Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. 210-236. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

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The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with escape of immune defenses. Human macrophage galactose-type lectin (hMGL, HML, CD301 or CLEC10A), a C-type lectin expressed by antigen presenting cells (APC), is a receptor of mucin-type TACAs, -GalNAc (Thomsen nouvelle antigen; Tn; CD175) and its 2,6-sialylated derivative (sTn; CD175s). To date, the relative contributions of these glycans, as well as underlying peptide backbone, and different degrees of valency, on binding thermodynamics and kinetics with hMGL remains elusive. In order to discern the subtle utility of these distinct features, chemical syntheses of the MUC1, HGVTSAPDTRPAPGSTAPPA tandem repeat sequence, and its site-specific serine (Ser) and threonine (Thr) glycosylated analogs were carried out. Circular dichroism (CD) spectroscopy experiments detected increasing structural order of the Thr glycopeptides compared to its nonglycosylated analogs. Isothermal titration calorimetry (ITC) data analysis of lectin binding to the Thr glycopeptides invariably showed enthalpy-driven processes. Affinity enhancement of the Thr glycopeptides for hMGL occurred relative to free GalNAc, revealing an increasing trend in affinity by one order of magnitude, for mono- (KD = 6-8 μM) to triglycosylated (KD = 600 nM) MUC1 peptides. To delineate the relevance of the solvent structure in the protein carbohydrate recognition process, experiments in D2O were performed, exposing enthalpy-entropy compensation differences. KinITC analysis highlighted prolonged complex lifetimes. Furthermore, atomic force microscopy (AFM) based dynamic single-molecule force spectroscopy (SMFS) provided molecular level insight into the energy landscapes governing recognition of the MUC1(Tn)-hMGL complexes. In summary, our results suggest that contact with hMGL critically depends on the type of TACA, nature of the vicinity surrounding the glycan, and its density. This highlights the importance and current efforts in design of prophylactic and therapeutic cancer vaccines with special emphasis on the synthetic glycopeptide vaccines. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

The aim of this thesis was the development of the method for the determination of eight monosaccharides commonly found in glycoproteins by capillary electrophoresis. Namely, it was determination of glucose, galactose mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, N-acetylneuraminic acid and xylose. Total length of silica capillary with inner diameter of 10 m was 50.0 cm and effective length was 35.0 cm. Background electrolyte was compound of sodium hydroxide of 50 mmol/l concentration, disodium phosphate of 22.5 mmol/l concentration and cetyltrimethylamoniumbromide of 0.2 mmol/l concentration. Samples were injected hydrodynamically with pressure of 5 kPa for 70 s, driving voltage was -30 kV and the pressure of 270 kPa was applied to the outlet vial during the separation; capacitively coupled contactless conductivity detector was used to detect the analytes. The limits of detection were between 5 and 7 mg/l and the limits of quantification were between 16 and 22 mg/l. Repeatability of peak areas and migration times related to 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid as an internal standard showed values of relative standard deviation lower than 4 %. Conditions for hydrolysis of oligosaccharides to monosaccharides were determined as 4M hydrochloric acid and 100 řC, hydrolysis...

In this project, separation of trypsin digested transferrin (Tf) has been studied, using a RP HPLC- UV system equipped with a C18 column. 0.1% TFA/MQ-water and 90% MeOH were used as mobile phase A and mobile phase B, respectively. For economic reasons, the protein cytochrome c (cyt-C) was used to optimize the digestion procedure and LC system, before analysis of Tf. Four digestion methods were applied for analyzing cyt-C and Tf. The first method was digestion with no denaturing, reducing or alkylating agent. The other digestion methods used urea or heating as a denaturing agent, and lastly dithiothreitol (DTT) and iodoacetamide (IAA) as reducing and alkylating agent, respectively. The results from HPLC-UV showed that a gradient elution with a high concentration of organic solvent is favorable for the separation of cyt-C peptides. MALDI-MS was used to identify peptides, and the outcomes showed that denaturation by heat before digestion gave the best results. / I detta projekt har separation av trypsin-klyvt transferrin (Tf) studerats, med användning av ett RP HPLC-UV system, som bestod av en C18 kolonn. 0,1% TFA/MQ-vatten och 90% MeOH användes som mobilfas A respektive mobilfas B. Av ekonomiska skäl användes proteinet cytokrom c (cyt-C) före analys av Tf för att optimera klyvningsprocessen och LC systemet. Fyra klyvningsmetoder studerades för analysering av cyt-C och Tf. Den första metoden innehöll inget denaturerande, reducerande eller alkylerande medel. De andra klyvningsmetoderna innehöll urea eller värme som denaturerande medel, och slutligen ditiotreitol (DTT) och jodacetamid (IAA) som reducerande respektive alkylerande medel. Resultaten från HPLC-UV visade att en gradienteluering med en hög koncentration av den organiska lösningen är gynnsam för separationen av peptiderna från cyt-C. MALDI-MS användes för att identifiera peptiderna, och resultaten visade att denaturering med värme före klyvning gav bäst resultat.

Titre de l'écran-titre (visionné le 11 janvier 2024) / La chimie des glucides a connu un essor dans les dernières décennies avec le développement de nouvelles méthodes de synthèses, de réactions et la compréhension des rôles essentiels des glucides dans différents systèmes biologiques. Ces nouvelles connaissances ont mené à la mise en marché de nouveaux médicaments pour le traitement de maladies et d'infections. Malgré qu'ils soient peu nombreux, plusieurs sont considérés comme essentiels par l'Organisation Mondiale de la Santé. Les études effectuées sur les glycopeptides et les glucides fluorés ont permis de nombreuses avancées pour le développement de sondes moléculaires à base de glucides et l'apprentissage de processus biologiques. Dans un objectif d'explorer le potentiel biologique des glycopeptides, une nouvelle méthode de synthèse de glycopeptides $C$-terminaux a été développée par une réaction d'aminolyse de peptides sur résine oxime. L'optimisation de la réaction a été effectuée à l'aide de composés modèles et la robustesse a été testée pour de nombreux groupements fonctionnels. Cet avancement a permis la préparation d'une chimiothèque d'analogues des lincosamides, une classe d'antibiotiques glucidiques. De plus, comme les lincosamides possèdent une activité antipaludique intéressante, des analogues hybrides entre la clindamycine, la chloroquine et la mortiamide D ont été synthétisés. Ces nouvelles molécules ont aussi été testées contre différentes souches du parasite $Plasmodium falciparum$ pour déterminer leur potentiel antipaludique. Puisque les réactions de glycosylation sont cruciales dans le développement de nouveaux médicaments à base de glucides, une nouvelle approche d'alkylation de la position anomérique de glucides fluorés a été développée. Lors de l'exploration de cette méthode, des analogues fluorés de l'étoposide, un agent anticancéreux, ont été synthétisés. Aussi, la synthèse d'un ʟ-nucléoside fluoré a été revisitée à partir du 2-désoxy-2-fluoro-ᴅ-galactopyranose; la clévudine (ʟ-FMAU). Cette dernière a été synthétisée en moins d'étapes et avec de meilleurs rendements. Deux étapes clés ont permis un accès facile au motif ʟ-arabinose : une cyclisation promue à l'iode et un clivage oxydatif. / Carbohydrate chemistry had a boom in the last decades with the development of new synthetic methods, reactions, and understanding of the essential roles of carbohydrates in various biological systems. This new knowledge has led to the marketing of new drugs for the treatment of diseases and infections. Although there are few, several are considered essential by the World Health Organization. The research carried out on glycopeptides and fluorinated carbohydrates has led to many advances for the development of carbohydratebased molecular probes and the learning of biological processes. With an objective to explore the biological potential of glycopeptides, a new method for the synthesis of $C$-terminal glycopeptides has been developed by an aminolysis reaction of peptides on oxime resin. Reaction optimization was performed using model compounds and robustness was tested for many functional groups. This advancement helps in the preparation of lincosamide analogs, a class of carbohydrate antibiotics. Moreover, as lincosamides possess interesting antimalarial activities, hybrid analogues between clindamycin, chloroquine and mortiamide D have been synthesized. These new molecules were also tested against different strains of the $Plasmodium falciparum$ parasite to determine their antimalarial potential. As glycosylation reactions are crucial in the development of new carbohydrate drugs, a new approach for alkylation of the anomeric position of fluorinated carbohydrates has been developed. While exploring this method, fluorinated analogues of etoposide, an anti-cancer agent, were synthesized. Also, the synthesis of a fluorinated ʟ-nucleoside has been revisited from 2-deoxy-2-fluoro-ᴅ-galactopyranose. Clevudine (ʟ-FMAU) was synthesized in fewer steps and in better yields with two key steps that allowed easy access to the ʟ-arabinose motif: molecular iodine-promoted cyclization and oxidative cleavage.

Staphylococcus aureus est une bactérie pathogène opportuniste de l’Homme et de l’animal qui représente l’une des premières causes d’infections nosocomiales. A l’heure actuelle, la sévérité de ces infections est accentuée par l’augmentation des multi-résistances aux antibiotiques qui en font un problème majeur de santé public. La découverte et l’étude des ARN régulateurs (ARNrég) exprimés par le staphylocoque doré a permis de mettre en évidence leurs rôles de régulateurs de l’étape de colonisation et d’infection (quorum sensing, virulence, résistance aux antibiotique, adaptation environnementales). Au cours de ma thèse, je me suis intéressée à l’étude de deux ARNrég, SprD impliqué dans la virulence de la bactérie sur modèle animal murin et SprX régulant la résistance aux glycopeptides. Dans le but de comprendre les bases moléculaires de leurs rôles dans la bactérie, nous avons souhaité identifier et valider l’ensemble des cibles directes à ces deux ARN. Dans un premier temps, nous nous sommes intéressés à l’étude des nouvelles cibles de SprX. Pour cela, nous avons mis en place une approche in vivo de validation des cibles des ARNrég basée sur la technique des gènes rapporteurs. Nous avons ainsi pu valider deux nouvelles cibles de SprX, les ARNm ecb et purC impliqués respectivement dans l’évasion du système immunitaire et dans le métabolisme des purines. SprX interagit au niveau du site d’initiation de la traduction de ces deux ARNm, et inhibe leurs traductions. Par la suite, nous avons souhaité rechercher de nouvelles cibles pour SprD. Nous avons pu adapter à S. aureus une technique d’identification de nouvelles cibles des ARNrég. L’ensemble des résultats obtenus permettent de préciser les rôles déjà connus des deux ARNrég et offrent de nouvelles possibilités concernant l’étude et la validation des cibles potentielles. / Staphylococcus aureus is an opportunistic pathogen that causes a wide variety of nosocomial and community-acquired infections. Its adaptive capacities, multiple antibiotic resistances and high virulence makes it a major public health issue. The discovery and study of small regulatory RNAs (sRNAs) expressed by S. aureus showed their implication in the switch colonization to host infection. During my phD, I focused on the study of two sRNAs: (i) SprD, involved in staphylococcal virulence and (ii) SprX which regulates glycopeptides resistance. To understand the molecular basis of their roles in staphylococcal physiology, we searched for their direct targets using two different techniques. For SprX, we set up an in vivo technique to validate mRNA targets previously predicted using bioinformatics tools. This method based on the use of a fluorescent reporter gene allowed us to validate two novels SprX targets, the ecb and purC mRNAs involved in immune evasion system and purine metabolism, respectively. We further showed that SprX interacts at the ribosome binding sites preventing translation initiation. In another chapter of my phD project, we adapted the MAPS technique to S. aureus and applied it to the search of SprD direct partners by affinity purification of sRNA-mRNA duplexes. Overall, the results obtained during my thesis allowed us to precise the roles of SprD and SprX and will offer novel possibilities for the staphylococcal community to identify and/or validate of sRNA targets.

Paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis. Aproximadamente 10 milhões de pessoas que vivem em áreas endêmicas estão infectadas com P. brasiliensis e até 2% destas podem desenvolver a PCM. Períodos extensos de quimioterapia são necessários e problemas de recidiva da doença são frequentes. Vacinas anti-PCM ainda não estão disponíveis para uso em humanos, porém, nos últimos anos, testes em modelo animal mostram que estratégias vacinais são viáveis. Formulações vacinais baseadas na proteína gp43 e no peptídeo P10 específico para células T CD4+ mostram-se promissores como antígenos vacinais. No presente trabalho, testamos as propriedades de uma formulação vacinal terapêutica baseada no peptídeo P10 combinado com flagelina de S. enterica, associada ou não a quimioterapia, como tratamento da PCM experimental. Os resultados indicaram que animais tratados apenas com a vacina, combinada ou não com os quimioterápicos, não demonstraram uma redução significativa da carga fúngica no pulmão. No entanto granulomas mais organizados foram observados no pulmão dos animais que receberam a formulação vacinal. / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Approximately 10 million people living in endemic areas may be infected with this fungus and up to 2% of them may develop the disease. Extended periods of chemotherapy are often necessary to treat PCM and relapses occur frequently. PCM vaccines are not yet available for use in humans, but in recent years tests in animal models showed that vaccine strategies are viable. Vaccine formulations based in protein gp43 and a derived peptide P10, representing a CD4+ T cell-specific epitope, have showed promising results as vaccine antigens. In the present work, we tested the properties of a vaccine formulation based on the P10 peptide admixed with the S. enterica flagellin, in combination or not with chemotherapy, as a PCM therapeutic treatment. Treated mice, combined or not with chemotherapy, showed no significant reduction in lung fungal burden. Nonetheless, more organized lung granulomas were observed in animals that received the vaccine formulation.

Paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis. Aproximadamente 10 milhões de pessoas que vivem em áreas endêmicas estão infectadas com P. brasiliensis e até 2% destas podem desenvolver a PCM. Períodos extensos de quimioterapia são necessários e problemas de recidiva da doença são frequentes. Vacinas anti-PCM ainda não estão disponíveis para uso em humanos, porém, nos últimos anos, testes em modelo animal mostram que estratégias vacinais são viáveis. Formulações vacinais baseadas na proteína gp43 e no peptídeo P10 específico para células T CD4+ mostram-se promissores como antígenos vacinais. No presente trabalho, testamos as propriedades de uma formulação vacinal terapêutica baseada no peptídeo P10 combinado com flagelina de S. enterica, associada ou não a quimioterapia, como tratamento da PCM experimental. Os resultados indicaram que animais tratados apenas com a vacina, combinada ou não com os quimioterápicos, não demonstraram uma redução significativa da carga fúngica no pulmão. No entanto granulomas mais organizados foram observados no pulmão dos animais que receberam a formulação vacinal. / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Approximately 10 million people living in endemic areas may be infected with this fungus and up to 2% of them may develop the disease. Extended periods of chemotherapy are often necessary to treat PCM and relapses occur frequently. PCM vaccines are not yet available for use in humans, but in recent years tests in animal models showed that vaccine strategies are viable. Vaccine formulations based in protein gp43 and a derived peptide P10, representing a CD4+ T cell-specific epitope, have showed promising results as vaccine antigens. In the present work, we tested the properties of a vaccine formulation based on the P10 peptide admixed with the S. enterica flagellin, in combination or not with chemotherapy, as a PCM therapeutic treatment. Treated mice, combined or not with chemotherapy, showed no significant reduction in lung fungal burden. Nonetheless, more organized lung granulomas were observed in animals that received the vaccine formulation.

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