A nicotina é um alcalóide extraído da planta do tabaco e age como agonista em neurônios e outras células que expressam receptores nicotínicos. Essa substância é capaz de atravessar a barreira hematoencefálica e mimetizar os efeitos da acetilcolina endógena ao interagir com os diferentes subtipos de receptores. O hipocampo e o cerebelo são regiões encefálicas ricas em receptores nicotínicos como o alfa7 e o alfa42 que estão também envolvidos na progressão de doenças neurodegenerativas, como a doença de Alzheimer, e neuropsiquiátricas, como a esquizofrenia, ambas apresentando um contexto inflamatório. O lipopolissacarídeo (LPS) é um indutor inflamatório que age via receptor toll-4 (TLR-4) ativando, dentre outros, o fator de transcrição NF-capaB. Vários estudos têm demonstrado tanto in vitro quanto in vivo que a exposição crônica à nicotina inibe a resposta imune. Uma vez que o fator de transcrição NF-capaB regula a expressão gênica de diversos fatores que medeiam a plasticidade e o desenvolvimento neuronal e inflamação, nosso objetivo foi avaliar os efeitos da nicotina crônica sobre a via de sinalização induzida pelo LPS. Sendo assim, avaliou-se a translocação nuclear do NF-capaB, a expressão de indutores inflamatórios (TNF, IL-1 e iNOS) e também do receptor TLR-4 tanto no hipocampo quanto no cerebelo. Além disso, foram determinados os níveis plasmáticos das citocinas TNF, IL-1, MIP-1alfa, CINC2alfa/. Nossos resultados mostram que, perifericamente, o LPS induziu a produção das citocinas plasmáticas, mas a nicotina não foi capaz de reverter seus efeitos. Já no cerebelo e no hipocampo, a nicotina crônica (0,1 e 1,0 mg/kg) interferiu significativamente na translocação do NF-capaB induzida pelo LPS, levando inclusive a um bloqueio importante da expressão do mRNA de TNF, IL-1, iNOS no hipocampo, e de IL-1 no cerebelo. Ademais, no hipocampo, a nicotina também interferiu na expressão do receptor TLR-4. A mecamilamina, antagonista heteromérico não-seletivo não foi capaz de reverter os efeitos da nicotina sobre o NF-capaB no hipocampo, mas o fez no cerebelo. Já o antagonista seletivo de receptor alfa 7, metililaconitina, bloqueou a ação da nicotina no hipocampo. / Nicotine is an alkaloid extracted from Tobacco plants, and it acts as an agonist in neurons and other cells that express nicotinic receptors. This substance crosses the blood brain barrier and mimics endogenous acetylcholine in interacting with various receptor subtypes. The hippocampus and cerebellum are brain regions rich in nicotinic receptors including 7, 42 and other subtypes, involved in neurodegenerative and neuropsychiatric disorders, including Alzheimer disease and schizophrenia, respectively, in a neuroinflammatory context. The lipopolysaccharide (LPS) is a known inflammatory inducer that exerts its effects through the Toll-like receptor 4 (TLR-4) and activates the transcription factor NF-capaB. In the present work, we evaluate the effects of chronic nicotine on the nuclear translocation of NF-capaB, on the expression of NF-capaB regulated inflammatory inducers (TNF, IL-1 and iNOS) and on the LPS-induced TLR4 mRNA expression in response to chronic nicotine treatment in hippocampus and cerebellum. In addition, we accessed the production of peripheral cytokines (TNF, IL-1, MIP-1, CINC 2/). Our results demonstrate that LPS induced the production of peripheral cytokines but nicotine treatment did not interfere in their plasma levels. However, in cerebellum and hippocampus, chronic nicotine 1.0 and 0.1 mg/kg interfered in the LPS-induced NF-capaB leading to a deficient mRNA expression of its related genes TNF, IL-1 and iNOS in hippocampus and of IL-1, in cerebellum. Chronic nicotine (0.1 mg/kg, s.c.) significantly interfered in the TLR4 mRNA expression in LPS-challenged animal group in hippocampus. The non-selective heteromeric antagonist mecamylamine (1.0 mg/kg s.c.) reverted the nicotine effects over NF-capaB nuclear translocation in cerebellum, but not in hippocampus. On the other hand, the 7 antagonist methyllycaconitine (MLA) (5.0 mg/kg i.p.) reverted the nicotine effects over NF-capaB in hippocampus but not in cerebellum. Nicotine prevents LPS effects, both in cerebellum and hippocampus, once it reduced significantly the nuclear translocation of NF-capaB and the expression of related pro-inflammatory cytokines. Besides, the nicotinic effects seem to be mainly mediated by heteromeric receptors in cerebellum and through 7 receptors in hippocampus.

Les principaux objectifs de ce travail de recherche étaient (i) l'étude de la composition lipidique de quatre bactéries Gram-négatives hydrocarbonoclastes Marinobacter hydrocarbonoclasticus, Marinobacter aquaeolei, Acinetobacter calcoaceticus et Pseudomonas oleovorans en fonction de la source de carbone, (ii) la détermination des différents voies métaboliques de dégradation des hydrocarbures comportant une chaîne alkyle et (iii) mieux connaître la composition en Β-hydroxy acides lipopolysaccharidiques de leurs membranes extérieures. La première partie de ce travail présente une description qualitative et quantitative des lipides "non liés", labiles en milieu basique et labiles en milieu acide de M. hydrocarbonoclasticus en fonction de l'hydrocarbure (alcanes normaux et ramifiés, phényl et cyclohexyl-alcanes, et 1-alcène) utilisé comme source unique de carbone et d'énergie. Ces résultats ont montré que la structure chimique des lipides identifiés suit celle de l'hydrocarbure fourni: apparition de proportions significatives et dans certains cas très importantes d'acides gras issus de la dégradation de ces composés. Les Β-hydroxy acides des LPS sont moins sensibles au changement de la structure de l'hydrocarbure. Le n-Β-12:0 est toujours dominant alors que les Β-hydroxy acides provenant de la dégradation des hydrocarbures peuvent être incorporés aux LPS mais ne constituent que des composés mineurs. La présence d'intermédiaires métaboliques de chaque hydrocarbure dans les lipides "non liés" a permis la détermination des différents voies métaboliques de dégradation. La deuxième partie de ce travail porte sur la composition lipidique des bactéries M. aqueolei, A. calcoaceticus et P. oleovorans en fonction de la source de carbone (acétate ou alcane). Cette partie a montré le degrés de variabilité des acides gras chez les bactéries Gram-négatives M. aquaeolei et A. calcoaceticus: substitution des acides gras normaux pairs par des acides gras de même parité et même squelette que l'alcane utilisé. Chez P. oleovorans l'utilisation d'alcanes courts se manifeste aussi bien sur la distribution des acides gras membranaire que sur la composition en Β-hydroxy acides des LPS. Enfin, les Β-hydroxy acides issus des quatre bactéries étudiées sont essentiellement normaux pairs de C10 à C14 dont le composé prédominant est dépendant de l'espèce bactérienne.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 137-158.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 5 uppsatser.

Os receptores do tipo Toll (TLRs) são as primeiras proteínas do sistema imune a identificarem distúrbios, reconhecem patógenos como bactérias, fungos e vírus. Como o processo inflamatório possui um importante papel em diversas doenças, os TLRs foram considerados potenciais alvos em estratégias terapêuticas, incluindo o tratamento de câncer. No entanto, o papel dos TLRs permanece ambíguo. Esse estudo teve como objetivos analisar os níveis de expressão dos TLRs presentes em membrana plasmática, TLRs (TLR1, TLR2, TLR4, TLR5, TLR6) em astrocitomas de diferentes graus de malignidade (grau II-IV), tumor mais prevalente do Sistema Nervoso Central (SNC). Nós demonstramos que a expressão dos TLRs foi mais alta em amostras de astrocitomas comparadas com tecido cerebral não-neoplásico, por qRT-PCR. A expressão gênica e proteica foi observada em células de linhagem de glioblastoma (GBM) U87MG e A172, mostrando sua presença em células tumorais. Foi observada expressão associada entre os heterodímeros TLR1- TLR2. Em GBMs, o subtipo mesenquimal mostrou maior nível de expressão dos TLRs comparados aos subtipos clássico e proneural. Com o objetivo de identificar o papel dos TLRs nas células tumorais, foi selecionado dentre os TLRs o que apresentou maior nível de expressão, o TLR4, e realizamos ensaios funcionais estimulando a U87MG com LPS, um agonista natural para TLR4. A taxa de proliferação da célula tratada com LPS foi similar a não tratada. No entanto, foi observado a ativação do NF-kB após 12hrs do estímulo com LPS. Quando a sinalização do receptor foi inibida por um composto químico (VGX-1027), o nível de proliferação da U87MG decaiu. Adicionalmente, análise in silico revelou uma forte associação dos TLRs hiperexpressos com aumento da expressão de genes relacionados à sinalização do ciclo celular, inflamassoma e ripoptossoma. O que sugere serem os TLRs alvos para complementação do tratamento do câncer / Toll-like receptors (TLRs) are the first to identify disturbances in the immune system, recognizing pathogens such as bacteria, fungi, and viruses. Since the inflammation process plays an important role in several diseases, TLRs have been considered potential therapeutic targets, including treatment for cancer. However, TLRs\' role in cancer remains ambiguous. This study aims to analyze the expression levels of plasmatic cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) in different grades (II-IV) of human astrocytoma, the most prevalent tumor of CNS. We demonstrated that TLR expressions were higher in astrocytoma samples compared to non-neoplastic brain tissue, by qRT-PCR. The genes and proteins expressions were observed in U87MG and A172 GBM cell lines, proving their presence in the tumor cells. Associated expressions between the known heterodimers TLR1-TLR2 were found in diffusely infiltrative astrocytoma. In GBM, the mesenchymal subtype showed higher levels of TLR expressions in relation to classical and proneural subtypes. Aiming to indentify the role of TLRs in tumor cells, we chose the highest TLR expressed in GBM cells, the TLR4, and performed functional assays stimulating U87MG-GBM cell line with LPS, a natural agonist for TLR4. The proliferation rate was similar in treated and non-treated cell with LPS. However, NF-kB activation was detected after 12hrs of LPS stimulation. When TLR4 signaling pathway was inhibited by a chemical compound (VGX-1027) a decrease in the proliferation rate was observed. Additionally, in silico analysis revealed a strong association of TLRs upregulation with increased expression level of genes related to cell cycle, inflammasome and ripoptosome pathways, further highlighting TLRs as interesting targets for cancer complementary treatment

Abstract Toll-like receptors (TLRs) are transmembrane proteins involved in the recognition of specific microbial structures and thus the activation of signaling cascades of innate immunity. Regulation of the innate immune response is a complex biological process involving the combined synergistic and antagonistic effects of distinct signaling mediators. Although TLR signaling has been widely studied in recent years, there remain many unexplored unique features of each TLR signaling pathway. The present study evaluated the activation and regulation of TLR4 and TLR2 signaling with the aim of better understanding the molecular mechanisms that control these inflammatory signaling pathways. In the present study, the signal transduction mechanisms of TLR4 and TLR2 in response to Escherichia coli LPS and Staphylococcus aureus LTA were evaluated in mouse macrophages. The inductions, interactions, and activations of the signaling molecules and mediators in the TLR pathways were studied by using several molecular biology and protein chemistry methods. In addition, the role of TLR4 and TLR2 in the regulation of the hepatic inflammatory reaction during endotoxemia was studied. Mouse macrophages were found to induce central proinflammatory mediators in response to LPS and LTA stimulation. Specific roles for PI 3-kinase and Btk were described. These kinases were found to be activated by LPS and LTA; moreover, PI 3-kinase and Btk were found to form specific interactions with TLRs and their intracellular signaling mediators. In addition, a unique IRF2 signaling pathway for LTA-induced TLR2 was found, resulting in the activation of signal transducers and activators of transcription (Stats) and IFN-α secretion. The secreted IFN-α was shown to regulate the LTA-induced inflammatory responses, thereby combining the LTA-induced IRF proteins into NF-κB pathway. The present study provides insight into the signal transduction mechanisms of TLRs. The understanding of these molecular mechanisms that control the activation of TLR signaling cascades will in the future help to predict predisposition and outcome in infectious diseases, and to control the course of disease at an earlier stage. / Tiivistelmä Toll:n kaltaiset reseptorit (TLR) ovat solukalvon proteiineja, jotka tunnistavat taudinaiheuttajien eli patogeenien spesifisiä rakenteita johtaen elimistön puolustusjärjestelmän, immuniteetin, aktivoitumiseen. Immuniteetin säätely on monimutkainen biologinen prosessi, joka tapahtuu kudosten, solujen ja erilaisten synnynnäiseen immuniteettiin liittyvien molekyylien vuorovaikutuksina. Tulehdusvasteen säätelyssä tasapaino positiivisten ja negatiivisten säätelysignaalien välillä on erittäin tärkeää, jotta autoimmuunisairauksien, akuuttien tai kroonisten tulehdusten sekä infektiosairauksien synty voitaisiin välttää. Tämän tutkimuksen tavoitteena oli saada lisätietoa TLR2 ja TLR4 proteiinien säätelemistä signaalireiteistä, niiden vasteista tiettyjä patogeenirakenteita vastaan ja ymmärtää paremmin synnynnäisen immuniteetin puolustusmekanismeja. Patogeenirakenteiden aiheuttamaa tulehdusvastetta tutkittiin pääosin soluviljelymallissa. Lisäksi selvitettiin immuunivasteen luonnetta fysiologisessa kokonaisuudessa ja sen korrelaatiota solutasolla nähtyihin vasteisiin käyttäen in vivo hiirimallia. Tutkimus tehtiin käyttäen useita molekyylibiologian ja proteiinikemian menetelmiä proteiini- ja mRNA-ekspressioiden sekä proteiini-interaktioiden tutkimiseen ja erilaisten aktiivisuuksien määrityksiin. Tulehdusvastetta tutkittiin etenkin sytokiinivastetta määrittämällä ja signaaliketjujen toimintaa analysoitiin estämällä spesifisesti niiden toimintaa. Tarkoituksena oli selvittää, mitkä tekijät ovat välttämättömiä kyseisten tulehdusta aiheuttavien bakteerien tunnistuksessa ja puolustusreaktiossa niitä vastaan. Tutkimuksessa havaittiin kahden kinaasin, PI 3-kinaasin ja Brutonin tyrosiinikinaasin, liittyvän oleellisesti TLR signaalireitteihin. Nämä TLR:ien stimulaation seurauksena aktivoituneet kinaasit muodostivat spesifisiä sidoksia TLR:ien ja niiden signaaliketjuihin liittyvien solunsisäisten signaalivälittäjien kanssa. Lisäksi TLR2 signaalireitillä havaittiin aktivoituvan tekijöitä, jotka johtivat interferoni-α välitteiseen tulehdusvasteen säätelyyn. TLR signaalireittien selvittäminen auttaa ymmärtämään tulehdussairauksien patofysiologiaa ja voi siten tulevaisuudessa johtaa parempien hoitomenetelmien kehittämiseen.

Abstract The response of the innate immune system is triggered through Toll-like transmembrane receptors (TLR) that recognize a variety of microbial products. TLR4 is the principal mediator for Gram negative bacterial endotoxin (LPS), whereas TLR2 mediates the response to Gram positive bacteria, mycobacteria, and yeast. Stimulation of TLR activates complex cascades leading first to the production of inflammatory mediators, such as proinflammatory cytokines IL-1 α/β and TNF-α. Overproduction of inflammatory cytokines as well as failure in the activation of innate immunity are detrimental to the host. Excess inflammatory stimulation leads to a septic shock, which may cause multi-organ failure and even death. The lack of any innate response exposes the host to overwhelming bacterial infections. Appropriate regulation of the innate immune response could be a target for attempts to find therapeutics to septic shock. This experimental study focuses on functional activation of the signaling receptors TLR4 and TLR2 upon a LPS challenge. An acute inflammation model was used for both in vivo and in vitro experiments. LPS was used to stimulate a mouse macrophage cell line. It was administred intraperitoneally or intra-amniotically to non-pregnant or time-mated mice. The basal and induced mRNA expression levels and the protein production of TLRs as well as the mRNA expression of several inflammatory mediators were studied. The present study showed that the expression of TLR4 and TLR2 is strain and tissue-specific. At the mRNA level, the levels of TLR4 expression limited the extent of the acute cytokine response. The quality of the cytokine response was modulated by protein aggregates formed by TLR4 on the cell surface. The LPS challenge caused a marked increase in the expression of TLR2 mRNA but not the protein; the significance of this remains to be studied. The study further showed that the expression of TLRs is regulated during the perinatal period, and that the acute cytokine response to LPS in the lung develops during antenatal differentiation. The present study provides information about how the activation of TLR regulates the acute inflammatory response and further helps to elucidate new targets for the anti-inflammatory strategies in controlling inflammatory events. / Tiivistelmä "Toll-like"-reseptorit (TLR) ovat solukalvon proteiineja, jotka spesifisesti tunnistavat erilaisia bakteerirakenteita. Infektiossa tällainen bakteerirakenne sitoutuu reseptoriin ja seurauksena solussa käynnistyy synnynnäinen immuunivaste eli tulehdusvälittäjäaineiden tuotto. Liiallinen tulehdusvälittäjäaineiden tuotto voi johtaa septiseen shokkiin eli verenmyrkytykseen, elinvaurioihin ja jopa kuolemaan. Septisen shokin synty voisi olla estettävissä immuunivasteen voimakkuuden tarkoituksenmukaisella säätelyllä. Väitöskirjassa on tutkittu, miten TLR4 ja TLR2 aktivoituvat bakteeri-infektiossa, tarkoituksena selvittää, säätelevätkö reseptorit immuunivasteen käynnistystä ja voimakkuutta solussa. Tutkimuksessa todettiin, että TLR4:n ja TLR2:n geenien ilmentymistä säädellään eri tavoin eri hiirikannoilla ja eri kudoksissa. TLR4-tason nousu aiheutti voimakkaamman immuunivasteen, kun taas reseptorin matala esiintymistaso laski immuunivasteen voimakkuutta. Lisäksi TLR4:aan solukalvolla sitoutuvat muut proteiinit vaikuttivat immuunivasteen laatuun. Tutkimuksessa todettiin myös, että TLR:n määrä sikiön keuhkoissa rajoittaa keuhkojen immuunivasteen kehittymistä. Tutkimus antaa tietoa siitä, miten TL-reseptorien aktivaatio säätelee synnynnäistä immuunipuolustusta ja selventää mahdollisuutta kontrolloida immuunivasteen voimakkuutta vaikuttamalla TL-reseptoriin.

As espécies Klebsiella pneumoniae, Escherichia coli e Pseudomonas aeruginosa são responsáveis por infecções neonatais hospitalares. Lipopolissacarídeo (LPS) é o principal indutor de respostas inflamatórias. Os objetivos foram avaliar a transferência placentária de IgG reativa ao LPS de K. pneumoniae, E. coli O111, O26 e O6 e P. aeruginosa empregando ELISA para dosar IgG em soro materno e de cordão de 29 neonatos pré-termos e 32 a termo; analisar IgM total e específica no soro materno; e investigar a influência das patologias apresentadas pelas mães na transferência placentária. Concentrações de IgG total foram reduzidas em pré-termos como esperado, porem índices de transferência placentária de IgG total e IgG anti-LPS foram sistematicamente reduzidos quando comparados aos neonatos a termo. Níveis de IgM total e anti-LPS foram equivalentes em mães de ambos os grupos. As patologias das mães influenciaram os níveis de IgM no grupo de mães de pré-termos. Estes resultados indicam uma imunidade adquirida deficiente pelo grupo pré-termo aumentando os riscos de infecção. / Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa species are responsible for neonatal nosocomial infections. Bacterial lipopolysaccharide (LPS) is the major inducer of the inflammatory responses. The aims were to evaluate the placental transfer of IgG reactive to LPS present in K. pneumoniae, in E. coli O111, O26 and O6 and in P. aeruginosa employing ELISA to detect IgG in maternal and cord sera from 29 preterm and 32 term neonates; to analyze total and specific IgM on the mothers sera; and to investigate the influence of the pathologies presented by some mothers in the placental transfer. Total IgG concentrations were reduced in preterm neonates as expected, but placental transfer indexes of total and anti-LPS IgG were systematically reduced when compared with term neonates. Total and anti-LPS IgM levels were equivalent on mothers of both groups. The mothers pathologies influenced only the IgM levels in the preterm mothers group. These results indicate a deficient acquired immunity by the preterm group increasing the risk of infection.