Two recently developed theories of electronic energy transfer/migration were for the first time applied to real protein systems for extracting molecular distances. The partial donor-donor energy migration (PDDEM) is an extension to the previously developed donor-donor energy migration (DDEM, F Bergström et al PNAS 96, 1999, 12477) which allows using chemically identical but photophysically different fluorophores in energy migration experiments. A method based on fluorescence quenching was investigated and applied to create an asymmetric energy migration between fluorophores which were covalently and specifically attached to plasminogen activator inhibitor type 2 (PAI-2). It was also shown experimentally that distance information can be obtained if the fluorescence relaxation for photophysically identical donors, exhibits multi-exponential relaxation. An extended Förster theory (EFT) that was previously derived (L. B.-Å. Johansson et al J. Chem. Phys., 1996, 105) ha been developed for analysis of donor-acceptor energy transfer systems as well as DDEM systems. Recently the EFT was also applied to determine intra molecular distances in the protein plasminogen activator inhibitor type 1 (PAI-1) which was labelled with a sulfhydryl specific derivative of BODIPY. The EFT explicitly accounts for the time-dependent reorientations which in a complex manner influence the rate of electronic energy transfer/migration. This difficulty is related to the “k2-problem”, which has been solved. It is also shown experimentally that the time-correlated single-photon counting (TCSPC) data is sensitive to the mutual configuration between the interacting fluorophores. To increase the accuracy in the extracted parameters it is furthermore suggested to collect the fluorescence data under various physico-chemical conditions. It was also shown that the Förster theory is only valid in the initial part of the fluorescence decay.

No description available.

No description available.

Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice. A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained. This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules. / QC 20100719

Performing simulations of large-scale bio-molecular systems has long been one of the great challenges of molecular biophysics. Phenomena, such as the folding and conformational rearrangement of proteins, often takes place over the course milliseconds-to-seconds. The methods of traditional molecular dynamics used to simulate such systems are on the other hand typically limited to giving trajectories of nanosecond-to-microsecond duration. The failure of traditional methods has thus motivated the development of many special purpose techniques that propose to capture the essential characteristics of systems over conventionally inaccessible timescales. This dissertation first focuses on presenting a set of advances made on one such technique, Milestoning. Milestoning gives a statistical procedure for recovering long trajectories of the system based on observations of many short trajectories that start and end on hypersurfaces in the system’s phase space. Justification of the method’s validity typically relies on the assumption that trajectories of the system lose all memory between crossing successive milestones. We start by giving a modified milestoning procedure in which both the memory loss assumption is relaxed and reaction mechanisms are more easily extracted. We follow with numerical examples illustrating the success of new procedure. Then we show how milestoning may be used to compute an experimentally relevant timescale known as the transit time (also known as the reaction path time). Finally, we discuss how time reversal symmetry may be exploited to improve sampling of the trajectory fragments that connect milestones. After discussing milestoning, the dissertation shifts focus to a different way of approaching the problem of simulating long timescales. We consider two polymers models that are sufficiently simple to permit numerical integration of the desired long trajectories of the system. In some limiting cases, we see their simplicity even permits some questions about the dynamcis to be answered analytically. Using these models, we make a series of experimentally verifiable predictions about the dynamics of unfolded polymers. / text

Sporadic reports of significant under-ice phytoplankton production indicate a critical knowledge gap of a key component of the Arctic ecosystem. In this thesis I examine the following research objectives in an effort to improve the understanding of under-ice phytoplankton production: (1) to determine the biophysical processes controlling the timing of under-ice phytoplankton production, and (2) to compare and contrast both the timing of the under-ice bloom and the controlling processes between multiple years of data. Data for objective (1) were collected during the three-year Arctic-ICE field campaign (2010-2012) near Resolute Bay, NU in the central Canadian Arctic. Additional data from the region were collected from open source databases and peer-reviewed literature for a dataset that spanned from early 1960 to the present, supporting the analysis to meet objective (2). Two separate under-ice phytoplankton blooms were observed during the three-year Arctic-ICE campaign. It was found that phytoplankton blooms conformed well to the critical depth hypothesis in the Canadian Archipelago under first-year ice, where snow and ice melt both increased light transmission and shoaled the surface mixed layer which, in turn, placed phytoplankton within a favourable light environment for positive net production underneath the ice cover. Factors such as timing of melt water drainage and water column mixing greatly affected bloom onset. From the historical analyses, I was able to show that under-ice phytoplankton blooms have regularly occurred under landfast ice from at least the 1960’s. Significant correlations between the timing of bloom onset with melt onset related variables (i.e., air temperature reaching 0 ºC and complete snow melt) suggested a strong link to climate change. In fact, the ii analysis supported that since the mid 1990s bloom onset has been occurring earlier, and is likely related to decreasing trends in day of complete snow melt, maximum ice thickness, and snow depth. Overall, this thesis has helped improve our understanding of the under-ice spring phytoplankton bloom, showing that under-ice production has been a regular occurrence in the Canadian Arctic. The results also support that timing of the spring phytoplankton blooms could be shifting earlier in response to the warming Arctic and its changing icescape. Such a shift could also have important consequences on the Arctic marine food web, influencing the transfer of energy through the food chain. Therefore, it is of utmost importance to continue future observational programs of the under-ice pelagic environment focussed on the late spring melt period to better understand how the system could change with further perturbations.

The spatiotemporal organization of membrane proteins is implicated in cellular trafficking, signalling and reception. It was proposed that biological membranes partition into lipid rafts that can promote and control the organization of membrane proteins to localize the mentioned processes. Lipid rafts are thought to be transient (microseconds) and small (nanometers), rendering their detection a challenging task. To circumvent this problem, multi-component artificial membrane systems are deployed to study the segregation of lipids at longer time and length scales. In this thesis, multi-component Droplet Interface Bilayers (DIBs) were imaged using fluorescence and interferometric scattering microscopy. DIBs were used to examine and manipulate microscopic lipid domains and to observe, for the first time, transient nanoscopic lipid domains. The techniques and results described here will have important implications on future research in this field.

Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane  barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.

Cell biophysical properties are a new class of biomarkers that can characterize cells into subgroups that indicate differences in phenotypes that may correlate with disease and cell state. Microfluidic biophysical cell sorters are platforms that utilize these newly developed biomarkers to expand biomedical capabilities for improvements in cell state detection and characterization. Cell biophysical properties are important indicators for cell state and function because they point to differences in cell structures, such as cytoskeletal arrangement and nuclear content. In particular, some diseases, such as cancer and malaria, can cause significant changes in cell biophysical properties. Therefore, cell biophysical properties have the potential to be used for disease diagnostics. Microfluidic systems which can interrogate these biophysical properties and exploit changes in biophysical properties to separate cells into subpopulations will provide important biomedical capabilities. In this combined theoretical and experimental investigation, we explore a new type of cell sorter which utilizes differences in biophysical properties of cells. These biophysical properties that can be utilized to sort cells include size, elasticity and viscosity. We invented a microfluidic system for continuous, label-free cell separation that utilizes variations in cell biophysical properties. A microfluidic channel is decorated by periodic diagonal ridges that are designed to compress flowing cells in rapid succession. The physical compression, in combination with hydrodynamic secondary flows induced by the ridged microfluidic channel, translates each cell perpendicular to the channel axis in proportion to its biophysical properties. Through careful experimental and computational studies, we found that the cell trajectories in the microfluidic cell sorter correlated to these biophysical properties. Furthermore, we examine the effect of channel design parameters under various experimental conditions to derive cell separation models that can be used to qualitatively predict cell sorting outcome. A variety of biophysical measurement tools, including atomic force microscopy and high-speed optical microscopy are used to directly characterize the heterogeneous population of cells before and after separation. Taken together, we describe the physical principles that our microfluidic approach can be effectively used to separate a variety of cell types. The major contribution is the creation and characterization of a novel microfluidic cell- sorting platform that utilizes cell biophysical properties to enrich cells into phenotypic subtypes. This innovative approach opens new ways for conducting rapid and low-cost cell analysis and disease diagnostics through biophysical markers.

In this research, a recent version of the Boreal Ecosystem Productivity Simulator (BEPS), called BEPS-TerrainLab, was adapted to northern peatlands and evaluated using observations made at the Mer Bleue bog located near Ottawa, Ontario, and the Sandhill fen located near Prince Albert, Saskatchewan. The code was extended and modified with a major focus on the adequate representation of northern peatlands' multi-layer canopy and the associated processes related to energy, water vapour and carbon dioxide fluxes through remotely-sensed leaf area index (LAI) maps. An important prerequisite for the successful mapping of LAI based on remote sensing imagery is the accurate measurement of LAI in the field with a standard technique such as the LAI-2000 plant canopy analyzer. As part of this research, a quick and reliable method to determine shrub LAI with the LAI-2000 instrument was developed. This method was used to collect a large number of LAI data at the Mer Bleue bog for the development of a new remote sensing-based methodology using multiple endmember spectral unmixing that allows for separate tree and shrub LAI mapping in ombrotrophic peatlands. A slight modification of this methodology allows for its application to minerotrophic peatlands and their surrounding landscapes. These LAI maps were used to explicitly represent the tree and shrub layers of the Mer Bleue bog and the tree and shrub/sedge layers of the Sandill fen within BEPS-TerrainLab. The adapted version of BEPS-TerrainLab was used to investigate the in fluence of mescoscale topography (Mer Bleue bog) and macro- and mesoscale topography (Sandhill fen) on wetness, evapotranspiration, and gross primary productivity during the snow-free period of 2004. This research suggests that future peatland ecosystem modelling efforts at regional and continental scales should include a peatland type-specific differentiation of macro- and mesoscale topographic effects on hydrology, to allow for a more realistic simulation of peatlands' soil water balance. This is an important prerequisite for the reduction of currently existing uncertainties in wetlands' contribution to North America's carbon dioxide and methane annual fluxes from an ecosystem modelling perspective.