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Two different methods were used to obtain a potential energy surface for the Arco molecule. One involved choosing a functional form for the repulsion and dispersion energies whose parameters were determined by a fit to experimental data. A physically justified potential that agreed with experiment could not be obtained. The other method was based on calculating ab initio interaction energies at different configurations of the molecule and interpolating between them. The resulting surface was scaled in the energy and the co-ordinates. Improved agreement was achieved for most observed bound states. Errors in the surface may have been due to an inadequate density of ab initio energies. So, how the molecular configurations chosen for interaction energy calculations affected the potential obtained was investigated. Both the co-ordinate system and the interpolation scheme also significantly affected the quality of surface obtained. The best compromise between accuracy and number of configurations, was points distributed on a regular grid in elliptic co-ordinates with Gauss-Legendre quadrature points in the angular co-ordinate. This information was employed to obtain a potential energy surface for the weakly bound HeOCS molecule which was in close agreement with experiment. A co-ordinate and energy scaling, guided by experiment, was applied to the ab initio surface. Only three scaling parameters were required due to the high quality of the initial surface.

Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.

A range of lanthanide compounds incorporating soft bridging ligands or alkoxide ligands have been synthesised and their magnetic properties investigated. These two classes of compound have shown promise as single molecule magnets but have not been widely studied; this thesis aims to expand on this area of research. Softer bridging ligands are found to slightly increase superexchange interactions between metal centres compared to harder bridging ligands. The introduction to this thesis covers the basic properties of the lanthanides, paying special attention to their chemistry with soft donor ligands and alkoxide ligands. Also included is an introduction to the field of single-molecule magnetism and the role of lanthanide complexes in the study of this behaviour. In Chapter 2, four complexes are reported: the phosphine adducts [Cp'3Ln(H2PMes)] and the phosphide-bridged trimers [(Cp'2)Ln(μ-PHMes)]3 (Ln = Er, Gd). Their structures and magnetic properties are characterised. In Chapter 3, the novel dodecametallic thiolate-bridged lanthanide macrocycles [(Cp'2Ln)3({μ-SCH2}3CMe)]4 (Ln = Dy, Y, Gd) are reported and characterised by X-ray crystallography, NMR spectroscopy and magnetometry. [(Cp'2Dy)3({μ-SCH2}3CMe)]4 is shown to be a single-molecule magnet with Ueff = 69 cm-1. In Chapter 4, the novel thiolate-bridged lanthanide dimers [Cp'2Ln(μ-SCH2{C4H7S2})]2 (Ln = Dy, Y, Gd) are reported, showing sulfur-sulfur bonding leading to ring cyclisation of the bridging ligand [MeC(CH2S)3]3-. These complexes are characterised by X-ray crystallography, NMR spectroscopy and magnetometry. Extra NMR spectroscopic studies were performed to investigate the mechanism of ring closure on the bridging ligand. [Cp'2Dy(μ-SCH2{C4H7S2})]2 is shown to be a single-molecule magnet with Ueff = 87 cm-1. In Chapter 5, four new lanthanide siloxide clusters incorporating alkali metals are reported: the trigonal bipyramidal [Dy2K3(OSiMe3)9]; the octahedral [Dy2K4(OSiMe3)10]; the bi-capped cuboid [Y4K6O6(OSiMe3)12]6-; and the [Dy3K8O3(OSiMe3)12]- 'burger' cluster. All clusters are structurally characterised by X-ray crystallography and [Dy2K4(OSiMe3)10] is magnetically characterised. The synthetic rationalisation for formation of these diverse structures is investigated.

A key component of neural circuit formation is the elaboration of complex dendritic arbors, the pattern of which constrains inputs to the neuron and thus, the information it processes. As such, many neurodevelopmental disorders such as autism and Down, Rett, and Fragile X Syndromes are associated with reduced forebrain dendrite arborization. Identifying molecules involved in regulating dendrite arborization and neural circuitry formation therefore, is a start to understanding these disorders. Nearly 70 cadherin superfamily adhesion molecules are encoded by the Pcdha, Pcdhb, and Pcdhg gene clusters. These so-called clustered protocadherins (Pcdhs) are broadly expressed throughout the nervous system, with lower levels found in a few non-neuronal tissues. Each neuron expresses a limited repertoire of clustered Pcdh genes, a complicated process controlled by differential methylation and promoter choice. The clustered Pcdh proteins interact homophilically in trans as cis-multimers, which has the potential to generate a combinatorially explosive number of distinct adhesive interfaces that may give neurons unique molecular identities important for circuit formation. Functional studies of animals in which clustered Pcdhs have been deleted or disrupted demonstrate that these proteins play critical roles in neuronal survival, axon and dendrite arborization, and synaptogenesis. Additionally, they have been implicated in the progression of several cancers, suggesting that basic studies of their function and signaling pathways will have important future clinical applications. Recent work has shown that γ-Pcdhs can regulate the Wnt signaling pathway, a common tumorigenic pathway which also play roles in neurodevelopment, but the molecular mechanisms remain unknown. I determined that γ-Pcdhs differentially regulate Wnt signaling: the C3 isoform uniquely inhibits the pathway while 13 other isoforms upregulate Wnt signaling. Focusing on γ-Pcdh-C3, I show that the variable cytoplasmic domain (VCD) is critical for Wnt signaling inhibition. γ-Pcdh-C3, but not other isoforms, physically interacts with Axin1, a key component of the canonical Wnt pathway. The C3 VCD competes with Dishevelled for binding to the DIX domain of Axin1, which stabilizes Axin1 at the membrane and leads to reduced phosphorylation of Wnt co-receptor Lrp6. I also present evidence that the Wnt pathway can be modulated up (by γ-Pcdh-A1) or down (by γ-Pcdh-C3) in the cerebral cortex in vivo, using conditional transgenic alleles. Studies have implicated γ-Pcdhs as a whole, in many neurodevelopmental processes but little is known if distinct roles exists for individual isoforms. By using a specific C3-isoform knockout mouse line engineered in collaboration with Dr. Robert Burgess of The Jackson Laboratory, I was able to uncover a unique role for the C3-isoform in the regulation of dendrite arborization. Mice without γ-Pcdh-C3 exhibit significantly reduced dendrite complexity in cortical neurons. This phenotype was recapitulated in cultured cortical neurons in vitro, which can be rescued by reintroducing the C3-isoform. The ability of γ-Pcdh-C3 to promote dendrite arborization cell-autonomously was abrogated when Axin1 was depleted with an shRNA, indicating that this process by which γ-Pcdh-C3 regulates dendrite arborization is mediated by its interaction with Axin1, which I had previously demonstrated. Together, these data suggest that γ-Pcdh-C3 has unique roles distinct from other γ-Pcdhs, in the regulation of Wnt signaling and dendrite arborization, both of which are mediated by interaction with Axin1.

Several Monte Carlo simulations of single-molecule fluorescence systems are developed to help evaluate and improve ongoing experiments. In the first simulation, trapping of a single molecule in a nanochannel is studied. Molecules move along the nanochannel by diffusion and electrokinetic flow. Single-molecule fluorescence signals excited by two spatially offset laser beams are detected and the direction of the flow is adjusted to try to equalize the signals and center the molecule between the beams. An algorithm is evaluated for trapping individual molecules in succession by rapidly reloading the trap after a molecule photobleaches or escapes. This is shown to be effective for trapping fast-diffusing single-chromophore molecules in succession within a micron-sized confocal region while accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware. In the second simulation, trapping of a molecule in a two-dimensional fluidic device consisting of sub-micron-separated glass plates is studied. Two different illumination schemes for sensing the molecule's position are compared: (i) a single continuous laser spot circularly scanned at 40 KHz or 240 KHz in the plane of the device; and (ii) four pulsed laser spots arranged in a square and temporally alternated at 304 MHz In either case, the times of detected photons are used by algorithms to control the electrokinetic flow in two dimensions to compensate diffusion and achieve single-molecule trapping. However each scheme is found to have limitations, as circular scanning produces a modulation in the fluorescence signal and in the autocorrelation function, whereas the four-pulse scheme becomes ineffective if the fluorescence lifetime of the molecule is greater than the time between laser pulses, The third simulation investigates appropriate conditions for detection of single molecules flowing through an array of fluidic channels for an application to high-throughput screening for pharmaceutical drug discovery. For parallelized single-molecule detection, illumination is provided by a continuous laser focused to a line intersecting all channels and fluorescence is imaged to a single row of pixels of an electron-multiplying CCD with sufficient gain for single-photon detection. The simulation separately models each channel to determine laser, flow, and camera operating conditions suitable for efficient detection.

An apparatus that combines single molecule fluorescence, optical trapping and bright-field microscopy is presented. Given the spread over orders of magnitude of the light intensities for the different techniques, special considerations in choosing the spectral regions for each were taken. Moreover, imaging single molecules in a background of intense light from the infra red laser used for the optical trap has been shown to result in enhanced photo-bleaching due to two-photon processes. A scheme for fast time-sharing was implemented in which the fluorescence excitation light and the trap light alternate in fast succession. This eliminates two-photon effects and enables stable manipulation using the optical trap. The simultaneous use of the bright-field imaging in differential interference contrast arrangement enables observation of refractile objects in the sample over large distances. The apparatus was designed for future use in studies of molecular motor regulation. However, to demonstrate the functionality of the system, the rotational diffusion of a micro-sphere fluorescently labelled at one spot was measured. / text

Single molecule wide-field polarization fluorescence imaging is an experimental method to determine the self-assembly of molecules dispersed in a thin film. Through a combination of wide-field imaging and confocal spectroscopy, the effect of synthetic structure of the oligomeric PPV-based materials was investigated to understand the effect of conjugation length, role of hydrogen bonding side chains, and influence of regioregularity on controlling chromophore folding. By studying alkoxy-linked and alkyl-linked bis(2-ethylhexyl)-p-phenylene vinylene (BEH-PPV) units of varying lengths (three, five, or seven), it was determined that conjugation length controlled the extent of molecular ordering and emission properties. Comparison of the experimental results to molecular dynamics simulations performed by collaborators confirmed that the materials became increasingly ordered as conjugation length increased. Further regulation of the assembly can be obtained through inclusion of hydrogen bonding side chains as seen in the altered amine and carboxylic acid alkoxy-linked trimer BEH-PPV in contrast to the bulky side chain tert-butyl trimer. The study of regio-regular (RR) and regio-random (RRa) alkoxy-linked pentamer poly(2-methoxy-5-(2’-ethylhexyloxy)-p-phenylenevinylene) (MEH-PPV) illustrates the limited effect of regioregularity of the side chains on self-assembly. Through synthetic structure, it is possible to design highly ordered materials through control of conjugation length and selection of side chains. / text

This work reports the development and spectroscopic studies of blinking-suppressed compact quantum dots. It is shown that a linearly graded alloy shell can be grown on a small CdSe core via a precisely controlled layer-by-layer process, and that this graded shell leads to a dramatic suppression of QD blinking both in organic solvents and in water. A substantial portion (over 25%) of the resulting QDs essentially does not blink (more than 99% of the time in the bright or “on” state). Theoretical modeling studies indicate that this type of linearly graded and relatively thin shells can not only minimize charge carrier access to surface traps, but also reduce accumulated lattice strains and defects at the core/shell interface, both of which are believed to be responsible for carrier trapping and QD blinking. Further, the biological utility of blinking-suppressed QDs by using both polyethylene glycol (PEG)-based and multidentate capping ligands is evaluated, and the results show that their optical properties are maintained regardless of surface coatings or solvating media, and that the blinking-suppressed QDs can provide continuous trajectories in live cell receptor tracking studies.

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