Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.

In recent years, localization-based super-resolution imaging has been developed to overcome the diffraction limit of far-field fluorescence microscopy. Photoswitchable probes are a hallmark of this technique. Their fluorescence can be modulated between an emissive and dark state whereby the sequential, nanoscale measurement of individual fluorophore positions can be used to reconstruct an image at higher spatial resolution. Despite the importance of photoswitchable probes for localization-based super-resolution imaging, both a mechanistic and quantitative understanding of the essential photoswitching properties is lacking for most fluorophores. In this thesis, we begin to address this need. Furthermore, we demonstrate the development of new probes and methodologies for both multicolor and live-cell super-resolution imaging. Chapter 2 describes our mechanistic insights into the photoswitching of a common class of dyes called carbocyanines. Red carbocyanines, such as Cy5, enter a long-lived dark state upon illumination with red light in the presence of a primary thiol. We show that the dark state is a covalent conjugate between the thiol and dye and that this dark state recovers by illumination with ultraviolet light. We also speculate on possible reactivation mechanisms. Our mechanistic studies may ultimately lead to the creation of new probes with improved photoswitching properties. Chapter 3 details our quantitative characterization of the photoswitching properties of 26 organic dyes, including carbocyanines and several other structural classes. We define the essential properties of photoswitchable probes, including photons per switching event, on/off duty cycle, photostability, and number of switching cycles, and demonstrate how these properties dictate super-resolution image quality. This rigorous evaluation will enable more effective use of probes. In Chapters 4 and 5, we focus on expanding the super-resolution toolbox with novel strategies for multicolor and live-cell imaging. Chapter 4 discusses two approaches we have developed for multicolor super-resolution imaging, which distinguish probes based on either the color of activation or emission light. These tools allow multiple cellular targets to be resolved with high spatial resolution. Lastly, Chapter 5 introduces a method for targeted cellular labeling with photoswitchable probes using a small peptide tag, as well as a new sulfonate-protection strategy for intracellular delivery of high performing photoswitchable dyes.

The area of study is the Avery Drive drainage area. The objective of these projects is to alleviate frequent flooding at the Avery Drive neighborhoods. RC&A designed two options for improving the Avery Drive drainage area. Option one increases the capacity of the existing sewer system, and option two constructs a new storm drain system along Simon Street while keeping the existing system in place. Although both options offer preliminary solutions for alleviating flooding, the designs are unviable due to the lack of data that was available for the study. The following research will prove that RC&A fulfilled its contractual obligation of practicing due diligence by recommending further investigation to obtain valuable data for a complete and successful final design, rather than providing a solid recommendation based on existing data. / text

Wind loading considered in the design of overhead transmission lines is based on extreme values of synoptic wind, i.e. boundary layer wind originating from largescale meteorological pressure systems. Localized high intensity wind (HIW) storms such as tornadoes and downbursts are a different type of extreme wind frequently causing failures of overhead lines. This thesis covers the design aspects of overhead transmission lines when subject to localized HIW storms. A comprehensive review of the literature is included on the effects of such wind storms on lines and on mitigation measures. Furthermore, several options for the design of self-supporting transmission towers against localized HIW are discussed based on numerical simulations of several simple load cases on four examples of lattice structures.

Stormwater management systems reduce the impact of surface runoff in urban environments. The Toronto Green Standard (TGS) has been developed to further mitigate effects of urban runoff by mandating runoff control implementation in new developments at the site level. The intended effect of these measures is to reduce the quantity and improve the quality of water. Recent concepts of sustainable stormwater suggest that amenity is an equally important aspect of such a system. This study evaluated Best Management Practices suggested by the TGS for their performance and amenity characteristics. Design professionals selected for prior experience with the TGS were consulted to determine industry perceptions of the requirements, and how the standards could be altered to improve amenity value. Several concerns were identified which appear to impede the successful implementation of sustainable stormwater management solutions, to which recommendations are proposed which may help balance performance and amenity requirements in the TGS.

No description available.

More frequent and harsh storms coupled with sea level rise are affecting Canada’s sensitive coastlines. This research studies Isle Madame in Cape Breton, Nova Scotia which has been designated by Natural Resource Canada as a sea level rise vulnerable coastal community in Canada. The research models the spatial and temporal impacts of sea level rise from storm surge by focusing on identifying vulnerable areas in the community via geographical information systems (GIS) using ArcGIS, as well as modeling dynamic coastal damage via system dynamics using STELLA. The research evaluates the impacts in terms of the environmental, social, cultural, economic pillars that profile the coastal community for a series of modelled Storm Scenarios. This research synthesizes information from a variety of sources including the coastal ecology and natural resources, as well as human society and socioeconomic indicators included in the four mentioned pillars. The objective of the research is to determine vulnerable areas on Isle Madame susceptible to storm damage, and consequently, to improve local community knowledge and preparedness to more frequent harsh storms. This research therefore presents a dynamic model for the evaluation of storm impacts in Isle Madame designed with the goal to help the community ultimately to plan and implement a strategy to adapt to pending environmental change.

This thesis examines how residents perceived and coped with storm surge flooding in Tsawwassen, British Columbia. On February 4, 2006 the community experienced a storm surge flood that affected residents within the neighbourhoods of Beach Grove and Boundary Bay Village. This study identifies how residents perceived and coped with the flood, and what factors influenced how individuals perceived and coped. Qualitative methods were used for this study, and a total of 23 in-person interviews were completed. The findings of this research showed that how participants perceived the threat of storm surge flooding and how they coped with the flooding varied greatly. Government intervention; the influence of family, friends and neighbours; the perceived benefits of living in the area; experience; financial support; and perception of other hazards all influenced how the participants perceived and coped with the storm surge flood.

Thesis (M Eng) -- University of South Australia, 1993

Thesis (M Eng) -- University of South Australia, 1993