L’adénocarcinome pancréatique canalaire (PDAC) est un cancer de mauvais pronostic en partie en raison de la résistance aux traitements conventionnels de chimiothérapie ou de radiothérapie. Certaines substances bioactives issues de l’alimentation (BFCs), prooxydantes sont capables de potentialiser l’action cytotoxique des drogues conventionnelles dans le PDAC. Par ailleurs ces BFCs pourraient augmenter les cassures de l’ADN induites par les radiations ionisantes. Objectifs : évaluer l’action combinée de BFCs en association avec la chimiothérapie et la radiothérapie dans des modèles de PDAC et identifier les mécanismes impliqués dans la chimio/radiosensibilisation. Méthodes : La survie cellulaire a été évaluée in vitro en présence de BFCs en combinaison sur 4 lignées tumorales pancréatiques. La production d’espèces réactives de l'oxygène (ROS) a été mesurée par cytométrie en flux et par microscopie à fluorescence. Les mécanismes moléculaires ont été décryptés par western-blot (voies de signalisation) et cytométrie en flux (cycle cellulaire et apoptose). Enfin nous avons évalué l’association de BFCs en gavage avec la chimiothérapie par gemcitabine intrapéritonéale et avec la radiothérapie de 2 Gy par fraction in vivo, sur des greffes sous-cutanée de cellules CAPAN-2. Résultats : In vitro, le resvératrol (R), la capsaïcine (C) et le sulforaphane (S) étaient cytotoxiques avec effet inhibiteur significativement plus élevé en combinaison (R+C), (C+S) ou (R+C+S), sans effet sur les fibroblastes. Par ailleurs, les combinaisons potentialisaient l’action autrement limitée de la gemcitabine sur les cellules testées in vitro. In vivo, l’addition de R+C au traitement par gemcitabine à dose réduite a permis une régression tumorale équivalente à celle obtenue avec la gemcitabine à pleine dose. Par ailleurs, in vitro, l’ajout des BFCs seuls et combinés à la radiothérapie augmente de façon significative la toxicité cellulaire sur la lignée épithéliale CAPAN-2, comparé au traitement par radiothérapie seule comme aux traitements par BFCs seuls ou combinés. In vivo, la combinaison R+C associée à la radiothérapie a permis une diminution significative des volumes tumoraux comparée à la radiothérapie seule. L’étude des voies de signalisation a montré une augmentation de protéines pro-apoptotiques avec l’association R+C, en rapport avec une induction augmentée de ROS, mais aussi, de façon surprenante, une inhibition de la réparation de l’ADN par inhibition de la phosphorylation d’ATM. Ces deux effets combinés précipitaient la mort des cellules tumorales. Conclusions : Les combinaisons de R+C ont un effet chimiosensibilisant et radiosensibilisant dans un modèle préclinique de PDAC, avec des mécanismes moléculaires identifiés pertinents dans le contexte des thérapies testées. En associant les BFCs, à la chimioradiothérapie avec gemcitabine, on peut espérer une double potentialisation de la radiothérapie et de la chimiothérapie, en augmentant l’efficacité de la RT, et en diminuant la dose de gemcitabine associée pour une même efficacité et une meilleure tolérance du traitement.Mots clés : Cancer du pancréas, Substances bioactives issues de l’alimentation, radiothérapie, chimiothérapie, resvératrol, capsaïcine / Background: Pancreatic ductal adenocarcinoma (PDAC) is of poor prognosis in part because of resistance to conventional treatments such as chemotherapy or radiotherapy. Some naturally occurring bioactive food components (BFCs), with pro-oxidant properties are able to potentiate the cytotoxic action of conventional drugs in the PDAC. In addition, these BFCs could increase the DNA breaks induced by ionizing radiation. Objectives: To evaluate the combined action of several BFCs in combination with chemotherapy and radiotherapy in PDAC models and to identify mechanisms involved in chemo / radiosensitization at the cellular and molecular levels. Methods: Cell survival was evaluated in vitro in the presence of BFCs in combination on 4 pancreatic tumor cell lines. The production of reactive oxygen species (ROS) was measured by flow cytometry and fluorescence microscopy. Molecular mechanisms have been decrypted by western blot (signaling pathways) and flow cytometry (cell cycle and apoptosis). Finally, we evaluated the association of BFCs in gavage with intraperitoneal gemcitabine chemotherapy and with radiotherapy (2 Gy/fraction) in vivo, on subcutaneous CAPAN-2 cell xenografts. Results: In vitro, resveratrol (R), capsaicin (C) and sulforaphane (S) were cytotoxic with significantly higher inhibitory effect in combination (R + C), (C + S) or (R + C + S), without effect on fibroblasts. In addition, the combinations potentiated the otherwise limited action of gemcitabine on cells tested in vitro. In vivo, the addition of R + C to treatment with gemcitabine at reduced dose allowed tumor inhibition equivalent to that obtained with gemcitabine at full dose. Furthermore, in vitro, the addition of BFCs alone and in combination with radiotherapy significantly increases cellular toxicity on the CAPAN-2 epithelial line, compared to radiotherapy alone and BFCs alone or combined. In vivo, the combination R + C associated with radiotherapy allowed a significant decrease in tumor volumes compared to radiotherapy alone. The study of signaling pathways showed an increase of proapoptotic proteins with the association R + C and radiotherapy, in relation with an increased induction of ROS, but also, surprisingly, an inhibition of the repair of the DNA by inhibition of ATM phosphorylation. These two combined effects precipitated the death of the tumor cells. Conclusions: Combinations of R + C have a chemosensitizing and radiosensitizing effect in a preclinical model of PDAC, with identified molecular mechanisms relevant in the context of the therapies tested. By combining BFCs with radiochemotherapy with gemcitabine, we can hope for a double potentiation of radiotherapy and chemotherapy, by increasing the effectiveness of RT, and by reducing the dose of gemcitabine associated for similar efficiency and better tolerance treatment.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Recently, oxidative stress was indicated as modulator of nociceptive transmission at spinal cord, and that nitric oxide (NO) may activate the TRPV1 in vitro. As no protein thiol compounds (SH) are the main endogenous antioxidants, we decided to investigate the relation between endogenous and exogenous SH, as well as the NO-mediated TRPV1 activation at spinal cord in mice. We observed that the systemic (i.p.), intrathecal (i.t.), but not local (I.pl.) N-acetylcysteine (NAC) administration reduced the nociception, the decrease in spinal SH, raise of thiobarbituric reactive species (TBARS) and 3-nitrotyrosine (3-NT) levels induced by intraplantar capsaicin (CAP). Similarly, i.t. or i.p. NAC administration reduced the nociception (mechanical allodynia) and decrease in spinal SH induced by complete Freund adjuvant (CFA)-induced chronic inflammation. Reinforcing these results, we observed that buthionine-sulfoxamine (BSO), an inhibitor of glutathione synthesis, the main endogenous SH compound, induced a decrease in spinal SH levels, chemical and mechanical allodynia, thermal and mechanical hyperalgesia. To investigate the participation of NO in these processes, we induced spinal nociception (thermal hyperalgesia) by i.t. L-arginine (ARG substrate for endogenous NO synthesis) administration and intraplantar CAP administration. In both cases, the i.t. pre-treatment with NAC or L-NAME (NO synthesis inhibitor) prevented this nociception, as well as the decrease in spinal SH and the raise in nitrite/nitrate (NOx) levels, a stable metabolites of NO. These changes were also prevented by the pharmacological blockade of TRPV1 with the antagonist SB366791, the spinal defunctionalization (induced by i.t. high dose of CAP) and genetic knockdown (induced by repeated oligonucleotide antisense i.t. administration). Thus, we may conclude that SH compounds participate in the spinal nociceptive transmission by neutralizing NO, preventing spinal TRPV1 activation. Therefore, antioxidants as NAC may present antinociceptive effect by this process. / Recentemente, o estresse oxidativo foi indicado como possível modulador da neurotransmissão dolorosa, havendo também a constatação de que o óxido nítrico (NO) pode ativar, in vitro, o receptor de potencial transitório vaniloide 1 (TRPV1). Como os compostos tiólicos não proteicos (SH) são antioxidantes endógenos importantes, decidimos investigar a relação entre os tióis endógenos, exógenos e a possível ativação do receptor TRPV1 mediada pelo NO, na medula espinhal de camundongos. Observamos que a administração sistêmica (i.p.), intratecal (i.t.), mas não intraplantar (i.pl.) de N-acetilcisteína (NAC) reduz a nocicepção, ocasionando a diminuição dos níveis espinhais de tióis não proteicos (SH), e o aumento de radicais livres ao ácido tiobarbitúrico (TBARS) e 3-nitrotirosina (3-NT) induzido por capsaicina (CAP) intraplantar. Similarmente, a administração i.p. ou i.t. de NAC reverte tanto a nocicepção (alodínia mecânica) quanto à diminuição nos níveis espinhais de SH induzida pela administração intraplantar de adjuvante completo de Freund (CFA). Comprovando esses dados, observamos que a butionina-sulfoxamina (BSO), inibidor da síntese do principal composto tiólico endógeno (glutationa), induziu a diminuição nos níveis de SH espinhal. Além disso, o BSO também induziu a alodínia química e mecânica, e a hiperalgesia térmica e mecânica. Para investigar o papel do NO nessas alterações, induzimos a nocicepção (hiperalgesia térmica) pela administração intraplantar de CAP ou intratecal de L-arginina (ARG), substrato da enzima óxido nítrico sintase (NOS). Em ambos os casos, o pré-tratamento i.t. com NAC ou L-NAME (inibidor da NOS) inibiu essa nocicepção, bem como a diminuição nos níveis de SH e a elevação nos níveis dos metabólitos estáveis do NO nitrito/nitrato (NOx). Essas respostas também foram prevenidas pela inibição farmacológica (antagonista SB366791) e pela desfuncionalização (administração intratecal prévia de capsaicina) ou ablação gênica (administração repetida de oligonucleotídeo antissentido) do receptor TRPV1. Dessa forma, podemos concluir que o conteúdo tiólico não proteico participa da transmissão dolorosa neutralizando o NO formado e impedindo a ativação do TRPV1 espinhal. Sendo assim, antioxidantes como a NAC podem exercer seu efeito antinociceptivo por impedir esse processo de ativação.

Made available in DSpace on 2016-01-26T18:55:43Z (GMT). No. of bitstreams: 1 Marcia Miranda.pdf: 658506 bytes, checksum: 8d66993b7a46777717dad48d941ce4ef (MD5) Previous issue date: 2015-06-22 / Obesity is characterized by excessive accumulation of adipose tissue, of multifactorial origin. In pursuit of weight reduction, individuals use functional foods, for example the Capsiate, CH-19 extract sweet (Capsicum annuum L.), which contains capsinoids, it is a capsaicin analog, not pungent, with thermogenic properties. The capsiate exhibits potent activity against angiogenesis and vascular permeability induced by vascular endothelial growth factor. The study was conducted face lack studies on capsianóides in reproductive organs in the literature. The aim: was to evaluate the effect of capsiate on the histology of the uterus, ovary, adrenal gland and lipid profile of obese and non-obese rats. Material and methods: Sixty-four male Wistar rats were divided into four groups (n = 16): Control; Obese; Capsiate and Capsiate Obeso. Groups Control and Capsiate received commercial feed. Group Obese and Capsiate Obese received palatable high fat die. For groups Capsiate and Capsiate Obese it was administered by gavage 10 mg capsiate diluted in 0.5 ml of filtered water, 1 once day, for groups Control and Obese it was administered by gavage 0,5 ml of filtered water (placebo). After 12 weeks, the animals anesthetized, blood collected and euthanized. Reproductive organs and the adrenal glands they were stained with HE technique and submitted to morphological analysis and morphometric. Statistical analysis, it was verified by the Levene test, was employed (one-way ANOVA), contrasts with the Tukey method. Performed using SPSS 16.0 software and 5% significance level. Results: significant elevation in HDL Col in the groups Capsiate and Capsiate Obese. The thickness of the endometrium was lower (p <0.05) in Capsiate and was observed increase (p <0.05) in the number of secondary follicles and corpus luteum in group Capsiate Obese. The glomerular regions, crosslinked and medullary they had a lower thickness (p <0.05) in Capsiate group compared to the control. Conclusion: The capsiate reduces endometrial proliferation no obese rats, increases the number of corpus luteum in obese rats and induces an increase in HDL-cholesterol, but not prevented increase in body weight of rats. However, further studies are needed to evaluate the effect of capsiate in reproductive organs because of the shortage in the scientific literature. / Obesidade é caracterizada pelo acúmulo excessivo de tecido adiposo, de origem multifatorial. Em busca da redução de peso, indivíduos utilizam alimentos funcionais, como exemplo o Capsiate, CH-19 extrato doce (Capsicum annuum L.), que contém capsinóides, é um análogo de Capsaicina, não pungente, com propriedades termogênicas. O capsiate exibe potente atividade contra a angiogênese e permeabilidade vascular induzida pelo fator de crescimento endotelial vascular. O estudo foi desenvolvido face ausência de estudos sobre capsianóides em órgãos reprodutivos na literatura. O objetivo foi avaliar o efeito do capsiate na histologia do útero, ovário, glândula adrenal e perfil lipídico de ratas obesas e não obesas. Material e Métodos: Sessenta e quatro ratas Wistar divididas em quatro grupos (n=16): Controle; Obeso; Capsiate e Capsiate Obeso. Os Grupos Controle e Capsiate receberam ração comercial. Já Grupo Obeso e Capsiate Obeso receberam dieta palatável hiperlipídica. Para os grupos Capsiate e Capsiate Obeso foi administrado por gavagem 10 mg de capsiate diluído em 0,5 ml de água filtrada, 1 vez dia, para os grupos Controle e Obeso foi administrado por gavagem 0,5 ml de água filtrada (placebo). Após 12 semanas, os animais anestesiados, sangue coletado e eutanasiados. Órgãos reprodutivos e as glândulas adrenais foram corados pela técnica de HE e submetidos à análise morfológica e morfométrica. Análise estatística foi verificada pelo teste de Levene, empregou-se (ANOVA one-way), com contrastes pelo método de Tukey. Realizadas com auxílio do Software SPSS 16.0 e com nível de significância de 5%. Resultados: significativa elevação nos níveis de HDL Col nos grupos Capsiate e Capsiate Obeso. A espessura do endométrio foi menor (p<0,05) no grupo Capsiate e foi observado aumento (p<0,05) no número de folículo secundário e corpos lúteos no grupo Capsiate Obeso. As regiões glomerular, reticulada e medular apresentaram espessura menor (p<0,05) no grupo Capsiate comparado ao Controle. Conclusão: O capsiate reduz proliferação endometrial em ratas não obesas, aumenta o número de corpos lúteos em ratas obesas e induz o aumento do HDL-colesterol, porém não evitou aumento no peso corporal das ratas. Porém, novos estudos são necessários para avaliar o efeito do capsiate em órgãos reprodutivos, devido à escassez na literatura científica.

Made available in DSpace on 2016-07-18T17:53:15Z (GMT). No. of bitstreams: 1 Marcia Miranda.pdf: 658506 bytes, checksum: 8d66993b7a46777717dad48d941ce4ef (MD5) Previous issue date: 2015-06-22 / Obesity is characterized by excessive accumulation of adipose tissue, of multifactorial origin. In pursuit of weight reduction, individuals use functional foods, for example the Capsiate, CH-19 extract sweet (Capsicum annuum L.), which contains capsinoids, it is a capsaicin analog, not pungent, with thermogenic properties. The capsiate exhibits potent activity against angiogenesis and vascular permeability induced by vascular endothelial growth factor. The study was conducted face lack studies on capsianóides in reproductive organs in the literature. The aim: was to evaluate the effect of capsiate on the histology of the uterus, ovary, adrenal gland and lipid profile of obese and non-obese rats. Material and methods: Sixty-four male Wistar rats were divided into four groups (n = 16): Control; Obese; Capsiate and Capsiate Obeso. Groups Control and Capsiate received commercial feed. Group Obese and Capsiate Obese received palatable high fat die. For groups Capsiate and Capsiate Obese it was administered by gavage 10 mg capsiate diluted in 0.5 ml of filtered water, 1 once day, for groups Control and Obese it was administered by gavage 0,5 ml of filtered water (placebo). After 12 weeks, the animals anesthetized, blood collected and euthanized. Reproductive organs and the adrenal glands they were stained with HE technique and submitted to morphological analysis and morphometric. Statistical analysis, it was verified by the Levene test, was employed (one-way ANOVA), contrasts with the Tukey method. Performed using SPSS 16.0 software and 5% significance level. Results: significant elevation in HDL Col in the groups Capsiate and Capsiate Obese. The thickness of the endometrium was lower (p <0.05) in Capsiate and was observed increase (p <0.05) in the number of secondary follicles and corpus luteum in group Capsiate Obese. The glomerular regions, crosslinked and medullary they had a lower thickness (p <0.05) in Capsiate group compared to the control. Conclusion: The capsiate reduces endometrial proliferation no obese rats, increases the number of corpus luteum in obese rats and induces an increase in HDL-cholesterol, but not prevented increase in body weight of rats. However, further studies are needed to evaluate the effect of capsiate in reproductive organs because of the shortage in the scientific literature. / Obesidade é caracterizada pelo acúmulo excessivo de tecido adiposo, de origem multifatorial. Em busca da redução de peso, indivíduos utilizam alimentos funcionais, como exemplo o Capsiate, CH-19 extrato doce (Capsicum annuum L.), que contém capsinóides, é um análogo de Capsaicina, não pungente, com propriedades termogênicas. O capsiate exibe potente atividade contra a angiogênese e permeabilidade vascular induzida pelo fator de crescimento endotelial vascular. O estudo foi desenvolvido face ausência de estudos sobre capsianóides em órgãos reprodutivos na literatura. O objetivo foi avaliar o efeito do capsiate na histologia do útero, ovário, glândula adrenal e perfil lipídico de ratas obesas e não obesas. Material e Métodos: Sessenta e quatro ratas Wistar divididas em quatro grupos (n=16): Controle; Obeso; Capsiate e Capsiate Obeso. Os Grupos Controle e Capsiate receberam ração comercial. Já Grupo Obeso e Capsiate Obeso receberam dieta palatável hiperlipídica. Para os grupos Capsiate e Capsiate Obeso foi administrado por gavagem 10 mg de capsiate diluído em 0,5 ml de água filtrada, 1 vez dia, para os grupos Controle e Obeso foi administrado por gavagem 0,5 ml de água filtrada (placebo). Após 12 semanas, os animais anestesiados, sangue coletado e eutanasiados. Órgãos reprodutivos e as glândulas adrenais foram corados pela técnica de HE e submetidos à análise morfológica e morfométrica. Análise estatística foi verificada pelo teste de Levene, empregou-se (ANOVA one-way), com contrastes pelo método de Tukey. Realizadas com auxílio do Software SPSS 16.0 e com nível de significância de 5%. Resultados: significativa elevação nos níveis de HDL Col nos grupos Capsiate e Capsiate Obeso. A espessura do endométrio foi menor (p<0,05) no grupo Capsiate e foi observado aumento (p<0,05) no número de folículo secundário e corpos lúteos no grupo Capsiate Obeso. As regiões glomerular, reticulada e medular apresentaram espessura menor (p<0,05) no grupo Capsiate comparado ao Controle. Conclusão: O capsiate reduz proliferação endometrial em ratas não obesas, aumenta o número de corpos lúteos em ratas obesas e induz o aumento do HDL-colesterol, porém não evitou aumento no peso corporal das ratas. Porém, novos estudos são necessários para avaliar o efeito do capsiate em órgãos reprodutivos, devido à escassez na literatura científica.

Modulation of a synaptic transmission in the spinal cord dorsal horn plays a key role in nociceptive signalling, especially in states of pathological pain. The goal of this study was to develop a method for calcium imaging in spinal cord slices in vitro. This method allowed us to record changes of intracellular free calcium ions concentration (iCa2+ ), that are a major mediator of neuronal plasticity. In this work, we have focused on application of this method in a conventional fluorescence microscope and on the role of different neuromodulators of synaptic activity. Changes of iCa2+ induced by dorsal root electrical stimulation were recorded altogether in 744 dorsal horn (lamina I and II) neurons. In the first series of experiments, stimulation protocols activating preferentially A and A + C dorsal root fibers were used and long-term stability of the calcium responses was verified. The dorsal root stimulation induced in the neurons fast and delayed type of calcium response. Application of AMPA and NMDA receptors antagonists, CNQX (50μM) and MK801 (45μM), reduced the calcium response amplitude and confirmed the importance of glutamate receptors in synaptic activation. In several experiments the effect of capsaicin a TRPV1 receptors agonist, application was tested. Application of even low...

Capsaicin, prostaglandin E2 (PGE2) and polydopamine (PDA) were used to target macrophage and myofibroblast activity to reduce biomaterial-induced fibrosis. The lifetime and efficacy of implantable biomedical devices are determined by the foreign body response. Immediately after implantation, proteins nonspecifically adsorb onto the material and initiate inflammation. Macrophages recruited to the site can differentiate into M1 and M2 phenotypes and upregulate inflammation and fibrosis which interferes with the intended function. M1 macrophages secrete pro-inflammatory mediators that induce chronic inflammation and promote myofibroblast differentiation while M2 macrophages are wound healing cells that suppress inflammation and regulate fibroblast activity. The fibrotic tissue is developed by myofibroblasts which produce collagen in an unregulated fashion. Collagen thickening and biomaterial encapsulation decreases efficacy and sensitive of biomedical devices. We investigated the in vitro and in vivo effects of capsaicin, PGE2 and polydopamine surface modification on macrophages and myofibroblasts. Capsaicin and PGE2 reduced poly(lactic-co-glycolic) acid (PLGA)-induced fibrosis by promoting M2 macrophage phenotype to secrete anti-inflammatory IL-10 and suppressing myofibroblast marker α-smooth muscle actin (α-SMA). Capsaicin decreased collagen by 40% and upregulated IL-10 secretion by 35% while PGE2 reduced collagen by 55% after 14 days of implantation and 40% less collagen after 42 days. PDA was used to bind an anti-fibrotic compound to the surface of a poly(dimethyl siloxane) (PDMS-PDA) to reduce fibrosis. However, PDMS-PDA controls gave an unexpected result by reducing fibrosis to the same extent as anti-fibrotic compound bound PDMS- v PDA. PDA modification reduced cellularity by 50% and significantly decreased collagen thickness by 30%. Overall, our results showed that biomaterial-induced fibrosis can be reduced by promoting M2 macrophage activity and inhibiting myofibroblast differentiation. This research demonstrates three compounds that have potential to reduce fibrosis and extend the lifetime and efficacy of implantable biomedical devices. / Thesis / Master of Applied Science (MASc) / Capsaicin, prostaglandin E2 (PGE2) and polydopamine were used to reduce scar tissue development around implanted polymers. Biomedical devices implanted in the body can undergo severe scar tissue formation, or fibrosis, and fail. Fibrosis is described by the accumulation of collagen and encapsulation of an implanted polymer. Macrophages regulate fibrosis by secreting pro-fibrotic compounds and myofibroblasts produce unregulated amounts of collagen. In this thesis, capsaicin, PGE2 and polydopamine were incorporated into implants to target macrophage and myofibroblast activity and reduce fibrosis in mice. Capsaicin and PGE2, released from a degradable polymer, altered macrophages to secrete anti-fibrotic compounds and decreased collagen by 40% and 55%, respectively. Polydopamine surface modified implants gave an unexpected result and suppressed overall cell activity to reduce fibrosis by 30%. The research conducted shows the potential of these compounds to reduce fibrosis and extend the lifetime of implantable devices.

No description available.

This thesis examined how and why TRPV1 function is being modulated in sensory neuropathy and explored the potential of its rescue in the urinary bladder of STZ-­‐induced diabetic rats. Diabetes induced a rapid decline in TRPV1 function and changes in neurogenically mediated electrically-­‐evoked responses together with a gradual decline in muscarinic function. Diabetic bladder was also deficient in muscarinic and TRPV1 organ bath temperature-­‐induced changes but not in those affecting spontaneous contractile activity. Exposure to a potential neuropathy causative agent, methylglyoxal was studied and its mechanism of action explored through the use of TRPA1 ligands. Methylglyoxal exposure mimicked some of the effects of diabetes on TRPV1, neurogenic electrically evoked responses and muscarinic function. Methylglyoxal effects were seen to be partly through TRPA1 receptor activation but other as yet undefined pathways were also involved. Use of TRPA1 ligands revealed an unexpected complexity of the interaction of the TRPA1 receptor with TRPV1. Finally the potential of reversing the diminished TRPV1 response was examined through the use of three known sensitising agents, bradykinin, NGF and insulin. Bradykinin was the only agent seen to reverse the TRPV1 diminished response back up to to control equivalent levels and through the use of bradykinin selective ligands, it was seen that the dual activation of BK-­‐1 and BK-­‐2 receptor was necessary to rescue the TRPV1 response. The likely mechanism of action of bradykinin was through prostaglandin production as indomethacin blocked TRPV1 rescue. In the acute stage of diabetes, TRPV1 function is downregulated and may be caused by exposure to a neuropathy-­‐causing metabolite such as methylglyoxal. The TRPV1 function still retains plasticity at this acute stage because function could be enhanced back to control levels by bradykinin receptor activation : a potential for early therapeutic intervention.

Les kinines sont des peptides neuro- et vaso- actifs impliqués dans les processus hémodynamiques, inflammatoires et douloureux. Leurs effets biologiques sont produits par l’entremise de deux types de récepteurs couplés aux protéines G, soit B1 (B1R) et B2 (B2R). Le B1R est inductible, son expression est augmentée à la suite d’un dommage tissulaire ou de l’exposition à des endotoxines bactériennes (lipopolysaccharide bactérien (LPS)), à des cytokines pro-inflammatoires (interleukine-1β (IL-1β), facteur de nécrose tumorale-α (TNF-α)) ou à des espèces réactives oxygénées (ROS). Les travaux présentés dans cette thèse avaient pour objectif d’élucider et/ou de raffiner les connaissances sur 1) la localisation, 2) le mécanisme d’induction et 3) le rôle physiopathologique du B1R dans des modèles expérimentaux de douleur chez le rat. Nos données ont permis de démontrer pour la première fois que le B1R est augmenté de façon significative dans la moelle épinière du rat diabétique de type 1 où il est localisé sur les fibres sensorielles de type C, les astrocytes et les cellules de la microglie (1er article). Également, l’inhibition de l’activation des cellules de la microglie supprime les neuropathies diabétiques, l’expression de médiateurs pro-inflammatoires ainsi que l’activité pro-nociceptive du B1R (2e et 3e articles). Finalement, nous avons démontré que la stimulation systémique du TRPV1 par la capsaïcine induit une surexpression du B1R au niveau microgliale, via un mécanisme impliquant l’augmentation de la production de ROS et possiblement de cytokines (4e article). Ces données nous permettent de mieux comprendre les mécanismes impliqués dans l’expression et l’activité du B1R. Aussi, elles nous permettent d’imaginer de nouvelles stratégies pour prévenir l’induction du B1R (inhibition du TRPV1) ou son activité délétère (inhibition de l’activation des cellules de la microglie) dans la douleur inflammatoire et neuropathique. / Kinins are vaso- and neuro-active peptides involved in hemodynamic, inflammatory and pain processes. Their biological effects are mediated by two G Protein Coupled Receptors (GPCR), termed B2R (constitutive) and B1R (inducible). B1R is expressed following tissue damage or exposure to bacterial endotoxin (LPS), pro-inflammatory cytokines (IL-1β, TNF-α) and increased reactive oxygen species (ROS) levels. The objectives of this doctoral thesis were to define 1) the localisation, 2) the mechanism of induction and 3) the pathophysiological role of B1R in experimental models of pain in rat. Our data showed that B1R is significantly upregulated on sensory C fibers, astrocytes and microglia in spinal cord of type 1 diabetic rat (paper #1). Moreover, pharmacological inhibition of microglia reversed diabetic pain neuropathy, reduced levels of pro-inflammatory mediators and prevented B1R pro-nociceptive activity (papers #2 and 3). Finally, our data showed that systemic stimulation of TRPV1 with capsaicin upregulated B1R expression, mainly on microglia, through the increase of ROS and possibly cytokines (paper #4). Altogether, these data increased our knowledge related to B1R mechanism of induction and B1R activity. Also, these data shed light on new strategies to prevent B1R expression (TRPV1 blockade) and B1R deleterious activity (inhibition of microglia activation) in inflammatory and neuropathic pain.

Neste estudo efetuou-se uma análise comparativa da resposta inflamatória e estresse oxidativo produzido pela administração intratraqueal (i.tr.) das PED e/ou 1,2-NQ em vias aéreas de ratos. O efeito destes poluentes sobre a funcionabilidade dos macrófagos foi também avaliado. A injeção i.tr. das PED, numa dose incapaz de causar edema, promoveu efeito aditivo no edema e influxo de leucócitos induzido pela 1,2-NQ nas vias aéreas. O edema foi inibido por antagonistas de taquicininas ou capsaicina, mas o influxo de leucócitos não. O tratamento com estes poluentes aumentou a expressão gênica dos receptores TRPV1, taquicininas e TNFR1, as quais foram reduzidas, mas a iNOS aumentada, em animais depletados de neuropeptídeos. Concentrações elevadas de citocinas pró-inflamatórias foram encontradas no brônquio de ratos saudáveis e, mais intensamente, naqueles tratados com capsaicina. Níveis basais de 3-NT, IL-10 ou IFN-?? no brônquio não foram alterados pelos poluentes. A fagocitose de partículas de zimosan foi aumentada após a exposição de animais saudáveis e depletados de neuropeptídeos aos poluentes. A inflamação nas vias aéreas de ratos expostos ao MP do diesel é influenciada por concentrações de 1,2-NQ no ambiente, via mecanismo neurogênico. / Pharmacological approaches on the healthy side effects evoked by the interaction between environmental pollutants are poorly studied. Here we tested the hypothesis that the environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), and that involves a neurogenic-mediated mechanism. Intra-tracheal (i.tr.) injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in rat airways. DEP (at a dose unable to produce oedema) increased the 1,2-NQ-induced responses in the rat airways in a additive manner. This effect was reduced by L-732,138, an NK1 receptor antagonist, and in a lesser extent by the NK2 receptor SR48968. Capsaicin treatment also markedly reduced pollutants-induced oedema. Exposure to pollutants increased the TRPV1, NK1 and NK2 receptors gene expression in bronchus, an effect nearly abolished by capsaicin treatment. No evidence of increased 3-NT in main bronchus was found. Our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ, and takes place by neurogenic-mediated mechanisms involving up-regulation of TRPV1 and tachykinin receptors.