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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Polymeric nano and microparticles are important tools for an increasing variety of applications in the life sciences such as cellular delivery, sensing and imaging, with a fundamental requirement being particle functionalisation. Herein, the use of zeta potential measurements is described as a convenient tool to allow a variety of chemical reactions to be rapidly monitored on particles. To allow multifunctionalisations these particles need to be orthogonally modified. As part of this thesis, novel dual-functionalised aminomethyl and boronic acid particles were synthesised. These particles could be modified via amide formation and palladiummediated cross coupling, with applications demonstrated in cellular delivery and cellbased cargo release. The requirement for bright fluorescent particles for applications in the life sciences was addressed by the synthesis and analysis of particles prepared using polymerisable fluorescein derivatives. Although nanoparticles are a promising technology to solve a variety of problems, their behaviour in biological systems is not fully understood. Herein, the effects of the particle’s surface chemistry on cellular uptake and toxicity were investigated.

As a structural analogue of adenine (6-aminopurine), 2-aminopurine (2AP) is a powerful fluorescent probe, when substituted in DNA in place of the natural adenine. Time-resolved fluorescence measurements of 2AP-labeled oligonucleotides, together with steady-state spectroscopy give us an in-depth view of DNA-enzyme interactions, especially the conformational dynamics in solution phase. Herein, this technique has been extended to the study of the transient unzipping of DNA bases, to investigate the structure of three-way junction (3WJ), and the role of base unzipping in the mechanism of human flap endonuclease (FEN). Seven 2AP-labelled 3WJs were investigated, each containing only one 2AP base in place of adenine. In four of the 3WJs, 2AP was placed in the long duplex region of an arm; while in the other three 3WJs, 2AP was placed near or in the branch point. Comparative time-resolved fluorescence measurements on the 3WJs and corresponding ssDNA and dsDNA controls were made to study the base dynamics, in particular the possibility of unzipping in the vicinity of the branch point. In combination with single-molecule FRET measurements and molecular dynamics simulations, the local and global structure of a DNA 3WJ in solution could be unraveled. It was found to adopt a Y-shaped, pyramidal structure, in which the bases adjacent to the branch point are unzipped, despite the full Watson-Crick complementarity of the molecule. Human flap endonuclease (hFEN) is divalent metal ion-dependent phosphodiesterase. hFEN carries out structure-specific hydrolysis of 5’ bifurcated DNA endonucleolytically. Cleavage occurs at a position one nucleotide into the downstream duplex region. Previous structural, biochemical and modeling studies suggested a double-nucleotide unzipping mechanism at single/double strand junctions for scissile phosphate placement. To confirm this mechanism, 2AP time-resolved fluorescence spectroscopy was used to investigate nucleotide unzipping in hFEN substrates. 2AP was substituted at positions +1 and -1 (relative to the scissile phosphodiester) respectively, in double flap substrates. A series of hFEN mutants including Y40A, R100A, K93A, were used in this study. In the experiments, ssDNA, dsDNA substrates, DNA substrate-enzyme complexes were investigated in order to elucidate the enzyme-induced distortion of the substrate at the +1 and -1 positions. TseI is a thermophilic type II restriction enzyme which has ideal activity at an elevated temperature. It is able to recognise and cut the 5 bp palindromic sequence of 5’-GCWGC-3’ (W=A or T). A range of biophysical methods have been applied to investigate this enzyme, including size-exclusion chromatography; fluorescence anisotropy (Kd value determination); denaturing HPLC for DNA cleavage analysis on matched and mismatched substrates; fluorescence-based activity assay (KM, Vmax, kcat, specificity constant values determination); steady-state fluorescence measurements (DNA-enzyme interaction study). The DNA cleavage characteristics of TseI were fully studied and it was found that it cuts A:A and T:T mismatches in CAG and CTG repeats. This potentially makes it a useful tool for exploring unusual DNA structures containing super-long CAG and CTG repeats which are involved in the aetiology of some neurodegenerative diseases, such as Huntington’s disease (HD). EcoP15I is a type III restriction-modification enzyme whose recognition sequence is 5-CAGCAG-3’. Methyltransferase EcoP15I (M.EcoP15I) adds a methyl group to the second adenine, in the presence of cofactor S-adenosyl methionine (SAM). SDS-PAGE, densitometry and size-exclusion HPLC were applied to confirm that EcoP15I adopts a Res1Mod2 stoichiometry in solution. The large structural distortion of its substrate (base flipping) by M.EcoP15I was investigated by both steady-state and time-resolved fluorescence. Also, nine 120 mer DNA duplexes, each containing two reversely oriented recognition sites were used to study matched and mismatched sequence cleavage by R.EcoP15 and a cleavage pattern was revealed.

The study was conducted to develop a technique that measures the amount of gadolinium based contrast agent accumulated in a head tumour by x-ray fluorescence, while a patient is exposed to neutrons or during external beam radiotherapy planning. In this research, measurements of the gadolinium concentration in a vessel simulating a brain tumour located inside a head phantom, by the x-ray fluorescence method were taken, where the Magnevist contrast medium which has gadolinium atom, in the tumour vessel, was excited by a 36 GBq (0.97 Ci) 241Am source that emits gamma rays of 59.54 keV, in 35.7 % of it’s decays, resulting the emission of characteristic fluorescence of gadolinium at 42.98 keV that appeared in the X-ray fluorescence spectrum. A Cadmium Telluride (CdTe) detector was used to evaluate and make an analysis of the gadolinium concentration. Determinations of the gadolinium content were obtained directly from the detector measurements of XRF from gadolinium in the exposed tumour vessel. The intensity measured by the detector was proportional to the gadolinium concentration in the tumour vessel. These concentrations of gadolinium were evaluated for dose assessment. The positioning of the head phantom was selected to be in the lateral and vertex positions for different sizes of tumour vessels. Spherical tumour vessels of 1.0, 2.0, 3.0 cm and an oval tumour vessel of 2.0 cm diameter and 4.0 cm length, containing the gadolinium agent, contained concentration between 5.62 to 78.63 mg/ml. They were placed at different depths inside a head phantom at different positions in front of the detector and the source for the measurements. These depths ranged from 0.5 cm to 5.5 cm between the center of the tumour and interior wall of the head phantom surface. The total number of measurements in all four sizes of the tumour vessel was 478; 78 examinations of a 1.0 cm spherical tumour vessel, 110 examinations of a 2.0 cm spherical tumour vessel, 150 examinations of a 3.0 cm spherical tumour vessel and 140 examinations of a 2.0 x 4.0 cm ellipsoid tumour vessel. To measure the size and the shape of the tumour by the alternative radiographic method, a general x-ray machine with radiograph film was used. Based on that, the appropriate shape of concentration could be selected for therapy. The differences of optical density in the x-ray films showed that the noise was increased with low concentration of the Gd. Because radiographic film may be subjected to different chemical processes where the darkness will be affected, these measurements would be very hard to be quantitative. Accordingly it is difficult to use the film for Gd concentrations. The obtained data show that the method works very well for such measurements.

The US Navy spends around $75 million on maintenance and rework of corroded structures, especially in the ballast tanks of ships. The Navy will profit immensely with better surface coating and quality at source with real time inspection system. The approach taken to improve the coating is the use of optically active paint system. This kind of paint will fluoresce with an incident UV light. The fluorescence or glow can be used to detect holidays or defects on the surface. The inspection prototype developed uses a high end camera and intense UV light source. The paint and additive properties are characterized with the help of ultraviolet-visible spectroscopy (UV Vis) to study the behavior and to help formulate a theory. The holidays or missed spots in the painted surface will appear dark and non-fluorescing which is enhanced with the use of commercial filters.

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