Les glycanes présents à la surface des cellules cancéreuses sont souvent modifiés par rapport à ceux d’une cellule saine. Or ces antigènes glucidiques n’induisent pas de réponse immune efficace. La GalNAc est le premier sucre fixé lors de la O-glycosylation de type mucine et ainsi ce sucre entre dans la composition de nombreux antigènes tumoraux. Le but de notre travail était de préparer des analogues synthétiques de la GalNAc susceptibles d’être incorporés à la surface de cellules cancéreuses et dans les mucines synthétisées par les tumeurs, afin d’augmenter la réponse immune vis-à-vis des glycanes tumoraux. Nous avons synthétisé chimiquement des analogues de la GalNAc afin de les tester in vitro en tant que substrats de la voie de « sauvetage » de la GalNAc chez les mammifères et donc d’enzymes impliquées dans cette voie : une kinase (GK2) et une UDP-pyrophosphorylase (AGX1) humaines. Les meilleurs candidats ont permis la synthèse de différents UDP-sucres et une GalNAc-transférase (ppGalNAc T1) bovine a pu être utilisée in vitro pour transférer certains de ces analogues, à partir de leur forme activée en UDP-sucre, sur des peptides. Nous avons donc pu montrer que certains des analogues synthétisés étaient capables de s’intégrer dans la voie de sauvetage et d’être incorporés dans des peptides. Le pouvoir immunologique des glycoconjugués de type mucine ainsi formés a été étudié chez la souris après couplage de ces glycoprotéines à une protéine immunostimulante (KLH). D’autre part, des cellules de mammifères ont également été cultivées en présence de ces analogues afin de vérifier leur incorporation au niveau des glycoconjugués de la surface des cellules. / Glycans are often present at the cancerous cell surface in a modified form compared to healthy cells. However, these carbohydrate antigens don’t lead to an effective immune response. GalNAc is the first sugar attached to mucin type O-glycans and is thus a component of numerous tumor antigens. The aim of our work was to prepare synthetic GalNAc analogs able to be incorporated at the surface of cancer cell and into mucins synthesized by tumors in order to increase the immune response toward tumor glycans. We chemically synthesized GalNAc analogs to test them in vitro as substrates of enzymes involved in the mammalian GalNAc salvage pathway: a human galactokinase (GK2) and a human UDP-pyrophosphorylase (AGX1). The best candidates allowed the synthesis of the corresponding UDP-sugars further used to test the transfer of those analogs onto peptides using a bovine GalNAc transferase (ppGalNAc T1). We have shown that some synthetic analogs could be integrated in the GalNAc salvage pathway and O-linked to peptides. Immunological properties of the glycoconjugates thus formed were studied in mice after coupling to an immunostimulant protein (KLH). Moreover, mammalian cells were cultivated in the presence of these analogs in order to check their incorporation into glycoconjugates at the cell surface.

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Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.

Les glycanes présents à la surface des cellules cancéreuses sont souvent modifiés par rapport à ceux d'une cellule saine. Or ces antigènes glucidiques n'induisent pas de réponse immune efficace. La GalNAc est le premier sucre fixé lors de la O-glycosylation de type mucine et ainsi ce sucre entre dans la composition de nombreux antigènes tumoraux. Le but de notre travail était de préparer des analogues synthétiques de la GalNAc susceptibles d'être incorporés à la surface de cellules cancéreuses et dans les mucines synthétisées par les tumeurs, afin d'augmenter la réponse immune vis-à-vis des glycanes tumoraux. Nous avons synthétisé chimiquement des analogues de la GalNAc afin de les tester in vitro en tant que substrats de la voie de " sauvetage " de la GalNAc chez les mammifères et donc d'enzymes impliquées dans cette voie : une kinase (GK2) et une UDP-pyrophosphorylase (AGX1) humaines. Les meilleurs candidats ont permis la synthèse de différents UDP-sucres et une GalNAc-transférase (ppGalNAc T1) bovine a pu être utilisée in vitro pour transférer certains de ces analogues, à partir de leur forme activée en UDP-sucre, sur des peptides. Nous avons donc pu montrer que certains des analogues synthétisés étaient capables de s'intégrer dans la voie de sauvetage et d'être incorporés dans des peptides. Le pouvoir immunologique des glycoconjugués de type mucine ainsi formés a été étudié chez la souris après couplage de ces glycoprotéines à une protéine immunostimulante (KLH). D'autre part, des cellules de mammifères ont également été cultivées en présence de ces analogues afin de vérifier leur incorporation au niveau des glycoconjugués de la surface des cellules.

Fibroblast growth factor-23 (FGF23) is a novel, bone-produced hormone that regulates renal phosphate (Pi) reabsorption and calcitriol metabolism. Disorders of mineral and bone metabolism, such as autosomal dominant hypophosphatemic rickets (ADHR) and hyperostosis-hyperphosphatemia syndrome (HHS), witness the importance of well-balanced serum levels of FGF23. Patients with chronic kidney disease (CKD) are highly morbid due to Pi retention/hyperphosphatemia and calcitriol deficiency, which lead to elevated serum levels of parathyroid hormone (PTH) and secondary hyperparathyroidism (sHPT). As a response to hyperphosphatemia, CKD patients have also remarkably high serum FGF23 levels, which are associated with cardiovascular risk factors and increased mortality in CKD. The overall aim of this dissertation was to discern a possible role of FGF23 in parathyroid biology. Our in vitro experiments on isolated bovine parathyroid cells demonstrate that FGF23 directly and dose-dependently suppresses the PTH production and secretion, while increasing the expression of the 25-hydroxyvitamin D3-activating enzyme 1α-hydroxylase. We investigated possible expressional changes in the FGF23 receptor co-factor Klotho in hyperparathyroid disorders and found that Klotho expression is decreased or absent and inversely correlated to serum calcium (Ca) in adenomas of primary HPT (pHPT). In the hyperplastic parathyroid glands of sHPT, Klotho expression declines in parallel with the kidney function and correlates with the glomerular filtration rate. Moreover, Klotho expression is suppressed by Ca and FGF23, increased by calcitriol, but unaffected by Pi and PTH in vitro. Finally, we identified a novel missense mutation in the gene encoding GALNT3, which is normally involved in the post-translational glycosylation of FGF23, as the cause of aberrant FGF23 processing in a patient with HHS. In summary, we provide evidence for a novel bone/parathyroid axis in which FGF23 functions as a direct, negative regulator of the PTH production. High extracellular Ca is a major determinant of the Klotho expression in pHPT, whereas the Klotho levels in sHPT may be attributed to a combination of the high FGF23 and Ca, and low calcitriol levels associated with CKD. Hence, the decreased Klotho expression in sHPT could explain the concomitantly high FGF23 and PTH levels, as well as the failure of FGF23 to prevent or mitigate the development of sHPT in CKD.