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The family of Sialic-acid-binding ImmunoGlobulin-like LECtins, known as Siglecs, play a vital role in the hematopoietic, immune and nervous systems of many mammals, including humans. These membrane-bound proteins mediate cell to cell adhesion and signalling, and act as regulatory systems for a variety of biological functions. All functional Siglecs bind to sialic acid (Neu5Ac) moieties displayed on the terminus of cell surface glycan structures. Although the Siglec family displays remarkably distinct selectivity for different glyosidic linkages (such as Neu5Ac-α2,6 or Neu5Ac-α2,3) and modifications (such as Neu5Ac or Neu5Gc), the carbohydrate binding pocket of the V-set domain is highly conserved. There are two major groups of Siglecs; the conserved Siglecs, including Siglec-1 (Sialoadhesin, Sn), Siglec-2 (CD22), Siglec-4 (myelin-associated glycoprotein, MAG) & Siglec-15, and the CD33-related Siglecs. The research detailed within this PhD thesis is predominately focused on the conserved Siglecs, in particular Siglec-2, Siglec-1 and Siglec-4. Since the discovery of the Siglec family, their role in a wide variety of disease states has been well established. Siglec-2 for example, is found on B cells which are known to play a significant role in autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, and systemic lupus erythematosus. Additionally, some non-Hodgkins lymphomas and certain leukemias involve B cells which express Siglec-2. Siglec-1 (primarily expressed on macrophages) is known to bind to Neu5Ac terminating glycans on the surface of pathogens including Neisseria meningitidis, Campylobacter jejuni, Trypanosoma cruzi and HIV-1. This binding can lead to the phagocytosis of the pathogen or, as is the case with HIV, further the spread of the virus to other cells. Siglec- 4 functions as an essential part of the maintenance of myelinated axons as well as an inhibitor of neurite outgrowth and axon regeneration. It has been demonstrated in vitro that blocking Siglec-4 binding stimulates axon outgrowth, which could pave the way for therapies that allow axon regeneration in patients with nerve injuries. It has been hypothesised that synthetic Siglec ligands with high affinity and selectivity could be used as potential therapeutics for these diseases. In the 19 years since the X-ray crystal structure of Siglec-1 was first resolved, many research groups have ventured to synthesise Siglec inhibitors, with the majority of these based upon the natural ligand, Neu5Ac. The research in this thesis seeks to explore the C4 position of Neu5Ac and how modifications at this position can influence the binding affinity of Siglecs to this novel class of Siglec ligands. Chapter 1 provides a general introduction to sialic acids, lectins and, more specifically, the Siglecs. The structure and function of Siglec-2, Siglec-1 and Siglec-4 are then described in detail. Previous research conducted within the Siglec inhibitor space is also briefly reviewed. Finally, an overview of the chemical synthesis that was carried out throughout the research project is described. Although the chemistry of Neu5Ac has been exhaustively documented for some four decades, Neu5Ac derivatives that incorporate modifications at C2, C3 and C4 are relatively underrepresented in the literature. Chapter 2 describes the development of methodologies required to functionalise Neu5Ac at the C2 position via the 2,3-β-epoxide intermediate. The described versatile synthesis allows for the introduction of various anomeric aglycon substituents and maintains the C3 hydroxyl group which could be further functionalised (for different research paths) or subsequently reduced to yield C3-anhydro derivatives. Chapter 3 describes the use of STD NMR to explore the interactions between novel C4- modified synthetic Siglec-2 ligands and the Siglec-2 protein. Initially we discovered that the addition of aromatic amides to the C4 position of Neu5Acα2Me improved Siglec-2 binding by up to 15-fold whilst also reducing Siglec-4 binding compared to the parent compound. By combining C4 modifications with the 9-biphenylcarboxamido (9-BPC) moiety described in the literature, we were able to enhance Siglec-2 binding by over 10,000-fold (compared to Neu5Acα2Me). Using STD NMR we were able to resolve the ligand binding epitope, which showed that the C4 and C9 substituents acted synergistically to enhance affinity. Furthermore, we used a novel whole cell STD NMR technique to visualise how synthetic ligands overcome cis-binding to recognise Siglec-2 in a cellular environment. This in turn led to the synthesis of potent Siglec-2 ligands that were functionalised at the anomeric position (C2), in addition to C4 and C9. Finally, we wanted to further explore the role that C2 and C4 modifications play in enhancing affinity and, more importantly, selectivity. The research outlined in Chapter 4 involves the synthesis and biological evaluation of a suite of C4-modified benzyl glycosides and C2-modified meta-nitrophenylcarboxamido (4-mNPC) Neu5Ac derivatives. Addition of the 9-BPC functionality to a select number of these compounds vastly improved Siglec-2 binding affinity to nanomolar inhibitory concentrations. Selectivity towards Siglec-2 could potentially be controlled by modifying the C2 and C4 substituents, however binding to Siglec-4 and Siglec-1 could not be abolished completely. Preliminary research in multivalent ligands and drug delivery systems is described in Chapter 5, along with the thesis conclusions.

The aim of this work was to define the possible occurrence of hematological changes during the course of a chronic ingestion of 137Cs. A mouse model was used, with ingestion through drinking water with a cesium concentration of 20 kBq l -1. Ingestion started in parent animals before mating, and 137Cs intake and its effect on the hematopoietic system was studied in offspring at various ages between birth and 20 weeks. 137Cs content was measured in various organs, indicating that 137Cs was distributed throughout the organism including lymphohematopoietic organs, i.e., femurs, spleen and thymus. However, we did not observe any effect on the hematopoietic system, whatever the parameter used. In fact, blood cell counts, mononuclear cell counts and progenitor frequency in bone marrow and spleen, and Flt3-ligand, Erythropoietin, G-CSF and SDF-1 concentration in plasma remained unchanged when compared to control animals. Moreover, phenotypic analysis did not show any change in the proportions of bone marrow cell populations. These results indicate that, although 137Cs was found in all organs implicated in the hematopoietic system, this did not induce any changes in bone marrow function. © Springer-Verlag 2010.