GLYTUNES

GLYTUNES ESR Projects

Host Institution: University of Naples Federico II (Naples, Italy) - UNINA
Primary Supervisor: Alba Silipo
Planned duration: 36 months
Subject Area: Structural and chemical biology; Glycomics and Glycosciences

Introduction: Pathogens exploitation of inhibitory Siglecs: Pathogens can shield their envelope glycans with self-ligands, with the aim of dampening host immune responses. Feared human pathogens such as membrane-enveloped viruses and bacteria such as GBS, C. jejuni, H. influenzae, P. aeruginosa, N. meningitides and gonhorreae, F. nucleatus, are coated with sialic acid containing ligands that can be potentially recognized by inhibitory Siglecs, this allowing to escape host immune responses by averting detection and immune elimination, promoting successful bacterial colonization and tolerance.

Aims: Science: The project aims to assess the structure and function of cell envelope components isolated from bacterial sialylated envelope glycans. ii) determine conformation and 3D structure of isolated microbial glycans. Training: Expertise in glycomics, microbial glycans structural and chemical biology, organic chemistry, carbohydrate chemistry and biochemistry, analytical chemistry, isolation, purification and characterization of microbial glycans (LPS, EPS, CPS….), NMR spectroscopy and MS spectrometry, computational methods for building 3D complex carbohydrate structures; stage(s) in the industry.

Expected Results: Formation of the ESR with a solid knowledge in glycomics, glycosciences, structural biology of glycoconjugates. Definition of the structure to function relationships of bacterial cell envelope compounds. Establishment of the molecular mimicry of host glycans from different microbial strains. Stage in the industry; PhD thesis.

Planned secondment(s): 3-month secondments at: 1) QIB: approaches to identify Siglecs receptors recognized by different isolated microbial glycans; 2) VUMC: Immunological approaches toward the understanding of immunopotential of isolated envelope components 3) ICENI: developments of multivalent glycomimetics based on the structure of isolated microbial glycans.

Enrolment in Doctoral degree(s): ESR1 will be enrolled at University of Naples Federico II

Host Institution: Quadram Institute Bioscience (Norwich, UK) - QIB
Primary Supervisor: Nathalie Juge
Planned duration: 36 months
Subject Area: Microbiology, glycobiology, cell biology, immunology

Introduction: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide and accounts for approximately 20% of all cancer-related deaths in developed countries. CRC has been associated with an enrichment of microbes in intestinal tissues with the most abundant species being Fusobacterium nucleatum. F. nucleatum potentiates intestinal tumorigenesis through immune suppression but the mechanisms underpinning its interaction with immune cells is unknown. We recently showed that F. nucleatum interact with Siglecs (sialic acid–binding immunoglobulin-like lectins), opening a new dimension in our understanding of how F. nucleatum promotes CRC progression and in the development of therapeutic strategies targeting Siglec axis.

Aims: Science: The project aims to (1) unravel the nature of the Fusobacterium nucleatum ligands of Siglecs and (2) their role in modulating host immune response and 3) test compounds derived from WP4 as potential glycomimetics inhibitors of the interaction between F. nucleatum and immune cells. Training: will be provided in molecular microbiology (anaerobic bacterial growth assays), cell biology (mammalian cells and gut-on-chips) and advanced bioimaging techniques (fluorescence and confocal microscopy; imaging flow cytometry), protein purification and protein-carbohydrate interactions (Octet, SPR, ITC). Training will include transferable skills including communication (to scientific and lay audiences), intellectual property impact of the research on human health and outreach activities.

Expected Results: The ESR will gain a solid knowledge in molecular microbiology, cell biology including gut-on-chip technology, biophysical and bioimaging techniques; Scientific outputs include the identification of F. nucleatum components interacting with Siglecs and new glycomimetics as inhibitors of F. nucleatum interaction with host Siglecs and host immune response; Experience in industry; PhD thesis.

Planned secondment(s): 3-month secondments at: 1) GIOTTO: Methodologies of Siglecs purification and characterization; 2) RAD: Development of glycoinhibitors targeting Fusobacterium strains 3) VUMC: Use of relevant cancer models on glycomimetics against Fusobacterium.

Enrolment in Doctoral degree(s): Enrolment in Doctoral degree(s): ESR2 will be enrolled at University of East Anglia

Host Institution: Center for Cooperative Research in Biosciences (Derio, Spain) - CICbioGUNE
Primary Supervisor: June Ereño-Orbea
Planned duration: 36 months
Subject Area: Glycobiology, structural biology, biophysics

Introduction: Siglecs expressed on innate immune cells and tumor-infiltrating T cells, have emerged as alternative immune checkpoint inhibitors. This family of receptors recognize sialic acid (Sia) containing glycans on the surface of cancer cells as self-associated molecular patterns. However, the molecular basis of Siglec binding to its ligands continues to be a puzzle. In this research project we plan to study the glycan binding specificity of Siglec receptors by a range of structural techniques including x-ray crystallography and NMR, and biophysical techniques (e.g. ITC and SPR). Understanding Siglec-sialic acid interactions from an atomic perspective will help us in better understanding their role in cancer development.

Aims: Science: 1) high-resolution analysis of the 3D structure of Siglec receptors at atomic resolution using X-ray crystallography. 2) molecular details of the interaction of the Siglecs with host glycans and cancer-associated glycoproteins using structural (X-ray crystallography, NMR, small-angle X-ray scattering (SAXS)) and biophysical (ITC, BLI, SPR) techniques. Training: Expression and purification of Siglecs and cancer-associated glycoproteins using human and bacterial cell lines. Crystallization of proteins and protein-glycan complexes, X-ray data collection and processing; computational methods; biophysical techniques (ITC, BLI, SPR).

Expected Results: Training of the ESR with a solid knowledge in structural biology and computational methods. Production and 3D structural details of Siglecs; production of cancer-associated glycoproteins; affinity determination and structural details of Siglecs binding to sialylated glycans and cancer-associated glycoproteins.

Planned secondment(s): 3-month secondments at: 1) GCU: Synthesis of sialylated glycans for recognition and binding studies; 2) UNINA: Structural biology/characterization of glycans; 3) VUMC: Explore biochemical approaches (ELISA or WB) to correlate the structural data with preliminary functional assays.

Enrolment in Doctoral degree(s): ESR3 will be enrolled at Universidad del País Vasco

Host Institution: VU University Medical Center (Amsterdam, The Netherlands) - VUMC
Primary Supervisor: Yvette van Kooyk and Fabrizio Chiodo
Planned duration: 48 months
Subject Area: Immunology, chemical biology

Introduction: Antigen presenting cells such as Dendritic cells are crucial in sensing pathogens resulting in specific T cell responses that correlate with the elimination of that pathogen. This can be achieved by innate receptors like glycan binding receptors CLRs and Siglecs, and TLRs that provide together an integrated signaling pattern that alter surface-markers, cytokine production and T cell responses. Unravelling these interactions is crucial for the proper activation of immunity.

Aims: Science: The immunological interactions between the sialylated exogenous and endogenous conjugates prepared by ESR1, ESR10 and ESR7, will be studied on human antigen-presenting cells (or injected in mice) (ESR4). Monocytes-derived dendritic cells, macrophages, and antigenpresenting cells isolated from human skin-biopsies will be stimulated with the sialylated compounds. The analysis of released cytokines, co-stimulatory surface-markers, immunological pathways and the intracellular routing of the sialylated compounds will be studied by flow cytometry, confocal microscopy and ELISA. The outcome of the dendritic cells/T-cells interaction will be also studied characterizing the type of T-cells generated (Th1, Th2 or Th17). In addition, the sialylated conjugates will be incubated with cells also in co-stimulation with Toll-like receptor (TLR) ligands like LPS, Pam3CSK4 etc., to study the cross-talk between Siglecs and TLRs. Training: The ESR will be trained on biochemistry, cellular immunology, flow cytometry, animal facilities and confocal microscopy. Also, the ESR will learn how to analyze big data with significant relevance. The ESR will learn how to write scientific publications, reports, posters and she/he will learn to present data at any type of audience (group meetings, department meetings conferences, press releases, etc.).

Expected Results: Formation of the ESR with a solid knowledge in cell biology and immunology; Science related: At the end of this project we will have a full understating on the immune-modulatory properties of sialylated conjugates. Some of them will be used to actively modulate DC, or to change the Th1/Th2 balance, important for different diseases (cancer, autoimmunity etc.). We will be able to have a full set of new immune modulators. A successful PhD thesis is expected from the ESR as well as the dissemination of the scientific works produced during the PhD training.

Planned secondment(s): 1) UNINA (3 months): To isolate and purify microbial sialylated glycans 2) ATLAS (4 months): to explore ATLAS cell screening assay to finally have in hands a selected library of sialylated compounds.

Enrolment in Doctoral degree(s): ESR4 will be enrolled at University Medical Center

Host Institution: University of Florence - Magnetic Resonance Center (Florence, Italy) - UNIFI-CERM
Primary Supervisor: Marco Fragai
Planned duration: 36 months
Subject Area: NMR spectroscopy and Structural Biology

Introduction: Solution NMR is a powerful tool for structural studies and an indispensable enabling technology for determining weak and transient macromolecule interactions as well as for characterizing functional processes. Recently, solid-state NMR is emerging as an important technique to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by solution NMR or other methods. The integration of NMR spectroscopy with other biophysical methodologies and computational methods is thus clearly the strategy of choice to investigate the structural determinants of Siglec-sialoglycans interaction.

Aims: Science: 1) NMR assignment of selected Siglecs in solution and at the solid-state NMR 2) Analysis of the Siglec-sialoglycans interactions to determine the binding specificity and affinity. 3) Determination of the key protein residues and functional surfaces driving the assembling of the Siglecsialoglycans complexes by NMR spectroscopy and using biophysical methods and site-directed mutagenesis data. 4) Characterization of the interaction between Siglecs and microbial envelope components isolated by ultrafast magic angle spinning (MAS) solid-state NMR spectroscopy. 5) Calculation of docking models to predict the structure of Siglec-sialoglycans complexes through the use of experimental NMR contacts and site-directed mutagenesis. Training: Expertise in i) preparation of biological sample for solid-state NMR spectroscopy; ii) NMR spectroscopy and methodologies for structural biology; iii) protocols to integrate NMR and site-directed mutagenesis data in docking calculation; iv) thermodynamic characterization of the binding between the selected Siglecs and sialoglycans. Secondments will provide a perspective of possible synergies that can be exploited by the ESR for his/her future career.

Expected Results: formation of the ESR with a solid knowledge in NMR-based and integrative structural biology of proteins and glycoproteins, molecular modeling and docking calculations. NMR assignment of Siglec proteins. Structural models of Siglec-sialoglycans complexes. Identification of the structural determinants of binding selectivity in Siglec-glycans complexes; Posters and publications on siglecs in relation to the their interaction with sialoglycans; Ph.D. thesis.

Planned secondment(s): 1) IBS, (4 months): Cryo-EM sample preparation and analysis and to relate the NMR analysis to Cryo-EM characterization; 2) ICENI, (4 months): glycans conjugation to nanoparticles/quantum dots in order to develop new strategies for the evaluation of protein-carbohydrates interactions based on solid-state NMR. 3) QIB, (3 months): Recombinant expression and purification of Siglecs from CHO cell lines.

Enrolment in Doctoral degree(s): ESR5 will be enrolled at University of Florence

Host Institution: Centre National De La Recherche Scientifique (Grenoble, France) - CNRS
Primary Supervisor: Irina Gutsche
Planned duration: 36 months
Subject Area: Structural biology, high resolution cryo-electron microscopy

Introduction: Siglecs are transmembrane receptors expressed on surfaces of innate immune cells. They recognise sialic acids used by vertebrates as signatures of “self” but also by some microbial pathogens that thereby modulate host immune responses. Aberrant interactions between Siglecs and their ligands lead to a variety of pathologies including infection, autoimmune diseases and cancer. Understanding the structural basic of Siglec-ligand binding is therefore a crucial step towards the rational design and development of novel therapeutic and diagnostic strategies against numerous diseases.

Aims: Science: The project aims to: i) Optimize biochemical conditions for obtaining dimers of the extracellular domain (ECD) of selected Siglecs, homogeneous both in terms of glycosylation and oligomeric state (secondment CIC-bioGUNE). (ii) Solve the structures of the best ECD dimers by single particle cryo-EM. iii) Attempt to produce transmembrane or full-length constructs in lipid nanodiscs or liposomes (secondment GIOTTO), with the goal to solve structures of native membrane-bound states by cryo-EM or cryo-ET. (iv) Analyse interaction of different soluble and membrane constructs with ligands (secondment UNIFI), define the most suitable targets for high resolution structural studies and solve the resulting structures by cryo-EM. Training: Purification and biochemical characterization of ECDs and transmembrane Siglecs; experimental cryo-EM and, if appropriate, cryo-ET; advanced cryo-EM 3D image processing, atomic model building and structure analysis.

Expected Results: Atomic structures of selected ECD dimers alone and in complex with a ligand. If feasible, first insights into the assembly of transmembrane Siglec constructs into model membranes and the effect of ligand binding. Structural analysis and definition of the molecular basis for the Siglec dimerisation, ligand binding and membrane insertion for therapeutic targeting. Secondment in industry.

Planned secondment(s): 3-month secondments at: 1) CIC-bioGUNE:Optimisation of biochemical preparation of ECD constructs homogeneous in terms of glycosylation and oligomeric state. 2) GIOTTO: Optimization of the expression and purification of transmembrane Siglecs. 3) UNIFI: Spectroscopic and biophysical analysis of the interactions of the corresponding Siglec constructs with ligands.

Enrolment in Doctoral degree(s): ESR6 will be enrolled at University Grenoble-Alps

Host Institution: Radboud University (Amsterdam, The Netherlands) - RAD
Primary Supervisor: Thomas Boltje
Planned duration: 48 months
Subject Area: Organic (carbohydrate) chemistry, medicinal chemistry, chemical biology

Introduction: Siglec receptors bind sialoglycans, glycans that contain sialic acid residues. The recognition of sialic acid residues by Siglecs depends on their structure, linkage type and the underlying glycan scaffold. Typically, Siglecs binding leads to immune suppression and represents an important mechanism to modulate immune system activity. However, various pathologies are associated with an imbalance in Siglec immune suppression and hence Siglec receptors are also promising therapeutic targets.

Aims: Science: The aim of this project is to develop new glycomimetics based on sialic acid to inhibit Siglec binding. To this end, new sialic acid glycomimetics will be designed, synthesized and tested in cellular assays. Finally, the developed inhibitors will be used in collaboration with project partners to evaluate the functional consequences of Siglec receptor inhibition. Training: Expertise in the synthesis, purification and characterization of sialic acid mimetics by means of organic chemistry, MS spectrometry and NMR spectroscopy. Basic cell biology skills such as cell culture and cellular assays. Secondments will provide a perspective of possible synergies that can be exploited by the ESR for his/her future career.

Expected Results: Formation of the ESR with a solid knowledge in chemical synthesis and chemical biology of sialic acid mimetics. Definition of the structure to function relationships of newly synthesized sialic acid mimetics for Siglec receptors. In collaboration with the ITN partners, functional use of the developed sialic acid mimetics in an immunological setting.

Planned secondment(s): 3-month secondments at: 1) QIB: Immunological evaluation of novel glycomimetics; 2) UNINA: Evaluation of glycomimetics binding via NMR 3) CICbioGUNE: X-ray crystallography, small-angle X-ray scattering (SAXS) of protein-glycomimetic complexes.

Enrolment in Doctoral degree(s): ESR7 will be enrolled at Radboud University

Host Institution: ATLAS Molecular Pharma (Bilbao, Spain) - ATLAS
Primary Supervisor: Oscar Millet
Planned duration: 36 months
Subject Area: High resolution NMR spectroscopy; drug discovery; protein-ligand interaction; molecular dynamics simulations; biophysics; pharmacology, pharmacological chaperones, protein isotope labeling

Introduction: In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered. We employ NMR spectroscopy, a very versatile and powerful technique to cover most of the stages in the process of drug discovery. We develop methodology and apply it to relevant biomedical problems, namely congenital metabolic disorders.

Aims: Science: The project aims to develop biophysical methods for the characterization of chemical libraries and their interactions with the target protein: i) To obtain an NMR based method to screen libraries of compounds that selectively associate to a predefined binding site. This will be applied to fluorinated glycomimetics that may compete with the natural ligands. ii) To provide innovative tools for the characterization of chemical libraries using NMR spectroscopy. Training: To train the ESRs in novel 19F-NMR methods for the characterization of protein-ligand interactions.

Expected Results: NMR methodology to the characterization of receptor-drug interactions at the thermodynamic and structural level, using NMR spectroscopy and other biophysical techniques. Application to several relevant systems associated to metabolic diseases. Specifically, New pulse sequences and/or methods for NMR-based drug discovery exploiting the potential and orthogonality of the ¹⁹F isotope. An integral training in NMR spectroscopy and the associated techniques, related to drug discovery.

Planned secondment(s): 1) GCU (3 months): To train on the synthesis and purification of complex glycan libraries; VUMC (4 months): To explore basic cellular experiments based on flow-cytometry to find a functional readout that will correlate with the structural data.

Enrolment in Doctoral degree(s): ESR8 will be enrolled at Universidad del País Vasco

Host Institution: ICENI Diagnostics (Norwich, UK) - ICENI
Primary Supervisor: Rob Field
Planned duration: 36 months
Subject Area: Organic chemistry, carbohydrate chemistry, protein chemistry, molecular diagnostics, assay development, lateral flow assay

Introduction: Sialic acid-Siglec interactions are keys for a wide variety of biological processes, from infection to immune response, with sialic acid being a crucial carbohydrate component of the extracellular matrix and responsible of critical contact point with the Siglec counterpart. The nature of the interaction is ruled by a high selectivity and dictated by the type of sialic acid, in terms of its linkage or modification of the core structure. Exploring new routes to access Sialic acid derivatives, both natural and un-natural, can open new avenues in the development of diagnostic tools and therapeutics.

Aims: Science: The project aims to develop a library of carbohydrate derivatives, based on known Siglec ligands but with enhanced affinity and improved discrimination between Siglec isoforms. This will be achieved through a combination of (i) chemical and enzymatic synthesis, with molecules set up for (ii) conjugation to nanoparticles/quantum dots or proteins to (iii) support biological evaluation through microscopic imaging, ELISA or lateral flow assays. The above will be integrated with (iv) experimental and computational evaluation of ligand structure and dynamics, in solution and complex with Siglec targets. Training: EExpertise in carbohydrate-based click chemistry, enzymatic synthesis, purification and characterization of carbohydrate derivatives, including by mass spectrometry and NMR spectroscopy. Secondments will provide academic and biological context for chemistry-based industry, to ensure full integration with the GLYTUNES program and to support broader chemical biology education in line with career development.

Expected Results: Definition of new Siglec ligands, with enhanced affinity and selectivity, for use in fundamental cell biology, as well as diagnostic or therapeutic applications. A well-trained ESR, equipped with skills and knowledge in carbohydrate and glycoconjugation chemistry, allied to the detection of carbohydrate-binding proteins.

Planned secondment(s): 1) VUMC (4 months): Immunological techniques and approaches to evaluate the novel compounds 2) UNIFI (4 months): Spectroscopic and biophysical approached to study the novel ligands interacting with Siglecs.

Enrolment in Doctoral degree(s): ESR9 will be enrolled at University of Manchester

Host Institution: GlycoUniverse (Potsdam, Germany) - GCU
Primary Supervisor: Kim Le Mai Hoang
Planned duration: 36 months
Subject Area: Glycomics and Glycosciences; Synthetic and Analytical Carbohydrate Chemistry; Enzymatic Synthesis; Automated Solid Phase Synthesis

Introduction: An understanding the molecular basis of carbohydrate-protein interactions can be gained by identifying the molecular features essential for molecular recognition. To this end, molecular editing through synthesizing a library of carbohydrates that present a variety of molecular features can help to understand exactly how that recognition works. In addition, synthetic chemistry can be used to introduce molecular labels that can act as a beacon to indicate when and where recognition takes place and to study the kinetics and thermodynamics of the interaction.

Aims: Science: The project aims to synthesize well-defined complex glycans to be used to assess their interactions with Siglecs proteins. Suitable monomeric building blocks will be assembled, making use of in-house expertise on identifying optimal protective group schemes and anomeric leaving groups. The glycans will be assembled by means of automated oligosaccharide synthesis using the automated synthesis platforms as well as chemoenzymatic methodologies to assemble sialic acids and analogues. Glycans of interest having various types of labels for biophysical studies (NMR, X-ray crystallography, Cryo-EM) will be prepared. Compounds representing specific portions of selected LPS/microbial glycans will be synthetized, to fully appreciate the molecular features essential for recognition and binding. Training: Synthetic organic chemistry; enzymatic chemistry; purification methodologies; automated glycan synthesis; carbohydrate synthesis.

Expected Results: To synthesize focused glycan libraries, Advanced sialylated glycans libraries, labelled or unlabeled, will be developed by a combination of organic and chemo-enzymatic approaches, the design of which will be guided in part by studies/epitopes identified by WP2 members; A well-trained ESR, equipped with skills and knowledge in PhD thesis.

Planned secondment(s): 1) CICbioGUNE (4 months): Interaction studies with complex glycans libraries; 2) VUMC (4 months): Studies of effects of glycans libraries on the host.

Enrolment in Doctoral degree(s): ESR10 will be enrolled at Freie Universität Berlin

Host Institution: Giotto Biotech (Sesto Fiorentino, Italy) - GIOTTO
Primary Supervisor: Tommaso Martelli
Planned duration: 36 months
Subject Area: Molecular Biology, Biochemistry, Biology, Chemistry, Strucutral Biology

Introduction: The definition of a good protocol for the expression and sample preparation for the selected Siglecs is mandatory for their study and analysis with in-vitro assays and also for their structural and interaction studies. Deep studies will be also focused on the definition of protocols for the expression of isotopically enriched samples (with 2H/13C/15N) for solution and solid-state NMR.

Aims: Science: Optimization of the expression and purification of the selected Siglecs for biophysical studies and for x-ray and cryo-EM structural characterization. Expression and purification of isotopically enriched samples (2H/13C/15N) of Siglec proteins for NMR studies. Biophysical characterization of Siglecs. Training: expertise in molecular biology and in the expression and purification of isotopically enriched proteins in E.coli; in biophysical techniques (FPLC, HPLC, CD, MALS-QELS).

Expected Results: formation of the ESR with a solid knowledge in molecular biology and in the expression, purification and biophysical characterization of recombinant proteins in E.coli expression system High yield production of isotopically enriched Siglecs-2, 9, -10 and -15 and of mutants for NMR and Cryo-EM studies; Posters and publications on siglecs in relation to the their interaction with sialoglycans ; Ph.D. thesis; stage in industry.

Planned secondment(s): 1) CICbioGUNE (6 months): to learn about expression and purification of proteins and glycoproteins using human cell lines; 2) QIB (4 months): to learn about methodologies (surface plasmon resonance and mass spectrometry) to assess protein-glycans interaction and heterologous expression approaches.

Enrolment in Doctoral degree(s): ESR11 will be enrolled at University of Florence

Introduction: Sialoglycans cross-talk/interaction with host immune Siglecs, description of 3D complexes. Molecular basis of recognition and binding of Siglecs to natural endogenous and exogenous substrates through ortogonal approaches (NMR spectroscopy, computational and biophysical techniques), with the aims to: get a detailed molecular picture of the Siglecs-glycans complexes, describe both protein and glycans key portions critical in the recognition and binding events; analyse the energetics and thermodynamics of the molecular interactions; fast screen possible binders with respect to a specific target receptor.

Aims: Science: Study of molecular recognition events in the Siglecs-sialoglycan interaction, comprehension of the binding properties and specificity of Siglecs receptors upon sialoglycans binding; analysis of sialoglycans and proteins region involved in the recognition and binding evente description of the and analysis of the binding regions by NMR spectroscopy, computational approaches and biophysical approaches. Analysis of 3D structures of Siglecs in complex with microbial sialoglycans;- the newly synthesized ligands will be analyzed in their interaction with target Siglecs and the ligands with improved binding affinity will be further developed. Training: in structural biology, NMR spectroscopy to define protein-ligand complexes, biophysical and computational techniques and their applications, with extensive impact in both pharmaceutical industry and academia environments applied to the structural characterization of the complexes of antimicrobials with bacterial envelope targets.

Expected Results: Learning how the conformation and presentation of epitopes is achieved by using NMR and other biophysical techniques; Learning how this presentation regulates the molecular recognition issues. The integrated approach based on NMR, docking and molecular modeling will profile the ligands’ epitope in their bound conformations and to provide consistent 3D-models of the interaction. With microbial ligands identification of key molecular actors of interactions between microbial ligands and Siglecs.

Planned secondment(s): 3-month secondments at: 1) UNIFI: Ultrafast HR-MAS NMR applied to complex systems. 2) RAD: to learn how to further develop ligands with improved binding affinity 2) ICENI: Without perturbing their binding properties, selected glycomimetics by adding a lipid chain suitable for the multivalent presentation.

Enrolment in Doctoral degree(s): ESR12 will be enrolled at University of Naples Federico II

Host Institution: University Medical Center (Amsterdam, The Netherlands) - VUMC
Primary Supervisor: Yvette van Kooyk and Fabrizio Chiodo
Planned duration: 48 months
Subject Area: Cancer immunology, glycosciences

Introduction: Tumor sialylation has great impact on tumor growth and immune defence. By altering sialylation tumors silence many different immune cells which as a consequence show deficient attack of the tumor. The molecular interplay between various Siglec receptors expressed on immune cells that sense sialic acids expression in the tumor environments needs to be unraveled. Moreover therapeutic strategies aimed to alter sialylation in the tumor microenvironment need to be tested, to restore immune activation and killing of the tumor.

Aims: Science: this ESR will be involved with the functional aspects developed by the colleagues in ESR 1-3-9-10 and 11. Aberrant (or modified) sialylation has been described in the cancer microenvironment as well as the expression (up or down-regulation) of different Siglecs. The ESR will use the Siglecs studied and expressed in WP2 to stain different cancer tissues trying to develop a glyco-cancer code (based on sialic acids) able to differentiate tumors at a different stage (or resistant to therapy). In addition, to study and visualize the sialic acids in the tumor microenvironment, the ESR will also track and study the location and ex. Training: The ESR will learn to explore different techniques to visualize glycans and glycans receptors. Confocal microscopy, mass cytometry, ELISA and tissues staining will give the ESR the basics to develop glyco-diagnostic tools and to study glyco-biomarkers.

Expected Results: PhD thesis the ESR will develop unique and unreported tools for diagnostics in cancer using glycans and glycans-receptors.

Planned secondment(s): 3-month secondments at: 1) CICbioGUNE: to study the expression of different Siglecs and glycoproteins. 2) GCU: to design and pre-screen glycomimetics targeting the Siglec-axis.

Enrolment in Doctoral degree(s): ESR13 will be enrolled at VU University Medical Center

Aims: Science: The project aims to: i) Identify new sialic acid biosynthesis inhibitors to abrogate Siglec binding and signaling. To this end, new sialic acid glycomimetics will be designed, synthesized and tested for activity in cellular assays. Active inhibitors will be used in functional assays in collaboration with various project partners. Training: Expertise in the synthesis, purification and characterization of sialic acid mimetics by means of organic chemistry, MS spectrometry and NMR spectroscopy. Basic cell biology skills such as cell culture and cellular assays. Secondments will provide a perspective of possible synergies that can be exploited by the ESR for his/her future career.

Planned secondment(s): 3-month secondments at: 1) QIB:) Evaluation of the activity of newly synthetized inhibitors on microbial cells; 3) VUMC:) as synthetic tools to interfere with the immunomodulatory events triggered in the TME as new therapeutic agents 2) GIOTTO:) NMR analysis of novel biosynthesis inhibitors.

Enrolment in Doctoral degree(s): ESR14 will be enrolled at Radboud University