La chaîne invariante forme un complexe nonamérique avec les molécules classiques du CMH de classe II. HLA-DM et HLA-DO, des molécules non-classiques de classe II, sont aussi impliquées dans la présentation des peptides antigéniques aux lymphocytes T. Ces molécules chaperones de la présentation antigénique modulent la capacité d’une cellule à présenter des antigènes par les moloécules classiques du CMH de classe II. La régulation transcriptionnelle des molécules chaperones, tout comme celle des autres molécules du CMH de classe II, est assurée par le transactivateur CIITA. La molécule HLA-DR peut être régulée négativement de manière post-traductionnelle par ubiquitination grâce à l’enzyme E3 ubiquitine ligase MARCH1. Celle-ci est induite par l’interleukine-10 dans les monocytes. L’objectif de ce projet était de déterminer si l’ubiquitination par MARCH1 peut aussi réguler l’expression des molécules chaperones de la présentation antigénique. Les expériences furent réalisées dans le contexte de co-transfections en cellules HEK293T. L’expression des molécules fut évaluée par immunomarquages et cytométrie de flux. Il a été montré que l’isoforme p33 de la chaîne invariante est régulé négativement en présence de MARCH1 à partir de la surface cellulaire, causant ainsi sa dégradation. Tel que démontré par l’utilisation d’un mutant dépourvu de queue cytoplasmique, cette dernière région n’est pas indispensable à ce phénomène. Une hypothèse est qu’une molécule non-identifiée, associée à Ii, serait ubiquitinée par MARCH1, l’entraînant dans sa régulation négative. Il fut déterminer que cette molécule n’était pas CXCR2, un récepteur pouvant être impliqué, avec la chaîne invariante et CD44, en tant que récepteur de MIF (Macrophage Inhibitory Factor). Il fut aussi montré que HLA-DO peut être ciblé par MARCH1 mais ceci ne semble pas être un phénomène dominant; l’expression des complexes DO/DM n’étant pas affectée bien qu’ils entrent en interaction avec MARCH1. L’expression de HLA-DM n’est pas affectée par MARCH1. Il n’a toutefois pas été déterminé hors de tout doute si MARCH1 peut modifier DM; des résultats obtenus avec une queue cytoplasmique de DM possédant une lysine laissant suggérer qu’il est possible que MARCH1 interagisse avec DM. Dans l’ensemble, les travaux démontrent que l’ubiquitination par MARCH1 joue un rôle dans la régulation post-transcriptionnelle de la chaîne invariante p33 mais pas HLA-DO et HLA-DM. / The invariant chain, which form a nonameric complex with the classical MHC class II molecules. HLA-DM and HLA-DO (non-classical class II molecules) are involved in the presentation of antigens to T lymphocytes. The chaperons molecules of the antigenic presentation can modulate the capacity of the cells to present antigens. The transcriptional regulation of the chaperons and all of the other molecules linked to the MHC is assured by the CIITA transactivator. Little is know of the post-transcriptional mechanisms, other than the fact that HLA-DR molecule can be down-regulated by ubiquitination due to E3 ubiquitin ligase MARCH1. MARCH1 is induce by interleukin-10 in monocytes. The goal of this project is to figure out if ubiquitination by MARCH1 can also regulate the expression of the antigenic presentation chaperons. The experiences were performed in the context of co-transfections in HEK293T cells and the expression of the diverses molecules was evaluated by cell stainings and FACS analysis. The p33 isoform of the invariant chain was found to be down-regulated and degraded in the presence of MARCH1. The invariant chain cytoplasmic tail is not completely essential to this phenomenon; a non-identified molecule, associated with Ii, is probably ubiquitinated by MARCH1 and is then down-regulated, together with Ii. It was shown tha CXCR2, a reeptor involved with the invariant chain and CD44 in the reception of the MIF signal, is not that molecule. HLA-DO can ben targetd by MARCH1 but this does not seem to be a general phenomenon; the expression of the DO/DM complexes remaning unaffected even with the interaction of those complexes with MARCH1. Therefore, a certain protection seem to be provided by HLA-DM to HLA-DO. The expression of HLA-DM itself is not affected by the presence of MARCH1. However, it was not cleary demonstrated if MARCH1 can modify DM. Some results obtained with a cytoplasmic tail of DM comprising an additional lysine suggest that there is a possibility that MARCH1 interact with DM. Generally, the work presented here show that ubiquitination by MARCH1 is involved in post-transcriptionnal regulation of the p33 isoform of the invariant chain but not in the regulation of HLA-DO and HLA-DM.

Os objetivos do presente trabalho consistem na síntese de dois polímeros - poli(2,1,3-benzotiadiazol-4,7-ilenovinileno-alt-9,9-n-dioctil-2,7-fluorenilenovinileno) (PBTDV-alt-PDO27FV) e poli[4,7-(2,1,3-benzotiadiazolileno)-alt-(2,5-dioctilóxi-1,4- fenilenovinileno)] (PBTDV-alt-PDOPPV) - cinco oligômeros - 4,7-bis(2-(9,9-n-dioctil-9H-fluoren-2-il)vinil)2,1,3-benzotiadiazol (FBF); 2,7-bis(2-(2,1,3-benzotiadiazol-4-il)vinil)9,9-n-dioctil-9H-fluoreno (BFB); 4-(2-(9,9-n-dioctil-9H-fluoren-2-il)vinil)2,1,3-benzotiadiazol (FB); 1,2-bis(9,9-n-dioctil-9H-fluoren)2-vinileno (FF) e 2,2\'-(2,2\'-(9,9-n-dioctil-9H-fluoreno)2,7-bis(vinileno)bis(9,9-n-dioctil-9H-fluoreno) (FFF) - e três small molecules - 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(6-fluoro-4-(5\'-hexil-[2\'-tiofeno-2\'-tiazol]-5-il)benzo[c][1,2,5]tiadiazol (G37FBT); 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(6-fluoro-4-(2\'-isobutil-[5\',2\'-ditiazol])-5-il)benzo[c][1,2,5]tiadiazol (J1) e 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(5\'-hexil-[2,2\'-bitiofeno]-5- bis(il)benzo[c][1,2,5]tiadiazol) (J2) - seguida da aplicação desses materiais em dispositivos fotovoltaicos e magnetorresistivos e em sensores de gás. Os polímeros e oligômeros preparados são derivados de poli(p-fenilenovinileno) e contêm unidades de fluoreno e/ou 2,1,3-benzotiadiazol, tendo sido empregada, na etapa final de cada síntese, reação de Wittig visando ao acoplamento e à formação das duplas vinilênicas. As small molecules, preparadas por meio de sucessivas formações de organo-estananas e reações de Stille, também foram sintetizadas com sucesso e em rendimentos apreciáveis. A espécie J2 não foi preparada com sucesso, obtendo-se, em contrapartida, um análogo dessa molécula, que foi devidamente isolado e caracterizado. No que tange à aplicação dos materiais em dispositivos fotovoltaicos, os resultados até então obtidos mostraram-se inconclusivos, sendo digno de nota, no entanto, que o estudo continua sendo conduzido por colaboradores. O trabalho de aplicação dos materiais em dispositivos magnetorresistivos apresentou resultados interessantes para os oligômeros FBF, BFB, FFF e FF, visto que dispositivos fabricados a partir das referidas espécies mostraram variação em seus valores de corrente elétrica quando submetidos a campo magnético de 200 mT. Por fim, foram desenvolvidos narizes eletrônicos com duas finalidades distintas: (a) identificação de diferentes espécies de madeira de interesse ambiental e (b) estudo da influência da atividade física no indivíduo por meio da análise do suor. Os sensores de gás foram preparados por meio da deposição de finos filmes de materiais orgânicos conjugados dopados sobre a superfície de eletrodos interdigitados. No estudo acerca da identificação de espécies de madeira, a análise de leave-one-out revelou 100 % de taxa de acerto na diferenciação entre as espécies angelim, cedro-rosa, imbuia e perobinha. O estudo da influência da atividade física no indivíduo também se mostrou promissor na medida em que identificou os diferentes estágios do exercício físico com taxa de acerto de 93 %, também definida pela técnica estatística de leave-one-out. / The present work consists on the synthesis of two polymers - poly(2,1,3- benzothiadiazole-4,7-ylenevinylene-alt-9,9-n-dioctyl-2,7-fluorenylenevinylene) (PBTDV-alt-PDO27FV) and poly[4,7-(2,1,3-benzothiadiazole-ylene)-alt-(2,5-dioctyloxy-1,4-phenylenevinylene)] (PBTDV-alt-PDOPPV) - five oligomers - 4,7-bis(2-(9,9-n-dioctyl- 9H-fluorene-2-yl)vinyl)2,1,3-benzothiadiazole (FBF); 2,7-bis(2-(2,1,3- benzothiadiazole-4-yl)vinyl)9,9-n-dioctyl-9H-fluorene (BFB); 4-(2-(9,9-n-dioctyl-9H-fluorene-2-yl)vinyl)2,1,3-benzothiadiazole (FB); 1,2-bis(9,9-n-dioctyl-9H-fluorene)2- vinylene (FF) and 2,2\'-(2,2\'-(9,9-n-dioctyl-9H-fluorene)2,7-bis(vinylene)bis(9,9-n-dioctyl-9H-fluorene) (FFF) - and three small molecules - 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(6-fluoro-4-(5\'-hexyl-[2\'-thiophene-2\'- thiazole]-5-yl)benzo[c][1,2,5]thiadiazole (G37FBT); 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(6-fluoro-4-(2\'-isobutyl-[5\',2\'-dithiazole]-5-yl)benzo[c][1,2,5]thiadiazole (J1) and 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(5\'-hexyl-[2,2-bithiophene]-5-bis(yl)benzo[c][1,2,5]thiadiazole) (J2) - followed by the application of such materials in organic photovoltaics (OPV), magnetoresistive devices and gas sensors. The polymers and oligomers are all poli(p-phenylenevinylene)s derivatives containing 2,1,3-benzothiadiazole and/or substituted fluorene units in the main chain. Those materials\' preparation comprises a coupling Wittig reaction as the key step, through which the vinylenic bonds are formed. The small molecules mentioned above were synthesized by successive organostannane preparations followed by cross-coupling Stille reactions, leading to the target-compounds in considerably high yields. Although the route is a very useful methodology for synthesizing organic conjugated small molecules, J2 was not successfully prepared. The reaction led to a J2 analogue instead, that was properly isolated and characterized. The application of the cited materials in organic photovoltaics (OPVs) is still in progress, since the study has shown inconclusive results so far. The study of the organic magnetoresistive properties of the synthesized materials has led to interesting results for the oligomers FBF, BFB, FFF and FF. The devices containing those compounds in their active layers presented considerable variations in their electric current values when submitted to a magnetic field of 150 mT magnitude. Finally, electronic noses for two different applications were developed: (a) the identification of wood species that can be easily mistaken and (b) the study of exercise physiology through analyses of sweat samples. The gas sensors were prepared by the deposition of doped organic conjugated materials thin films onto the surface of intedigitated electrodes. The study on the differentiation of several species of wood showed 100 % rate of hits determined by leave-one-out statistics analysis. The study on the exercise physiology was also promising since it allowed the identification of the three different stages of the physical exercise with a 93 % rate of hits, also determined by leave-one-out methods. Thus, both studies suggest that the electronic nose can be a powerful tool to study many different targets in which the release of volatile compounds is involved

Introdução: Hanseníase, doença crônica, incapacitante, causada pelo M.leprae, que afeta a pele e os nervos periféricos. Manifesta-se como doença espectral, que exibe dois polos imunologicamente distintos, denominados hanseníase virchowiana (lepromatous - LL), caracterizada por uma resposta celular ineficiente; e hanseníase tuberculoide (Tuberculoid - TT), com resposta imune celular efetiva. A ativação de células T requer a sinalização através de moléculas coestimulatórias expressas em células apresentadoras de antígeno e seus ligantes nas células T. As células T regulatórias (Treg) exercem importante papel no mecanismo de falha do controle da disseminação antigênica nas formas graves das doenças crônicas granulomatosas, mas seu real papel na hanseníase ainda não foi elucidado. Métodos: Estudamos a expressão das moléculas coestimulatórias e células Treg na resposta imune de pacientes nos diferentes espectros da doença. A expressão de células Treg foi quantificada por citometria de fluxo (CD4+CD25+FoxP3+) nas células mononucleares do sangue periférico de pacientes e controles (16 eram virchowianos, 12 tuberculoides e 6 controles) estimulados in vitro com o antígeno do M. leprae e com o mitógeno phytohemaglutinina, bem como nas lesões de pele por imunohistoquímica. A expressão de moléculas coestimulatorias (CD80, CD86, CD28, CTLA-4, PD-1, ICOS) foi avaliada por citometria de fluxo in vitro, a partir do estimulo de células mononucleares provenientes de 14 pacientes virchowianos, 14 tuberculoides e 10 indivíduos saudáveis expostos ao bacilo. A resposta linfoproliferativa, a produção de citocinas (IL10 e IFN-?) in vitro e a expressão in situ de IL-10, TGF? e CTLA-4 também foram determinadas. Resultados: Nós demonstramos que pacientes virchowianos apresentam deficiência na expressão da molécula CD86 na superfície de monócitos, e isso contribui para uma apresentação antigênica ineficiente, favorecendo o estado de anergia observado nesse grupo de pacientes. Observamos ainda, que o bloqueio da expressão dessa molécula, mas não do CD80 inibe a resposta linfoproliferativa ao M.leprae. Ainda no polo virchowiano anérgico, observamos redução da expressão de moléculas coestimulatórias de sinalização positiva (CD28, CD86) na superfície dos linfócitos. Em contraste, nos pacientes tuberculoides notamos um aumento da expressão de moléculas de sinalização negativa (CTLA-4 e PD-1), que pode representar uma provável modulação da resposta imune exacerbada, regulando dessa forma os efeitos deletérios da hiperreatividade celular. Notavelmente, os contatos também expressão em menor intensidade CD86 e CD28, mas não se observou expressão exacerbada de CTLA-4 ou PD-1, sugerindo que eles provavelmente desenvolvem resposta imune balanceada sem que haja necessidade de sinalização imunossupressora. Observamos ainda, que o antígeno do M.leprae induz uma significativa redução da resposta linfoproliferativa, mas um aumento significativo de células Treg no sangue e na pele dos pacientes virchowianos, quando comparado ao grupo tuberculoide. Em paralelo, notamos o aumento da expressão de moléculas anti-inflamatórias (IL-10 e CTLA-4) nas lesões cutâneas desses pacientes virchowianos. Conclusão: Nossos resultados sugerem que células Treg e moléculas coestimulatórias desempenham papel importante na patogênese da hanseníase, especialmente no polo virchowiano, em que favoreceria a anergia e a multiplicação bacilar / Introduction: Leprosy, caused by the bacillus Mycobacterium leprae, is a chronic, incapacitating disease that affects the peripheral nerves and skin. Leprosy manifests as a spectral disease, exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. T regulatory cells (Tregs) play an important role in the mechanism of host\'s failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated. The objective of this study was to investigate the influence of costimulatory molecules and Tregs cels on the immune responses of subjects along the leprosy spectrum. Patients and Methods: Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells (PBMC) of patients (16 lepromatous, 12 tuberculoids and controls (n = 6) stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The expression of the costimulatory molecules (CD80, CD86, CD28, CTLA-4, PD-1, ICOS) was evaluated in in vitro-stimulated peripheral blood mononuclear cells isolated from 14 lepromatous and 14 tuberculoid patients, and 10 healthy individuals exposed to the bacillus. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-g (IFN-g) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-b (TGF-b), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. Results: We show that lepromatous patients have defective monocytes\' CD86 expression, likely contributing to the impairment of the antigen presentation process and to their anergy. Accordingly, anti-CD86 blocking monoclonal antibody, but not anti-CD80 antibody, inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the lepromatous pole anergy, there was reduced T-cells\' expression of the positive signaling costimulatory molecules CD28 and CD86 in these patients. Tuberculoid patients, on the other hand, had increased expression of the negative signaling CTLA-4 and PD-1 molecules, probably representing a means of modulating exacerbated immune response and avoiding immunopathology. Interestingly, contacts exhibited proper CD86 and CD28 expression, but not exacerbated CD152 and PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signaling. We also observed that M. leprae antigens induced significantly lower lymphoproliferation but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced lymphoproliferation and Treg frequencies were not significantly different between the three groups. Tregs were also more frequent in situ in multibacillary patients, and this was paralleled by increased expression of the anti-inflammatory molecules IL-10 and CTLA-4, but not TGF. Conclusion: Our results suggest that Tregs and costimuatory molecules play a major role in the pathogenesis of leprosy, especially the lepromatous pole, in which they would act to favor the anergy and the unrestricted multiplication of the bacilli

Os objetivos do presente trabalho consistem na síntese de dois polímeros - poli(2,1,3-benzotiadiazol-4,7-ilenovinileno-alt-9,9-n-dioctil-2,7-fluorenilenovinileno) (PBTDV-alt-PDO27FV) e poli[4,7-(2,1,3-benzotiadiazolileno)-alt-(2,5-dioctilóxi-1,4- fenilenovinileno)] (PBTDV-alt-PDOPPV) - cinco oligômeros - 4,7-bis(2-(9,9-n-dioctil-9H-fluoren-2-il)vinil)2,1,3-benzotiadiazol (FBF); 2,7-bis(2-(2,1,3-benzotiadiazol-4-il)vinil)9,9-n-dioctil-9H-fluoreno (BFB); 4-(2-(9,9-n-dioctil-9H-fluoren-2-il)vinil)2,1,3-benzotiadiazol (FB); 1,2-bis(9,9-n-dioctil-9H-fluoren)2-vinileno (FF) e 2,2\'-(2,2\'-(9,9-n-dioctil-9H-fluoreno)2,7-bis(vinileno)bis(9,9-n-dioctil-9H-fluoreno) (FFF) - e três small molecules - 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(6-fluoro-4-(5\'-hexil-[2\'-tiofeno-2\'-tiazol]-5-il)benzo[c][1,2,5]tiadiazol (G37FBT); 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(6-fluoro-4-(2\'-isobutil-[5\',2\'-ditiazol])-5-il)benzo[c][1,2,5]tiadiazol (J1) e 7,7\'-(4,4-bis(2-etil-hexil)-4H-silolo[3,2-b-:4,5-b\']ditiofen-2,6-diil)bis(5\'-hexil-[2,2\'-bitiofeno]-5- bis(il)benzo[c][1,2,5]tiadiazol) (J2) - seguida da aplicação desses materiais em dispositivos fotovoltaicos e magnetorresistivos e em sensores de gás. Os polímeros e oligômeros preparados são derivados de poli(p-fenilenovinileno) e contêm unidades de fluoreno e/ou 2,1,3-benzotiadiazol, tendo sido empregada, na etapa final de cada síntese, reação de Wittig visando ao acoplamento e à formação das duplas vinilênicas. As small molecules, preparadas por meio de sucessivas formações de organo-estananas e reações de Stille, também foram sintetizadas com sucesso e em rendimentos apreciáveis. A espécie J2 não foi preparada com sucesso, obtendo-se, em contrapartida, um análogo dessa molécula, que foi devidamente isolado e caracterizado. No que tange à aplicação dos materiais em dispositivos fotovoltaicos, os resultados até então obtidos mostraram-se inconclusivos, sendo digno de nota, no entanto, que o estudo continua sendo conduzido por colaboradores. O trabalho de aplicação dos materiais em dispositivos magnetorresistivos apresentou resultados interessantes para os oligômeros FBF, BFB, FFF e FF, visto que dispositivos fabricados a partir das referidas espécies mostraram variação em seus valores de corrente elétrica quando submetidos a campo magnético de 200 mT. Por fim, foram desenvolvidos narizes eletrônicos com duas finalidades distintas: (a) identificação de diferentes espécies de madeira de interesse ambiental e (b) estudo da influência da atividade física no indivíduo por meio da análise do suor. Os sensores de gás foram preparados por meio da deposição de finos filmes de materiais orgânicos conjugados dopados sobre a superfície de eletrodos interdigitados. No estudo acerca da identificação de espécies de madeira, a análise de leave-one-out revelou 100 % de taxa de acerto na diferenciação entre as espécies angelim, cedro-rosa, imbuia e perobinha. O estudo da influência da atividade física no indivíduo também se mostrou promissor na medida em que identificou os diferentes estágios do exercício físico com taxa de acerto de 93 %, também definida pela técnica estatística de leave-one-out. / The present work consists on the synthesis of two polymers - poly(2,1,3- benzothiadiazole-4,7-ylenevinylene-alt-9,9-n-dioctyl-2,7-fluorenylenevinylene) (PBTDV-alt-PDO27FV) and poly[4,7-(2,1,3-benzothiadiazole-ylene)-alt-(2,5-dioctyloxy-1,4-phenylenevinylene)] (PBTDV-alt-PDOPPV) - five oligomers - 4,7-bis(2-(9,9-n-dioctyl- 9H-fluorene-2-yl)vinyl)2,1,3-benzothiadiazole (FBF); 2,7-bis(2-(2,1,3- benzothiadiazole-4-yl)vinyl)9,9-n-dioctyl-9H-fluorene (BFB); 4-(2-(9,9-n-dioctyl-9H-fluorene-2-yl)vinyl)2,1,3-benzothiadiazole (FB); 1,2-bis(9,9-n-dioctyl-9H-fluorene)2- vinylene (FF) and 2,2\'-(2,2\'-(9,9-n-dioctyl-9H-fluorene)2,7-bis(vinylene)bis(9,9-n-dioctyl-9H-fluorene) (FFF) - and three small molecules - 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(6-fluoro-4-(5\'-hexyl-[2\'-thiophene-2\'- thiazole]-5-yl)benzo[c][1,2,5]thiadiazole (G37FBT); 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(6-fluoro-4-(2\'-isobutyl-[5\',2\'-dithiazole]-5-yl)benzo[c][1,2,5]thiadiazole (J1) and 7,7\'-(4,4-bis(2-ethyl-hexyl)-4H-silolo[3,2-b-:4,5-b\']dithiophene-2,6-diyl)bis(5\'-hexyl-[2,2-bithiophene]-5-bis(yl)benzo[c][1,2,5]thiadiazole) (J2) - followed by the application of such materials in organic photovoltaics (OPV), magnetoresistive devices and gas sensors. The polymers and oligomers are all poli(p-phenylenevinylene)s derivatives containing 2,1,3-benzothiadiazole and/or substituted fluorene units in the main chain. Those materials\' preparation comprises a coupling Wittig reaction as the key step, through which the vinylenic bonds are formed. The small molecules mentioned above were synthesized by successive organostannane preparations followed by cross-coupling Stille reactions, leading to the target-compounds in considerably high yields. Although the route is a very useful methodology for synthesizing organic conjugated small molecules, J2 was not successfully prepared. The reaction led to a J2 analogue instead, that was properly isolated and characterized. The application of the cited materials in organic photovoltaics (OPVs) is still in progress, since the study has shown inconclusive results so far. The study of the organic magnetoresistive properties of the synthesized materials has led to interesting results for the oligomers FBF, BFB, FFF and FF. The devices containing those compounds in their active layers presented considerable variations in their electric current values when submitted to a magnetic field of 150 mT magnitude. Finally, electronic noses for two different applications were developed: (a) the identification of wood species that can be easily mistaken and (b) the study of exercise physiology through analyses of sweat samples. The gas sensors were prepared by the deposition of doped organic conjugated materials thin films onto the surface of intedigitated electrodes. The study on the differentiation of several species of wood showed 100 % rate of hits determined by leave-one-out statistics analysis. The study on the exercise physiology was also promising since it allowed the identification of the three different stages of the physical exercise with a 93 % rate of hits, also determined by leave-one-out methods. Thus, both studies suggest that the electronic nose can be a powerful tool to study many different targets in which the release of volatile compounds is involved

Introdução: Hanseníase, doença crônica, incapacitante, causada pelo M.leprae, que afeta a pele e os nervos periféricos. Manifesta-se como doença espectral, que exibe dois polos imunologicamente distintos, denominados hanseníase virchowiana (lepromatous - LL), caracterizada por uma resposta celular ineficiente; e hanseníase tuberculoide (Tuberculoid - TT), com resposta imune celular efetiva. A ativação de células T requer a sinalização através de moléculas coestimulatórias expressas em células apresentadoras de antígeno e seus ligantes nas células T. As células T regulatórias (Treg) exercem importante papel no mecanismo de falha do controle da disseminação antigênica nas formas graves das doenças crônicas granulomatosas, mas seu real papel na hanseníase ainda não foi elucidado. Métodos: Estudamos a expressão das moléculas coestimulatórias e células Treg na resposta imune de pacientes nos diferentes espectros da doença. A expressão de células Treg foi quantificada por citometria de fluxo (CD4+CD25+FoxP3+) nas células mononucleares do sangue periférico de pacientes e controles (16 eram virchowianos, 12 tuberculoides e 6 controles) estimulados in vitro com o antígeno do M. leprae e com o mitógeno phytohemaglutinina, bem como nas lesões de pele por imunohistoquímica. A expressão de moléculas coestimulatorias (CD80, CD86, CD28, CTLA-4, PD-1, ICOS) foi avaliada por citometria de fluxo in vitro, a partir do estimulo de células mononucleares provenientes de 14 pacientes virchowianos, 14 tuberculoides e 10 indivíduos saudáveis expostos ao bacilo. A resposta linfoproliferativa, a produção de citocinas (IL10 e IFN-?) in vitro e a expressão in situ de IL-10, TGF? e CTLA-4 também foram determinadas. Resultados: Nós demonstramos que pacientes virchowianos apresentam deficiência na expressão da molécula CD86 na superfície de monócitos, e isso contribui para uma apresentação antigênica ineficiente, favorecendo o estado de anergia observado nesse grupo de pacientes. Observamos ainda, que o bloqueio da expressão dessa molécula, mas não do CD80 inibe a resposta linfoproliferativa ao M.leprae. Ainda no polo virchowiano anérgico, observamos redução da expressão de moléculas coestimulatórias de sinalização positiva (CD28, CD86) na superfície dos linfócitos. Em contraste, nos pacientes tuberculoides notamos um aumento da expressão de moléculas de sinalização negativa (CTLA-4 e PD-1), que pode representar uma provável modulação da resposta imune exacerbada, regulando dessa forma os efeitos deletérios da hiperreatividade celular. Notavelmente, os contatos também expressão em menor intensidade CD86 e CD28, mas não se observou expressão exacerbada de CTLA-4 ou PD-1, sugerindo que eles provavelmente desenvolvem resposta imune balanceada sem que haja necessidade de sinalização imunossupressora. Observamos ainda, que o antígeno do M.leprae induz uma significativa redução da resposta linfoproliferativa, mas um aumento significativo de células Treg no sangue e na pele dos pacientes virchowianos, quando comparado ao grupo tuberculoide. Em paralelo, notamos o aumento da expressão de moléculas anti-inflamatórias (IL-10 e CTLA-4) nas lesões cutâneas desses pacientes virchowianos. Conclusão: Nossos resultados sugerem que células Treg e moléculas coestimulatórias desempenham papel importante na patogênese da hanseníase, especialmente no polo virchowiano, em que favoreceria a anergia e a multiplicação bacilar / Introduction: Leprosy, caused by the bacillus Mycobacterium leprae, is a chronic, incapacitating disease that affects the peripheral nerves and skin. Leprosy manifests as a spectral disease, exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. T regulatory cells (Tregs) play an important role in the mechanism of host\'s failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated. The objective of this study was to investigate the influence of costimulatory molecules and Tregs cels on the immune responses of subjects along the leprosy spectrum. Patients and Methods: Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells (PBMC) of patients (16 lepromatous, 12 tuberculoids and controls (n = 6) stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The expression of the costimulatory molecules (CD80, CD86, CD28, CTLA-4, PD-1, ICOS) was evaluated in in vitro-stimulated peripheral blood mononuclear cells isolated from 14 lepromatous and 14 tuberculoid patients, and 10 healthy individuals exposed to the bacillus. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-g (IFN-g) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-b (TGF-b), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. Results: We show that lepromatous patients have defective monocytes\' CD86 expression, likely contributing to the impairment of the antigen presentation process and to their anergy. Accordingly, anti-CD86 blocking monoclonal antibody, but not anti-CD80 antibody, inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the lepromatous pole anergy, there was reduced T-cells\' expression of the positive signaling costimulatory molecules CD28 and CD86 in these patients. Tuberculoid patients, on the other hand, had increased expression of the negative signaling CTLA-4 and PD-1 molecules, probably representing a means of modulating exacerbated immune response and avoiding immunopathology. Interestingly, contacts exhibited proper CD86 and CD28 expression, but not exacerbated CD152 and PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signaling. We also observed that M. leprae antigens induced significantly lower lymphoproliferation but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced lymphoproliferation and Treg frequencies were not significantly different between the three groups. Tregs were also more frequent in situ in multibacillary patients, and this was paralleled by increased expression of the anti-inflammatory molecules IL-10 and CTLA-4, but not TGF. Conclusion: Our results suggest that Tregs and costimuatory molecules play a major role in the pathogenesis of leprosy, especially the lepromatous pole, in which they would act to favor the anergy and the unrestricted multiplication of the bacilli

L'eau joue un rôle fondamental dans la photochimie du milieu interstellaire (MIS), à travers la formation d'espèces très réactives comme OH. Les radicaux OH peuvent par la suite interagir avec d'autres molécules hydrogénées pour reformer H2O par abstraction d'hydrogène: R-H + OH → R* + H2O. Dans le cadre de ce travail de thèse, nous avons étudié l'influence des photons VUV sur des analogues de glace interstellaire. Nous montrons que l'incorporation d'une petite quantité d'eau dans NH3 et CH4 glaces augmente considérablement la formation de radicaux réactifs comme NH2 et CH3 pendant le processus de photolyse et que le chauffage des glaces binaires irradiées telles que NH3-H2O et CH4-H2O conduit à la formation de NH2OH et d'espèces alcooliques plus complexes comme le propanol et le métoxyméthanol. Nous avons également entamé d'autres études en parallèle sur le l'évolution thermique des glaces de NH2OH d'une part et la formation de propanol par voies énergétiques (irradiation VUV) et non énergétique (réaction d'addition H) d'autre part afin de tenter d'expliquer la non-détection des ces espèces organiques dans le milieu interstellaire. L'étude des glaces mixtes irradiées NH3-CH4-H2O a montré la formation à basse température d'espèces plus exotiques en combinant les spectrométries IR et de masse. Nous avons réussi à identifier des composés organiques très complexes déjà détectés ou activement recherchés dans le MIS. / Water plays a fundamental role in the photochemistry of the interstellar medium (ISM), through OH radical formation. OH radicals can interact with other H-containing species to form H2O through a hydrogen abstraction reaction: R-H + OH → R* + H2O. In this work, we have investigated the VUV processing on different interstellar ice analogs. We show that the incorporation of small amount of water in NH3 and CH4 ices greatly increases the formation of reactive NH2 and CH3 radicals during the photolysis processing. Thermal treatments of irradiated NH3-H2O and CH4-H2O ices lead to the formation of NH2OH and larger alcoholic species such as propanol and metoxymethanol. Further studies of thermal processing of NH2OH ice and formation of propanol through energetic (VUV irradiation) and non-energetic (surface H-addition reaction) processing were carried out in the context of this thesis in order to try explaining their non-detection in the interstellar medium. The study of the irradiated mixed NH3-CH4-H2O ices showed the formation of more exotic species by combining the IR and mass spectrometries. We managed to identify very large complex organic compounds already detected or tensively sought in the ISM.

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

La chaîne invariante forme un complexe nonamérique avec les molécules classiques du CMH de classe II. HLA-DM et HLA-DO, des molécules non-classiques de classe II, sont aussi impliquées dans la présentation des peptides antigéniques aux lymphocytes T. Ces molécules chaperones de la présentation antigénique modulent la capacité d’une cellule à présenter des antigènes par les moloécules classiques du CMH de classe II. La régulation transcriptionnelle des molécules chaperones, tout comme celle des autres molécules du CMH de classe II, est assurée par le transactivateur CIITA. La molécule HLA-DR peut être régulée négativement de manière post-traductionnelle par ubiquitination grâce à l’enzyme E3 ubiquitine ligase MARCH1. Celle-ci est induite par l’interleukine-10 dans les monocytes. L’objectif de ce projet était de déterminer si l’ubiquitination par MARCH1 peut aussi réguler l’expression des molécules chaperones de la présentation antigénique. Les expériences furent réalisées dans le contexte de co-transfections en cellules HEK293T. L’expression des molécules fut évaluée par immunomarquages et cytométrie de flux. Il a été montré que l’isoforme p33 de la chaîne invariante est régulé négativement en présence de MARCH1 à partir de la surface cellulaire, causant ainsi sa dégradation. Tel que démontré par l’utilisation d’un mutant dépourvu de queue cytoplasmique, cette dernière région n’est pas indispensable à ce phénomène. Une hypothèse est qu’une molécule non-identifiée, associée à Ii, serait ubiquitinée par MARCH1, l’entraînant dans sa régulation négative. Il fut déterminer que cette molécule n’était pas CXCR2, un récepteur pouvant être impliqué, avec la chaîne invariante et CD44, en tant que récepteur de MIF (Macrophage Inhibitory Factor). Il fut aussi montré que HLA-DO peut être ciblé par MARCH1 mais ceci ne semble pas être un phénomène dominant; l’expression des complexes DO/DM n’étant pas affectée bien qu’ils entrent en interaction avec MARCH1. L’expression de HLA-DM n’est pas affectée par MARCH1. Il n’a toutefois pas été déterminé hors de tout doute si MARCH1 peut modifier DM; des résultats obtenus avec une queue cytoplasmique de DM possédant une lysine laissant suggérer qu’il est possible que MARCH1 interagisse avec DM. Dans l’ensemble, les travaux démontrent que l’ubiquitination par MARCH1 joue un rôle dans la régulation post-transcriptionnelle de la chaîne invariante p33 mais pas HLA-DO et HLA-DM. / The invariant chain, which form a nonameric complex with the classical MHC class II molecules. HLA-DM and HLA-DO (non-classical class II molecules) are involved in the presentation of antigens to T lymphocytes. The chaperons molecules of the antigenic presentation can modulate the capacity of the cells to present antigens. The transcriptional regulation of the chaperons and all of the other molecules linked to the MHC is assured by the CIITA transactivator. Little is know of the post-transcriptional mechanisms, other than the fact that HLA-DR molecule can be down-regulated by ubiquitination due to E3 ubiquitin ligase MARCH1. MARCH1 is induce by interleukin-10 in monocytes. The goal of this project is to figure out if ubiquitination by MARCH1 can also regulate the expression of the antigenic presentation chaperons. The experiences were performed in the context of co-transfections in HEK293T cells and the expression of the diverses molecules was evaluated by cell stainings and FACS analysis. The p33 isoform of the invariant chain was found to be down-regulated and degraded in the presence of MARCH1. The invariant chain cytoplasmic tail is not completely essential to this phenomenon; a non-identified molecule, associated with Ii, is probably ubiquitinated by MARCH1 and is then down-regulated, together with Ii. It was shown tha CXCR2, a reeptor involved with the invariant chain and CD44 in the reception of the MIF signal, is not that molecule. HLA-DO can ben targetd by MARCH1 but this does not seem to be a general phenomenon; the expression of the DO/DM complexes remaning unaffected even with the interaction of those complexes with MARCH1. Therefore, a certain protection seem to be provided by HLA-DM to HLA-DO. The expression of HLA-DM itself is not affected by the presence of MARCH1. However, it was not cleary demonstrated if MARCH1 can modify DM. Some results obtained with a cytoplasmic tail of DM comprising an additional lysine suggest that there is a possibility that MARCH1 interact with DM. Generally, the work presented here show that ubiquitination by MARCH1 is involved in post-transcriptionnal regulation of the p33 isoform of the invariant chain but not in the regulation of HLA-DO and HLA-DM.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Obtaining enantio pure drug molecules is a long standing challenge in asymmetric synthesis implying that the identification of enantiomers and the determination of enantiomeric purity from a racemic mixture are of profound importance. In achieving this target NMR spectroscopy has proven to be an excellent analytical tool. It is well known that normal achiral NMR solvents do not distinguish the spectra of enantiomers. On the other hand, the conversion of substrates to diastereomers using one of the enantiopure chiral auxiliaries, such as, chiral solvating agent, chiral derivatizing agent and chiral lanthanide shift reagent, circumvents this problem. The imposition of diasteomeric interactions circumvents this problem. There is a pool of chiral auxiliaries available in the literature, each of which is specific to molecules of certain functionalities and has its own advantages and limitations. These classical methods have two limitations as they demand the presence of a targeted functional group in the chiral molecule and utilize only chemical shifts to visualize enantiomers. On the other hand in chiral anisotropic medium, due to differential ordering effect, the order-sensitive NMR observables, viz. chemical shift anisotropies (∆σi), dipolar couplings (Dij) and for nuclei with spin >1/2 the quadrupolar couplings (Qi) have enormous power of exhibiting different spectrum for each enantiomer permitting their discrimination. Numerous weakly ordered chiral aligning media have been reported in the literature. Nevertheless there is a scarcity of water compatible medium. Research work presented in this thesis is focused on various aspects, such as, the discovery of new chiral aligning medium for the enantiodiscrimination of water soluble chiral molecules, potential utility of DNA liquid crystal for discrimination of amino acids, on-the-fly monitoring of enzyme kinetics and the preparation of novel composite liquid crystals, hydrogels and thin films. The derived results are discussed in different chapters. Chapter 1 provides a brief introduction to NMR spectroscopy with special emphasis on the conceptual understanding of the tensorial interaction parameters, such as chemical shifts, scalar and dipolar couplings, quadrupolar couplings, effect of r.f pulses, basic introduction to 2D NMR experiments. Subsequently, a broad overview of the enantiomers, specification of their configurations, chirality without stereogenic carbon, chirality in molecules containing different atoms, are discussed. Following this a brief introduction to liquid crystals and their properties, their classification, their orientation in the magnetic field, order parameter are also discussed. The description on the chiral liquid crystals, the differential ordering effect, employment of the orientation dependent NMR interactions, utility of 2H NMR experiments for the visualization of enantiomers and the measurement of enantiomeric composition has been set out in brief. Chapter 2: As far as the organo soluble chiral molecules is concerned (in solvents such as, chloroform, dioxane, tetrahydrofuran and dimethylformamide), it has been well established that an ideal choice of chiral liquid crystal for enantiodiscrimination is poly-�-benzyl-L-glutamate (PBLG). Nevertheless, there is a scarcity of weak aligning medium for water soluble chiral molecules. This chapter introduces the chiral liquid crystal derived from the polysaccharide xanthan, which has numerous applications. The detailed discussion on the preparation of polysaccharide xanthan mesophase is given. The appearance of the mesophse is established by detecting the quadrupole split doublet of dissolved water. Subsequently enantiodiscrimination power of this new medium has been investigated on deuterated D/L-Alanine and (R/S)-β-butyrolactone. For such a purpose the selective 2D-SERF (SElective ReFocussing) experiment has been employed. It has been convincingly demonstrated that the medium has wide applicability for the discrimination of enantiomers, enantiotopic directions in prochiral molecules, measurement of enantiomeric excess and the RDCs in medium sized molecules. The new medium is sustainable over a wide range of temperature and concentration of ingredients, the mesophase is reversible, reproducible, easy to prepare besides being cost effective. It is possible to have the controlled tuning of the degree of order for specific application. Chapter 3: In this chapter the real discriminatory potential of DNA liquid crystalline phase has been explored. It is unambiguously established that; i) the fragmented DNA liquid crystal is able to differentiate between enantiomers of structurally different chiral amino acids; ii) the T1 (2H) values for L/D (alanine) is nearly equal indicating the similar dynamics for both the enantiomers, thus permitting the measurement of ee from the integral areas of the peaks of the contours of 2D spectrum; iii) the enantiotopic discrimination in prochiral compounds has also been successfully explored. Furthermore the analyses of NMR results yielded fruitful information on the analytical potential of DNA chiral liquid crystal, such as, (a) the chiral discrimination is effective on a large range of amino acids with spectral differences ΔΔʋQ‘s and ΔʋQ‘s varying from 80 to 338 Hz, and 50 to 900 Hz respectively; (b) the discrimination phenomenon remain active irrespective of the structure and the electronic nature (polarity) of the fourth substituent around the stereogenic center; (c) compared to an alkyl moiety, the presence of a terminal –OH or –SH group seems to slightly increase both the degree of alignment of the solute and the enantiodiscrimination efficiency compared to alanine; (d) The enantiodiscrimination can be detected easily not only on CD3 and CD groups, but also on CD2 sites exhibiting inequivalent diastereotopic directions; (e) discriminations with rather large differential ordering effect were obtained even for the sites that are situated far away from the asymmetric center; (f) The relative position of quadrupolar doublets from one 2H site to another can be reversed with regard to the absolute configuration (L/D). Chapter 4: Racemases recognize a chiral substrate such as (L-Alanine) and convert it into its enantiomer, i.e., (D-Alanine) and vice versa. Alanine racemase plays a vital role for certain bacteria, providing D-Alanine for peptidoglycan cell-wall biosynthesis. Elucidating the mechanism of enzymatic racemization is crucial for designing new inhibitors that may be useful as a novel class of antibiotics. This requires techniques to discriminate L-and D-Alanine and follow their concentrations as a function of time, so that one can determine the kinetic parameters and study the effect of inhibitors. In this chapter the utility of DNA liquid crystal media for in situ and real-time monitoring of the interconversion of L-and D-alanine-d3 by alanine racemase from Bacillus stearothermophilus has been demonstrated. The enantiomeric excess has been measured at different time intervals to monitor the enzymatic racemization at different time intervals in pseudo 2D NMR. The study unambiguously ascertains the reliability and robustness of utility of NMR in chiral anisotropic phase for monitoring the enzymatic racemization. The method thus provides new mechanistic insight and a better understanding of enzymatic reactions, in particular for alanine racemase. Chapter 5: In continuation with the development of weakly ordered liquid crystals, this chapter reports the spontaneous formation of composite graphene oxide (GO)/double stranded DNA (dsDNA) liquid crystals at higher concentrations of ingredients, and hydrogels at lower concentrations of ingredients, the process of which involves simple mixing in an aqueous phase has been demonstrated. The liquid crystalline phases and hydrogels have been characterized using optical polarized microscopy (OPM), scanning electron microscopy (SEM), Raman spectroscopy and 2H NMR spectroscopy. The observation of strong birefringence in the optical polarized microscope gives evidence for the formation of GO/dsDNA liquid crystals. The strong interaction between the dsDNA and GO was confirmed using Raman spectroscopic analysis. Furthermore, GO/dsDNA thin films have also been prepared and characterized using SEM and OPM. The GO/dsDNA thin film was prepared and its liquid crystal nature was established using OPM and 2H NMR. Importantly, the GO/dsDNA hydrogels were formed without any heat treatment to unwind dsDNA molecules and the porosity of hydrogels can be controlled by changing concentration of the dsDNA. This novel multifunctional composite liquid crystals and hydrogels of GO/dsDNA thus opens up new avenues for many applications like security papers, optical devices such as circular polarizers, reflective displays and drug delivery as well as tissue engineering using GO composite hydrogels.