Cyclooxygenases (COXs) are of important therapeutic value as they are the target site of aspirin-like drugs. Here I report nine new COX-1 splice variants in chapter 1, which I characterized with regard to heme-binding and other properties. Inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) are co-inducible in many tissues following mitogenic and proinflammatory stimulation. In chapter 2, I investigate the physical and enzymatic properties of human COX-2 and iNOS and demonstrate that, despite reports to the contrary by another laboratory, they do not interact. The only reported COX-1 splice variant to exhibit cyclooxygenase activity has been isolated from dog brain and is termed COX-3. It contains an in-frame insertion of intron 1. However the existence of human COX-3 remains questionable since intron 1 is out of frame. Two putative in-frame human COX-3 isozymes, COX-1b2 and COX-1b3, (herein designated as COX-3-72 and COX-3-50) have been reported in the literature, but only one of them, COX-3-72, has been characterized. In chapter 3, COX-3-50 and COX-3-72 are reported to be over-expressed and determined to be active cyclooxygenases. COX-3-72 and, to a greater extent, COX-3-50, were stimulated by rofecoxib at physiological concentrations. A similar rofecoxib-stimulated COX activity is observed in quiescent A549 cells. Immunoblot and immunoprecipitation analysis suggest that human platelet and potentially A549 cells, contain a COX-3-50 like protein. Lonicera japonica is used as an anti-inflammatory treatment in traditional Chinese medicine. Its working mechanism is not well known. In chapter 4, I report that extracts from this herb inhibit COX-2 by three mechanisms: direct inhibition, transcriptional and post-transcriptional down regulation. COX-1 and COX-2 are similar to each other in their crystallographic structures. One of the most striking differences is that there are eight amino acids immediately following the signal peptide in COX-1 which are not found in COX-2. The function of this sequence is unknown. In chapter 5, I found that deletion of these amino acids decreased COX-1 Vmax by approximately 4-fold, but had little effect on other properties of the enzyme. Selecting bacteria transformed with recombinant plasmids is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. In appendix I, I describe an ultra fast plasmid screening method. This new method was frequently used in the experiments performed in chapters 2-6.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2007" Includes bibliographical references.

Chronic inflammation has been linked to cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Although both COX isoforms have similar structure and function, they are encoded by different genes and show distinct expression patterns. COX-1 is expressed in most cells and tissues and remains constant under most physiologic conditions to play a housekeeping role whereas the COX-2 form is inducible and usually only expressed in response to various inflammatory stimuli. COX enzymes may be involved in both tumor establishment and maintenance of existing tumors. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. Prevention may be the best approach to reduce ovarian cancer. Ovarian cancer is the fifth leading cause of cancer death among women and the most lethal gynecological malignancy. There are at least 3 well established risk factors for ovarian cancer: age, family history and environmental factors. Ovarian cancer is mainly seen in older women when their ovaries are not reproductively functional. Close to half of the women with ovarian cancer (48%) are in the age group of 65 or older. Epidemiological and preclinical studies indicate that increased dietary intake of omega-3 fatty acids (OM-3FAs) reduces the incidence and growth of various cancers. Thus, increasing the consumption of OM-3FAs may be a nontoxic way to prevent or suppress ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Fish oil is a source of OM-3FAs which may be effective in prevention of ovarian cancer. The main OM-6FA, Linoleic Acid (LA), is a direct precursor of the Arachidonic Acid (AA). Alpha-linolenic acid (ALA) is the main OM-3FA found in flax oil, whereas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the OM-3FAs in fish oil. ALA is elongated to form EPA and DHA in the intestine. Celecoxib is a non-steroidal anti-inflammatory (NSAID) drug that selectively inhibits COX-2. There are evidences showing that Celecoxib has some anti-cancer properties. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. Our first objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by Immunohistochemistry, western blot, quantitative real-time PCR and LC-MS-MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. Since no effect on ovarian cancer incidence was detected after feeding the 2. 5 year old hens with 10% flaxseed for 1 year, we designed a long-term study using 6 month old hens. Our objectives were: 1) to examine the expression of COX enzymes and PGE2 levels in ovaries and correlate them to ovarian cancer and aging 2) to determine if long-term consumption of a flaxseed enriched diet decreases ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE2 concentration. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4 years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured by western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE2 concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE2 concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE2. Furthermore, elevated PGE2 in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. The results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE2 and expression of COX-2 were diminished in ovaries of hens fed flaxseed. Our findings suggest that the lower levels of COX-2 and PGE2 are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer. Based on our previous findings, targeting COX expression and prostaglandin biosynthesis by dietary intervention using OM-3FAs and selective COX inhibitor can be an effective approach to prevent or suppress ovarian cancer. Thus, we conducted a series of studies to assess effect of fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined or flax oil and Celecoxib combined on COX-1 and COX-2 expression, PGE2 concentrations, proliferation and apoptosis in normal and cancerous ovaries of laying hens. This study had not been performed in hens before, thus the first step was to find the optimum doses. In order to do so, we utilized one year old hens, divided them to groups of 6 hens, and fed them different doses of fish oil (50, 100, 175, 375 and 700 mg/kg), flax oil (100, 250, 500, 1000 and 1500 mg/kg) or Celecoxib (35, 65 and 100 mg/kg) for three weeks. The OM3-FAs andomega-6 fatty acids contents of egg yolks were determined by gas chromatography. Proliferation, apoptosis,COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, western blot, quantitative real-time qPCR and ELISA, respectively. The results indicated that 100 mg/kg fish oil was the most effective dose in reducing COX enzymes and PGE2, and increased apoptosis and reduced proliferation in ovaries. The lower doses of fish oil incorporated more OM-3FAs into yolks, reduced OM-6FAs and increased the egg laying frequency but did not affect EP4 expression. Unlike fish oil, the highest dose of flax oil (1500 mg/kg) caused the most significant reduction in COX expression and PGE2 concentration. Celecoxib was not perfectly selective in targeting COX-2, however, treating the hens with 65 mg/kg Celecoxib resulted in the most significant amelioration of PGE2 levels in ovaries. Using the optimum doses of fish oil, flax oil and Celecoxib, we aimed to investigate if these components can alter ovarian cancer end-points in normal and cancerous hen ovaries. There is an adverse relation between ovulation and health of ovaries. Thus, 3-4 year old hens were monitored for egg laying frequency and the hens with the least ovulation rate were selected for health assessment. The hens presenting poor health were scanned using ultrasound and if tumor mass and/or ascites were detected, they were chosen for this study. The hens with normal and cancerous ovaries were divided to groups and were fed fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined, or flax oil and Celecoxib combined for 42 days. The results showed that fish oil and flax oil increased the incorporation of OM-3FAs into egg yolks in both normal and cancerous ovaries of hens. Fish oil reduced COX-1 and COX-2 in normal and cancerous ovaries. Fish oil, flax oil and Celecoxib reduced the COX-2 expression in ovaries. Combination of fish oil and Celecoxib and flax oil and Celecoxib decreased COX and PGE2 more than each of these treatments alone. The cancerous ovaries of hens treated with fish oil, flax oil, Celecoxib, and flax oil and Celecoxib combined increased the percentage of apoptotic cells compared to cancerous ovaries of control hens. The cancerous ovaries of hens treated with fish oil and Celecoxib had the highest number of apoptotic cells indicating that the combination of fish oil and Celecoxib is more effective than fish oil or Celecoxib alone. To our knowledge the present study provides the first insight into the efficacy of fish oil, flax oil, Celecoxib, alone or combined on the reduction of COX enzyme expression, PGE2 concentration and apoptosis in the normal and cancerous ovaries and further demonstrates the utility of the hen model for ovarian cancer. Our studies provided new insight into the potential mechanism of action of flaxseed, fish oil, flax oil and Celecoxib in the reduction of ovarian cancer and will establish the foundation for clinical trials to test the efficacy of dietary intervention for the prevention and suppression of ovarian cancer in women.

Dans la première partie nous décrivons la synthèse et l'évaluation biologique de nouveaux inhibiteurs de l'agrégation plaquettaire, composés dont la structure a été établie en partant du 12-HETE et du 13-HODE. Dans la seconde partie nous développons de nouvelles méthodologies pour la préparation de molécules contenant des motifs CF₂R. Tout d'abord une stratégie très flexible a été mise au point pour la préparation de composés gem-difluorobisaryliques et de leurs analogues hétéroaromatiques. Elle est basée sur l'emploi d'intermédiaires gem-difluoropropargyliques faciles d'accès. Par une séquence de Diels-alder-aromatisation on obtient les molécules cibles de la première série. Pour la seconde, des réactions de cycloaddition dipolaire 1,3 ont été utilisées. A partir de ces intermédiaires, des chimiothèques ciblées de molécules fluorées ont été préparées. Nous nous sommes intéressés ensuite à la synthèse de composés fluorés fonctionnalisés et chiraux à travers des réactions d'organocatalyse asymétrique. A partir d'énals gem-difluorés des réactions de Diels-Alder et des additions 1,4 asymétriques ont été réalisées avec succès. / The first part of the thesis deals with the synthesis and biological evaluation of new platelets aggregation inhibitors, based on 12-HETE, 13-HODE and their analogues. In the second part we are interested in novel methodologies for the preparation of CF₂-containing molecules : First, a flexible strategy for the synthesis of gem-difluoro-bisarylic derivatives and heteroaromatic analogues was designed based on the easy synthesis and the reactivity of gem-difluoro propargylic intermediates, which by Diels-Alder cycloaddition and 1,3-dipolar cycloadditions afforded respectively the bisarylic and mixed arylic heteroarylic scaffolds. In addition, two small libraries were constructed around a bisarylic scaffold as representative examples. Second, we were interested in the synthesis of optically active functionalized molecules containing a gem-difluoro group, using asymmetric organocatalysis protocols. After preparation of the gem-difluoro enals, from their difluoropropargylic precursors, asymmetric organocalytic Diels-Alder cycloaddition and 1,4-conjugated additions were successfully performed.

Objective: To appraise whether adverse events following oral implant placement may be associated with peri-operative use of non-steroidal anti-inflammatory drugs (NSAIDs). Methods: All patients with recorded implant failures between 1979 and 2012 in the Implant Prosthodontics Unit were contacted to solicit additional information about potential peri-operative use of NSAIDs. Results: From a total of 168 patients with 292 implant failures between 1979 and 2012, 122 consented to participate and had intact records. Just over half (56.6%) reported no peri-operative NSAID usage. However, compared to patients who did not use peri-operative NSAIDs, four times as many had complicated medical histories and twice as many patients taking NSAIDs suffered multiple implant failures. Conclusions: Patients with a variety of systemic diseases may be adversely affected by the inhibitory effect of NSAIDs on bone healing. Further prospective clinical studies are warranted to clarify this potential causative relationship in humans.

Objective: To appraise whether adverse events following oral implant placement may be associated with peri-operative use of non-steroidal anti-inflammatory drugs (NSAIDs). Methods: All patients with recorded implant failures between 1979 and 2012 in the Implant Prosthodontics Unit were contacted to solicit additional information about potential peri-operative use of NSAIDs. Results: From a total of 168 patients with 292 implant failures between 1979 and 2012, 122 consented to participate and had intact records. Just over half (56.6%) reported no peri-operative NSAID usage. However, compared to patients who did not use peri-operative NSAIDs, four times as many had complicated medical histories and twice as many patients taking NSAIDs suffered multiple implant failures. Conclusions: Patients with a variety of systemic diseases may be adversely affected by the inhibitory effect of NSAIDs on bone healing. Further prospective clinical studies are warranted to clarify this potential causative relationship in humans.

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.

<p>Karakterizacija metanolnih ekstrakata jedanaest vrsta samoniklih bokvica (rod<em> Plantago L.</em>) obuhvatala je fitohemijski skrining i ispitivanje antioksidantne i antiinflamatorne aktivnosti. Primenom LC-MS/MS tehnike odreĊen je sadržaj odabranih sekundarnih biomolekula. Antioksidantna aktivnosti ekstrakata (sposobnost neutralizacije slobodnih radikala, redukcioni potencijal i inhibicija lipidne peroksidacije) ispitana je primenom spektrofotometrijskih metoda. U cilju odreĊivanja antiinflamatornog potencijala, razvijena je<em> in vitro </em>metoda za praćenje aktivnosti trombocitne ciklooksigenaze-1 i 12-lipoksigenaze. Svi ispitani ekstrakti pokazali su znaĉajnu biolo&scaron;ku aktivnost.</p> / <p>Characterization of methanol extracts of eleven<em> Plantago L.</em> species included phytochemical screening and evaluation of antioxidant and anti-inflammatory activity. The content of several secondary metabolites was determined by LC-MS/MS technique. Antioxidant activity of extracts (radical scavenger capacity, reduction potential and inhibition of lipid peroxidation) was examined by spectrophotometric methods. With the intention to evaluate anti-inflammatory activity, an<em> in vitro method</em> was developed to measure activity of platelet cyclooxygenase-1 and 12-lipoxygenase. All examined extracts showed noticeable biological activity.</p>

The first part of the thesis deals with the synthesis and biological evaluation of new platelets aggregation inhibitors, based on 12-HETE, 13-HODE and their analogues. In the second part we are interested in novel methodologies for the preparation of CF₂-containing molecules : First, a flexible strategy for the synthesis of gem-difluoro-bisarylic derivatives and heteroaromatic analogues was designed based on the easy synthesis and the reactivity of gem-difluoro propargylic intermediates, which by Diels-Alder cycloaddition and 1,3-dipolar cycloadditions afforded respectively the bisarylic and mixed arylic heteroarylic scaffolds. In addition, two small libraries were constructed around a bisarylic scaffold as representative examples. Second, we were interested in the synthesis of optically active functionalized molecules containing a gem-difluoro group, using asymmetric organocatalysis protocols. After preparation of the gem-difluoro enals, from their difluoropropargylic precursors, asymmetric organocalytic Diels-Alder cycloaddition and 1,4-conjugated additions were successfully performed.

The anti-inflammatory properties of four medicinal plants were investigated. These plant extracts were subjected to screening for their possible effects as antioxidative, analgesic, and anti-inflammatory agents. In the antioxidant activity, the Plantago lancelota extracts resulted in an IC50 value of 0.4 mg/mL compared to the positive control quecertin with IC50 0.04 mg/mL Plantago lanceolata inhibited COX-2 activity with IC50 values of 0.41 mg/mL. However, the COX-1 inhibition indicated an IC50 of 68.99 mg/mL. The lipoxygenase assay indicated that Plantago lanceolata was the most active plant species with an IC50 value of 4.86 mg/mL compared to the positive control (quecertin) with an IC50<2mg/mL. The nitric oxide assay of the plant extracts indicates a dose-dependent activity of our plant extracts. Likewise the cell viability result indicated a good activity at dose 100 mg/mL. / Life and Consumer Sciences / M. Sc. (Life Sciences)