L’objectif de cette thèse a été d’optimiser la réponse immunitaire anti-tumorale induite par les cellules dendritiques (DC) et les cellules iNKTs, en réponse à la prise en charge du KRN7000 (a-galactosyl-céramide) par la molécule CD1d située sur les DCs. Le premier axe de travail visait à synthétiser de nouveaux analogues du KRN7000, en fonctionnalisant la position C6 du sucre et en greffant un groupement phényl sur l’une des chaînes grasses. Les études in vitro ont montré que les modifications apportées par rapport au KRN7000 n’ont pas altéré la prise en charge des molécules obtenues par les DCs. Dans tous les cas, une sécrétion de cytokines a pu être observée. Des études complémentaires visant à décrire le profil cytokinique in vivo sont en cours. Le second axe a consisté en la mise au point d’une stratégie de vectorisation du KRN7000 afin de favoriser sa présentation aux DCs, en l’associant à des molécules d’intérêt comme un peptide spécifique d’une tumeur, une molécule de ciblage des DCs ou des ligands des TLRs. Dans les conditions utilisées, le phénomène d’anergie induit classiquement par l’administration répétée du KRN7000 n’a pas pu être levé. Cependant, nous avons montré d’une part que le KRN7000 vectorisé dans les liposomes est toujours pris en charge par les cellules dendritiques, et d’autre part qu’une réponse immunitaire se traduisant par la production de cytokines par les cellules iNKTs est induite. / The aim of this project was to optimize the anti-tumor immune response induced by dendritic and iNKTs cells, in response to KRN7000 (a-galactosyl-ceramide), which interacts with CD1d molecule situated on DCs. At first we synthesized new analogues of KRN7000, by functionalizing the C6 position of the carbohydrate moiety and by grafting a phenyl group on one of the fatty chain. In vitro studies indicated that chemical modifications of KRN7000 did not alter its interaction with CD1d. In all cases, cytokine secretion was observed. Further studies are in progress to describe the in vivo cytokine profile. In a second step, we developed liposomal constructs incorporating KRN7000 to optimize its presentation to DCs. Some constructions containing KRN7000 were able to associate a peptide, a targetting molecule of DCs or TLR ligands. Even if the anergy phenomenon induced by repeated administrations of KRN7000 could not be regulated by the use of liposomes, we have shown that encapsulated KRN7000 is still supported by DCs and that an immune response resulting in cytokines secretion by iNKT cells is induced.

Objective: While most allergic responses to food are directed against protein epitopes and occur within 30 minutes of ingesting the allergen, recent studies suggest that delayed reactions may occur, sometimes mediated by IgE antibodies directed against carbohydrate moieties. The objective of this review is to summarize the clinical features and management of delayed hypersensitivity reactions to mammalian meat mediated by IgE antibodies to galactose-alpha 1,3-galactose (alpha-gal), an oligosaccharide.Methods: A PubMed search was conducted with MeSH terms: galactosyl-(1,3) galactose, oligosaccharides, cetuximab, allergy/hypersensitivity, and anaphylaxis. Reported cases with alpha-gal-mediated reactions were reviewed. This research study was approved by the Institutional Review Board of East Tennessee State University.Results: Thirty-two cases of adults presenting with red-meat induced allergy thought to be related to oligosaccharides have been reported in the literature so far, making this a rare and evolving syndrome. Most of these patients demonstrated delayed reactions to beef, as was seen in the case reported by us in this manuscript. IgE specific to alpha-gal was identified in most patients with variable response to skin testing with beef and pork. Inhibition studies in some cases showed that the IgE antibodies to beef were directed towards alpha-gal in the meat rather than the protein. The patients often reported history of tick bites, the significance of which is unclear at present. Reactions to cetuximab, a monoclonal antibody, are mediated by a similar mechanism, with IgE antibodies directed against an alpha-gal moiety incorporated in the drug structure.Conclusion: Alpha-gal is an oligosaccharide recently incriminated in delayed anaphylactic reactions to mammalian meats such as to beef, pork, and lamb. It appears that anaphylactic reactions to the anti-cancer biological agent, cetuximab, may be linked mechanistically to the same process. More studies are required to understand the underlying molecular basis for these delayed reactions in specific, and their broader implications for host defense in general.

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

L'objectif de cette thèse a été d'optimiser la réponse immunitaire anti-tumorale induite par les cellules dendritiques (DC) et les cellules iNKTs, en réponse à la prise en charge du KRN7000 (a-galactosyl-céramide) par la molécule CD1d située sur les DCs. Le premier axe de travail visait à synthétiser de nouveaux analogues du KRN7000, en fonctionnalisant la position C6 du sucre et en greffant un groupement phényl sur l'une des chaînes grasses. Les études in vitro ont montré que les modifications apportées par rapport au KRN7000 n'ont pas altéré la prise en charge des molécules obtenues par les DCs. Dans tous les cas, une sécrétion de cytokines a pu être observée. Des études complémentaires visant à décrire le profil cytokinique in vivo sont en cours. Le second axe a consisté en la mise au point d'une stratégie de vectorisation du KRN7000 afin de favoriser sa présentation aux DCs, en l'associant à des molécules d'intérêt comme un peptide spécifique d'une tumeur, une molécule de ciblage des DCs ou des ligands des TLRs. Dans les conditions utilisées, le phénomène d'anergie induit classiquement par l'administration répétée du KRN7000 n'a pas pu être levé. Cependant, nous avons montré d'une part que le KRN7000 vectorisé dans les liposomes est toujours pris en charge par les cellules dendritiques, et d'autre part qu'une réponse immunitaire se traduisant par la production de cytokines par les cellules iNKTs est induite.

A doença de Chagas é considerada a terceira doença parasitária tropical de maior incidência no mundo, só superada pela malária e esquistossomose, e seu agente causador é o protozoário flagelado Trypanosoma cruzi. O parasita expressa uma enzima de superfície denominada trans-sialidase de Trypanosoma cruzi (TcTS), responsável pela transferência do ácidos siálicos de células do hospedeiro para moléculas de ?-galactose terminais presentes em glicoproteínas de sua superfície. As moléculas de glicoproteína sialiladas estão envolvidas na adesão e subsequente penetração do parasita em células hospedeiras. O papel fundamental da TcTS no reconhecimento e na invasão de células hospedeiras, bem como sua ausência em seres humanos, torna esta enzima um alvo potencial a ser estudado. A TcTS é específica em catalisar, preferencialmente, a transferência de ácido siálico para moléculas de mucina, originando ligações ?-2,3 com unidades de ?-galactose aceptoras na superfície do parasita. Considerando a importância da unidade de galactose e da função carboxila do ácido siálico para interações no sítio ativo de TcTS, priorizamos na síntese de derivados galactosil-triazolo-benzenossulfonamidas com diferentes substituintes, visto que o grupo sulfonamida é bioisóstero do ácido carboxílico, na busca de potenciais inibidores de TcTS. Os derivados galactosiltriazolo- benzenossulfonamidas 45-51 foram preparados via estratégia de click chemistry, por reação de ciclo-adição azido-alcino catalisada por Cu(I) (CuAAC), a partir do intermediário de galactose contento função amino terminal 30 e os derivados aril azidas 38-44. Após etapa de desacetilação, os produtos obtidos 52-58 foram testado em TcTS por ensaio fluorimétrico in vitro para avaliação de sua atividade inibitória. Os resultados obtidos são interessantes e bastante promissores, principalmente com os obtidos com o produto 58 (contendo o grupo galactosiltriazólico ligado a sulfapiridina), que apresentou atividade inibitória promissora (81%) na concentração de 1,0 mM, abrindo perspectivas para a síntese de um maior número de derivados galactosil-triazolo-benzenossulfonamidas com diferentes substituintes em R, para o estabelecimento de estudos de relação estruturaatividade. Adicionalmente, os compostos 53-55 foram testados em ensaios in vitro para avaliação de sua atividade tripanocida e citotóxica, e apresentaram atividade tripanocida máxima de 50%, normalmente nas concentrações de 500 a 250 ?M, com destaque para o derivado 55, contendo o grupo galactosil-triazólico ligado a sulfamerazina, que apresentou atividade moderada, mas superior ao benznidazol nas concentrações mais baixas (15,0 - 1,9 ?mol.L-1). Por outro lado, de acordo com os resultados do ensaio de citotoxicidade, a atividade citotóxica foi observada apenas nas concentrações mais elevadas, similar ao benznidazol. / Chagas disease is considered the third most common tropical parasitic disease worldwide, after malaria and schistosomiasis, and its causer is the flagellate protozoan, Trypanosoma cruzi. The parasite expresses a surface enzyme known as Trypanosoma cruzi trans-sialidase (TcTS), responsible for the transference of sialic acid from host cell to ?-galactose terminal molecules present in surface glycoproteins. Sialylated glycoproteins molecules are involved in adhesion and further penetration of parasite in host cell. Due to TcTS primordial role in recognizing and invasion of host cells, as well as its absence in humans, this enzyme becomes a potential target to be investigated. TcTS is specific on catalyzing, specially, transference of sialic acid to mucin molecule giving ?-2,3 bond with ?-galactose moiety in parasite surface. Considering the importance of the galactose moiety and the function of carboxylic in sialic acid for interactions in TcTS enzyme, we prioritized the synthesis of galactosyl-triazol-benzenesulfonamides derivatives with different substituents since sulfonamide group is bioisoster of carboxylic acid, in attempt to produce potential inhibitors of TcTS. The galactosyl-triazol-benzenesulfonamides derivatives 45-51 were prepared via click chemistry reaction (Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)) from galactose intermediate with terminal amino group 30 and aril azides derivatives 38-44. After removing acetyl group, the inhibiting activity of products 52-58 were evaluated in TcTS fluorimetric in vitro assay. We found very promising results, specially with 58 (containing galactosyl-triazolic group bonded to sulfapyridine), wich showed 81% of inhibitory activity in 1,0mM solution, bringing expectations for synthesis of greater number of galactosyl-triazolbenzenesulfonamides derivatives with different substituents in R, to establish studies of structure relationship activity. Additionally, trypanocidal and cytotoxic activity of compounds 53-55 were tested and showed maximum activity of 50%, commonly in concentrations of 500 to 250 ?M, specially compound 55, containing galactosyltriazolic group bonded to sulfamerazine, with showed moderate activity, but higher then benznidazol in lower concentrations (15,0 - 1,9 ?mol.L-1). On the other hand, according to cytotoxicity results, activity were observed only in higher concentrations, as for benznidazol.

A doença de Chagas é considerada a terceira doença parasitária tropical de maior incidência no mundo, só superada pela malária e esquistossomose, e seu agente causador é o protozoário flagelado Trypanosoma cruzi. O parasita expressa uma enzima de superfície denominada trans-sialidase de Trypanosoma cruzi (TcTS), responsável pela transferência do ácidos siálicos de células do hospedeiro para moléculas de ?-galactose terminais presentes em glicoproteínas de sua superfície. As moléculas de glicoproteína sialiladas estão envolvidas na adesão e subsequente penetração do parasita em células hospedeiras. O papel fundamental da TcTS no reconhecimento e na invasão de células hospedeiras, bem como sua ausência em seres humanos, torna esta enzima um alvo potencial a ser estudado. A TcTS é específica em catalisar, preferencialmente, a transferência de ácido siálico para moléculas de mucina, originando ligações ?-2,3 com unidades de ?-galactose aceptoras na superfície do parasita. Considerando a importância da unidade de galactose e da função carboxila do ácido siálico para interações no sítio ativo de TcTS, priorizamos na síntese de derivados galactosil-triazolo-benzenossulfonamidas com diferentes substituintes, visto que o grupo sulfonamida é bioisóstero do ácido carboxílico, na busca de potenciais inibidores de TcTS. Os derivados galactosiltriazolo- benzenossulfonamidas 45-51 foram preparados via estratégia de click chemistry, por reação de ciclo-adição azido-alcino catalisada por Cu(I) (CuAAC), a partir do intermediário de galactose contento função amino terminal 30 e os derivados aril azidas 38-44. Após etapa de desacetilação, os produtos obtidos 52-58 foram testado em TcTS por ensaio fluorimétrico in vitro para avaliação de sua atividade inibitória. Os resultados obtidos são interessantes e bastante promissores, principalmente com os obtidos com o produto 58 (contendo o grupo galactosiltriazólico ligado a sulfapiridina), que apresentou atividade inibitória promissora (81%) na concentração de 1,0 mM, abrindo perspectivas para a síntese de um maior número de derivados galactosil-triazolo-benzenossulfonamidas com diferentes substituintes em R, para o estabelecimento de estudos de relação estruturaatividade. Adicionalmente, os compostos 53-55 foram testados em ensaios in vitro para avaliação de sua atividade tripanocida e citotóxica, e apresentaram atividade tripanocida máxima de 50%, normalmente nas concentrações de 500 a 250 ?M, com destaque para o derivado 55, contendo o grupo galactosil-triazólico ligado a sulfamerazina, que apresentou atividade moderada, mas superior ao benznidazol nas concentrações mais baixas (15,0 - 1,9 ?mol.L-1). Por outro lado, de acordo com os resultados do ensaio de citotoxicidade, a atividade citotóxica foi observada apenas nas concentrações mais elevadas, similar ao benznidazol. / Chagas disease is considered the third most common tropical parasitic disease worldwide, after malaria and schistosomiasis, and its causer is the flagellate protozoan, Trypanosoma cruzi. The parasite expresses a surface enzyme known as Trypanosoma cruzi trans-sialidase (TcTS), responsible for the transference of sialic acid from host cell to ?-galactose terminal molecules present in surface glycoproteins. Sialylated glycoproteins molecules are involved in adhesion and further penetration of parasite in host cell. Due to TcTS primordial role in recognizing and invasion of host cells, as well as its absence in humans, this enzyme becomes a potential target to be investigated. TcTS is specific on catalyzing, specially, transference of sialic acid to mucin molecule giving ?-2,3 bond with ?-galactose moiety in parasite surface. Considering the importance of the galactose moiety and the function of carboxylic in sialic acid for interactions in TcTS enzyme, we prioritized the synthesis of galactosyl-triazol-benzenesulfonamides derivatives with different substituents since sulfonamide group is bioisoster of carboxylic acid, in attempt to produce potential inhibitors of TcTS. The galactosyl-triazol-benzenesulfonamides derivatives 45-51 were prepared via click chemistry reaction (Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)) from galactose intermediate with terminal amino group 30 and aril azides derivatives 38-44. After removing acetyl group, the inhibiting activity of products 52-58 were evaluated in TcTS fluorimetric in vitro assay. We found very promising results, specially with 58 (containing galactosyl-triazolic group bonded to sulfapyridine), wich showed 81% of inhibitory activity in 1,0mM solution, bringing expectations for synthesis of greater number of galactosyl-triazolbenzenesulfonamides derivatives with different substituents in R, to establish studies of structure relationship activity. Additionally, trypanocidal and cytotoxic activity of compounds 53-55 were tested and showed maximum activity of 50%, commonly in concentrations of 500 to 250 ?M, specially compound 55, containing galactosyltriazolic group bonded to sulfamerazine, with showed moderate activity, but higher then benznidazol in lower concentrations (15,0 - 1,9 ?mol.L-1). On the other hand, according to cytotoxicity results, activity were observed only in higher concentrations, as for benznidazol.