Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-11T17:48:42Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Chrisjacele Dissertação.pdf: 1102106 bytes, checksum: 157102cd60a6a7f996ddbb9302ae91b9 (MD5) / Made available in DSpace on 2016-10-11T17:48:42Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Chrisjacele Dissertação.pdf: 1102106 bytes, checksum: 157102cd60a6a7f996ddbb9302ae91b9 (MD5) Previous issue date: 2012-02-15 / Facepe / Diabetes mellitus (DM) caracteriza um grupo de doenças metabólicas resultante de defeitos na secreção e/ou ação da insulina que levam à hiperglicemia. As plantas do Gênero Bauhinia, pertencentes à Família das Fabaceae, conhecidas como “pata-de-vaca” são utilizadas pela medicina popular para o tratamento de diabetes em várias regiões do mundo, tais como África, Ásia bem como América Central e do Sul; estudos prévios já demonstraram atividade hipoglicemiante de extrato folhas de Bauhinia monandra. Lectinas são proteínas ou glicoproteínas, de origem não imune, que reconhecem e se ligam a carboidratos de forma especifica e reversível. Uma lectina presente em folhas da B. monandra (BmoLL, B. monandra Leaf Lectin), galactose específica, foi purificada através de cromatografia de afinidade em gel de guar. Apenas uma banda foi revelada por eletroforese em gel de poliacrilamida, PAGE, para proteínas nativas. Os efeitos da ação hipoglicemiante do tratamento de BmoLL purificada em camundongos NOD (NON OBESE DIABETIC), foi avaliado a na utilização de três grupos de animais, cada grupo com (n=4), intraperitonealmente, por 21 dias. O grupo tratado com BmoLL recebeu 60mg/kg/dia, o grupo controle para diabetes não tratado recebeu veículo, água. Grupo controle para valores normoglicêmicos foram camundongos que não desenvolveram diabetes. No grupo tratado os picos hiperglicêmicos foram vistos antes do tratamento, em uma média de glicose (307,50 ± 83,30 mg/dL), equivalente ao grupo diabéticos não tratados 364,5±106,24 mg/dl. No final do tratamento, 21dias, o grupo tratado (137,50 ± 68,26mg/dL) atingiu valores equivalentes ao grupo normoglicêmico (110,50 ± 16,66mg/dL). O controle nos níveis glicêmicos no grupo tratado mostrou apenas diferença estatística significante (p= 0,002) em comparação com o grupo que recebeu apenas veículo. Já o grupo de camundongos não diabéticos, ou seja, normoglicêmicos quando comparado com o grupo tratado não apresentou diferença estatistica significante (p= 0,12) os testes foram realizados pelo teste “t” de “Student”. Observando as consequências secundarias a hiperglicemia apresentadas pelo grupo controle para diabetes não tratado, não foram apresentadas pelo grupo tratado com BmoLL nem pelo grupo controle de níveis normoglicêmicos; conclui-se que a ação do tratamento com BmoLL ao ser capaz de controlar os níveis glicêmicos a valores normais de forma continua minimizou também as complicações secundárias a hiperglicemia, durante todo o período de 21 dias. Necessários estudos posteriores para entendimento dos mecanismos pelos quais a BmoLL tem seu efeito hipoglicemiante. / Diabetes mellitus ( DM ) features a group of metabolic disorders resulting from defects in the secretion and/or insulin action leading to hyperglycemia. The plants of the genus Bauhinia, belonging to the family Fabaceae, known as "paw-of-cow" are used in folk medicine for the treatment of diabetes in many parts of the world such as Africa, Asia and Central and South America; previous studies have demonstrated hypoglycemic activity of leaf extract of Bauhinia monandra. Lectins are proteins or glycoproteins of non- immune origin which recognize and bind carbohydrates specifically and reversibly form. A lectin present in the leaves of B. monandra (BmoLL , B. monandra Leaf Lectin), specifically galactose was purified by affinity chromatography on guar gel. Only one band was revealed by polyacrylamide gel PAGE for native proteins. The effects of treatment with hypoglycemic action BmoLL purified NOD (non obese DIABETIC) was evaluated using the three groups of animals, each group (n=4) intraperitoneally for 21 days. The treated group received 60mg/kg/day BmoLL, the control group received untreated diabetes vehicle, water. Values for the control group were normoglycemic mice did not develop diabetes. In the group treated hyperglycemic peaks were seen before treatment, at an average glucose (307.50 ± 83.30mg/dL), equivalent to the untreated diabetic group 364.5 ± 106.24mg /dl. At the end of treatment, 21 days, the treated group (137.50 ± 68.26mg/dL) achieved equivalent to the normoglycemic group (110.50 ± 16.66mg/dL) values. The control on blood glucose levels in the treated group showed only statistically significant difference (p=0.002) compared with the group that received only vehicle. The group of non-diabetic mice, ie, normoglycemic compared with the treated group showed no statistically significant difference (p=0.12) tests were performed by the "t" of " Student" test. Observing the secondary consequences hyperglycemia presented by the control group to untreated diabetes, were not presented by the group treated with BmoLL nor the normoglycemic control group levels, it is concluded that the action of treatment with BmoLL to be able to control blood glucose levels to normal values continuously also minimized the complications secondary to hyperglycemia during the whole period of 21 days. Required further studies to understand the mechanisms by which BmoLL has its hypoglycemic effect .

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-11-24T14:57:09Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Chrisjacele Tese CCB Bioquimica e Fisiologia.pdf: 1964201 bytes, checksum: 106af1b3d737e6027907a88876429988 (MD5) / Made available in DSpace on 2016-11-24T14:57:09Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Chrisjacele Tese CCB Bioquimica e Fisiologia.pdf: 1964201 bytes, checksum: 106af1b3d737e6027907a88876429988 (MD5) Previous issue date: 2015-02-23 / Facepe / As plantas do gênero Bauhinia (Família Fabaceae) são encontradas na África, Ásia bem como América Central e do Sul. No Brasil, elas são conhecidas como “pata-de-vaca“ e as suas folhas são usadas popularmente em forma de chá (infusão) para o tratamento de diabetes, como anti-inflamatório e como analgésico. Dentre diversos compostos purificados de plantas estão as lectinas, as quais são proteínas ou glicoproteínas que se ligam a carboidratos, por reconhecimento específico e reversível. Das folhas da espécie Bauhinia monandra foi purificada uma lectina galactose específica (BmoLL, B. monandra Leaf Lectin) através de cromatografia de afinidade em gel de guar. Diante das atividades descritas para as lectinas e do uso das plantas do gênero Bauhinia na medicina popular, foi levantada a hipótese de que as atividades biológicas relatadas pela população B. monandra têm como princípio ativo a lectina BmoLL. Desta forma o objetivo do presente estudo foi investigar propriedades toxicológicas, hipoglicemiante, anti-inflamatória e antinociceptiva da lectina. BmoLL não apresentou toxicidade para camundongos, uma vez que não induziu a morte nem perda de peso a 2000 mg/kg. Além disso, ela não afetou a sobrevivência de Artemia salina nas concentrações testadas (250-1.000 μg/mL). A atividade hipoglicemiante da BmoLL foi avaliada utilizando em camundongos NOD (non obese diabetic) como modelo experimental. Camundongos NOD diabéticos foram tratados com BmoLL (60 mg/kg/dia) por administração intraperitoneal durante 21 dias. O efeito da lectina foi comparado com os dados obtidos para um grupo diabético não-tratado e e um grupo não-diabético (normoglicêmico). No final do experimento, os animais tratados com BmoLL exibiram uma redução do nível de glicose no sangue (de 307,5 ± 83,3 mg/dL para 137,5 ± 68,3 mg/dL), atingindo valores semelhantes ao do grupo normoglicêmico (110,5 ± 16,7 mg/dL). O grupo diabético não-tratado apresentou concentração de glicose sanguínea ≥500 mg/dL. Além disso, as concentrações de triglicéridos e VLDL-colesterol no sangue foram significativamente reduzidas (p<0,05) pelo tratamento com BmoLL, em comparação com os animais diabéticos não tratados. Para avaliação da atividade antiinflamtória, foi realizado primeiramente o método de edema de pata induzido por carragenina. BmoLL reduziu a inflamação significativamente (p<0,05) em 47% (30 mg/kg) e 60,5% (60mg/kg). O grupo controle, tratado com ácido acetilsalicílico, apresentou 70,5% de redução. Na avaliação da atividade atiinflamatória por ensaio de peritonite, a migração de leucócitos foi significativamente reduzida (p<0,05) no grupo tratado com BmoLL, sendo o resultado semelhante ao obtido com grupo tratado com ácido acetilsalicílico. Na avaliação da atividade antinociceptiva BmoLL, os tratamentos com doses de 15, 30 e 60 mg/kg reduziram significativamente (p<0,05) o número de contorções abdominais (induzidas por ácido acético) em 43,1%, 50,1% e 71,3%, respectivamente. No ensaio de placa quente, a BmoLL em doses de 30 e 60 mg/kg, mostrou um efeito antinociceptivo significativo (p<0,05). Em conclusão, BmoLL é uma molécula interessante para ser estudada mais profundamente quanto a suas aplicações farmacológicas, desde que não apresentou toxicidade aguda para camundongos e demonstrou as atividades hipoglicemiante, anti-inflamatória e antinociceptiva, não apresentou toxicidade para A. salina nem para camundongos. / Plants of Bauhinia genus are found in Africa, Asia as well as in Central and South America. In Brazil, they are know as “pata-de-vaca” and their leaves are popularly used as tea (infusion) to treat diabetes, as anti-inflamatory and as analgesic. Among the compounds isolated from plants, there are the lectins, which Consist in protein or glycoproteins that bind carbohydrates by reversible and specific recognizement. From the leaves of Bauhinia monandra, it was isolated a galactose-specific lectin (BmoLL, B. monandra Leaf Lectin) by affinity chromatography on guar gel. In face of the biological activities described for lectins and the use of Bauhinia plants in folk medicine, it was hypothesized whether the biological activities reported by population for B. monandra have the lectin BmoLL as active principle. In this sense, the aim of this work was to investigate toxicological, hypoglycemic, anti-inflammatory, and antinociceptive properties of the lectin. BmoLL did not show toxicity to mice since it did not induce death and weight loss at 2,000 mg/kg. In addition, it did not affect the survival of Artemia salina at the tested concentrations (250-1,000 μg/mL). The hypoglicemic activity of BmoLL was evaluated using NOD (non obese diabetic) mice as experimental model. Diabetic NOD mice were treated with BmoLL (60 mg/kg/day) by intraperitoneal administration during 21 days. The lectin effect was compared with data obtained for a non-treated diabetic group and a non-diabetic (normoglycemic) group. At the end of the experiment, animals treated with BmoLL showed reduction in blood glucose level (dfrom 307.5 ± 83.3 mg/dL to 137.5 ± 68.3 mg/dL), reaching values similar to those from normoglycemic group (110.5 ± 16.7 mg/dL). Non-treated diabetic group showe blood glucose ≥500 mg/dL. In addition, the concentrations of triglycerides and VLDL-cholesterol in blood were also significantly (p<0.05) reduced by the treatment with BmnoLL, in comparison with non-treated diabetic animals. For the evaluation of the anti-inflammatory activity, it was first performed the carragenan-induced paw edema method. BmoLL reduced the inflammation significantly (p<0.05) in 47% (30 mg/kg) and 60.5% (60 mg/kg). The control group, treated with acetylsalycilic acid, showed a 70.5% reduction. In the evaluation of anti-inflammatory activity by peritonitis model, the leukocyte migration was significantly (p<0.05) reduced in the group treated with BmoLL, being the result similar to that obtained for the group trated with acetylsalycilic acid. In the evaluation of the antinociceptive activity of BmoLL, the treatments with doses of 15, 30, and 60 mg/kg reduced significantly (p<0.05) the number of abdominal writhings (induced by acetic acid) in 43.1%, 50.1% and 73.1%, respectively. In hot plate assay, BmoLL at 30 and 60 mg/kg showed a significant antinociceptive effect. In conclusion, BmoLL is an interesting molecule to be more deeply studied for its pharmacological applications since it did not show acute toxicity to mice and showed the biological activities described in this work.

Orientadores: Anibal Eugenio Vercesi, Fernanda Ramos Gadelha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:24:39Z (GMT). No. of bitstreams: 1 Fernandes_MarianaPinheiro_D.pdf: 41725038 bytes, checksum: ed76a74896573daa3ea8087e075ca302 (MD5) Previous issue date: 2010 / Resumo: Lectinas são proteínas ou glicoproteínas que reconhecem glicoconjugados de superfície celular. Neste trabalho, nós investigamos se a lectina de sementes de Cratylia mollis (Cramoll 1,4) tem efeitos tóxicos em Trypanosoma cruzi. Cramoll 1,4 reconheceu glicoconjugados presentes na superfície celular dos parasitas, levando a aglutinação de epimastigotas e tripomastigotas, de uma maneira dose dependente (1-50 µg/ml). Além disso, Cramoll 1,4 diminuiu a proliferação de epimastigotas; 93% de inibição foi obtida com 50 µg/ml. A incubação de células (1.25 x 108 /ml) na presença de Cramoll 1,4 (50 |ig/ml) e Ca2+ (10µM) por 1 h induziu permeabilização de membrana plasmática seguida por influxo de Ca2+ e cúmulo de Ca2+ mitocondrial, resultado que se assemelha ao efeito clássico da digitonina. Cramoll 1,4 estimulou (cinco vezes) a produção de espécies reativas de oxigênio (EROs) mitocondrial, diminuiu de maneira significativa o potencial elétrico de membrana mitocondrial (??m) e prejudicou a fosforilação de ADP. A geração de EROs induzida por Cramoll 1,4 foi diminuída de forma significativa por EGTA. A permeabilização de membrana plasmática por digitonina (20 µM) em meio contendo Ca2+ também estimulou a geração de EROs mitocondrial, mas numa proporção menor que Cramoll 1,4. A velocidade de respiração desacoplada em epimastigotas não foi afetada pelo tratamento com Cramoll 1,4 + Ca2+ mas a respiração de repouso induzida por oligomicina foi 65% maior nas células tratadas do que nos controles. Experimentos usando frações mitocondriais de T. cruzi (MTc) mostraram que, em contraste com a digitonina, a lectina diminuiu de forma significativa o ??m por um mecanismo sensível a EGTA. Em concordância com os resultados da permeabilização de membrana plasmática e prejuízo da fosforilação oxidativa pela lectina, experimentos de microscopia de fluorescência usando iodeto de propídeo revelaram que Cramoll 1,4 induziu morte de epimastigotas por necrose. Experimentos feitos com mitocôndrias isoladas de fígado de rato (MFR) mostraram que Cramoll 1,4 induziu transição de permeabilidade mitocondrial, dependente de Ca2+, com aumento na produção de EROs. Em contraste ao que foi observado em MFR, o efeito de Cramoll 1,4 em MTc é insensível a ciclosporina A, um inibidor clássico do poro de transição de permeabilidade mitocondrial. Nós mostramos que a toxicidade de Cramoll 1,4 em epimastigotas parece resultar de uma ação conjunta nas membranas plasmática e mitocondrial do parasita, sobrecarga de Ca2+ mitocondrial e produção de EROs / Abstract: Lectins are proteins or glycoproteins that recognize cell surface glycoconjugates. In this work, we investigated whether Cratylia mollis seed lectin (Cramoll 1,4) has toxic effects on Trypanosoma cruzi. Cramoll 1,4 recognized glycoconjugates present on the cell surfaces of parasites, leading to agglutination of both epimastigotes and trypomastigotes in a dose-dependent manner (1-50 µg/ml). In additional, Cramoll 1,4 decreased epimastigote proliferation; 93% inhibition was attained at 50 µg/ml. Incubation of cells (1.25 x 108 /ml) in the presence of 50 µg/ml Cramoll 1,4 and 10 Ca2+ for 1 h induced plasma membrane permeabilization followed by Ca2+ influx and mitochondrial Ca2+ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (??m) and impaired ADP phosphorylation. ROS generation induced by Cramoll 1,4 was significantly diminished by EGTA. Plasma membrane permeabilization by 20 digitonin in a Ca2+ -containing medium also stimulated mitochondrial ROS generation but at a lower rate than Cramoll 1,4. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca2+ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions (MTc) showed that, in contrast to digitonin, the lectin significantly decreased ??m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigote death by necrosis. Experiments performed with rat liver mitochondria (RLM) showed that Cramoll 1,4 induced Ca2+-dependent mitochondrial permeability transition increasing the ROS production. In contrast to RLM, the Cramoll 1,4 effect in MTc is insensitive to cyclosporin A, a classic inhibitor of mitochondrial permeability transition pore. We showed that Cramoll 1,4 toxicity to T. cruzi epimastigotes seems to result from a concerted action on the parasite's plasma and mitochondrial membranes, mitochondrial Ca2+ overload and ROS production / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / InteraÃÃes entre a resposta imune inata e adquirida participam na fisiopatologia de doenÃas auto-imunes. Embora infecÃÃes estejam associadas ao desenvolvimento de artrites crÃnicas, à possÃvel que exposiÃÃo a alguns germes, como helmintos e fungos, potencialmente influencie a prevalÃncia e/ou gravidade de doenÃas imunomediadas. Lectinas derivadas de plantas, por aÃÃo em receptores de resposta inata, podem modular inflamaÃÃo. NÃs investigamos o efeito dos extratos de Ascaris suum (AS) e de Coccidioides posadasii (CP) e de uma lectina isolada da Dioclea violacea (Dviol) na artrite induzida por zymosan (AZy). Ratos Wistar e camundongos Swiss receberam 1 mg ou 0,1 mg de zymosan intra-articular (i.art.), respectivamente. Grupos foram prÃ-tratados (30 min) com os extratos de AS (0,25 - 2,5 mg/animal; i.p ou p.o.), CP (1 - 100 Âg/animal; i.art., i.p. ou p.o.) ou Dviol (0,3 - 30 Âg i.art. ou 1 - 6 mg/Kg e.v.). Grupo nÃo-tratado (NT) recebeu Zy (i.art.) e veÃculo. Animais naive receberam apenas salina (i.art.) e veÃculo. A hipernocicepÃÃo foi avaliada atravÃs do teste de incapacitaÃÃo articular em s / 1min. O lavado articular foi usado para anÃlise do influxo celular (IC), nÃveis de nitrito e citocinas. A sinÃvia foi utilizada para histopatologia. O conteÃdo de glicosaminoglicanos (GAG) da cartilagem foi quantificado para medir dano estrutural. O extrato de AS, seja i.p. ou p.o., inibiu de forma dose-dependente a hipernocicepÃÃo e o IC na AZy em relaÃÃo ao grupo NT (P<0,01), bem como reverteu o dano articular avaliado pela quantificaÃÃo de GAG e a sinovite vista à histologia. A administraÃÃo do extrato de AS, reduziu significantemente os nÃveis de nitrito, inteleucina-1&#946; (IL-1&#946;) e IL-10, mas nÃo de fator de necrose tumoral alfa (TNF-&#945;), em relaÃÃo ao NT. Em camundongos, o extrato de AS reduziu os nÃveis de IL-10, mas nÃo de IL-1&#946; ou TNF-&#945;. O tratamento com o extrato de CP, seja i.p. ou p.o., inibiu significantemente a hipernocicepÃÃo e o IC na AZy, em relaÃÃo ao NT, no entanto, nÃo reverteu a lesÃo articular medida pela quantidade de GAG e histologia. A administraÃÃo da Dviol, em animais naive promoveu IC significante, embora apenas a maior dose (30Âg) promoveu hipernocicepÃÃo. Na AZy, a injeÃÃo i.art. da Dviol reduziu o IC e hipernocicepÃÃo de forma dose-dependente, em relaÃÃo ao NT (P<0,01). A administraÃÃo da Dviol (i.v.) reduziu ambos hipernocicepÃÃo e IC na AZy, em relaÃÃo ao NT (P<0,01). O efeito da Dviol foi revertido quando essa lectina foi prÃ-incubada com manose 1 M. Os dados mostram que um extrato de AS promove melhora funcional e protege do dano estrutural na AZy, que sÃo associados com reduÃÃo na liberaÃÃo de NO e citocinas i.art. Esse efeito independe da espÃcie e ocorre por via oral. Um extrato do fungo CP tem aÃÃo anti-inflamatÃria na AZy. Uma lectina isolada da Dviol reduz IC e hipernocicepÃÃo na AZy, provavelmente por acoplamento a um receptor de manose. Em conjunto, os resultados mostram que substÃncias que agem em receptores de resposta inata modulam a inflamaÃÃo articular imunomediada. / The interactions between innate and acquired immune responses participate in the pathophysiology of the autoimmune diseases. Though infections are associate with the development of the chronic arthritis it is possible that exposure to some germs as helminthes and fungi influences potentially the prevalence and/or gravity of the immune diseases. Lectins derivate of the plants can modulate the inflammation by action in receptors of the innate response. We investigated the effect of extracts from Ascaris suum (AS), Coccidioides posadasii (CS) and a lectin isolated from Dioclea violacea (Dviol) in zymosan-induced arthritis (ZyA). Wistar rats and Swiss mice received 1 mg or 0.1 mg zymosan intra-articular (i.art.), respectively. Groups were pretreated (30 min) with AS (0.25 - 2.5 mg/animal; i.p. or p.o.) CP (1 - 100 Âg/animal; i.art. i.p. or p.o) or Dviol (0.3 - 30 Âg; i.art. or 1 - 6 mg/kg; i.v.). Non-treated group (NT) received Zy (i.art.) and the vehicle. Naive animals received just saline (i.art.) and the vehicle. The hypernociception was evaluated through articular incapacitation test in s/1min. The joint exudate was used for evaluation of cell influx (CI), nitrite and cytokine levels. The synovium was used for histopatology. The glycosaminoglycan (GAG) content of the cartilage was quantificated for the measured of the structural damage. The AS extract both i.p. and p.o. significantly and dose-dependently inhibited CI and hypernociception in ZyA as compared to NT (P<0.01) as well as reverted articular damage assessed by quantification of the GAG and by synovitis observed in the histology. The administration of the AS extract reduced significantly levels of nitrite, interleukin-1&#946; (IL-1&#946;) and IL-10, but not tumor necrosis factor alpha (TNF-&#945;) as compared to NT. In mice, it reduced IL-10 but not IL-1&#946; and TNF- &#945;. The treatment with CP extract both i.p. and p.o. inhibited hypernociception and CI in ZyA as compared to NT, but not reverted articular injury measured by GAG and histology. The administration of the Dviol in naÃve animals promoted CI significant, though just the highest dose (30 &#61549;g) promoted hypernociception. In ZyA, Dviol (i.art.) reduced the CI and hypernociception dose-dependently (P<0.01). The administration of Dviol (i.v.) significantly reduced both the hyperalgesia and CI in ZyA as compared to NT (P<0.01). The effect of the Dviol was reverted when it was pre-incubated with mannose (1M). The date show that AS extract promote functional improve and protect of the articular damage in ZyA that are associate with reduction of the NO and cytokine (i.art.) liberation. This effect is species independent and functions orally. An extract of the fungi CP has anti-inflammatory activity in ZyA. A lectin isolated of the Dviol reduces CI and hypernociception in ZyA probably by coupling the mannose receptor. Together the results show that substances that act in receptors of the innate response modulate the immunomediate articular inflammation.

Diante da importância das lectinas no sistema imunológico avaliamos o papel da Nattectina, lectina tipo C identificada no veneno de Thalassophryne nattereri, no desenvolvimento das respostas imunes inata e específica. A Nattectina induziu peritonite em camundongos, caracterizada pelo influxo de neutrófilos e macrófagos, acompanhada da liberação de PGE2, LTB4, IL-1<font face=\"symbol\">b, IL-6, KC, MCP-1, IL-10 e IL-12p70. A resposta imune específica induzida pela Nattectina foi caracterizada pela produção de anticorpos específicos IgG, IgG1 e principalmente IgG2a com síntese de IL-10 e IFN-<font face=\"symbol\">g pelas células esplênicas re-estimuladas in vitro. A incubação de células dendríticas imaturas com a Nattectina gerou maturação destas células com aumento da expressão de moléculas MHC classe II, CD40, CD80, CD86 e expressão de MMP-2 e MMP-9 distribuídas no núcleo e no citoplasma celular, produção das citocinas IL-10 e IL-12p70 e eficiente apresentação antigênica. Concluímos que a Nattectina é capaz de induzir inflamação e resposta imune específica do tipo Th1 mediante a ativação de células dendríticas. / Due to the importance of the lectins in the immunological system we evaluated the role of Nattectin a C-type lectin identified in the venom of Thalassophryne nattereri on development of the innate and specific immune responses. Nattectin induced a significant cellular recruitment into peritoneal cavity of mice, mainly by influx of neutrophils, followed by macrophages, with synthesis of PGE2, LTB4, IL-1<font face=\"symbol\">b, IL-6, KC, MCP-1, IL-10, and IL-12p70. The specific immune response induced by Nattectin was characterized by the production of specific antibodies IgG, IgG1 and mainly IgG2a with IL-10 and IFN-<font face=\"symbol\">g synthesis by splenic cells. Incubation of immature dendritic cells with Nattectin resulted in maturation with up-regulation of MHC class II, CD40, CD80, CD86, and expression of MMP-2 e MMP-9 distributed in nucleus and cytoplasm. Mature dendritic cells produced and release IL-10 and IL-12p70 and present the antigen efficiently. We concluded that Nattectin is able to induce inflammation and Th1 specific immune response through the activation of dendritic cells.

Pseudomonas aeruginosa (PA) est la troisième bactérie impliquée dans les maladies nosocomiales et est la principale cause de mortalité des patients atteints de la mucoviscidose. PA est résistante à la plupart des traitements antibiotiques. Trouver de nouvelles stratégies thérapeutiques est devenu un enjeu majeur de santé publique, l’une d’entre elles est l’inhibition de facteurs de virulence. Parmi ceux-ci, les lectines sont des protéines impliquées dans l’adhésion et la formation de biofilm via des interactions avec des sucres (PA-IL, PA- IIL, FliC, FliD, PilA, PilY1 et CupB6).Le but de ce travail est donc de trouver des leurres moléculaires ayant une forte affinité pour ces lectines. Ceux-ci sont des motifs saccharidiques présentés de façon multivalente : glycoclusters. De part leur grande diversité structurale et leur faible quantité, un outil de criblage innovant a été développé qui consiste en une lame de verre microstructurée : le glycocluster-microarray. Les glycoclusters sont immobilisés de manière ordonnée par DNA Directed Immobilization (DDI). Deux méthodes de criblage ont été développées grâce à cet outils : 1) le criblage en solution et par compétition d’une bibliothèque de motifs saccharidiques et 2) le criblage d’une bibliothèque de glycoclusters immobilisés sur le microarray. Avec cet outil, des protocoles de mesures d’IC50 et de Kd ont aussi été fiabilisés pour caractériser les meilleurs candidats inhibiteurs des lectines. Le glycocluster- microarray présente l’avantage de n’utiliser qu’une très faible quantité de matériel (quelques picomoles) et permet de réaliser diverses analyses en parallèle.Afin de valider cet outil, une étude sur l’impact de la densité de surface en glycocluster a été menée. Le criblage de plus de 150 motifs saccharidiques a permis de sélectionner ceux ayant une forte affinité pour les lectines. L’analyse sur microarray complétée par de la modélisation moléculaire d’une bibliothèque de glycoclusters, possédant ces motifs et différentes topologies, valences et propriétés (aromaticité, charge,…), a permis d’identifier les paramètres clés dirigeant les relations structure-affinité. Une activité anti-biofilm chez PA a été démontrée avec les meilleurs glycoclusters ciblant PA-IL.Tester l’activité in vivo, chez l’animal, des meilleurs candidats est une voie à explorer. Cibler d’autres lectines comme celles présentes sur le flagelle et les pili de PA et notamment impliquées dans son adhésion précoce est aussi une voie à développer. Pour cela, des tests préliminaires ont été présentés et d’autres sont en cours faisant appel à l’utilisation de bactéries entières ainsi qu’à une détection sans marquage des lectines. / Summary: Pseudomonas aeruginosa (PA) is the third pathogen involved in nosocomial diseases and the major cause of mortality of cystic fibrosis patients. PA develops resistance to antibiotics treatments. And so, developing new therapeutic strategies is a public health issue. One of the promising strategies is to inhibit virulence factors involved in the adhesion and the biofilm formation of PA. Some of these virulence factors are lectins which interact with sugars (PA-IL, PA-IIL, FliC, FliD, PilA, PilY1 and CupB6).The goal of this work is to find molecular decoys which have a strong affinity for these lectins. These are saccharidic units with a multivalent display: glycoclusters. An innovative screening tool has been developed: the glycocluster-microarray, to study lectin/glycocluster interactions. It is a microstructured glass slide where glycoclusters are immobilized by DNA Directed Immobilization (DDI). Two screening methods have been developed with this microarray: 1) the screening in solution and by competition of a saccharidic units library and2) the screening of a glycoclusters library immobilized on the microarray. Protocols of IC50 and Kd measurements have also been developed with this tool to characterize the best lectins inhibitors. This tool allows to use few amount of material (few picomoles) and to do parallel analysis.To validate the microarray, a study of the impact of glycoclusters surface density has been done. The screening of more than 150 saccharidic units allowed the selection of the ones that display the best affinity forlectins. The analysis, on microarray and molecular simulations, of the glycoclusters library displaying thesesaccharidic units and several topologies, valences and properties (aromaticity, charge,…) enable to identify key parameters of structure-affinity relationships. An anti-biofilm activity has been observed for the best glycoclusters targeting PA-IL.Testing in vivo activity of these best candidates will be explored. Targeting others lectins such as the ones on the flagella and pili of PA and involved in the early adhesion needs also to be developed. To this end, preliminary tests have been showed and some are in progress.

Le trypanosome sud américain, Trypanosoma cruzi, transmis par un insecte hématophage de type triatome est le protozoaire connus pour causer la maladie de Chagas chez l’Homme. Le cycle de vie de ce parasite alterne à la fois sur le type d’hôte, insecte ou hôte vertébré, et sur la forme :trypomastigote pour la forme quiescente et amastigote et épimastigote pour les formes prolifératives. Concernant la forme, seuls les parasites épimastigotes évoluent et prolifèrent dans le tube digestif des triatomes et possèdent un système endocytaire actif nécessaire à leur besoin énergétique. Toutefois, cette endocytose est restreinte à deux sites membranaires, la poche flagellaire et le cytostome, à partir desquels se créent des cargos endocytaires. Ces cargos endocytaires fusionnent ensuite avec un réseau vésiculaire endosomal qui délivre son contenu dans des réservosomes, compartiments similaires aux lysosomes.Chez le trypanosome africain (Trypanosoma brucei brucei), l’endocytose ne se réalise qu’au niveau de la poche flagellaire. Certaines protéines appartenant à cette voie endocytaire sont modifiées par de longues chaines de résidus poly-N-acétyllactosamine (pNAL) de manière post-traductionnelle. Initialement, il a été proposé que ces résidus puissent agir en tant que signal de tri dans le processus d’endocytose chez ces parasites.En nous basant sur les travaux qui ont été réalisés chez le trypanosome africain, nous nous sommes proposés d’approfondir les connaissances sur la voie endocytaire du trypanosome sud américain (Trypanosoma cruzi) qui est beaucoup moins étudié. Pour ce faire, à l’aide de deux lectines, la tomatolectine et la lectine de Griffonia simplicifolia qui présentent respectivement une affinité pour les résidus pNAL et les résidus N-acétylglucosamine (GlcNAc) en fin de chaine, nous avons pu enrichir et caractériser par LC-MS², 173 glycoprotéines putatives dont plus de 13% sont localisées dans la voie endocytaire. Parmi les protéines identifiées, en plus des nombreuses hydrolases lysosomiales, nous avons pu identifier une lectine de type C localisée dans la partie antérieure des parasites, au niveau des principaux sites endocytaires. Cette dernière possédant de nombreux résidus en commun avec les récepteurs de type scavengers, elle pourrait donc jouer un rôle important dans la fixation et l’endocytose de certains nutriments.Nos travaux ont ainsi permis d’établir que similairement aux trypanosomes africains, Trypanosoma cruzi possèdent des glycoprotéines modifiées par des N-glycanes contenant des pNAL. Nos travaux ont également permis d’établir que ces résidus s’associent préférentiellement aux glycoprotéines de la voie endocytaire (au niveau du cytostome et du réservosome) de la forme épimastigote. L’ensemble des résultats obtenus durant cette thèse tendent à montrer que les résidus pNAL des glycoprotéines présentes dans la voie endocytaire ont été conservées entre les deux parasites étudiés (Trypanosoma cruzi et Trypanosoma brucei brucei). / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La tuberculose (TB) est une maladie très répandue qui provoque la mort de plus d'un million de personnes dans le monde chaque année. Cela représente un véritable problème de santé publique qui nécessite le développement de nouveaux médicaments et traitements afin de mieux lutter contre cette maladie destructrice. Dans ce contexte, il est important de comprendre la relation entre l'agent étiologique, Mycobacterium tuberculosis (Mtb) et le système immunitaire. Les mycobactéries sont recouvertes de structure glycolipidiques qui pourraient être reconnus par des récepteur spécifiques appelés lectine de type C. Ici, nous avons étudié le rôle de deux lectines récemment identifiées qui n'ont pas été étudié dans un contexte de TB, CL-LK et DCIR. Au cours des dernières années, de nouvelles lectines solubles ont été identifiées et parmi elles ; CL-K1 a été définie par un criblage de divers ligands glycosylés, comme étant un potentiel récepteur de motifs présents sur la paroi de Mtb. Dans le sang, CL-K1 est associé avec une autre lectine soluble du nom de CL-L1, formant un complexe du nom de CL-LK. Par des expériences in vitro, notamment via des approches biochimique et d'analyses cytométriques, nous avons pu confirmer que CL-LK peut se lier à Mtb et encore plus particulièrement à la coiffe mannose présent sur le lipoarabinomannane, un constituant majeur de la mycomembrane. Les souris déficientes en CL-K1, une sous unités de CL-LK, ne présentent pas d'altération particulière de susceptibilité à Mtb. Cependant, nous avons pu mettre en évidence que la quantité de CL-LK dans le sérum de patient atteint d'une TB active est réduite comparé aux patients contrôles, et que cela corrèle de manière inverse à la réponse immune induite par le pathogène. Ces résultats indiquent que CL-LK pourrait présenter un intérêt comme élément de futur diagnostics ou suivi de traitement. Dans une seconde partie de la thèse, nous nous sommes concentré sur la lectine DCIR dans la réponse immune envers l'agent étiologique de la TB. DCIR est exprimé dans les lésions pulmonaires de macaques infectés par Mtb à la fois durant les infections asymptomatiques et dans les infections conduisant à une TB active. In vitro, une analyse globale de l'expression génique couplée à des validations par RT-qPCR ont pu révéler que suite à une infection par Mtb, l'expression d'un grand nombre de gènes répondant à l'interféron (IFN) de type I est inhibée dans les cellules dendritiques issues de moelle osseuses de souris déficientes en DCIR comparées à celles issues de souris sauvages. Cette inhibition corrèle avec une phosphorylation excessive de SHP2 dans les cellules dépourvues de DCIR, suivies d'une déphosphorylation de STAT1, laquelle est impliquée dans la régulation de la réponse à l'IFN de type I. L' IFN de type I est connu comme pouvant inhiber la production d' IL-12 dans les cellules dendritiques, et en effet, nous avons pu démontrer que l'inhibition de la signalisation relative à l'IFN de type I dans les cellules dendritiques déficientes en DCIR est associée avec une augmentation de la production d'IL12p70 ainsi qu'à une plus grande capacité de ces cellules à stimuler la prolifération de lymphocytes Th1 producteur d'IFN?. Les souris déficientes en DCIR contrôlent mieux l'infection que les souris sauvages, ce qui corrèle également avec une plus grande production d'IL12p70, une plus forte réponse des lymphocytes Th1, ainsi qu'à une augmentation de l'inflammation et de l'infiltration cellulaire dans les poumons des souris déficientes. Cette inflammation excessive est caractérisée par une augmentation de production du TNF-a et de iNOS dans les poumons. En conclusion, nos résultats révèlent une nouvelle voie moléculaire par laquelle les lectines peuvent moduler l'équilibre entre l'inflammation induite par l'infection et le contrôle du pathogène par l'intermédiaire de la modulation de la signalisation relative à l'IFN de type I dans les cellules dendritiques. / Tuberculosis (TB) is a wide-spread disease which causes the death of more than one million of people around the world each year. This represents a really harmfull healthcare and new drugs and treatments are always in development to better fight this destructive illness. In this context, it is crucial to understand the relation between the etiological agent, Mycobacterium tuberculosis (Mtb) and the immune system. Mycobacteria are recovered by some glycolipid structure which could be recognized by specific receptor called C-Type Lectin. Here we investigated the role of two recently highlights C-type lectins which haven't been studied in a tuberculosis context, CL-LK (Collectin Liver Kidney) and DCIR (Dendritic Cell (DC) ImmunoReceptor). In the recent years, novel soluble lectins were identified and belong these; CL-K1 (Collectin Kidney 1) was identified by a glycan array as a potential receptor in the sugar complex recognition of M.tb. In blood, CL-K1 is linked with another soluble lectin called CL-L1 (Collectin Liver 1) and form a complex CL-LK molecule. With some in vitro experiment, notably with biochemistry approach and cytometer analysis, we were able to confirm that CL-LK can bind Mtb and more particularly the mannose residue presents on the Lipoarabinomannan, a major constituent of the mycomembrane. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to Mtb. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes. In a second part of this thesis, we have focused our interest on the C-Type lectin DCIR in immunity to the tuberculosis agent, Mtb. DCIR is expressed in pulmonary lesions in Mtb-infected non-human primates during both symptomless infection and active TB disease. In vitro, global gene expression profiling coupled to RT-qPCR validation revealed that upon Mtb infection, the expression of a number of interferon (IFN)-responsive genes was impaired in DCIR-deficient murine bone marrow-derived DCs, compared to in wild-type cells. This inhibition correlated with an excessive phosphorylation of Src homology 2 domain tyrosine phosphatase (SHP)2 in DCIR-KO cells, followed by a subsequent dephosphorylation of Signal transducer and activator of transcription1(STAT1) which is crucially involved in the regulation of the type I IFN.Type I IFNs are known to inhibit interleukin (IL)-12 production in DCs, and indeed, we found that impaired IFN signaling in DCIR-deficient DCs was associated with an increased production of IL-12p70 and an increased ability of Mtb-infected cells to stimulate IFN?-producing Th1 lymphocyte proliferation. DCIR-deficient mice controlled Mtb infection better than wild-type animals, which correlated with an increased production of IL-12p70, an increased proliferation of Th1 lymphocytes, and an increased inflammation and cell infiltration in the lungs of DCIR-KO animals. This excessive inflammation is characterized by an increased production of tumor necrosis factor alpha (TNFa) and inducible nitric oxide synthase (iNOS) in the lungs. Taken together, our results reveal a novel pathway by which a C-Type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs.

Les interactions sucre-lectine jouent un rôle très important dans de nombreux processus biologiques comme les infections par des virus ou des bactéries. Toutefois, ces interactions étant faibles, la présentation de manière multivalente des résidus saccharidiques est nécessaire pour obtenir une augmentation significative des constantes d'association. Une technique basée sur l'utilisation de glycooligonucléotides et d'une puce à ADN utilisée comme plateforme d'ancrage a permis d'étudier l'affinité d'un grand nombre de composés envers les lectines PA-IL et PA-IIL de Pseudomonas aeruginosa et la lectine BambL de Burkholderia ambifaria. Les glycooligonucléotides ont été synthétisés, à partir de blocs de construction synthétisés en aval, en utilisant la chimie des acides nucléiques supportée et automatisée (phosphoramidites et H-phosphonate) ainsi que des réactions de « click chemistry » (la cycloaddition 1,3-dipolaire catalysée par le cuivre (I) ou le couplage thiol par addition de type Michael ou par substitution nucléophile d'un dérivé bromoacetamide).Les glycoclusters ayant montrés une bonne affinité envers les lectines cibles ont été sélectionnés et resynthétisés en solution sans l'étiquette ADN à l'échelle de la centaine de milligrammes. Les glycoclusters ainsi synthétisés en deux ou trois étapes avec une seule purification ont pu être évalués par quatre techniques d'analyse des interactions (HIA, ELLA, SPR et ITC) en présence des lectines PA-IL, PA-IIL et BambL. Nous avons trouvé un tétragalactocluster et un tétrafucocluster possédant une forte affinité envers la lectine PA-IL et BambL respectivement avec des valeurs de Kd de 157 nM et 43 nM. / Carbohydrate-lectin interactions play a key role in various biological processes such as infection by viruses or bacteria. As these interactions are weak, the multivalent association of carbohydrate is necessary to increase the binding constant. We used glycooligonucleotide and DNA chip to study the affinity of diverse compounds to PA-IL and PA-IIL lectins of Pseudomonas aeruginosa and Bambl lectin of Burkholderia ambifaria. Glycooligonucleotides were synthesized with previously prepared building blocks, using automated supported nucleic acid chemistry (phosphoramidites and H-phosphonate) and “Click chemistry” (copper (I) catalyzed 1,3-dipolar cycloaddition, thiol coupling by Michael addition and nucleophilic substitution of bromoacetamide derivative).Glycoclusters showing the better affinities toward the lectins have been synthesized to a hundred milligrams scale in solution without the DNA tag. The synthesis processes in two or three steps and only one final purification. Their interactions with the lectins PA-IL, PA-IIL and BambL were studied by several assays (HIA, ELLA, SPR and ITC). A tetragalactocluster and a tetrafucocluster showed high affinity toward respectively the lectin PA-IL (Kd = 157 nM) and the lectin BambL (Kd = 43 nM).

No description available.