T cell immunoglobulin and mucin (Tim) family molecules are cell membrane proteins with four functional Tim family members in mouse, and three in human. They are preferentially expressed on immune cells with Tim1 on Th2 cells, Tim3 on Th1 cells and Tim4 on antigen-presenting cells (APCs). They have several roles, including regulating immune responses and mediating phagocytosis of dead cells. However, little is known about them beyond these two species, and nothing outside mammals. To investigate the Tim family in the chicken, the genes were identified and cDNAs cloned. Differently to mammals, the chicken genome only contains genes for Tim1 and Tim4. Chicken Tim1 (chTim1) has similar mRNA expression patterns to those of mammalian Tim1 in lymphoid tissues and immune cells. Interestingly, chTim4 has at least four splice variants – an extra short isoform (chTimeS) lacking exons 5, 6, 7 and 8, a short isoform (chTim4S) without exons 3, 4 and 5, a long isoform (chTim4L) with all exons and an extra long isoform (chTim4eL), which is similar to chTim4L but with a longer exon 3. The chTim4S is a homologue of mammalian Tim4 with constitutive expression in lymphoid tissues and immune cells; other chTim4 variants showed inducible or cell-specific expression patterns. Like mammalian Tim4, chTim4S is expressed by APCs; but differently to mammals, chTim4S is also expressed by γδ T cells, suggesting a unique role for chTim4 in this population of T cells. The biological activities of the chicken Tim family molecules were also investigated using chTim-Ig fusion proteins. Like mammals, chTim1 and chTim4S fusion proteins can specifically recognise phosphatidylserine (PS), an indicator of apoptotic cells, suggesting they are PS receptors. Pre-incubation with PS blocked binding of the chTim4S fusion protein to PS-exposing apoptotic cells. Physiologically, recognition of PS by the chTim proteins mediates apoptotic cell clearance, which was demonstrated using chTim-transfected fibroblast cells (3T3), which significantly increased their uptake of apoptotic cells compared with untransfected cells. The chTim4-Ig fusion protein also had costimulatory activity on chicken T cells. Monoclonal antibodies against the chTim proteins were generated. They specifically recognise their own antigen tested intensively by different immunological assays. ChTim4L is expressed intracellularly in freshly-isolated splenocytes rather than on the surface, whereas PMA-stimulated splenocytes express chTim4S and chTim4L on the cell surface. Like mammals, chicken splenic macrophages also express chTim4S and chTim4L. Both of them are also expressed by bone marrow-derived macrophages but not bone marrow-derived DCs. The chTim1 protein was detected at high levels in bursal cells and splenocytes by western blot analysis using polyclonal anti-chTim1 serum, which is consistent with its mRNA expression pattern through qRT-PCR analysis, suggesting B and T cells may express chTim1, consistent with its expression in mammals. Mammalian Tim1 is expressed on Th2 cells, its ligand, Tim4, on APCs; the interaction between them drives Th2 cell proliferation. The knowledge from this study will help to further dissect how the chicken’s Th2 responses are regulated through cell surface molecules.

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This thesis will present the synthesis, characterization and magnetic measurements of lanthanide complexes with varying nuclearities (Ln, Ln2, Ln3 and Ln4). EuIII, GdIII, TbIII, DyIII, HoIII and YbIII have been selected as the metal centers. Eight polydentate Schiff-base ligands have been synthesized with N- and mostly O-based coordination environments which chelate 7-, 8- or 9-coordinate lanthanide ions. The molecular structures were characterized by single crystal X-ray crystallography and the magnetic properties were measured using a SQUID magnetometer. Each chapter consists of crystal structures and magnetic measurements for complexes with the same nuclearity. There are eight DyIII SMMs in this thesis which are discrete molecules that act as magnets below a certain temperature called their blocking temperature. This phenomenon results from an appreciable spin ground state (S) as well as negative uni-axial anisotropy (D), both present in lanthanide ions owing to their f electron shell, generating an effective energy barrier for the reversal of the magnetization (Ueff). The ab initio calculations are also included for the SMMs with high anisotropic energy barriers to understand the mechanisms of slow magnetic relaxation in these systems.

The versatility of N-heterocyclic carbenes (NHCs) is demonstrated by numerous practical applications in homogeneous transition metal catalysis, organocatalysis and materials science. There remains a paucity of electropositive metal NHC complexes and so this chemistry is poorly developed with respect to that of the late transition metal and main group elements. This thesis describes the synthesis of new alkoxy-tethered NHC proligands, their use in the synthesis of reactive metal amide and metal alkyl complexes, and finally small molecule activation using these complexes. Chapter One introduces NHCs and discusses their use as supporting ligands for early transition metal and f-block complexes. Small molecule activation using organometallic complexes is examined alongside the use of electropositive metal NHC complexes in catalysis. Chapter Two contains the synthesis and characterisation of new alkoxy-tethered NHC proligands and a variety of electropositive MII (M = Mg and Zn), MIII (M = Y, Sc, Ce and U) and MIV (M = Ce and U) amide complexes. X-ray diffraction studies and a DFT study are used to probe the extent of covalency in the bonding of the MIV complexes. Chapter Three investigates the reactivity of the amide complexes prepared in Chapter Two. The MII complexes are shown to be initiators for the polymerisation of raclactide into biodegradable polymers. The MIII complexes are used to demonstrate additionelimination reactivity of polar substrates across the M-Ccarbene bond which allows the formation of new N-E (E = Si, Sn, P or B) bonds. Treatment of the UIII silylamide complex U(N{SiMe3}2)3 with CO results in the reductive coupling and homologation of CO to form an ynediolate core -OC≡CO- and the first example of subsequent reactivity of the ynediolate group. The MIV complexes are used to examine the potential for forming MIV cationic species and alkyl complexes. Chapter Four examines the synthesis of MIII (M = Ce and Sc) aminobenzyl complexes and MIII (M = Y, Sc and U) neosilyl and neopentyl alkyl complexes. The addition-elimination reactivity discussed in Chapter Three is extended to include C-E bond formation (E = Si, Sn, P, B, I or C). Chapter Five provides overall conclusions to the work presented within this thesis. Chapter Six gives experimental and characterising data for all complexes and reactions in this work.

Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.

In this work a system-level approach was taken to the single-molecule fluorescence microscopy of living cells. This primarily involved the unification of relevant information within appropriately structured artefacts that were used to inform and enhance experimentation. Initially the diversity of emerging single-molecule techniques was reviewed and presented with a novel article structure to suit the purpose of designing an experiment (Harriman and Leake 2011). Techniques were grouped by the type of information they could access, rather than the standard organisation centred on the techniques themselves. A bespoke microscope was conceived and built with reference to knowledge and tools from the fields of Architecture and Systems-Engineering. The microscope layout would enable multiple experiment types through independent control of multiple illumination beams. A technique was developed enabling the prescription of evanescent field penetration depth for each incident beam. The various empirical and theoretical results that are used to understand and modify a microscopy experiment were integrated into an internally consistent simulation model (Harriman and Leake. 2013). This was used to inform the selection of experimental components and parameters and ultimately acquire higher data quality as measured by functions such as signal-to-noise ratio (SNR). The combined experimental system of microscope and simulation model was applied in two live-cell investigations. In Escherichia coli, the spatial distribution of membrane bound proteins was investigated and a novel technique was applied to the analysis of colocalisation. Results indicate that NADH dehydrogenase and ATP synthase follow uncorrelated trajectories. This supports the hypothesis of spatial decoupling of molecules that energise the membrane and molecules that use membrane energy. In human carcinoma cells, the mechanism of ligand-receptor binding was investigated. Data was collected prior to and periodically after the addition of ligands, and fluorescence images were acquired of both ligands and receptors. Analyses based on single particle tracking are currently being carried out by a collaborator to extract information on stoichiometry and dynamics at the single-molecule level.

Thesis advisor: Marc L. Snapper / Chapter 1 Review of methodology developments in the area of selective tosylation of alcohols. Chapter 2 Development of a catalytic enantioselective tosylation of alcohols with an amino-acid-based organocatalyst. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.