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Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport. / Biology

A number of microcantilever systems of fundamental importance are explored using theoretical and numerical methods to quantify and provide physical insights into the dynamics of experimentally accessible systems that include a variety of configurations and viscous fluids. It is first shown that the correlated dynamics of both a laterally and vertically offset cantilever pair can be accurately predicted by numerical simulations. This is verified by comparing the correlated dynamics yielded by numerical simulations with experimental measurement. It is also demonstrated that in order to obtain these accurate predictions, geometric details of the cantilever must be included in the numerical simulation to directly reflect the experimental cantilever. A microrheology technique that utilizes the fluctuation-dissipation theorem is proposed. It is shown that by including the frequency dependence of the fluid damping, improvements in accuracy of the predictions of the rheological properties of the surrounding fluid are observed over current techniques. The amplitude spectrum of a 2-D cantilever in a power-law fluid is studied. The resulting amplitude spectrum yielded a curve similar to an overdamped system. It is observed that the amplitude and noise spectrum yield the same qualitative response for a 2-D cantilever in a shear thinning, power-law fluid. The correlated dynamics of a tethered vertically offset cantilever pair is investigated. It is shown that for a range of stiffness ratios, which is the ratio of the spring constant of the tethering relative to the cantilever spring constant, the change in the correlated dynamics of a Hookean spring tethered cantilever pair can be seen in the presence of fluid coupling. The dynamics of a spring-mass tethered, vertically offset cantilever pair is qualitatively studied by simplifying the model to an array of springs and masses. The resulting study found that the correlated dynamics of the displacement of mass of the tethered object yielded newly observed features and characteristics. It is shown that the curve shape of the cross-correlation of the displacement of the mass of the tethered object is similar to that of the auto-correlation of the displacement of the mass representing a step forced cantilever. The cross-correlation of the displacement of the mass of the tethered object, however, is found to be significantly more dependent on the stiffness ratio than the auto-correlation of the displacement of the mass representing a cantilever for t > 0. At t = 0, it is observed that the mass of the tethered object yields the same finite value for the cross-correlation for all studied values of the stiffness ratio. This characteristic is a result of the symmetry of the studied spring-mass system. / Ph. D.

Yes / Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10−5. A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach. / This study was supported by a grant from the Swedish Research Council to T.S.