藥物。 / Breast cancer is one of the most prevalent cancers affecting women. The majority of breast tumor growth occurred in the post-menopausal period are estrogen dependent. Aromatase (CYP19) catalyzes the rate-limiting step in the synthetic reaction of estrogen and aromatase inhibitors (AIs) are contemporary treatment for estrogen-positive breast cancer. However, estrogen-lowering drugs may promote osteoporosis. Our objective of this study further identified some alternatives for AIs. / Flavonoids are polyphenolic compounds that are ubiquitously distributed in plants. We have previously found that the flavanone hesperetin can inhibit the activity of aromatase and suppress aromatase-expressing breast tumor growth. In this project, we investigated the potential interaction between hesperetin and the AI letrozole in a mouse model. Our results showed that hesperetin could inhibit the tumor growth and reduce bone loss induced by letrozole. Similarly, another flavonoid luteolin also inhibited aromatase and prevented bone deterioration as observed in this project. In cells stably transfected with CYP19 (MCF-7aro), luteolin inhibited the aromatase activity with an IC50 value of 3μM. In addition, 5μM luteolin significantly reduced CYP19 mRNA expression in MCF-7 cells. Luciferase reporter assay revealed that luteolin could suppress CYP19 transcription at promoter regions I.3 and II. Western analysis illustrated that JNK signaling pathway was involved and deactivation of AP-1 could be the underlying molecular mechanism. Subsequently, we examined the effect in vivo. Our results showed that luteolin could inhibit the MCF-7aro tumor growth and improved bone loss induced by letrozole. / Cyclooxygenase (COX) is an enzyme responsible for the conversion of arachidonic acid into prostaglandins. It is over-expressed in breast cancer tissue and an increased expression of COX-2 was also observed in the xenograft model employed in this project. In the last study we evaluated the importance of COX-2 in breast tumor growth in this model. Our data showed that celecoxib and aspirin could significantly suppress the tumor growth without changing the plasma estrogen level. Western analysis illustrated that COX-2, Cyclin A, Bcl-xL and ER were reduced in celecoxib- and aspirin- treated tumor samples and miR-98, miR-222 and miR-145 were altered by celecoxib or aspirin. / After all, this project demonstrated that hesperetin, luteolin and COX-inhibitors could be potential chemopreventive or co-therapeutic agents. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Fengjuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 131-148). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / LIST OF ABBREVIATIONS --- p.V / TABLE OF CONTENTS --- p.VII / CHAPTER 1 --- p.1 / GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Types of Breast Cancer --- p.3 / Chapter 1.2 --- Nuclear Receptor Signaling Pathways in Breast Cancer --- p.5 / Chapter 1.3 --- Estrogen and Breast Cancer --- p.7 / Chapter 1.4 --- Estrogen and Bone Health --- p.8 / Chapter 1.5 --- Estrogen Biosynthesis and Aromatase --- p.10 / Chapter 1.6 --- Tissue Specific Promoter for Aromatase Expression --- p.13 / Chapter 1.7 --- Nuclear Receptors and Aromatase Promoter Regulation --- p.15 / Chapter 1.8 --- Signaling Pathway and Aromatase Expression --- p.17 / Chapter 1.9 --- Cell Cycle in Breast Cancer --- p.20 / Chapter 1.10 --- Cell Apoptosis --- p.23 / Chapter 1.11 --- Treatment of breast cancer --- p.25 / Chapter 1.12 --- Phytoestrogens --- p.29 / Chapter 1.13 --- Aim of My Study --- p.32 / CHAPTER 2 --- p.33 / MATERIALS AND METHODS --- p.33 / Chapter 2.1 --- Chemicals and Materials --- p.33 / Chapter 2.1.1 --- Chemicals --- p.33 / Chapter 2.1.2 --- Plasmids --- p.33 / Chapter 2.2 --- Cell Culture --- p.33 / Chapter 2.3 --- Aromatase Activity Assay --- p.34 / Chapter 2.4 --- Quantitative Real Time PCR --- p.36 / Chapter 2.4.1 --- RNA Isolation and cDNA Synthesis --- p.36 / Chapter 2.4.2 --- Quantitative Real Time PCR Assay --- p.37 / Chapter 2.4.3 --- MiRNA Quantitative Real Time PCR Assay --- p.38 / Chapter 2.5 --- Western Blot --- p.39 / Chapter 2.6 --- Measurement of Promoter Activity --- p.41 / Chapter 2.6.1 --- Plasmid Preparation --- p.41 / Chapter 2.6.2 --- Transient Transfection and Dual-Luciferase Assay --- p.42 / Chapter 2.7 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.43 / Chapter 2.7.1 --- Nuclear protein extraction --- p.43 / Chapter 2.7.2 --- Electrophorectic Mobility Shift Assay --- p.44 / Chapter 2.8 --- Animal Experiment Design --- p.45 / Chapter 2.8.1 --- Animal Model for Hesperetin Study --- p.45 / Chapter 2.8.2 --- Animal Model for Luteolin Study --- p.46 / Chapter 2.8.3 --- Animal Model for Cycooxygenase Inhibitors Study --- p.48 / Chapter 2.8.4 --- Serum Estradiol Determination --- p.49 / Chapter 2.8.5 --- Analysis of serum lipoproteins --- p.49 / Chapter 2.8.6 --- Bone Image Acquisition and Region of Interest Selection --- p.50 / Chapter 2.9 --- Statistical Analysis --- p.50 / CHAPTER 3 --- p.51 / The citrus flavonone hesperetin prevents letrozole- induced bone loss in a mouse model of breast cancer --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Murine Body Weight and Liver Weight --- p.54 / Chapter 3.2.2 --- Effect of Hesperetin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.55 / Chapter 3.2.3 --- Hesperetin Reduced Plasma Estradiol Concentration --- p.58 / Chapter 3.2.4 --- PS2 mRNA Expression in Tumor --- p.59 / Chapter 3.2.5 --- Uterine Wet Weight --- p.60 / Chapter 3.2.6 --- Hesperetin Prevent Bone Deterioration Induced by Letrozole --- p.61 / Chapter 3.3 --- DISCUSSION --- p.63 / CHAPTER 4 --- p.66 / dIETARY FLAVONOID LUTEOLIN ON cyp19 transcription in the breast cancer cells mcf-7 --- p.66 / Chapter 4.1 --- Introduction --- p.66 / Chapter 4.2 --- Results --- p.68 / Chapter 4.2.1 --- Inhibitory Effect of Luteolin on Aromatase Activity --- p.68 / Chapter 4.2.2 --- Luteolin Reduced Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.3 --- Effect of Luteolin on Promoter I.3/II Activity of CYP19 in MCF-7 Cells --- p.71 / Chapter 4.2.4 --- The Effect of Luteolin on Truncation CYP19 Gene Reporter Assay --- p.72 / Chapter 4.2.5 --- Luteolin Reduced AP-1 Binding in Promoter I.3/II DNA Fragment --- p.74 / Chapter 4.2.6 --- Inhibitory Effect of Luteolin on Protein Kinase Signaling --- p.76 / Chapter 4.3 --- Discussion --- p.78 / CHAPTER 5 --- p.83 / interaction OF LUTEOLIN and letrozole in a postmenopausal breast cancer model --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Results --- p.86 / Chapter 5.2.1 --- Luteolin and letrozole treatment had no effect on mouse body weight and liver weight --- p.86 / Chapter 5.2.2 --- Effect of luteolin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.88 / Chapter 5.2.3 --- Luteolin reduced plasma estradiol concentration --- p.91 / Chapter 5.2.4 --- Luteolin Counteracted Uterine Weight Reduction under Letrozole Treatment --- p.92 / Chapter 5.2.5 --- Luteolin Prevented Bone Deterioration Induced by Letrozole --- p.93 / Chapter 5.2.6 --- The Effect of Luteolin on Plasma TC and TG --- p.95 / Chapter 5.2.7 --- Luteolin Increased HDL Level and Reduced the Ratio of LDL/HDL --- p.97 / Chapter 5.2.8 --- Effect of Luteolin on Cell Cycle and Apoptotic Protein Expression --- p.99 / Chapter 5.3 --- DISCUSSION --- p.104 / CHAPTER 6 --- p.107 / cyclooxygenase inhibitors suppresse breast tumor growth in NUDE MICE --- p.107 / Chapter 6.1 --- Introduction --- p.107 / Chapter 6.2 --- Results --- p.109 / Chapter 6.2.1 --- Celecoxib and aspirin treatment had no effect on mouse body weight and liver weight --- p.109 / Chapter 6.2.2 --- Effect of celecoxib and aspirin on Xenograft Growth in Ovariectomized Mice --- p.111 / Chapter 6.2.3 --- Celecoxib and aspirin had no effect on plasma estradiol concentration --- p.113 / Chapter 6.2.4 --- Celecoxib and Aspirin Had no Effect on Uterine Weight --- p.114 / Chapter 6.2.5 --- Protein expression of COX-2, Cell cycle-related and cell Apoptotic Genes --- p.115 / Chapter 6.2.6 --- Detection of Related miRNA Expression Level in Tumors --- p.118 / Chapter 6.2.7 --- c-Myc mRNA Expression Level were Regulated in Tumors --- p.121 / Chapter 6.3 --- DISCUSSION --- p.124 / CHAPTER 7 --- p.127 / SUMMARY --- p.127 / REFERENCE --- p.131

To increase diversity of enzymes and proteins, cells mix and match exonic and intronic regions retained in mature mRNAs by alternative splicing. An estimated 94% of all multi-exon genes express one or more alternatively spliced transcripts generating proteins with similar or modified functions. Cyclooxygenase is a signaling enzyme that catalyzes the rate-limiting step in the synthesis of diverse bioactive lipids termed prostaglandins. Prostaglandins are involved in myriad physiological and pathopysiological processes including vasoregulation, stomach mucosal maintenance, parturition, pain, fever, inflammation, neoplasia and angiogenesis and are inhibited by aspirin-like drugs known as NSAIDs. In 2002 an alternatively spliced, intron-1 retaining variant of COX-1 was cloned from canine brain tissue. This new variant, termed COX-3 or COX-1b, is an enzymatically active prostaglandin synthase expressed at relatively high levels in a tissue and cell type dependant manner in all species examined. In humans and most rodent species intron-1 is 94 and 98 nucleotides long respectively. Retention of the intron in these species introduces a frameshift and is predicted to result in translation of a very small 8-16kD protein with little similarity to either 72kD COX-1 or COX-2, calling into question the role of this variant. In this dissertation, I present my results from cloning and ectopically expressing a complete and accurate COX-3 cDNA from both rat and human. I confirmed that COX-3 mRNA encodes multiple large molecular weight cyclooxygenase-like proteins in the same reading frame as COX-1. Translation of these proteins relies on several recoding mechanisms including cap-independent translation initiation, alternative start site selection, and ribosomal frameshifting. Using siRNA and Western blotting I have identified some of these proteins in tissues and cells. Two COX-3 encoded proteins are active prostaglandin synthase enzymes with activities similar to COX-1 and represent novel targets of NSAIDs. Other COX-3 proteins have unknown function, but their size and cellular location suggest potential roles as diverse as cytosolic enzymes and nuclear factors.

No description available.