Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-14T12:01:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) / Made available in DSpace on 2016-07-14T12:01:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) Previous issue date: 2015-07-29 / FACEPE / O Sildenafil é um inibidor potente e seletivo da fosfodiesterase-5 (PDE5). Este fármaco foi aprovado para uso terapêutico na disfunção erétil e, atualmente, vem sendo usado também no tratamento da hipertensão pulmonar. Embora mantenha um excelente nível de segurança e perfil de tolerabilidade, poucos estudos avaliaram os possíveis efeitos colaterais do tratamento crônico com Sildenafil sobre o sistema reprodutor masculino, especialmente na próstata visando o relaxamento da uretra e alívio dos Sintomas do Trato Urinário Inferior (STUI). Desta forma, avaliamos o efeito do tratamento, em camundongos C57Bl/6, através de análise morfológica, ultraestrutural e expressão molecular de guanilato ciclase solúvel-sGC, Óxido nítrico sintase endotelial-eNOS, Antígeno prostático específico-PSA e Fator de crescimento transformador beta-TGF- no tecido prostático. Foi-se observado que o tratamento com Sildenafil não induz danos evidentes na próstata. Além disso, tem sido demonstrado que Sildenafil tem eficácia terapêutica em doenças inflamatórias crônicas, podendo apresentar uma eficácia terapêutica potencial em diferentes doenças. Entre elas, uma atenção especial tem sido dada para as patologias relacionadas ao trato urogenital masculino, como a Hiperplasia Prostática Benigna (HPB), Câncer de próstata e Prostatites. A inflamação tem sido considerada como um fator etiológio da HPB e STUI. Assim, foi proposto um modelo de lesão prostática com injeção intrauretral de LPS (1mg/ml), em camundongos machos Swiss e C57Bl/6, desenvolvido durante 3, 7, 10 e 14 dias. A análise dos resultados mostrou que a indução intrauretral com lipopolisacarideo-LPS atua como importante agente da HPB, além de promover o aumento de fatores de crescimento (FGF-7 e FGF- β), α-actina e citocinas pró-inflamatórias (IL-1, IL-6, IL-17), tanto no estroma como no epitélio. Uma vez que o Sildenafil tem potencial anti-inflamatório, este estudo se propôs a analisar a ação do Sildenafil em um modelo de lesão prostática experimental, induzido por injeção intrauretral de LPS em camundongos C57BL/6. O tratamento com Sildenafil (25mg/kg) dos animais com prostatite apresentaram redução significativa de -actina, COX-2, NFK- B, IL-6, IL-17 e FGF-7. Por não induzir danos na próstata , o Sildenafil, por não induzir a longo prazo danos evidentes na próstata pode representar uma estratégia farmacológica para o tratamento de doenças inflamatórias crônicas do trato urogenital. / Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5). This drug has been approved for therapeutic use in erectile dysfunction and currently has been also used to treat pulmonary hypertension. While maintaining an excellent level of safety and tolerability profile, few studies have evaluated the possible side effects of chronic treatment with Sildenafil on the male reproductive system, especially in prostate aimed at relaxing the urethra and relief of symptoms of Lower Urinary Tract (LUTS). Therefore, we assessed the treatment effect in C57Bl/6 mice, using morphological analysis, ultrastructural and molecular expression of soluble guanylate cyclase-sGC, endothelial nitric oxide synthase, eNOS, prostate-specific antigen PSA and transforming growth factor beta TGF- in prostate tissue. It is observed that the treatment with sildenafil does not induce apparent damage to the prostate. Furthermore, Sildenafil has been shown to have therapeutic efficacy in chronic inflammatory diseases, which have a potential therapeutic efficacy in various diseases. Among them, special attention has been given to the pathologies related to the male urogenital tract, such as Benign Prostatic Hyperplasia (BPH), prostate cancer and Prostatitis. Inflammation has been considered as a factor etiológio of BPH and LUTS. Thus, it has been proposed a prostatic intraurethral injection injury model of LPS (1mg/ml) in male Swiss mice and C57Bl /6 developed for 3, 7, 10 and 14 days. The results showed that the intraurethral induction with lipopolysaccharide-LPS acts as an important agent of HPB, and to promote the increase of growth factors (FGF-7 and FGF- ), -actin and proinflammatory cytokines (IL- 1, IL-6, IL-17), both in the stroma and the epithelium. Since Sildenafil has potential anti-inflammatory, this study aimed to analyze the action of Sildenafil in an experimental prostate injury model induced by intraurethral injection of LPS in C57Bl/6 mice. Treatment with sildenafil (25 mg/kg) of animals with prostatitis showed a significant reduction of -actin, COX-2 NFK-kB, IL-6, IL-17 and FGF-7. By not induce damage to the prostate, Sildenafil, not to induce long term damage evident in the prostate may represent a pharmacologic strategy for the treatment of chronic inflammatory diseases of the urogenital tract.

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<p>Oxygen (O2) plays a crucial role in the energy metabolism of complex multicellular life on earth. Due to the small and finite energy stores in the body, fine-tuned changes within the body are required to meet metabolic demand during skeletal muscle contractions, such as during exercise and activities of daily living. The skeletal muscle microcirculation is one of the last steps in the O2 transport pathway from the lungs to muscle cells and represents the largest surface area for O2 and substrate exchange. When skeletal muscle O2 uptake increases during contractions to meet metabolic demand, there must be an increase in muscle O2 delivery. To achieve these elevations in O2 delivery, vessel (arteriole) diameter in the microcirculation is increased, known as vasodilation. This process in the skeletal muscle microcirculation is regulated by several factors, such as neurohumoral, mechanical, endothelial, paracrine, and metabolic influences, which are imperative in properly regulating O2 delivery at rest and during muscular contractions. Two vasodilatory pathways of interest in this dissertation are the cyclooxygenase (COX) and nitric oxide (NO) vasodilatory pathways.</p> <p>The primary aim of my dissertation studies was to determine the mechanisms that modulate skeletal muscle oxygenation in health and to define the impact of a potentially effective intervention, whole-body chronic heat therapy (HT), to treat heart failure with preserved ejection fraction (HFpEF). In Chapter 2, we report that acute selective COX-2 inhibition had no effect on resting or exercising skeletal muscle microvascular oxygenation, pulmonary oxygen uptake, or exercise tolerance in healthy young humans. In Chapter 3, we report that NO, via phosphodiesterase type 5 inhibition, regulates myocyte O2 transport at rest and during recovery from muscle contractions in healthy young rats. In Chapter 4, we show that whole-body chronic HT promotes central and peripheral adaptations, which impact positively exercise tolerance in a pre-clinical rat model of HFpEF. Specifically, whole-body chronic HT had beneficial influences on exercise tolerance, skeletal muscle oxygenation from rest to contractions (driven, at least in part, by enhanced NO bioavailability), body composition, and cardiac function. Chapter 5 is a summary of the results and limitations of the projects presented in Chapters 2-4, with a brief discussion of potential future research directions. </p>

Les substances pharmaceutiques font partie du groupe des contaminants émergents du fait de leur intérêt récent dans les études environnementales comparativement à des polluants étudiés depuis plus longtemps tels que les pesticides. Elles correspondent aux principes actifs des médicaments et, à ce titre, sont responsables des propriétés pharmacologiques des médicaments. Ce sont donc des molécules biologiquement actives qui peuvent agir sur les organismes vivants présents dans les écosystèmes impactés. L’origine des substances pharmaceutiques dans l’environnement est variable mais les principales sources sont liées à leur utilisation en médecine humaine ou vétérinaire. Une fois consommées, les substances pharmaceutiques sont excrétées dans les urines ou les fèces et se retrouvent dans les eaux usées (consommation humaine) ou dans les déchets d’élevage (consommation vétérinaire). Dans le premier cas, elles peuvent être rejetées directement dans le milieu, ou indirectement, avec les eaux usées traitées ou les boues résiduaires, après traitement dans les stations d’épuration (STEP). Dans le deuxième cas, elles atteignent directement le milieu lorsque les animaux sont élevés en prairie ou indirectement lorsque les déchets d’élevage sont épandus sur les sols agricoles pour les fertiliser. Ces travaux de thèse se sont attachés à étudier l’origine et le devenir de ces substances dans ces 2 cas de figure. Ainsi en se basant sur des critères de consommation, de présence dans l’environnement par rapport à des études antérieures, de toxicité et d’écotoxicité, d’originalité et de disponibilité des composés standards de référence, 32 puis 78 molécules appartenant aux classes thérapeutiques des antibiotiques, des anticancéreux, des béta-bloquants, des anti-VIH et des inhibiteurs de phosphodiestérase de type 5 (PDE 5) ont été étudiées dans 2 continuums : i) effluents hospitaliers - eaux usées brutes et traitées – eaux de surface, et ii) eaux usées brutes et traitées - eaux de surface - eaux de captage souterraines. En s’appuyant sur les mêmes critères de sélection, le devenir de 7 antibiotiques a été étudié dans des lisiers porcins dans des filières simples de traitement du lisier (fosse de stockage), dans des filières complexes de traitement du lisier (système de traitement ressemblant à des mini STEP) et dans des mésocosmes en conditions contrôlées. Pour pouvoir réaliser l’ensemble de ces études, des protocoles analytiques mettant en œuvre une étape d’extraction par SPE (Solide Phase Extraction) ou d’extraction ASE (Extraction Accélérée par Solvant) puis de purification par SPE et d’analyse par LC/MS/MS (Chromatographie en phase liquide couplée à la spectrométrie de masse en tandem) ont été développés. Ces protocoles, en remplissant des critères de qualité tels que des limites de détection et de quantification compatibles avec des analyses environnementales (de l’ordre du ng/l à la dizaine de ng/l), une bonne linéarité, précision, justesse et performance, ont permis d’analyser la phase dissoute des échantillons d’eaux et la phase dissoute et solide des échantillons de lisiers. Il ressort des analyses des échantillons aqueux que : i) les béta-bloquants, les anti-VIH et les antibiotiques appartenant aux familles des macrolides, des fluoroquinolones et des sulfonamides, sont les molécules les plus représentatifs de la contamination du milieu naturel parmi les classes étudiées ; ii) les rejets de STEP sont une source majeure de la contamination des systèmes aquatiques ; iii) les eaux usées sont davantage contaminées en hiver qu’en été ; et iv) les eaux de surface sont davantage contaminées en été qu’en hiver. / Pharmaceutical substances belong to the group of emerging contaminants due to their recent interest in environmental studies in comparison with pollutants who have been studied for a longer time like pesticides. They correspond to the active ingredient of drugs and by this mean are responsible for their pharmacological properties. Consequently they are biologically active molecules that can act on living organisms present in impacted ecosystems. The origin of pharmaceuticals in the environment is variable but the main sources are related to their use in human and veterinary medicine. Once consumed, pharmaceutical substances are excreted in urine or feces and are found in wastewater (human consumption) or animal manure (veterinary consumption). In the first case, they can be discharged directly in the environment, or indirectly, with treated wastewater or sludge from sewage treatment plants (SWTP). In the second case, they directly reach the environment when animals are bred on grassland or indirectly when livestock wastes are spread on agricultural soils as fertilizer. This PhD work has been focused on the study of the origin and fate of pharmaceutical substances in these 2 cases. Thus according to consumption data, occurrence in the environment reported in previous studies, toxicity and ecotoxicity data, originality and availability of reference standard compounds, 32 then 78 molecules belonging to 5 different therapeutic classes (antibiotics, antineoplastics, beta-blockers, anti-HIV, phosphodiesterase type 5 inhibitors (PDE 5 inhibitors)) were studied in 2 continuums : i) hospital wastewater effluents – raw and treated wastewater – surface water, and ii) raw and treated wastewater – surface water – ground water. Based on the same selection criteria, the fate of 7 antibiotics was studied in pig manure in simple manure storage facilities (storage tank), in aerobic manure treatment facilities (treatment system like in small SWTP) and in mesocosms under controlled conditions. In order to achieve all these studies, analytical protocols implementing an extraction step by SPE (Solid Phase Extraction) or an ASE extraction (Accelerated Solvent Extraction) followed by a SPE purification and an analytical step by LC / MS / MS (liquid chromatography tandem mass spectrometry) have been developed. These protocols, by filling out quality criteria such as limits of detection and quantification compatible with environmental analysis (ng/l to dozen of ng/l), good linearity, precision, accuracy and performance, were used to analyze the dissolved phase of water samples and dissolved and solid phases of pig manure samples. The water samples analysis shows : i) beta-blockers, anti-HIV and antibiotic belonging to the families of macrolides, fluoroquinolones and sulfonamides are the most representative molecules of the environmental contamination from the classes studied; ii) SWTP releases are a major source of aquatic systems’ contamination; iii) wastewaters are more contaminated in winter than in summer; and iv) surface water are more contaminated in summer than in winter. The pig manure samples analysis shows : i) the levels of contamination of manure by antibiotics are high, from a few µg/l to mg/l; ii) the manure level of contamination is not related to the physiological stage of pigs; iii) the interest to store manure before spreading in order to reduce the antibiotics contamination is not highlighted; iv) oxytetracycline, tetracycline, tylosin and marbofloxacin are mainly present in the solid phase whereas sulfadiazine, lincomycin and monensin are mainly present in the liquid phase of manure; v) the separation of solid and liquid phases reduce manure contamination in aerobic treatment facilities; and vi) antibiotics degradation is mainly aerobic.Key words: ,