Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-08-12T16:15:37Z No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-23T15:31:26Z (GMT) No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / Made available in DSpace on 2016-08-23T15:31:26Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / CAPES; CNPq; FAPESB; National Institute of Health; / A prevalência de infecção por helmintos é negativamente associada à prevalência de alergia, e alguns trabalhos têm demonstrado uma inversa associação entre infecções por helmintos e gravidade de asma. Neste trabalho, foi avaliada a resposta imune específica para alérgeno 1 de Dermatophagoides pteronyssinus (Der p 1) em asmáticos infectados com helmintos de uma área endêmica no município do Conde-Bahia (Grupo 1), comparando-se com a resposta em asmáticos não infectados de uma área não endêmica, Salvador-Bahia (Grupo 2). Os indivíduos foram selecionados utilizando-se questionário ISAAC modificado e exames parasitológicos de fezes por meio da técnica de Kato-Katz. Foram realizados testes cutâneos de leitura imediata com aeroalérgenos, dosagem de IgE específica para Der p 1, coleta de amostras de poeira domiciliar para estudo da fauna acarina, além da avaliação da resposta imunológica pela produção de citocinas por células mononucleares do sangue periférico estimuladas, in vitro, com Der p 1. A dosagem de citocinas no sobrenadante das culturas foi realizada pelo método de ELISA, a expressão de IL-4 pela técnica de RT-PCR e a fonte produtora de IL-10 foi avaliada utilizando-se citometria de fluxo. Foi observada menor positividade aos testes cutâneos de leitura imediata com aeroalérgenos nos asmáticos infectados por helmintos, incluindo S. mansoni (grupo 1), quando comparados com asmáticos não infectados. Embora a positividade aos testes cutâneos tenha sido baixa no grupo de asmáticos infectados, em 40,6% dos indivíduos foi observada IgE específica para o Der p 1 acima de 0,70 KU/L (classe II). Com relação à produção de citocinas, observou-se que células de asmáticos infectados pelo S. mansoni produziram baixos níveis de IL-5 em culturas estimuladas com Der p 1, quando comparados à produção desta citocina por células de indivíduos não infectados (p<0,0001). Da mesma forma, a razão da densitometria de IL-4/HPRT no grupo de infectados foi mais baixa do que no grupo de não infectados (p<0,05). Em contraste, os níveis de IL-10 foram altos em cultura de células de asmáticos infectados por S. mansoni e baixos ou indetectáveis no grupo controle (p=0,0018). Observou-se uma associação positiva entre carga parasitária de S. mansoni e níveis de IL-10 em culturas estimuladas com Der p 1 (= 0,3212, p=0,012). A adição de IL-10 recombinante humana (rhIL-10) às culturas de asmáticos não infectados resultou em diminuição na produção de IL-5 (p<0,05). Os indivíduos do grupo 1 foram tratados com anti-helmínticos e houve redução da produção de IL-10 específica para Der p 1 (p<0,05), associada à piora dos sintomas de asma após o tratamento. Por fim, os dados mostraram que, em asmáticos infectados com S. mansoni, a IL-10 foi produzida principalmente por células T CD8+ e monócitos e, no grupo de não infectados, esta citocina foi produzida basicamente por monócitos. Este estudo sugere que a IL-10 é uma citocina supressora da resposta imune inflamatória do tipo Th2 em asmáticos infectados por helmintos, incluindo S. mansoni e que as células T CD8+ constituem uma importante fonte produtora desta citocina nos indivíduos infectados.

Le lymphocyte B (LB) des patients lupiques est réfractaire à tous les procédés décrits à ce jour pour activer une fonction régulatrice B (Breg). Il constitue de ce fait un modèle intéressant d’étude de la déficience Breg chez l’Homme et soulève de nombreuses interrogations. Est-il possible de restaurer un défaut d’activation de la régulation LB? Si oui est-il possible d'agir à temps et le plus efficacement possible, et comment s'y prendre? Ou au contraire, est-ce un état irréversible de la cellule B? Ce travail de thèse a pour objectif principal de répondre à cette problématique essentielle à notre compréhension du Breg. Grace à un polypeptide de synthèse le Glatiramer acetate (GA), nous montrons que la restauration de la fonction régulatrice d’un Breg chez les patients lupiques est possible. Le compartiment LB mémoire fixe fortement le GA et la pré-sensibilisation par le GA permet d’augmenter le potentiel régulateur des LB mémoires mais n’affecte aucunement celui des LB matures. Le GA exerce deux actions majeures sur le LB mémoire. D’une part, il génère une meilleure capacité d’inhibition de la prolifération T, dont le mécanisme est associé à un contact cellulaire impliquant les molécules HLA-DR. D’autre part, il favorise un contrôle plus efficace de la polarisation Th1 qui est très probablement associé à sa capacité à induire la production d’IL-10 dans ces LB. Enfin, le GA modifie le phénotype des LB mémoires puisque l’expression de CD5, IL-21R, ou encore PD-1 est significativement augmentée, autant de molécules impliquées dans la fonction suppressive et dans la production d’IL-10. En conclusion, nous montrons qu’amplifier une fonction régulatrice et surtout la restaurer lorsqu’elle est défaillante chez les malades, est parfaitement possible in vitro. Face à l’engouement suscité par le développement de procédés favorisant l’expansion des Bregs chez la souris à des fins thérapeutiques, l’enjeu est aujourd’hui d’être en mesure d’extrapoler de telles démarches chez l’Homme. Ce travail, avec toute la modestie requise, contribuera à faire naître un nouvel élan vers de telles perspectives. / B cell in systemic lupus erythematous (SLE) is unresponsive to all methods described to date, to activate B cells regulatory (Breg) function. Therfore, it is an interesting model to study the Breg deficiency in Human, and highlights many questions: is there a way to restore a defect of the Breg activation ? If yes, how can we act more efficiently ? Or in contrast, is it an irreversible state of the B cell? Glatiramer Acetate (GA) is a synthetic polypeptide used in the treatment of multiple sclerosis. We show that Breg activity of SLE B cells can be restored after stimulation with GA. Interestingly, memory B cells bound high level of FITC-conjuated GA in contrast to mature B cells. We desmonstrate that GA can increased specifically the regulatory activities of memory B cells. GA exerts two major actions on the memory B cells. It generates an improved capacity of inhibition of the T cell proliferative response, whose mechanism is associated with a cellular contact involving HLA-DR molecules. In addition, GA supports a more effective control of the Th1 polarization which is most likely associated with its capacity to induce the production of IL-10 in these B cells. Finaly, GA modifies the memory B cell phenotype since the expression CD5, IL-21R, or PD-1 is significantly increased, all molecules involved in the suppressive function and the IL-10 production. In conclusion, our results show for the first time that amplification of Breg function and additionally its restoration when it is defective in patients, can be perfectly achieved in vitro. Currently, while the development of new process supporting the expansion of Bregs in the mouse model exist, the challenge is to extrapolate such methods in human. Through the control of their regulatory potential, regulatory B cells could be the targets of novel therapeutic approach in autoimmune diseases. This study might open up new horizons in this field.

Data compiled by the Centers for Disease Control demonstrates that African American women, in particular young people between the ages of 14-25, have an increased incidence of infection with the sexually transmitted pathogen Neisseria gonorrhoeae. Estradiol is a key regulatory hormone in female reproductive function. It has been studied extensively in the cardiovascular field, and has been linked to breast and endometrial cancers in women. However, its impact on infectious diseases is largely unknown. Given what is known about the effect of estradiol on immunologic and inflammatory disorders in women, I hypothesize that estradiol alters the infectivity of Neisseria gonorrhoeae in the female reproductive tract by altering the host inflammatory immune response. This may explain a risk factor for increased rates of infection in some populations. I sought to develop a relevant in vitro model. After screening a number of candidate cell lines, I selected the human endometrial adenocarcinoma cell line, Ishikawa. These cells express specific estrogen receptors and respond to exogenous estrogen stimulation. Estrogen treatment of Ishikawa cells did not have an impact on the invasion of N. gonorrhoeae, nor did it impact bacterial growth. However, gonococcal induced chemokine secretion was reduced by estrogen, as measured by interleukin-8 secretion. I conclude that estrogen blunts the inflammatory response to Neisseria gonorrhoeae without altering bacterial infectivity.

?rea de concentra??o: Farmacologia de produtos naturais e plantas medicinais. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:08:56Z No. of bitstreams: 2 michaelle_geralda_santos.pdf: 2124437 bytes, checksum: 4efd2da996375e9bd402d5e8df0ae10f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:10:40Z (GMT) No. of bitstreams: 2 michaelle_geralda_santos.pdf: 2124437 bytes, checksum: 4efd2da996375e9bd402d5e8df0ae10f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T13:11:27Z (GMT) No. of bitstreams: 2 michaelle_geralda_santos.pdf: 2124437 bytes, checksum: 4efd2da996375e9bd402d5e8df0ae10f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-01-07T13:11:27Z (GMT). No. of bitstreams: 2 michaelle_geralda_santos.pdf: 2124437 bytes, checksum: 4efd2da996375e9bd402d5e8df0ae10f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2014 / A Eriosema crinitum (Kunth) G. Don ? utilizada no Vale do Jequitinhonha como planta anti-inflamat?ria na forma de decoc??o de suas ra?zes. Tendo em vista a complexidade do processo inflamat?rio, o qual envolve a ativa??o de c?lulas do sistema imune e produ??o de diversos mediadores, o objetivo desse estudo foi realizar uma triagem fitoqu?mica e avaliar a poss?vel atividade anti-inflamat?ria, in vitro, do extrato diclorometano-etan?lico de ra?zes de E. crinitum, atrav?s da avalia??o do efeito do mesmo sobre a prolifera??o de linf?citos, a produ??o de citocinas e da ciclooxigenase 2 (COX-2). A triagem fitoqu?mica do extrato foi realizada por meio de rea??es cromog?nicas, fluorog?nicas e de precipita??o, seguidas da an?lise em cromatografia l?quida de alta efici?ncia acoplada a detector de arranjo de diodos (CLAE-DAD). A citotoxicidade do extrato sobre hem?cias, ap?s 4 horas de cultura, e sobre leuc?citos do sangue perif?rico humano, ap?s 24 horas ou 5 dias de cultura, foi avaliada por meio do teste de atividade hemol?tica e pelo m?todo de exclus?o com azul de Tripan, respectivamente. Na avalia??o do efeito do extrato sobre a prolifera??o de linf?citos, por citometria de fluxo, c?lulas mononucleares do sangue perif?rico humano (PBMC) foram incubadas, por 5 dias, em meio de cultura contendo o mit?geno Fitohemaglutinina (PHA), na presen?a ou aus?ncia do extrato nas concentra??es de 25 ?g/mL, 12,5 ?g/mL e 6,25 ?g/mL. Essas mesmas culturas foram realizadas no tempo de 36 horas para an?lise da citocina IL-2, por ELISA. Para an?lise da produ??o das citocinas TNF-? e IFN-?, culturas de sangue total foram estimuladas com Miristato Acetato de Forbol (PMA) e tratadas com o extrato nas concentra??es de 50 ?g/mL, 25 ?g/mL e 12,5 ?g/mL. Essas concentra??es foram utilizadas em culturas de sangue total estimuladas com lipopolissacar?deo (LPS) para investiga??o do efeito do extrato sobre a express?o de COX-2. A produ??o de citocinas e de COX-2 foi analisada por citometria de fluxo. Os resultados evidenciaram a presen?a de terpenos e das subclasses dos flavonoides: flavonas e flavon?is no extrato. O extrato, nas concentra??es iguais ou inferiores a 50 ?g/mL, n?o foi t?xico para culturas celulares de 24 horas e, para culturas de 5 dias, as concentra??es n?o t?xicas foram iguais ou inferiores a 25 ?g/mL. Quanto ? a??o anti-inflamat?ria, o extrato n?o inibiu a produ??o das citocinas TNF-? e IFN-? e tamb?m n?o foi capaz de reduzir a express?o de COX-2. No entanto, o extrato inibiu a prolifera??o de linf?citos e suas subpopula??es T CD4 e TCD8, tendo uma efic?cia em rela??o ? Dexametasona de 71,65%, 53,14% e 167,94% para linf?citos totais, T CD4 e T CD8, respectivamente. O extrato tamb?m reduziu os n?veis de IL-2 nas culturas estimuladas com PHA. Esses achados sugerem que o extrato apresenta um efeito inibidor, principalmente sobre a prolifera??o de c?lulas T CD8, associado ? inibi??o na produ??o de IL-2. Diante dos resultados apresentados, os princ?pios ativos presentes na planta parecem exercer suas fun??es anti-inflamat?rias regulando a prolifera??o de linf?citos T, principalmente os linf?citos T CD8. Essa fun??o poderia ser explorada em desordens de natureza inflamat?ria-linfoproliferativa, bem como preven??o da rejei??o de transplantes de ?rg?os. Para tal, ensaios adicionais s?o necess?rios para investigar e identificar os constituintes ativos e os mecanismos moleculares envolvidos na a??o inibit?ria apresentada pelo extrato. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT The Eriosema crinitum (Kunth) G. Don is used in Jequitinhonha Valley as anti-inflammatory plant in the form of decoction of its roots. Given the complexity of the inflammatory process, which involves the activation of immune cells and production of several mediators, the aim of this study was to perform a phytochemical screening and evaluate the potential anti-inflammatory activity, in vitro, the dichloromethane-ethanol extract of the roots of E. crinitum by assessing the effect of the same on lymphocyte proliferation, production of cytokines and cyclooxygenase-2 (COX-2). The phytochemical screening of the extract was performed using chromogenic reactions, fluorogenic and precipitation followed by analysis in high efficiency liquid chromatography coupled with a diode array detector (HPLC-DAD). The cytotoxicity of the extract after 24 hours or 5 days of culture on erythrocytes and leukocytes in human peripheral blood was evaluated by the test of hemolytic activity and the method of exclusion with Trypan blue, respectively. In evaluating of the effect of the extract on the proliferation of lymphocytes by flow cytometry, mononuclear cells from human peripheral blood (PBMC) have been incubated for 5 days in a culture medium containing the mitogen Phytohemagglutinin (PHA) in the presence or absence of extract at concentrations of 25 ?g/mL, 12.5 ?g/mL and 6.25 ?g/mL. These cultures too were performed in the time of 36 hours for analysis of IL -2 by ELISA. To analyze the production of cytokines TNF-? and IFN-?, whole blood cultures were stimulated with Phorbol Myristate acetate (PMA) and treated with the extract at concentrations of 50 ?g/mL, 25 ?g/mL and 12.5 ?g/mL. These concentrations were used in whole blood cultures stimulated with lipopolysaccharide (LPS) to investigate the effect of the extract on the expression of COX-2. Cytokine production and COX-2 was analyzed by flow cytometry. The results showed the presence of terpenes and subclasses of flavonoids: flavones and flavonols in extract. The extract, in concentration equal or greater than 50 ?g/mL was not toxic to cell cultures after 24 hours and in culture after 5 days, non- toxic concentrations were equal to or greater than 25 ?g/mL. In relation to the anti-inflammatory action, the extract did not inhibit the production of TNF-? and IFN-? and also was not able to reduce the expression of COX-2. Moreover, the extract inhibited the proliferation of lymphocytes and their subpopulations CD4 and CD8, and its efficacy compared to dexamethasone was 71.65%, 167.94% and 53.14% for total lymphocytes, CD4 and CD8, respectively. The extract also reduced the levels of IL-2 in cultures stimulated with PHA. These findings suggest that the extract has an inhibitory effect, especially on the proliferation of CD8 T cells associated with the inhibition of IL-2. Considering the presented results, the active molecules present in the plant seem to play their anti-inflammatory functions by regulating the T lymphocytes proliferation,?mainly T CD8 lymphocytes. This function could be exploited in inflammatory lymphoproliferative disorders, as well as in the prevention of transplant rejection. Further trials are needed to investigate the active constituents and the molecular mechanisms involved in the inhibitory action displayed by the extract.

?rea de concentra??o: Neuroimunoendocrinologia. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T18:24:18Z No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T18:24:49Z (GMT) No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-01-04T18:24:49Z (GMT). No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / A osteartrite de joelho (OAj) ? uma doen?a que afeta principalmente os idosos e pode levar a grandes limita??es f?sicas e funcionais. No entanto, os efeitos espec?ficos da terapia por exerc?cios, especialmente a caminhada sobre o sistema imunol?gico, s?o desconhecidas. Portanto, este estudo teve como objetivo analisar o efeito de 12 semanas de caminhada (3x / semana) no perfil de leuc?citos, nos n?veis plasm?ticos de interleucina (IL-6), do fator de necrose tumoral alfa (TNF-?), dos receptores sol?veis de TNF-? (sTNFR1 e sTNFR2), e do fator neurotr?fico derivado do c?rebro (BDNF) a partir de plasma retirado do sangue perif?rico de mulheres idosas com OAj. Al?m disso, as avalia??es cl?nicas e funcionais (teste de WOMAC, teste de caminhada de 6 minutos, SF-36, a percep??o da dor) foram realizadas. Dezesseis mulheres (idade: 67 ? 4 anos, ?ndice de massa corporal: 28,07 ? 4,16 kg/m2) participaram de um programa de caminhada (36 sess?es de fisioterapia) e de um esfor?o f?sico (01 sess?o de fisioterapia). As vari?veis foram avaliadas antes e ap?s 12 semanas de treinamento com dura??o (30-55 min) e intensidade (70-80% da FCm?x) progressivamente maiores. As amostras de sangue coletadas foram analisadas com um contador de c?lulas, cit?metro de fluxo e pelo m?todo ELISA. As sess?es de fisioterapia resultaram em um aumento de 47% da qualidade de vida (p <0,05) e um aumento de 21% no VO2max (p <0,0001) em mulheres idosas com OAj. Al?m disso, houve uma redu??o nas c?lulas T CD4 + (antes 46,59 ? 7%, depois 44,58 ? 9%, p = 0,0189) e uma intensidade de fluoresc?ncia mais elevada para CD18 + CD4 + (antes 45,30 ? 10, depois de 64,27 ? 33, p = 0,0256) e CD18 + CD8 + (antes: 64,2 ? 27, depois de 85,02 ? 35, p = 0,0130). A ET aumentou a concentra??o plasm?tica de sTNR1; no entanto, diminuiu a concentra??o de plasma de sTNFR2, quando comparado com os n?veis em repouso de pacientes. O exerc?cio agudo afetou diferencialmente os n?veis de sTNFR1 dependente de quando as amostras foram tomadas, antes e ap?s o treinamento aer?bico. No entanto, os n?veis de sTNFR2 n?o foram afetados pelo treinamento. O exerc?cio agudo aumentou os n?veis de BDNF apenas antes do per?odo de treinamento de 12 semanas (p <0,001). Al?m disso, houve aumento das concentra??es plasm?ticas de BDNF (p <0,0001) e melhora em par?metros cl?nicos (funcional p <0,001; percep??o da dor p <0,01). A varia??o dos n?veis de receptores sol?veis correlacionou-se com a melhora funcional; no entanto, os marcadores inflamat?rios de osteoartrite (IL-6 e TNF-?) n?o foram afetados pelas 36 sess?es de fisioterapia. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT Osteoarthritis of the knee (kOA) is a disease that mainly affects the elderly and can lead to major physical and functional limitations. However, the specific effects of exercise therapy (ET), specially walking and particularly on the immune system, are unknown. Therefore, this study aimed to analyze the effect of 12 weeks of walking (3x/week) on the leukocyte profile, levels of interleukin 6 (IL-6), tumor necrosis factor alpha, (TNF-?), soluble forms of the TNF-? receptor (sTNFR1 and sTNFR2), and brain-derived neurotrophic factor (BDNF) from plasma taken from the peripheral blood of elderly women with kOA. Additionally, clinical and functional assessments (WOMAC test, 6-min walk, SF-36, pain perception) were performed. Sixteen women (age: 67 ? 4 years, body mass index: 28.07 ? 4.16 kg/m2) participated in a walking program and physical exertion. The variables were assessed before and after 12 weeks of training with a progressively longer duration (30-55 min) and higher intensity (70-80% of HRmax). The blood samples were collected for analysis with a cell counter, the Scan Fac flow cytometer and were measured by ELISA. The ET resulted in a 47% enhancement of the self-reported quality of life (p <0.05) and a 21% increase in the VO2max (p <0.0001) in elderly women with kOA. Furthermore, there was a reduction in CD4+ cells (before 46.59?7%, after 44.58?9%, p=0.0189) and a higher fluorescence intensity for CD18+CD4+ (before 45.30 ? 10, after 64.27 ? 33, p=0.0256) and CD18+CD8+ (before: 64.2 ?27, after 85.02 ?35, p=0.0130). Aerobic training increased the plasma concentration of sTNR1; however, it decreased the plasma concentration of sTNFR2, when compared with levels of resting patients. Acute exercise differentially affects the levels of sTNFR1 dependent on when the samples were taken, before and after aerobic training. However, the levels of sTNFR2 were not affected by training. The acute exercise increased the levels of BDNF only before the 12-week training period (p<0.001). Moreover, the training augmented the plasma concentrations of BDNF (p<0.0001) and improved clinical parameters (functional p<0.001; pain perception p<0.01). The variation in the levels of soluble receptors correlated with functional improvement; however, the inflammatory osteoarthritis markers (IL-6 and TNF-?) were unaffected by the walking exercises, in physical therapy.

This thesis deals with an italian writer Luigi Pirandello and with one of his most famous novel: Il fu Mattia Pascal. The main aim is to find the examples of Pirandellos humour in this novel and to prove, that the novel is rightly called one of his first humour compositions. At the beginning of the thesis I introduce the writer and his works generally. Than I focus on his essay LUmorismo, which is important for analysis of the novel. In this essay Pirandello summarized the concept of his conception of humour. On the basis of the essay I analyse the novel.

This thesis was focused on the pro-inflammatory cytokines IL-1beta and IFN-gamma. Absolute and differential leukocyte count was also monitored. The experiment was conducted at 8 clinically healthy heifers, hybrids of Holstein and Czech Pied that have been housed by tethering in stalls and fed with a standard diet. The inflammatory reaction was induced by lipopolysaccharide (LPS; 5 ug in 20 ml PBS), as a control a phosphate buff-ered saline (PBS) was used. Results were measured at 1, 2, 3 and 7 days after stimulation of mammary gland by above-mentioned factors. Concentration of each cytokine was detected by a sandwich ELISA using commercially available kits. At 1 day after stimulation of mammary gland by LPS and PBS an average number of leukocytes, which was statistically significantly higher in the case of stimulation by LPS (P <0.01), was detected. After 7 days there was a significant decrease in the total number of leukocytes. There has also been a shift in the differential leukocyte count. Most abundant cell type were neutrophils, whose number was higher in the case of stimulation by LPS. Between day 1 and day 7 after challenge, there was a gradual reduction in the proportion of neutrophils. In the same period an increase in the proportion of macrophages and lymphocytes was detected. Concentration of IL-1beta also increased, 1 day after the activation a striking increase has been detected. In following days there was gradual decline of IL-1beta concentration almost to the level prior to treatment of the mammary gland. In the case of IFN-gamma similar pattern in the form of strong growth and a subsequent gradual decline in concentration to the original values was detected. There was found positive correlation between the increase in IL-1beta and IFN-gamma concentration and a shift in the differential leukocyte count in favor of neutrophils, which confirmed the important role of these pro-inflammatory cytokines in the establishment of inflammatory response and the mobilization of the components of natural and specific immunity.

La BPCO se traduit rapidement par l'apparition d'une susceptibilité aux infections liées aux atteintes des mécanismes de défense du poumon. Les travaux antérieurs de l’équipe montrent qu'une altération de la réponse IL-17/IL-22 et de la fonction des cellules dendritiques (DC) participe au développement de l’exacerbation de la BPCO par les bactéries. Les mécanismes responsables de ce défaut de réponse ne sont pas élucidés. Au cours de cette thèse, nous nous sommes intéressés aux points suivants :1-Mécanismes cellulaires responsables du défaut de production d'IL-17 et d'IL-22 au cours de l'infection.Les cellules présentatrices d'antigène (APC) et en particulier, les DC jouent un rôle essentiel dans la réponse antimicrobienne, par leur fonction de phagocytes et par l’activation et la polarisation de cellules immunitaires innées et adaptatives. Sur des modèles murins d’exacerbation de la BPCO par Streptococcus pneumoniae ou Haemophilus influenzae non typable (NTHi), nous avons réalisé des tris de macrophages, DC et monocytes inflammatoires du poumon par cytométrie en flux. Ces analyses montrent que les cellules APC pulmonaires présentent des altérations fonctionnelles aboutissant à une limitation de leur capacité à polariser la réponse Th17 de Lymphocytes T CD4+. Une analyse transcriptomique est également effectuée sur les ARN des APC triées afin de préciser les altérations fonctionnelles de ces cellules par rapport aux souris contrôles.2-Rôle des cytokines IL-20 dans la susceptibilité à l'infection et l'exacerbation de la BPCO Myles et al ont montré en 2013 que les cytokines IL-20 (IL-19, IL-20 et IL-24) jouent un rôle délétère dans la réponse immunitaire cutanée contre Staphylococcus aureus par un mécanisme impliquant une inhibition indirecte d’IL-17 produite par les cellules T. La fonction des DC peut être affecté par l'environnement cytokinique. Comme les cytokines IL-20 sont surexprimées chez les souris BPCO, notre objectif a été de définir leur rôle au cours de l'exacerbation de la BPCO et l'impact de ces cytokines sur les DC dans ce contexte.Dans notre modèle d’exacerbation de la BPCO, nous avons bloqué cette voie en neutralisant l'IL-20RB qui est commune aux 2 récepteurs de ces cytokines afin d’étudier leur impact sur l'exacerbation et sur la réponse immune associée, notamment la réponse IL-17/IL-22. En parallèle, nous avons analysé la modulation de la fonction des DC humaines par ces cytokines dans un contexte d'infection bactérienne. Nos résultats montrent que le traitement avec l'anticorps bloquant anti-IL-20RB permet de bloquer le développement de l'exacerbation de la BPCO en diminuant la charge bactérienne et l'inflammation associée. Cet effet est associé à une diminution importante de la mobilisation des DC dans le poumon mais sans affecter sur la réponse IL-17/IL-22. In vitro, les cytokines IL-20 sont produites par les DC dans un contexte infectieux. De plus, ces cytokines sont capables de diminuer l'activation des ces cellules par les bactéries et de réduire leur capacité à activer les Lymphocytes T dans ce contexte.3-Capacité d'un immunostimulant à restaurer la réponse IL-17/IL-22, et à limiter le développement de l'exacerbation.L’utilisation d’immunostimulant dont la flagelline (agoniste du TLR-5, principale composante du flagelle bactérien) est souvent proposé comment pouvant promouvoir la réponse immunitaire des muqueuses et en particulier la réponse IL-17/IL-22. Nous avons analysé la capacité de cet agoniste du TLR-5 à améliorer la réponse à S. pneumoniae et NTHi dans notre modèle d’exacerbation de la BPCO.Le traitement par la flagelline permet de limiter les conséquences de l'infection bactérienne chez les souris BPCO en diminuant l'inflammation et les lésions pulmonaires associées. L'effet de ce ligand de TLR est au moins en partie dépendant de la production d'IL-22._A terme, ces données permettent d'envisager de nouvelles options thérapeutiques pour le traitement des exacerbations de la BPCO. / Patients with COPD often presented a susceptibility to respiratory infections. Previous works in our lab have showed that a defect of IL-17/IL-22 response and an altered dendritic cell (DC) function is involved in COPD exacerbation with bacteria. However, the mechanism responsible for this defect is not elucidated yet. In order to better define these mechanisms and to develop new therapeutic approaches against COPD exacerbation, we focused on the following points during this PhD project:1-Cellular mechanisms responsible of the defect of IL-17 and IL-22 production during infection in COPD.Antigen presenting cells (APC) particularly, DC are essential in antimicrobial immune response since they are professional phagocytes able to engulf and kill bacteria, but also by their essential role in the polarization of innate and adaptive immune responses. We worked on COPD exacerbation mice model infected with Streptococcus pneumoniae or Nontypeable Haemophilus influenzae (NTHi). APC including macrophages, DC and inflammatory monocytes were sorted by flow cytometry for phenotype and functional analysis. We found an altered function of these lung APC showing limited capacity of Th17 polarization. Transcriptional analysis on total RNA from sorted APC is performed to decipher the mechanisms involved in this functional alteration regarding to control mice.2-The role of IL-20 cytokines in the susceptibility to infection during COPD exacerbation.Myles et al showed in 2013 that IL-20 cytokines (IL-19, IL-20 and IL-24) are deleterious in skin immune response against Staphylococcus aureus. Indeed, these IL-20 cytokines indirectly inhibited IL-17 produced by T cells. The function of DC is also controlled by the presence of cytokines in the microenvironment. Because IL-20 cytokines are overexpressed in COPD, we aimed to determine their role during COPD exacerbation and the impact on DC.We used IL-20RB (common subunits of the 2 receptors) neutralizing antibodies to blocked IL-20 cytokines in COPD exacerbation mice model. We analyzed the impact of this treatment on the immune response, more particularly IL-17/IL-22 response. In addition, we analyzed the modulation of human monocyte derived DC (MDDC) function by IL-20 cytokines in the context of bacterial infection.Our results shows that treatment with IL-20 RB neutralizing antibodies limited COPD exacerbation by reducing the bacterial burden and the associated inflammatory response. This process was associated to reduced number of DC in the lung without impacting IL-17 and IL-22 production. In vitro, MDDC produced IL-20 cytokines upon bacterial infection. Additionally, these cytokines impaired MDDC activation following bacterial infection, which was associated to a reduced capacity of MDDC to activate T lymphocytes.3-The possibility to restore IL-17 and IL-22 response with immuno-stimulants in order to limit the development of COPD exacerbation.Flagellin (a TLR-5 agonist, the main component of bacterial flagellum) is an immuno-stimulant often used to promote mucosal immune response. This activity is related to its ability to promote IL-17 and IL-22 production. In this PhD work, we analyzed the capacity of this TLR-5 agonist to improve the immune response during S. pneumoniae and NTHi infection in COPD exacerbation mice model.Flagellin treatment reduced the bacterial burden and limited the consequences of bacterial infection in COPD mice, by lowering the inflammation and the associated lung remodeling. We also found that the immunomodulatory effect of flagellin was at least partially IL-22 dependent.Finally, these data allow to identify new therapeutic tools potentially useful for the treatment of COPD exacerbations.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2013-02-08T13:31:12Z No. of bitstreams: 1 sabina_v_montero_ioc_bcm_0053_2011.pdf: 2783003 bytes, checksum: 58d0698eae0f2913066b128813d0d416 (MD5) / Made available in DSpace on 2013-02-08T13:31:12Z (GMT). No. of bitstreams: 1 sabina_v_montero_ioc_bcm_0053_2011.pdf: 2783003 bytes, checksum: 58d0698eae0f2913066b128813d0d416 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / Pacientes infectados pelo HIV-1 apresentam aumentada permeabilidade intestinal, a qual permite a passagem para a circulação sanguínea de produtos microbianos, fenômeno conhecido por translocação microbiana. Dentre os produtos translocados são encontrados vários ligantes dos receptores do tipo Toll (TRL). A ativação de TLR desencadeia uma complexa cascata de sinalização, induz a síntese de diversas citocinas, e modula a função de células dendríticas (CDs), macrófagos e linfócitos, células-alvo da infecção pelo HIV-1. Estudos experimentais mostram que a ativação de TLRs influencia a replicação do HIV-1, como, por exemplo, a ativação de TLR-4 e TLR-3 resulta em diminuição da replicação viral. No entanto, os estudos relacionados à ativação de TLR-2 e HIV-1 são escassos. Assim, em nosso estudo, resolvemos analisar o efeito da ativação de TLR-2 sobre a replicação do HIV-1 em PBMCs e macrófagos primários humanos infectados in vitro. Para isto, PBMCs e macrófagos obtidos de doadores saudáveis foram infectados pelo HIV-1 e em seguida expostos ao Zymosaqn ou Pam3CSK4, ambos ligantes de TLR-2, e a replicação viral foi avaliada pela detecção da proteína viral p24 nos sobredanantes de cultura. Vimos que tanto o Zymosan quanto o Pam3CSK4 inibem de forma potente (até 90%) a replicação do isolado Ba-L (trópico para CCR5) de HIV-1 em PBMCs e macrófagos, assim como isolados primários trópicos para CCR5 e CXCR4. o tratamento das células com os ligantes de TLR-2 antes da infecção também induziu a queda da replicação viral. Ambos os ligantes de TLR-2 induziram aumento da produção das β-quimiocinas CCL3, CCL4 e CCL5 em macrófagos e PBMCs, e de IL-10 em macrófagos. A imuno-neutralização das β-quimiocinas diminuiu expressivamente o seu efeito inibitório sobre a replicação do HIV-1,. sugerindo que estas moléculas participam da inibição da replicação do HIV-1 resultante da ativação de TLR2. no entanto, a neutralização do receptor de IL-10 não produziu resultados semelhantes. A expressão dos receptores celulares CD4, CCR5 e CXCR4 não foi alterada quando macrófagos e PBMCs foram tratados com Pam3CSK4. observamos, ainda, que a proteína quinase R (PKR) é ativada por Pam3CSK4 tanto em macrófagos quanto em PBMCs. Estes resultados mostram que a ativação de TLR-2 resulta em uma potente inibição da replicação do HIV-1 em PBMCs e macrófagos, e sugerem que as β-quimiocinas estão envolvidas neste fenômeno. Nossos achados ressaltam o papel anti-HIV-1 resultante da ativação de TLR-2, e indicam que novos estudos devem ser realizados para esclarecer, com maior profundidade, os mecanismos envolvidos neste processo / Patients infected with HIV-1 exhibit increased intestinal permeability, which allows passage into the bloodstream of microbial products, a phenomenon known as microbial translocation. Among the products are found translocated several ligands of Toll-like receptors (TRL). The activation of TLR triggers a complex cascade of signaling, inducing synthesis of different cytokines, and modulates the function of dendritic cells (DCs), macrophages and lymphocytes, target cells from infection by HIV-1. Experimental studies have shown that activation of TLRs influences the replication of HIV-1, for example, activation of TLR-4, TLR-3 results in decreased viral replication. However, studies related to the activation of TLR-2 and HIV-1 are scarce. Thus, in our study, we decided to analyze the effect of the activation of TLR-2 on HIV-1 replication in PBMCs and human primary macrophages infected in vitro. For this purpose, PBMC and macrophages obtained from healthy donors were infected with HIV-1 and then exposed to Zymosaqn or Pam3CSK4, both from the TLR-2 ligands, and viral replication was assessed by the detection of viral protein p24 in culture sobredanantes. We have seen that both Zymosan as potently inhibit Pam3CSK4 (up to 90%) replication isolated Ba-L (CCR5 tropic) HIV-1 PBMC and macrophages, as well as primary isolates tropic CCR5 and CXCR4. treating the cells with the ligands of TLR-2 prior to infection also induced decrease in viral replication. Both ligands of TLR-2 induced increased production of β-chemokines CCL3, CCL4 and CCL5 in PBMC and macrophages, and IL-10 in macrophages. The immuno-neutralization of β-chemokine significantly reduced their inhibitory effect on the replication of HIV-1. suggesting that these molecules participate in inhibiting the replication of HIV-1 resulting from activation of TLR2. However, the neutralization of IL-10 did not produce similar results. The expression of cell receptors CD4, CCR5 and CXCR4 was not altered when macrophages and PBMCs were treated with Pam3CSK4. noted further that protein kinase R (PKR) is activated by Pam3CSK4 in both macrophages and in PBMCs. These results show that activation of TLR-2 results in a potent inhibition of HIV-1 replication in PBMC and macrophages, and suggest that β-chemokines are involved in this phenomenon. Our findings highlight the role of anti-HIV-1 resulting from the activation of TLR-2, and suggest that further studies should be conducted to clarify, in greater depth, the mechanisms involved in this process

Submitted by MARCOS AURELIO RIBEIRO DA SILVA (marcosars@ufba.br) on 2016-01-15T17:25:17Z No. of bitstreams: 1 Dissertação de Mestrado - Denisar.pdf: 3263987 bytes, checksum: 526d1efae345d51d6e41a539652a0a6f (MD5) / Approved for entry into archive by MARCOS AURELIO RIBEIRO DA SILVA (marcosars@ufba.br) on 2016-01-15T17:29:22Z (GMT) No. of bitstreams: 1 Dissertação de Mestrado - Denisar.pdf: 3263987 bytes, checksum: 526d1efae345d51d6e41a539652a0a6f (MD5) / Made available in DSpace on 2016-01-15T17:29:22Z (GMT). No. of bitstreams: 1 Dissertação de Mestrado - Denisar.pdf: 3263987 bytes, checksum: 526d1efae345d51d6e41a539652a0a6f (MD5) / CAPES / PALMITO, Denisar dos Santos. Camundongos C57Bl/6 e A/j apresentam resposta inflamatória diferenciada após a imunização com Staphylococcus aureus e desafio em modelo de bolsão de ar. Dissertação (Mestrado) – Instituto Multidisciplinar de Saúde, Universidade Federal da Bahia, Vitória da Conquista, 2015. Introdução/Objetivo: Staphylococcus aureus é o principal agente etiológico de infecções bacterianas em humanos, sendo relatado como o agente causador de patologias como endocardite e sepse. O presente trabalho teve por objetivo avaliar quais os possíveis componentes do sistema imune poderiam atuar de maneira protetora contra a infecção por S. aureus em camundongos imunizados intradermicamente. Materiais e métodos: Camundongos C57Bl/6 e A/j foram imunizados intradermicamente com amostras de S. aureus inativadas por calor e, posteriormente, foram desafiados com amostras viáveis em um modelo de bolsão de ar. Nos tempos de 6, 12 e 24 horas após o desafio, a eutanásia ocorreu e o perfil celular do infiltrado inflamatório, bem como a carga bacteriana, foram avaliados no lavado do bolsão de ar. No soro e no lavado foram quantificados anticorpos da classe IgG e a pele do bolsão foi avaliada por técnicas histopatológicas. Pela técnica de ELISA foram quantificadas citocinas presentes no lavado do bolsão, baço e medula. Resultados: Animais imunizados e desafiados com amostras resistentes no modelo de bolsão apresentaram recrutamento celular inflamatório constituído predominantemente por neutrófilos. Além disso, foi observada uma associação entre uma produção de anticorpos IgG e a redução da carga bacteriana em um modelo murino de imunização intradérmica. Ademais, animais imunizados apresentaram menor inflamação no sítio de infecção e uma maior produção local de IL-17A. Em relação as outras citocinas, não foram encontradas diferenças nos tecidos analisados. Discussão: S. aureus é capaz de induzir uma resposta sistêmica, assim como, recrutar células inflamatórias para o sítio de infecção. Porém, animais imunizados tendem a controlar melhor o processo inflamatório, aparentemente por uma correlação entre a produção de anticorpos IgG e a redução da carga bacteriana em um modelo murino de imunização intradérmica. A menor carga bacteriana nos animais imunizados se correlacionou, 10 também, com uma menor inflamação no sítio de infecção e uma maior produção local de IL-17A. Conclusão: A presença de anticorpos IgG2a se correlaciona com diminuição da carga bacteriana em animais imunizados intradermicamente com S. aureus, juntamente com a elevação de IL-17A no modelo de bolsão de ar. / PALMITO, Denisar dos Santos. C57Bl/6 and A/j mice presente diferente inflamatory response patterns after immunization with Staphylococcus aureus and challenge in air pouch model. Master Dissertation - Instituto Multidisciplinar de Saúde, Universidade Federal da Bahia, Vitória da Conquista, 2015. Introduction / Objective: Staphylococcus aureus is the major etiological agent of bacterial infections in humans and is reported as the causative agent of diseases such as sepsis and endocarditis. This study aimed to evaluate which components of the immune system could act protectively against S. aureus infection in mice immunized intradermally. Methods: C57Bl/6 and A/j mice were immunized intradermally with S. aureus strains inactivated by heat and then were challenged with viable strains in an air pouch model. At the times of 6, 12 and 24 hours after challenge, the euthanasia was performed and the cellular profile of the inflammatory infiltrate as well as the bacterial load were evaluated in the air pouch lavages. Serum and lavages of air pouch were quantified for IgG antibodies and the skin was evaluated by histopathological techniques. Cytokines present in air pouch, spleen and bone marrow were quantified by ELISA . Results: Animals immunized and challenged with strains resistant to the air pouch model presented an inflammatory cell recruitment constituted predominantly by neutrophils. Furthermore, it was observed an association between the production of IgG antibodies and reduced the bacterial load in our model of intradermal immunization. In addition, immunized animals showed lower inflammation at the site of infection and increased local production of IL-17A. Regarding the other cytokines, no differences were found in the tissues. Discussion: S. aureus is able to induce a systemic response as well as recruiting inflammatory cells into the infection site. Immunized animals showed a higher control the inflammatory process correlated with the production of IgG antibodies and a reduced bacterial burden in the inflammatory site. The lower bacterial load in animals immunized correlated also with lower inflammation in the site of infection and increased local production of IL-17A. Conclusion: The presence of IgG2a antibodies correlates with a decreased bacterial load in animals immunized intradermally with S. aureus along with the elevation of IL-17A