Les microglies, les cellules de l’immunité innée, résidentes du cerveau, sont impliquées dans la réponse inflammatoire cérébrale et le modelage des réseaux neuronaux au cours du développement. La perturbation de leurs activités par des stimuli environnementaux pouvant conduire à des altérations psychopathologiques, dans cette thèse, nous avons étudié le rôle des microglies dans les effets neurobiologiques et comportementaux d’un stimulus inflammatoire. Les travaux précédents ont révélé que l’administration de lipopolysaccharide (LPS), une endotoxine bactérienne, provoque des comportements de type dépressifs. Le rôle des microglies dans ces altérations a fait l’objet de la première étude de cette thèse (Chapitre 2). Afin de dépléter les microglies du cerveau, des souris adultes ont reçu par voie d’administration intra-hippocampique des liposomes contenant du clodronate, provoquant ainsi l’apoptose des microglies phagocytaires. L’administration de LPS active dans l’hippocampe la synthèse de cytokines pro-inflammatoires [interleukine (IL)-1b et facteur de nécrose tumorale (TNF)-a] et anti-inflammatoires (IL-10), et de l'indoleamine 2,3-dioxygénase, une enzyme impliquée dans le métabolisme du tryptophane aux activités pro-dépressives. La déplétion des microglies phagocytaires atténue les effets du LPS, à l’exception de l’IL-1b, dont l’expression est exacerbée. De plus, l’administration de clodronate prévient les effets du LPS sur les comportements de type dépressif. Dans leur ensemble, ces résultats ont révélé que les microglies phagocytaires sont impliquées dans les effets inflammatoires et comportementaux de type dépressif induits par le LPS. Nous avons ensuite étudié le rôle des microglies dans les effets comportementaux d’une inflammation maternelle précoce provoquée lors de la colonisation du cerveau fœtal par les microglies (Chapitres 3 & 4). Ainsi, nous avons administré au jour gestationnel (JG)9.5 du LPS à des souris gestantes et évalué la trajectoire développementale pré- et post-natale des microglies et du comportement de la progéniture (Chapitre 3). L’administration de LPS à JG9.5 provoque une réduction du pourcentage représenté par les microglies matures aux JG14.5 et 18.5 et des déficits comportementaux persistants à l’âge adulte avec un dimorphisme sexuel prononcé. Nous avons alors recherché à identifier les mécanismes moléculaires impliqués dans les effets du LPS administré à JG9.5, en étudiant la piste de l’action des cytokines inflammatoires (Chapitre 4). Pour cela, nous nous sommes focalisé sur l’IL-1b, la cytokine inflammatoire effectrice principale de l’activité microgliale. L’expression de l'IL-1b et des cytokines associées (IL-6, TNFa et IL-10) augmente dans le plasma maternel, le placenta et le cerveau fœtal, 2 et 4 heures après l’administration de LPS. Ces changements sont accompagnés d'un phénotype microglial immature à JG18.5 et d’une réduction de la population microgliale totale au jour postnatal (JPN)9. À l’âge adulte (JPN65), nous avons observé une modification morphologique de la microglie dans plusieurs structures cérébrales. Enfin, les souris adultes, prénatalement traitées au LPS, développent des altérations des comportements de type sociaux et des comportements répétitifs. Les altérations du nombre de microglies induites par le LPS sont corrélées aux troubles comportementaux, et ce, de façon spécifique en fonction du sexe des souris. Enfin, la co-administration de l'antagoniste du récepteur de l'IL-1 et de LPS chez les femelles gestantes au JG9.5 réduit, voire prévient les effets inflammatoires et comportementaux du LPS. [...] / Recent research on microglia has uncovered a multitude of activities that extends the role of these cells well beyond their traditional function as immune sentinels. The most prominent of these newly described activities is an intricate role in neuronal network remodeling notably upon environmental challenge or during brain development, the disruption of which can result in long lasting consequences relevant to several psychopathologies. We sought, in the current thesis, to identify some of the mechanisms involved. Our initial approach was to target the immune function of microglia, based on our previous findings linking systemic immunogenic challenge with lipopolysaccharide (LPS) in mice with the development of despair-like behavior/depression. Here, we sought to identify immune mediators activated in microglia following a single systemic challenge with LPS (Chapter 2). These studies were conducted in adult mice in which phagocytic microglia were depleted using a single injection of liposomal clodronate in the CA3 region of the hippocampus. LPS challenge significantly upregulated the expression of both pro-inflammatory [interleukin (IL)-1b and tumor necrosis factor (TNF)-a] and anti-inflammatory (IL-10) cytokines compared to saline treated animals. In addition, LPS highly increased the expression of indoleamine 2,3-dioxygenase (IDO), an important rate limiting enzyme for metabolizing tryptophan in the brain and an established indicator of the activation of this depression mediating pathway. Clodronate-mediated depletion attenuated all of these effects apart from IL-1b expression which was further exacerbated. Behavioral assessment of the mice demonstrated a significant LPS-induced increase of immobility in the forced swim test (FST), which was prevented by clodronate. This experimental approach provided a snapshot of the role of inflammation in the development of brain dysfunction mediated by microglia. In subsequent studies (chapter 3 & 4), and in order to perform a more comprehensive, longer-term investigation of microglia activity in neurodevelopment, we utilized a prenatal infection model using LPS to activate maternal immunity at a relatively early [Gestational Day (GD)9.5] time point when microglia colonize the fetal brain to assess the impact on microglial population during development and the subsequent behavior of the progeny (Chapter 3). The results demonstrated LPS reduced the percentage of mature microglial population at GD14.5 and GD18.5 representing mid to late gestation. In addition, prenatal LPS had a significant effect on the offspring’s neonatal as well as adult behavior, with a clear divergence along sex lines in adulthood. In the final study (Chapter 4), we sought to investigate the mechanisms underlying the changes we noted in microglial development and the sexually dimorphic behavioral deficits. For this, we focused on the role played by pro-inflammatory cytokines, particularly IL-1b which represents the main effector of microglial activation following infection or injury. Detailed analysis of the expression of IL-1b and other related cytokines (IL-6, TNF-a and IL-10) revealed an increased expression of these mediators in maternal plasma, placenta and fetal brain, 2 and 4 hours after the prenatal LPS treatment. These changes were accompanied with a decreased percentage of mature microglia in the brain of embryos at GD18.5 and of total microglia population at post-natal day (PND)9. In the adult offspring (PND65), we detected an increased density and altered microglial morphology in specific higher-order structures implicated in complex behaviors, as well as altered social preference and memory and increased repetitive actions. [...]

Coccidiosis vaccine associated side effects, oocyst shedding patterns, intestinal lesions, and mucosal gene expression in the turkey were studied. The first study examined vaccine associated side effects and oocyst shedding patterns under experimental conditions. Peak oocyst shedding occurred on days 5-6, 13-17, and 19-20 days post vaccination. Throughout the course of the study, several poults exhibited clinical coccidiosis. Based on body weights, growth was correlated with vaccine cycling. The second study examined coccidiosis vaccine induced lesions and changes in mucosal gene expression on day 5, 10, 13, 17, and 20 days post vaccination. Poults were gavaged the equivalent of 0x, 1/2x, 1x, and 2x the available vaccine dose. Intestinal sections adjacent to the Meckel's diverticulum, ileocecal junction, and middle of the ceca were collected for histological analysis and gene expression. Measurements from the tip of the villus to the base of the lamina propria, villus width, and the muscularis mucosae thickness were acquired from the histological sections. Interleukin-10, IL-1beta, and GAPDH gene expression were measured by extracting mRNA in the tissues and quantified using real-time RT-qPCR. Starting on day five post vaccination, the control group weighed significantly more than the group that received the 2x dose. Body weight and oocyst dose were inversely related through day 17. Intestinal measurements did not necessarily correlate with the vaccine dose, although there appears to be some correlation on day five. There were no significant changes in the mucosal gene expression of IL-10 and IL-1beta in the intestinal tissue adjacent to the Meckel's diverticulum throughout the course of the study. On day five post vaccination, IL-10 and IL-1beta were significantly upregulted in the ileocecal junction. Interleukin-10 was significantly upregulated on day 17 and IL-1beta was significanlty down regulated on day 20 in the ileocecal junction. Both IL-10 and IL-1beta were significantly upregulated in the ceca days 5, 10, and 13 post vaccination. Interleukin-10 was significnalty upregulated in the ceca on day 17 and significantly down regulated on day 20. Individual variation among poults in the same group merits further attention.

Made available in DSpace on 2016-03-22T17:26:46Z (GMT). No. of bitstreams: 1 capa final rosana.pdf: 15525 bytes, checksum: fab2d4afcfe9faedc9db870806e272cd (MD5) Previous issue date: 2012-07-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Objective: The purpose of this study was to clarify the effect of imune response in diferente mood episodes. Methods: This report is part of a cross-sectional population-based study including 1560 individuals 18 to 35 year-old living in the urban area of Pelotas, RS (Brazil). We randomly selected 241 subjects from the population-based study. The diagnostic of current depression, mania or mixed episode was made using the Mini International Neuropsychiatric Interview 5.0 (MINI). Serum levels of IL-1b were measured using a commercial available immunoassay kit. Results: In the present work we found 110 (45.6%) patients with no current mood episode, 15 (6.2%) patients in maniac episodes, 91 (37.8%) patients with current depression and 25 (10.4%) patients in a mixed states. Socio-demographic variables like ethnicity and years in school were not significantly different between the groups. Regarding the cytokine measurements, patients in maniac and depressive episodes had similar levels of IL-1b (8.85 and 8.90 pg/mL, respectively) when compared to the group with no mood episodes (10.24 pg/mL). However, patients in mixed states had an increase (13.19 pg/mL) in IL-1b levels (P=0.09) when compared to the other groups. Discussion: These results suggest that mixed mood episodes are associated with higher levels of IL-1b. This pro-inflammatory state might underlie the symptom severity and the poor outcome observed in these patients / Nos últimos anos as doenças psiquiátricas foram as doenças que mais aumentaram na população mundial (Andlin-Sobocki et al., 2005). Estima-se que os transtornos de humor serão o grupo de doenças com maior custo absoluto nos próximos anos, conjuntamente com as patologias demenciais (Andlin-Sobocki et al., 2005). Apesar dos recentes progressos alcançados, as bases etiológicas dos transtornos de humor permanecem pouco elucidadas e a terapia não tem sido totalmente eficaz, com apenas 60% dos pacientes sendo responsivos aos fármacos existentes no mercado (Gareri et al., 2000). Além disso, o diagnóstico dos transtornos de humor é feito basicamente pela observação clínica do paciente, sendo muitas vezes falho e ineficaz. Desta maneira, a identificação de marcadores biológicos preditivos ou com valor diagnóstico para estas doenças pode representar um importante avanço no diagnóstico e tratamento dos transtornos de humor

A hiperalgesia inflamatória é o processo pelo qual ocorre a sensibilização dos neurônios nociceptores aferentes primários por mediadores químicos inflamatórios, gerando assim uma diminuição do limiar nociceptivo e como consequência episódios de dor. Entre os principais mediadores envolvidos com a sensibilizacão das fibras nociceptivas periféricas está a prostaglandina E2 (PGE2), que é liberada como um produto final de uma cascata de mediadores inflamatórios. Dentro desta cascata de liberação hierárquica podemos destacar a interleucina -1? (IL)-1?, uma citocina importante na gênese da dor inflamatória, devido à sua capacidade de induzir a produção da enzima cicloxigenase-2 (COX-2), e consequentemente PGE2. O mecanismo de controle da produção da IL-1 ? envolvem dois passos intracelulares: a indução da expressão de uma forma protêica inativa (a pró-IL-1 ?) e a geração da forma biologicamente ativa (IL-1?) a partir da pró-IL-1 ?. Este último passo envolve a ação de uma cisteína-protease ativada em decorrência de um processo inflamatório, conhecida como Caspase-1, a qual cliva a pró-IL-1? em IL1?. Recentemente, nosso grupo demonstrou que a caspase-1 tem um papel importante na gênese da dor inflamatória, sendo crucial para a geração de IL-1? e consequentemente COX2/PGE2. Porém, não são conhecidos os mecanismos de ativação da caspase-1 na hiperalgesia inflamatória. Sabe-se que a ativação da Caspase-1 e clivagem da pro-IL-1? são dependentes de uma plataforma molecular intracelular denominada inflamassoma. Os principais inflamassomas ativadores de caspase-1 são formados pelas proteínas NLRP3, IPAF (NLRC4) e por sua molécula adaptadora ASC. O objetivo desse trabalho então foi avaliar a participação do inflamassoma na gênese da dor inflamatória. Nós identificamos que as moléculas IPAF e ASC, mas não o NLRP3, participa no desenvolvimento da hiperalgesia inflamatória mecânica e térmica induzida pela carragenina. Observou-se que estas moléculas são cruciais para a ativação da Caspase-1 e, consequentemente, para a produção da IL-1? ativa. Estes resultados evidenciam pela primeira vez um papel importante do inflamassoma no desenvolvimento da hiperalgesia inflamatória. / The inflammatory hyperalgesic is the process by which occurs the sensitization of nociceptors primary afferent neurons by inflammatory chemical mediators, that generating a decreased nociceptive threshold and result in episodes of pain. Among the main of nociceptive mediators involved with sensitization of peripheral nociceptive fibers are prostaglandin E2 (PGE2), which is released as a final product of a cascade of inflammatory mediators. Within this hierarchical cascade of release can highlight interleukin-1? (IL)-1?, a cytokine important in the genesis of inflammatory pain due to their ability to induce the production of the enzyme cyclooxygenase-2 (COX-2) and consequently PGE2. The control mechanism production of intracellular IL-1 ? involved two steps: induction of expression of a protein inactive form (pro-IL-1 ?) and the generation of the biologically active form from pro-IL-1 ? (IL-1?). This last step involves the action of a cysteine protease-activated due to an inflammatory process, known as Caspase-1, which cleaves pro-IL-1? to IL-1?. Recently our group has demonstrated that caspase-1 plays an important role in the genesis of inflammatory pain, crucial for the generation of IL-1? and consequently COX2/PGE2. However, there aren\'t known mechanisms of activation of caspase-1 in inflammatory hyperalgesic. It is known that the activation of Caspase-1 cleavage and pro-IL-1? are dependent on an intracellular molecular platform called inflammassome. The main inflammassome activators of caspase-1 proteins are formed by NLRP3, IPAF (NLRC4) and its adapter molecule ASC. The aim of this study was to evaluate the inflammassome participation in the genesis of inflammatory pain. We have identified molecules IPAF and ASC, but not NLRP3, is participate in the development of mechanical and thermal inflammatory hyperalgesic induced by carrageenan. It was observed that these molecules are crucial for the activation of Caspase-1 and thus for the production of active IL-1?. These results demonstrate for the first time an important role of the inflammassome in the development of inflammatory hyperalgesic.

A hiperalgesia inflamatória é o processo pelo qual ocorre a sensibilização dos neurônios nociceptores aferentes primários por mediadores químicos inflamatórios, gerando assim uma diminuição do limiar nociceptivo e como consequência episódios de dor. Entre os principais mediadores envolvidos com a sensibilizacão das fibras nociceptivas periféricas está a prostaglandina E2 (PGE2), que é liberada como um produto final de uma cascata de mediadores inflamatórios. Dentro desta cascata de liberação hierárquica podemos destacar a interleucina -1? (IL)-1?, uma citocina importante na gênese da dor inflamatória, devido à sua capacidade de induzir a produção da enzima cicloxigenase-2 (COX-2), e consequentemente PGE2. O mecanismo de controle da produção da IL-1 ? envolvem dois passos intracelulares: a indução da expressão de uma forma protêica inativa (a pró-IL-1 ?) e a geração da forma biologicamente ativa (IL-1?) a partir da pró-IL-1 ?. Este último passo envolve a ação de uma cisteína-protease ativada em decorrência de um processo inflamatório, conhecida como Caspase-1, a qual cliva a pró-IL-1? em IL1?. Recentemente, nosso grupo demonstrou que a caspase-1 tem um papel importante na gênese da dor inflamatória, sendo crucial para a geração de IL-1? e consequentemente COX2/PGE2. Porém, não são conhecidos os mecanismos de ativação da caspase-1 na hiperalgesia inflamatória. Sabe-se que a ativação da Caspase-1 e clivagem da pro-IL-1? são dependentes de uma plataforma molecular intracelular denominada inflamassoma. Os principais inflamassomas ativadores de caspase-1 são formados pelas proteínas NLRP3, IPAF (NLRC4) e por sua molécula adaptadora ASC. O objetivo desse trabalho então foi avaliar a participação do inflamassoma na gênese da dor inflamatória. Nós identificamos que as moléculas IPAF e ASC, mas não o NLRP3, participa no desenvolvimento da hiperalgesia inflamatória mecânica e térmica induzida pela carragenina. Observou-se que estas moléculas são cruciais para a ativação da Caspase-1 e, consequentemente, para a produção da IL-1? ativa. Estes resultados evidenciam pela primeira vez um papel importante do inflamassoma no desenvolvimento da hiperalgesia inflamatória. / The inflammatory hyperalgesic is the process by which occurs the sensitization of nociceptors primary afferent neurons by inflammatory chemical mediators, that generating a decreased nociceptive threshold and result in episodes of pain. Among the main of nociceptive mediators involved with sensitization of peripheral nociceptive fibers are prostaglandin E2 (PGE2), which is released as a final product of a cascade of inflammatory mediators. Within this hierarchical cascade of release can highlight interleukin-1? (IL)-1?, a cytokine important in the genesis of inflammatory pain due to their ability to induce the production of the enzyme cyclooxygenase-2 (COX-2) and consequently PGE2. The control mechanism production of intracellular IL-1 ? involved two steps: induction of expression of a protein inactive form (pro-IL-1 ?) and the generation of the biologically active form from pro-IL-1 ? (IL-1?). This last step involves the action of a cysteine protease-activated due to an inflammatory process, known as Caspase-1, which cleaves pro-IL-1? to IL-1?. Recently our group has demonstrated that caspase-1 plays an important role in the genesis of inflammatory pain, crucial for the generation of IL-1? and consequently COX2/PGE2. However, there aren\'t known mechanisms of activation of caspase-1 in inflammatory hyperalgesic. It is known that the activation of Caspase-1 cleavage and pro-IL-1? are dependent on an intracellular molecular platform called inflammassome. The main inflammassome activators of caspase-1 proteins are formed by NLRP3, IPAF (NLRC4) and its adapter molecule ASC. The aim of this study was to evaluate the inflammassome participation in the genesis of inflammatory pain. We have identified molecules IPAF and ASC, but not NLRP3, is participate in the development of mechanical and thermal inflammatory hyperalgesic induced by carrageenan. It was observed that these molecules are crucial for the activation of Caspase-1 and thus for the production of active IL-1?. These results demonstrate for the first time an important role of the inflammassome in the development of inflammatory hyperalgesic.

Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2016-10-19T16:18:10Z No. of bitstreams: 1 Dissertação CLARISSA RIBEIRO BASTOS.pdf: 4376745 bytes, checksum: 05e26e833e53a5a4c385cc5908d0bbcc (MD5) / Made available in DSpace on 2016-10-19T16:18:10Z (GMT). No. of bitstreams: 1 Dissertação CLARISSA RIBEIRO BASTOS.pdf: 4376745 bytes, checksum: 05e26e833e53a5a4c385cc5908d0bbcc (MD5) Previous issue date: 2016-02-26 / Introduction: Suicide is one of the ten leading causes of death worldwide, emerging from a complex interaction between genetic, environmental and psychosocial factors. In this aspect studies found that individuals with suicidal tendencies have elevations in the levels of pro-inflammatory cytokines and alterations in the functioning of the HPA axis. Objective: Investigate the association of the polymorphism in the CRHR1 gene (rs110402) with the suicidal behavior, as well as the genotype effects on IL-1β levels. Methods: Sample consisted in 171 individuals that participated in the study, from which 15 were at risk of suicide, 20 had attempted suicide and 136 were controls. We perform genotyping of individuals by real-time PCR and IL-1β levels measured by ELISA technique. Results: Individuals with a clinical diagnosis of attempted suicide carrying the A allele of SNP rs110402 showed increased IL-1β levels (19.3 IQR: 6.84 to 36.47) compared to the control group (4.91 IQR: 3.22 to 6.39; p = 0.027). Ratings isolated from the levels of this interleukin and genotypes of the rs10402 SNP showed no relationship with suicidal behavior. Conclusion: Our results suggest that alterations in CRHR1 gene can affect the levels of pro-inflammatory molecules, showing the complex interactions of biological markers in suicidal behavior. / Introdução: O suicídio é umas das dez principais causas de morte no mundo, emergindo de uma interação complexa entre fatores genéticos, ambientais e psicossociais. Neste aspecto, estudos identificaram que indivíduos com tendências suicidas apresentam elevações nos níveis de citocinas pró-inflamatórias e alterações no funcionamento do eixo HPA. Objetivo: Investigar a associação de um polimorfismo gene do CRHR1 (rs110402) com o comportamento suicida, bem como os efeitos do genótipo sobre os níveis de IL-1β. Métodos: A amostra foi composta de 171 indivíduos, e destes 15 apresentavam risco de suicídio, 20 já haviam tentado o suicídio e 136 eram controles. Realizamos a genotipagem dos indivíduos por PCR em tempo real e a dosagem de IL-1β pela técnica de Elisa. Resultados: Indivíduos com um diagnóstico clínico de tentativa de suicídio que carregavam o alelo A do SNP rs110402 mostraram aumento dos níveis de IL-1β (19,3 IQR: 6,84 - 36,47) em relação ao grupo controle (4,91 IQR: 3,22-6,39; p = 0,027). Avaliações isoladas em relação aos níveis dessa interleucina e aos genótipos desse polimorfismo não mostraram relação com o comportamento suicida. Conclusão: Nossos resultados sugerem que alterações no gene do CRHR1 podem afetar os níveis de moléculas pró-inflamatórias, mostrando as interações complexas de marcadores biológicos no comportamento suicida.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18425号 / 生博第305号 / 新制||生||40(附属図書館) / 31283 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 稲葉 ｶﾖ, 教授 米原 伸, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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