In recent years the glycan shield of the HIV-1 envelope spike (Env) has emerged as a potential target for microbicide and vaccine design. The densely packed glycans on its surface include an intrinsic population of under-processed oligomannose structures, and a number of lectins and broadly neutralising antibodies (bnAbs) have been isolated which are reactive to these ‘non-self’ glycan structures. The potential value of these agents in therapeutic or vaccine contexts depends upon the prevalence of their glycan targets in nature and their resilience to sequence mutation. Here the prevalence of oligomannose-type glycans on recombinant gp120 was demonstrated across a panel of isolates, revealing subtle cross clade differences. Alanine scanning of all potential N-glycosylation sites (PNGSs) within a model gp120 demonstrated the overall stability of the oligomannose population, but highlighted regions of glycan clusters where individual glycans act to limit the processing of their neighbours. This was formally demonstrated for the N332 ‘site of vulnerability’, where deletion of nearby glycosylation sites led to altered glycan processing at the N332 site. A panel of N332-dependent bnAbs was screened for their ability to tolerate such changes in glycan processing, with differing results. While some displayed promiscuous binding, others were more sensitive to glycan microheterogeneity. Site-specific glycosylation analysis of the PGT135 epitope revealed that an intolerance of certain glycoforms may explain its limited breadth. While a greater understanding of Env glycan microheterogeneity and bnAb promiscuity is required, these findings reveal insights into the architecture of the HIV-1 glycan shield that suggest it is a conserved and robust target for drug and vaccine design.

Iminosugars are known to behave as carbohydrate mimics in biological systems by virtue of their similar structures. However as the ring nitrogen prevents metabolism it means that iminosugars have the potential to become inhibitors of these systems. It is known, for example, that iminosugars can behave as mimics in the hydrolysis mechanism. This leads to possible medicinal applications of iminosugars. One such case is lysosomal storage disorders which arise as a result of a genetic defect which causes missense mutations coding for the N-acetylhexosaminidase enzymatic protein. N-Acetylhexosaminidases are a sub-member of the class of glycosidase enzymes. They are responsible for the cleavage of N-acetylhexosamine residues from glycoconjugates in the lysosome. Mutations in the gene coding for this protein lead to a deficiency in the enzymatic activity resulting in accumulation of unhydrolysed substrate in the lysosome. Lysosomal storage disorders have a phenotype of poor motor development and neurological problems. The infantile form usually leads to death before the age of five. An iminosugar mimic could give rise to a possible treatment for lysosomal storage disorders by acting as a molecular chaperone during protein folding, promoting correct folding by its intrinsic affinity for the native fold of the enzyme. Likewise in the treatment of cancer, the inhibitory ability of iminosugars has potential applications. In cancer, extracellular hydrolysis occurs which favours cancer cell survival. Macrophages, which attack and eliminate cancer cells, can be activated by macrophage activating factor (MAF) which displays an α-N-acetylgalactosamine residue that appears essential for the activation cascade. Cancer cells secrete an α-N-acetylgalactosaminidase enzyme that acts to decrease the potency of MAF, thus promoting cancer cell survival. Inhibition of cancer cell α-N-acetylgalactosaminidase may restore macrophage activation and generate potential therapeutics. Chapter 1 of this thesis contains extended discussion of the aforementioned, and related, diseases and the therapeutic applications of iminosugars. Some historically and biologically important iminosugars are described along with some current iminosugar drugs. Chapter 2 describes the synthetic strategies explored in an attempt to synthesise all the members of the 2-acetamido pyrrolidine iminosugars. An overview of the compounds synthesised towards this end by a past group member is given along with the work performed as part of this thesis to complete this goal. Both enantiomers with arabino- and ribo- stereochemistry and D-lyxo- were previously synthesised. The syntheses of both enantiomers with xylo- stereochemistry along with the L-lyxo- compound were completed as part of this thesis, from either D- or L-glucuronolactone and D-ribose, respectively. Chapter 3 details the synthetic strategy adopted to synthesise the enantiomer of D-DNJNAc, the first potent α-N-acetylgalactosaminidase inhibitor to be found. The synthesis towards another piperidine iminosugar, 6-deoxy DGJNAc, is presented in the second half of this chapter, along with two related compounds.

Diagnoses of tuberculosis, 'TB,' currently rely upon non-specific techniques such as X-ray exams and acid-fast microscopy. Improved diagnostics would preferably consider specific bacterial processes to provide real-time readouts of disease burden and response to chemotherapy. This dissertation presents the cell-wall incorporation of trehalose analogues (fluorescent and radioactive) by the mycobacterial antigen 85 enzymes as a novel method to label the causative bacteria of TB, Mycobacterium tuberculosis (Mtb). The trehalose mycolyltransesterase enzymes (antigens 85A, B, and C (Ag85)) serve as essential mediators of cell envelope function and biogenesis in Mtb. We show that the Ag85 enzymes display activities so broad that they allow added non-natural carbohydrate probes to be incorporated into Mtb growing in vitro and within macrophages. Design and synthesis of a library of structurally-diverse analogs of the sugar trehalose (Tre) revealed that Ag85-enzymes catalyze esterification of a wide variety of non-natural Tre structures, even stereoisomers and those appended with charged or bulky groups (Chapter 2). A novel mass-spectrometry based Ag85 enzyme assay was developed and employed to screen the library of compounds against all three isoforms of Ag85 (Chapter 3). This screen revealed that the Ag85 enzymes exhibit preference for dissacharides over monosaccharides and a broad tolerance for most modified trehalose compounds. This activity assay also afforded full kinetic analysis and the discovery of a novel, covalent inhibitor of the Ag85 enzymes. The Ag85 activity assay informed the design of a fluorescent trehalose-based compound (FITC-Tre), which is the first, non-toxic, selective, small molecule probe for mycobacterial infection. FITC-Tre was acylated with mycolyl esters by growing mycobacteria, anchoring the probe in the cell envelope resulting in fluorescent bacteria (Chapter 4). Adding FITC-Tre to Mtb-infected macrophages allowed selective, fluorescent tagging of Mtb in vivo (Chapter 5). Colocalization studies with antibodies against a variety of phagosomal associated components have hinted at the possibility of FITC-Tre as readout of cellular trafficking of bacteria. ¹⁸F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. ¹⁸F-trehalose shows promise as Mtb selective PET probe in an infected rabbit model of tuberculosis. Future work with these probes may allow for fluorescent tracking of the Mtb during the macrophage infection process, as well as the ability to label Mtb in infected tissue. The functional differences between the three isoforms of Ag85, A, B and C, are not well understood and may have implications for the survival and persistence of mycobacteria within humans. The differences in substrate specificity and catalytic activity between the Ag85 isoforms (discussed in Chapter 3) has been further investigated (Chapter 6). Mutation of three secondary site amino acids from Ag85C into Ag85B afforded nearly a twenty-fold gain in enzyme activity. Mutation of the equivalent Ag85B residues into Ag85C triggered nearly a twenty-fold loss in activity. Dissection of the roles of these three amino acids helps to explain the previously reported large differences in catalytic activity between Ag85A, B and C. Overexpression of Ag85A, B and C under tetracycline regulation revealed that these enzymes differentially modulate incorporation of mycolates into the cell wall. The Ag85 enzymes are not functionally redundant, and instead serve unique purposes in cell wall biosynthesis. In summary, this research has demonstrated that the broad substrate tolerance of Ag85 enzymes, coupled with their extracellular location, opens the door to probes of mycobacterial infection using many imaging modalities.

L’hépatite C est une maladie due à l’infection par le virus de l’hépatite C (VHC), qui se transmet par le sang. On considère que ce virus infecte 3 à 4 millions de personnes chaque année, sa prévalence pouvant aller jusqu’à 22% de la population. Ces dernières années, le développement de thérapies antivirales spécifiques du virus à agents antiviraux directs (AAD) a permis de faire fortement régresser l’infection dans les pays développés. Cependant, les traitements sont encore extrêmement couteux, et les mécanismes de l’infection sont toujours mal compris. En effet, le virus de l’hépatite C appartient au groupe des virus oncogènes, au même titre que celui de l’hépatite B ou le papillomavirus. Il n’a cependant jamais été démontré que le VHC induit des perturbations au niveau génétique des cellules infectées, et les processus viraux qui mènent à la transformation cellulaire sont inconnus. Le VHC est un virus qui infecte les hépatocytes, les cellules épithéliales du foie. Il a précédemment été montré que l’infection induit de fortes perturbations métaboliques dans ces cellules qui participent à la pathogenèse virale. Par ailleurs, l’infection induit également une accumulation anormale de tissu cicatriciel dans le foie (fibrose) pouvant sur le long terme conduire à des dérèglements fonctionnels et architecturaux de l’organe (cirrhose) qui deviennent irréversibles. L’ultime étape dans la progression de l’hépatite C est le carcinome hépatocellulaire (CHC) dans 1 à 5% des cas, et une large partie de ces patients meurent dans l’année suivant le diagnostic. Nous nous sommes particulièrement intéressés aux étapes précoces de la pathogenèse virale, en particulier l’établissement de la fibrose hépatique. Celle-ci résulte de l’accumulation de composés de la matrice extracellulaire (MEC) dans l’espace intercellulaire du foie, alimentée d’une part par la surproduction de ces composés, et d’autre part par la diminution de leur dégradation par leurs enzymes spécifiques. La MEC est un espace complexe qui contrôle la signalisation entre les cellules. Ceci dépend en grande partie de l’espace situé immédiatement au-dessus de la membrane plasmique, appelé manteau cellulaire ou glycocalyx. Cet espace est majoritairement composé de protéoglycanes comportant un corps protéique sur lequel sont ancrées de nombreuses chaînes de sucres sulfatés capables ou non de fixer les facteurs de signalisation présents dans la MEC pour les rapprocher ou non de leurs récepteurs. Après avoir démontré dans une première étude que l’infection par le VHC induisait une diminution de l’expression d’un composant majeur du glycocalyx dans les cellules infectées, nous nous sommes intéressés plus globalement à l’évolution de cet espace suite à l’infection. Nos travaux reposent sur des approches pluridisciplinaires regroupant biologie moléculaire, biochimie, et biologie cellulaire. Nous avons pu étudier en détail les composés du glycocalyx présents à la surface des cellules infectées ou non par le VHC, ainsi que les facteurs d’une des voies de leur biosynthèse. Ces études ont été réalisées dans un premier temps en modèle d’infection in vitro, mais également sur échantillons biologiques de patients. Nous avons observé un remaniement important des composés du glycocalyx à la surface des cellules infectées, quantitatif et qualitatif, corrélé à des altérations majeures dans la voie de biosynthèse de composants de cet espace. Nous avons constaté que la profondeur de ces altérations descendait à l’échelle du simple sucre (xylose) dont la concentration varie entre les conditions contrôle et infectée. Cette variation de quantité de xylose dans les cellules infectées semble être reflétée dans l’altération d’une importante voie de signalisation contrôlant la destinée cellulaire. Ce travail de thèse permet donc d’éclairer une partie de la pathogenèse virale du VHC encore jamais explorée, soulignant des problèmes majeurs de communication cellulaire dans les organes infectés / Hepatitis C is a chronic liver disease due to the infection by the hepatitis C virus (HCV), through exposure to contaminated blood. An estimated 3 to 4 million people are infected every year, with a viral prevalence rising up to 22% in the general population in Egypt. Over the past years, directacting antivirals (DAA) have emerged on the market, allowing a strong regression of the infection in developed countries. However these treatments are very expensive, and the underlying mechanisms of HCV infection remain ill-defined. HCV is an oncovirus, as are among others, the hepatitis B virus, human papilloma viruses, and herpes viruses. Contrary to those viruses, HCV does not seem to alter the genetic background of infected cells. Therefore, infectious mechanisms leading to cellular transformation are still unknown. HCV infects the epithelial cells of the liver, the hepatocytes. Infection leads to strong disruptions of glucide and lipid metabolism in these cells, contributing to HCV pathogenesis. Moreover HCV infection induces the accumulation of scarring tissue in the intercellular space of the liver, called fibrosis, which can evolve into cirrhosis with irreversible architectural and functional disorders. The ultimate step in hepatitis C progression is the development of an hepatocellular carcinoma (HCC) in 1-5% of cases, and many of these patients die the year following cancer diagnosis. We aimed at studying the early steps of viral pathogenesis, especially the establishment of liver fibrosis. This is the consequence of extracellular matrix (ECM) deposition in the intercellular space of the liver, fueled by both an oversecretion of ECM components and a lack of ECM degradation by specialized enzymes. The ECM is a complex compartment of the liver which controls molecular cellto- cell signaling. This greatly depends on what happens in the area situated right above the plasma membrane, called the cell coat or glycocalyx. This area mainly consists of proteoglycans, composed of a core protein on which long chains of sulfated sugars are anchored. These sugar chains are able to recognize and capture a myriad of signaling molecules in the ECM, in order to bring them closer to their receptors. Our first study demonstrated that one major proteoglycan was downregulated following HCV infection (Grigorov et al, 2017). We then decided to study on a more global scale how the glycocalyx evolved following this infection. Our work here is based on a multi-disciplinary approach combining molecular biology, biochemistry and cellular biology. We studied in details the glycocalyx components present at the surface of cells infected or not, as well as the expression of factors involved in their biosynthesis. This was first done in in vitro cellular models of infection, and extended where possible to biological samples from patients at various stages of chronic hepatitis. We observed a strong quantitative and qualitative reshuffling of the glycocalyx at the surface of infected cells, which correlated with major alterations in the biosynthesis of some proteoglycans. These abnormalities seemed to originate from the amount of a simple sugar, xylose, the main component of the biosynthesis of some proteoglycans. Indeed, intracellular concentrations of xylose were decreased in infected cells. This might bear a link to the observed alterations of a major signaling pathway controlling cell fate, which is partly regulated by xylose. This study sheds light on a previously unexplored aspect of HCV pathogenesis. Our results could contribute to explain the complications linked to this infection, since they underline major cellto- cell communication issues. Since most of the DAA-based therapies work badly once liver cirrhosis has settled, it could be interesting to combine antiviral treatments with anti-fibrotic agents in patients suffering from advanced hepatitis C

The HIV-1 envelope glycoprotein trimer (Env) is covered by an extensive array of glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints that limit the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific locations. These oligomannose-type glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, perhaps paradoxically, target it. In multiple studies, bNAbs have been shown to be capable of providing passive protection from viral challenge, making Env a focus of antibody-mediated vaccine design. Here, the development of a workflow for the semi-quantitative, site-specific N-glycosylation analysis of a soluble recombinant, native-like trimer mimic (BG505 SOSIP.664) is reported. The resulting data reveal a mosaic of dense clusters on the outer domain of Env and allow mapping the extremes of simplicity and diversity of glycan processing. Although individual sites usually minimally affect the global integrity of the glycan shield, examples are identified of how deleting certain glycans can subtly influence neutralization by bNAbs that bind at distant sites. Env is a trimer of heterodimers of gp120 and gp41, which is generated by cleavage of an endogenous protease. In this thesis, the detailed effect of protease cleavage on glycan processing is examined by comparing the site-specific N-glycosylation profiles of the native-like trimer mimic to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. Trimer-associated glycan remodeling forms a localized subdomain of the native mannose patch. Furthermore, the glycosylation analysis of further Env immunogens – a glycan-depleted trimer and a flexibly-linked, uncleaved trimer (both based on BG505 SOSIP.664) – provides important insights into the robustness of the HIV-1 glycan shield and the Env maturation pathway. Overall, this thesis reveals how structural constraints shape Env glycosylation and the network of bNAb-targeted glycans that should be preserved on recombinant vaccine candidates.

The aim of the study was to investigate the expression NDST transcripts in a wide variety of tissues using RNA-sequencing experimental data from five published studies, using two common in silico tools: the Tophat-Cufflink pipeline and the HTSeq-DEXSeq pipeline. We show that to detect NDST alternative transcripts, paired-end sequencing should be used with replicates of samples or conditions together with 100 base read length to allow for reliable detection of the low expressed transcripts in the NDST family.  As a demonstration project, we also characterized HS synthesized by the adrenal carcinoma (ACC) cell line H295R and determined expression of NDSTs in the cells and in ACC tumor samples. We could show that roughly 65% of newly synthesized proteoglycans isolated after metabolic 35S-sulfate labeling of the cells are made up of heparan sulfate (HS) with an average chain length of 45 kDa. The HS chains show a high frequency of N-sulfation and a high total degree of sulfation. Interestingly, disaccharide analysis demonstrated a three-time higher amount of stored chondroitin sulfate (CS) compared to HS in the ACC cell line.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A junção aderente (JA) é um dos principais componentes do complexo juncional apical. Esta juncão é um complexo multiprotéico em que a E-caderina, uma glicoproteína transmembrana, atua como principal mediadora da adesão célula-célula. Sua ancoragem ao citoesqueleto de actina ocorre via proteínas da família das cateninas. Modificações pós-traducionais da E-caderina, como fosforilação e glicosilação, podem modular a estabilidade e organização das JAs. Muitos estudos têm sugerido que no câncer a invasão e a metástase podem estar associadas a arranjos de glicanos na superfície celular. Em câncer colo-retal, o papel de alterações na expressão de glicanos sobre a estabilidade da adesão mediada por Ecaderina ainda não está claro. Neste estudo, investigamos a relação entre estas alterações e a estabilidade das JAs em células de câncer colo-retal. Nós utilizamos duas linhagens celulares com diferentes potenciais metastáticos, Caco-2 and HCT- 116, que constituem dois modelos de JAs: estáveis e instáveis, respectivamente. Ensaios de precipitação de lectinas e immunoblotting demonstraram que em HCT- 116, a linhagem mais invasiva, a E-caderina apresenta uma diminuição de glicanos reconhecidos pelas lectinas HPA e WGA, que reconhecem resíduos de Nacetilgalactosamina e, _-N-acetilhexosaminas e ácido siálico, respectivamente. Concomitantemente, em HCT-116 também foi observado um aumento de glicanos reconhecidos pelas lectinas L-PHA e E-PHA, que reconhecem respectivamente: acetilglicosamina_1,6-ligada formando N-glicanos tri- e tetraantenados e, Nacetilglicosamina bisectante _1,4-ligada formando N-glicanos biantenados. Ensaios de imunofluorescência mostraram que a presença desses glicanos reconhecidos por L-PHA, em HCT-116, é intensa na região de contato célula-célula quando comparada com células Caco-2, em que a marcação foi observada, predominantemente, na região apical. Além disso, a inibição completa da Nglicosilação por tunicamicina ou a inibição da síntese de N-glicanos complexos por swainsonina, aumentou a associação da E-caderina com o citoesquleto de actina em células HCT-116, mas não em Caco-2. Em células HCT-116, a inibição de Nglicosilação por tunicamicina produziu uma diminuição da atividade de ERK1/2 e a formação de adesão célula-célula foi mais evidente. Estes dados sugerem que alterações na expressão e localização subcelular de diferentes glicanos podem ser importantes eventos associados à perda da estabilidade das JAs em câncer coloretal. / The adherent junction (AJ) is one of the main components of the apical junctional complex. It is a multiprotein complex where E-cadherin, a transmembrane glycoprotein, acts as the main mediator of cell-cell adhesion in epithelium. This protein is anchored to the actin cytoskeleton via proteins of the catenin family. Posttranslational modifications of E-cadherin, such as phosphorylation and glycosylation, can modulate the assembly of AJs. Various studies have suggested that invasion and metastasis is associated to glycan patterns on the cell surface of tumor cells. In colorectal cancer the role of altered glycans expression and stability of E-cadherin-mediated adhesion is not clear. In this study we investigated the relation between changes of the glycans expression and AJs stability in colorectal cancer cells. We used two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116, both models of stable and unstable AJs, respectively. Lectin binding assays demonstrated that in HCT-116, the more invasive cell line, E-cadherin presents a decrease of the glycans recognized by HPA and WGA lectins, which recognize N-acetylgalactosamine and, _-N-acetylhexosamines and sialic acid, respectively. Conversely, in HCT-116, there was an increase of glycans recognized by E-PHA and L-PHA lectins, which recognize bisecting _1,4- branched and _1,6-branched N-acetylglucosamine, respectively. Immunofluorescence assays showed a stronger L-PHA binding on cell-cell contact regions of HCT-116 cells when compared with Caco-2. Furthermore, in HCT-116 cells a complete inhibition of N-glycosylation by tunicamycin or inhibition of complex N-glycans synthesis by swainsonine increased the association of E-cadherin with the actin cytoskeleton. Finally, it was possible to observe that the inhibition of N-linked glycosylation by tunicamycin, leaded to a decreasing of ERK1/2 phosphorylation concomitantly with the formation of more intimate cell-cell contacts. These findings suggest that altered expression and subcellular localization of different glycans can be important events associated to loss of AJs stability in colorectal cancer.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A junção aderente (JA) é um dos principais componentes do complexo juncional apical. Esta juncão é um complexo multiprotéico em que a E-caderina, uma glicoproteína transmembrana, atua como principal mediadora da adesão célula-célula. Sua ancoragem ao citoesqueleto de actina ocorre via proteínas da família das cateninas. Modificações pós-traducionais da E-caderina, como fosforilação e glicosilação, podem modular a estabilidade e organização das JAs. Muitos estudos têm sugerido que no câncer a invasão e a metástase podem estar associadas a arranjos de glicanos na superfície celular. Em câncer colo-retal, o papel de alterações na expressão de glicanos sobre a estabilidade da adesão mediada por Ecaderina ainda não está claro. Neste estudo, investigamos a relação entre estas alterações e a estabilidade das JAs em células de câncer colo-retal. Nós utilizamos duas linhagens celulares com diferentes potenciais metastáticos, Caco-2 and HCT- 116, que constituem dois modelos de JAs: estáveis e instáveis, respectivamente. Ensaios de precipitação de lectinas e immunoblotting demonstraram que em HCT- 116, a linhagem mais invasiva, a E-caderina apresenta uma diminuição de glicanos reconhecidos pelas lectinas HPA e WGA, que reconhecem resíduos de Nacetilgalactosamina e, _-N-acetilhexosaminas e ácido siálico, respectivamente. Concomitantemente, em HCT-116 também foi observado um aumento de glicanos reconhecidos pelas lectinas L-PHA e E-PHA, que reconhecem respectivamente: acetilglicosamina_1,6-ligada formando N-glicanos tri- e tetraantenados e, Nacetilglicosamina bisectante _1,4-ligada formando N-glicanos biantenados. Ensaios de imunofluorescência mostraram que a presença desses glicanos reconhecidos por L-PHA, em HCT-116, é intensa na região de contato célula-célula quando comparada com células Caco-2, em que a marcação foi observada, predominantemente, na região apical. Além disso, a inibição completa da Nglicosilação por tunicamicina ou a inibição da síntese de N-glicanos complexos por swainsonina, aumentou a associação da E-caderina com o citoesquleto de actina em células HCT-116, mas não em Caco-2. Em células HCT-116, a inibição de Nglicosilação por tunicamicina produziu uma diminuição da atividade de ERK1/2 e a formação de adesão célula-célula foi mais evidente. Estes dados sugerem que alterações na expressão e localização subcelular de diferentes glicanos podem ser importantes eventos associados à perda da estabilidade das JAs em câncer coloretal. / The adherent junction (AJ) is one of the main components of the apical junctional complex. It is a multiprotein complex where E-cadherin, a transmembrane glycoprotein, acts as the main mediator of cell-cell adhesion in epithelium. This protein is anchored to the actin cytoskeleton via proteins of the catenin family. Posttranslational modifications of E-cadherin, such as phosphorylation and glycosylation, can modulate the assembly of AJs. Various studies have suggested that invasion and metastasis is associated to glycan patterns on the cell surface of tumor cells. In colorectal cancer the role of altered glycans expression and stability of E-cadherin-mediated adhesion is not clear. In this study we investigated the relation between changes of the glycans expression and AJs stability in colorectal cancer cells. We used two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116, both models of stable and unstable AJs, respectively. Lectin binding assays demonstrated that in HCT-116, the more invasive cell line, E-cadherin presents a decrease of the glycans recognized by HPA and WGA lectins, which recognize N-acetylgalactosamine and, _-N-acetylhexosamines and sialic acid, respectively. Conversely, in HCT-116, there was an increase of glycans recognized by E-PHA and L-PHA lectins, which recognize bisecting _1,4- branched and _1,6-branched N-acetylglucosamine, respectively. Immunofluorescence assays showed a stronger L-PHA binding on cell-cell contact regions of HCT-116 cells when compared with Caco-2. Furthermore, in HCT-116 cells a complete inhibition of N-glycosylation by tunicamycin or inhibition of complex N-glycans synthesis by swainsonine increased the association of E-cadherin with the actin cytoskeleton. Finally, it was possible to observe that the inhibition of N-linked glycosylation by tunicamycin, leaded to a decreasing of ERK1/2 phosphorylation concomitantly with the formation of more intimate cell-cell contacts. These findings suggest that altered expression and subcellular localization of different glycans can be important events associated to loss of AJs stability in colorectal cancer.

La pigmentation de la peau résulte en grande partie de la présence de mélanine dans l’épiderme. Ce pigment est synthétisé par les mélanocytes puis transféré aux kératinocytes pour assurer une fonction photoprotectrice. Le transfert de mélanosomes nécessite une reconnaissance cellulaire entre mélanocytes et kératinocytes. L’interaction entre lectines et glycanes joue un rôle dans cette reconnaissance et peut constituer une cible d’intérêt pour le développement de produits à activité dépigmentante. Par ailleurs, les conditions du microenvironnement cutané, telles que le taux d’oxygène, sont cruciales pour l’homéostasie tissulaire. Les objectifs de ce travail de thèse, réalisé dans le cadre du projet FUI Glycoskin I, ont été : l’étude de la reconnaissance cellulaire entre mélanocytes et kératinocytes à travers l’interaction lectine-glycane, la caractérisation des mélanosomes sécrétés par les mélanocytes et la mise au point d’une méthode d’évaluation du transfert des mélanosomes aux kératinocytes pour tester l’activité de phosphoconjugués. D’autre part, nous avons étudié l’effet du taux d’oxygène sur le processus de mélanogénèse et sur l’interaction lectine-glycane. Nos résultats ont permis d’élucider les profils glycaniques et lectiniques à la surface des mélanocytes et des kératinocytes et de sélectionner des phosphoconjugués potentiellement inhibiteurs du transfert de mélanosomes aux kératinocytes. Nous avons mis au point un modèle d’évaluation du transfert de mélanosomes aux kératinocytes afin de tester l’effet inhibiteur des phosphoconjugués. Nous avons identifié un phosphosaccharide inhibiteur de la reconnaissance entre mélanosomes et kératinocytes. Par ailleurs, ce projet constitue la première étude de la pigmentation en physioxie. Nous avons montré qu’un travail en physioxie induit des modulations des profils glycaniques et lectiniques, ainsi qu’une stimulation de la mélanogénèse. Ces résultats montrent l’importance de se placer en physioxie lors de l’étude de la mélanogénèse in vitro afin de se rapprocher au maximum des conditions physiologiques du microenvironnement cutané lors de l’évaluation de composés actifs. / Skin pigmentation is mostly due to the presence of melanin in the epidermis. This pigment is produced by melanocytes and transferred to keratinocytes, to play a photoprotective role. Melanosome transfer requires cellular recognition between melanocytes and keratinocytes. Lectin-glycan interaction plays a role in this phenomena and can be an interesting target for developing depigmenting products. Besides, the cutaneous microenvironment conditions, such as oxygen level, are crucial for tissular homeostasis. The aims of this work, as part of the Glycoskin I FUI project, were : to study cellular recognition between melanocytes and keratinocytes through lectin-glycan interaction, to characterize melanosomes released from melanocytes and to develop a method for the evaluation of melanosome transfer to keratinocytes in order to assess phosphoconjugate activity. Also, we studied the effect of oxygen level on melanogenesis and lectin-glycan interaction. Our results allowed to elucidate lectin and glycan profiles on the surface of melanocytes and keratinocytes and to select phosphoconjugates potentially able to inhibit melanosome transfer. We developed a method to assess melanosome transfer in order to test phosphoconjugates inhibiting effect. We identified one phosphosaccharide able to inhibit melanocytes-keratinocytes recognition. Furthermore, this project is the first study of pigmentation in physioxia. We showed that physioxia induces modulations of lectin and glycan profiles and stimulated melanogenesis. These results show the importance of physioxia conditions when studying melanogenesis in vitro to approach cutaneous physiological microenvironment when evaluating active compounds.

Malaria caused by the protozan parasite Plasmodium is one of the most serious infectious diseases in the developing world. It is estimated that malaria causes an annual mortality rate of ~627,000. New drugs are urgently required, as the incidence of resistance is spreading rapidly. Glycosylphosphatidylinositol (GPI) anchored proteins decorate the merozoite surface and several of which, including merozoite surface proteins - 1 and -2 have previously been shown to be essential for erythrocyte invasion and parasite survival. Plasmodium GPI-anchors contain a glycan core consisting of four mannose residues. Therefore, the enzymes involved in the synthesis of activated mannose, guanidine diphosphomannose pyrophosphorylase (GDP-Man PP) and dolichol phosphate mannose synthase (DPMS), are thought to be crucial for GPI-anchor biosynthesis and as such potential drug targets. Double homologous recombination has been exploited to test whether PfGDP-Man PP and PfDPMS are essential during the erythrocytic portion of the parasite's life cycle. Additionally, overexpression parasite lines for both enzymes have been generated and have shown that the regulation of the two enzymes are intricately linked. Focused metabolomics by multi-reaction monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volumes of the intra-erythrocytic stages have been determined and show that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. Furthermore, the sub-cellular localisation of GDP-Man PP and DPMS has been determined by immunofluorescence assay. The recombinant expression of DPMS in E. coli allowed its active site residues to be probed as well as establishing a platform for inhibitors to be screened against the enzyme. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.