<p>The human interactome presents a goldmine of potentially powerful therapeutic targets, yet very few small molecule modulators of protein-protein interactions (PPI) have been identified. PPI pose a particular challenge for drug discovery, and one of the major obstacles to fully exploiting these interactions is a lack of appropriate technologies to screen for modulating compounds. This thesis aims to address the need for function- based approaches that target PPI by using magnetic beads (MB) and mass spectrometry (MS) to develop efficient assays to monitor these interactions and their modulation by small molecules. The work begins with the validation of a novel magnetic “fishing” assay, which uses affinity-capture MB to isolate intact complexes of a “bait” protein from solution. By monitoring the recovery of the secondary binding partner, this assay was used to functionally screen a library of 1000 compounds for small molecule modulators of a calmodulin/melittin (CaM/Mel) model system. The versatility of magnetic “fishing” is clearly demonstrated during a study of a more relevant CaM-based system, which uncovered a novel mode of interaction for the CaM-binding domain of transcription factor SOX9. In addition to the MB-based approach, a simple MS-based competitive displacement assay is developed to identify minimal inhibitory fragments of a target complex as indicators of potential ‘hot-spots’. The assay was used to probe a DNA repair complex of XRCC4/ligaseIV, and identified a short helix that can be used as a more defined target surface for future high-throughput screening and rational drug design. The functional MS-based assays herein are highly adaptable tools to monitor PPI, and will facilitate the study of these and other important biomolecular interactions.</p> / Doctor of Philosophy (PhD)

Pathogenic bacteria pose serious threats to public health and safety. They can cause illness, death, and substantial economic losses. The most widely used bacterial detection methods include cell culturing, antibody-based assays, and nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Unfortunately, these techniques are not well suited for point-of-care application, especially in the resource-limited regions of the world, as they require highly trained personnel to perform the test, they take a long time to complete (especially culturing), and they require sophisticated lab equipment. Thus, there is a great need for simpler, faster, and more accurate methods for bacterial detection. In this thesis, we present a simple, low-cost assay for detecting pathogenic bacteria that is based on the immobilization of a bacteria-specific RNA-cleaving DNAzyme (DNAzyme) onto a surface. If the target bacteria is present, a fluorescently labelled piece of DNA (FDNA) is released through the activity of the DNAzyme; if the target bacteria is not present, the FDNA remains attached to the surface as part of the DNAzyme construct. This method allows untrained users to determine whether a target bacteria is present by simply monitoring the fluorescence intensity in the liquid phase with a hand-held fluorimeter. The first step in this work was to experimentally evaluate different surfaces (including reduced graphene oxide and different beads) onto which the DNAzyme could be immobilized. These tests determined that agarose beads, covered with streptavidin, were ideally suited for DNAzyme immobilization. Next, we conducted a comparative evaluation of the kinetics/activity of the DNAzyme that had been immobilized onto the beads and the free DNAzyme in solution; the results of this evaluation revealed virtually identical reaction rates for the two cases, suggesting no loss of activity after immobilization. Finally, we explored how the DNAzyme sequence length influenced the assay. Specifically, we analyzed a full-length DNAzyme (Full DNAzyme) sequence and a truncated alternative (Short DNAzyme) and found that the full-length construct resulted in faster signal generation. Therefore, it was determined that the long version should be used in the assays. When coupled with a filtration step, the immobilization of biotinylated DNAzymes onto the surface of streptavidin-coated agarose beads enabled the sensitive detection of E. coli in both water samples and complex matrices, such as milk and apple juice. The bead-based assay was able to produce a strong fluorescence signal readout in as little as 2.5 min following contact with E. coli, and it was capable of achieving a detection limit of 1,000 colony-forming units (CFUs) without sample enrichment. As DNAzyme probes can be generated through in vitro selection to react to different bacteria, the RNA-cleavage based detection mechanism described in this work can be adapted for the detection of a wide range of bacterial targets. Overall, this research has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that is highly attractive for field applications, especially in resource-limited regions. / Thesis / Master of Applied Science (MASc)

New vigorous regulations have been established for decreasing the allowable formaldehyde emissions from nonstructural wood based composites. Two main sources of formaldehyde emission in non-structural wood based composites are adhesive and wood. Adhesives are quite well known and great efforts have been conducted to decrease their formaldehyde content; however formaldehyde emission from wood has received little attention and it is not completely understood. Wood-borne formaldehyde emission exists in a complex equilibrium in wood matrix. The reaction between formaldehyde and wood hydroxyl groups/water can hinder the complete formaldehyde extraction. In order to have a complete formaldehyde extraction, a stronger nucleophile than hydroxyl and water groups is needed. In this study cross-linked poly (allylamine) (PAA) beads were synthesized and used as a strong nucleophile to extract all the biogenic and synthetic free-formaldehyde within the woody matrix of never-heated and heat-treated Virginia pines; the results were compared to simple water extraction. A new formaldehyde capturing device was also developed using a serum bottle. Results showed that there was no advantage of using PAA beads over simple water extraction for extracting woody matrix free-formaldehyde. This means that simple water extraction can extract all the free-formaldehyde from the woody matrix. It was also found that thermal treatment resulted in generating more wood-borne formaldehyde. The other important finding was the new developed formaldehyde capturing device. The device was very promising for detecting wood-borne formaldehyde from very small pieces of wood (5-70 mg) and can be very useful in future studies. / Master of Science

Le détachement d'une goutte est un phénomène que nous observons quotidiennement. Il résulte de la rupture de l'interface entre le fluide dispersé en goutte et le fluide environnant. Cette rupture a fait l'objet de nombreuses études. Il est bien établi que sa dynamique est régie par une compétition entre la capillarité, l'inertie, et la viscosité du fluide. Ce manuscrit décrit l'influence sur la dynamique de rupture d'une modification des propriétés de l'interface entre deux fluides à l'aide d'agents de surface. Lorsque l'agent de surface est un surfactant (SDS), la dynamique d'amincissement peut se faire selon deux modes. Deux régimes linéaires en temps constituent le premier mode. Le second mode comporte trois régimes linéaires. Dans les deux cas, l'aminicissement commence par un premier régime, suivi d'un deuxième régime de pente plus forte. Lorsque le troisième régime existe, sa pente est inférieure à celle du second régime. La variation des pentes des régimes linéaires témoigne du comportement dynamique du surfactant à l'interface. La valeur de la tension interfaciale $\gamma$ extraite du premier régime linéaire correspond à la valeur à l'équilibre de la tension interfaciale du système, $\gamma_{eq}$. La vitesse d'amincissement plus élevée au cours du second régime est reliée à une dépletion partielle en surfactant de la zone d'amincissement maximal. Le ralentissement constaté pendant le troisième régime est lié au déplacement de cette zone vers une région plus riche en surfactant, où la tension $\gamma$ est plus faible. La dynamique d'amincissement du cou est très différente lorsque des polymères de poids moléculaire intermédiaire ($\sim$ 100 kDa) sont présents simultanément avec du SDS dans la phase continue. Lorsque $C_{SDS}$ est supérieure à 0,15 fois la concentration micellaire critique (CMC), le comportement est identique à celui observé en présence de surfactant seul. En dessous de 0,15 CMC, l'amincissement ralentit exponentiellement à l'approche de la rupture, et un phénomène de beads-on-a-string apparaît. Ces constatations sont analogues à celles faites lorsqu'une solution de polymères est menée à la rupture. Dans notre cas, les polymères sont uniquement à la surface du jet et non dans son volume! Une analyse des profils du cou au cours du temps démontre l'existence d'une auto-similarité à l'approche de la rupture. Bien que les systèmes étudiés soient plus complexes, ils présentent des caractéristiques qualitativement analogues à celles observées dans des systèmes de fluides simples. Toutefois, il existe une grande différence quantitative. / Droplet detachment is ubiquitous in everyday life. It results from the rupture of an interface separating two fluids. This rupture has been widely studied. It is now well established that it relies on a competition between capillary, inertial and viscous phenomena. In this manuscript, we report on the influence on the breakup dynamics of the presence of surface agents at the interface. When SDS is used as a surface agent, thinning can proceed in two ways. In the first mode, the dynamics of thinning are characterized by two linear-in-time regimes. The second mode is made of three linear-in-time regimes. In both cases, thinning starts with a first regime, followed by a steeper second regime. When a third regime exists, its slope is softer. Slope variation bears witness to a dynamical behaviour of the surfactants at the interface. The value for the interfacial tension $\gamma$ calculated from the slope of the first linear regime is in agreement with the equilibrium interfacial tension of the system, $\gamma_{eq}$. The higher thinning speed during the second regime is linked to a partial depletion in surfactant of the maximal thinning zone. The slowdown in the tihrd regime is related to a displacement of the thinning zone in a region of higher surfactant concentration, where $\gamma$ is lower. The thinning dynamics is very different when polymers are added to the surfactant solution. If $C_{SDS}$ is higher than 0.15 times the critical micellar concentration (CMC), a behaviour similar to the pure-surfactant case is observed. Below 0.15 CMC, an exponential slowdown is observed in the last instants, as well as a "`beads-on-a-string"' phenomenon. These observations are analogous to what is seen when a solution of polymers is led to breakup. In our case, polymers are not in the bulk; they are at the interface of the two fluids! Analysis of the profiles of the neck in both cases showed that profiles are self-similar. Qualitatively, they share features with profiles observed in the case of breakup of interfaces between simple fluids. Quantitatively, slopes and angles are different.

Les anticorps anti-HLA d’isotype IgG sont une cause de perte de greffon en transplantation d’organes.Les tests « single antigen » (SAFB) sont les outils les plus précis et sensibles pour l’identification desanticorps anti-HLA dirigés contre le donneur (DSA) dans le sérum des receveurs. Leur résultat semiquantitatif,la MFI, n’est pas parfaitement associé à l’issue clinique, ce qui pourrait être dû à plusieursraisons. Premièrement, nous avons montré que les SAFB de classe I détectent fréquemment des anticorpsanti-HLA dénaturé de classe I, incapables de se lier à la surface cellulaire et donc n’ayant pas designification clinique, alors qu’ils ont un impact négatif sur l’accès à la transplantation. Leuridentification a été réalisée par un traitement acide des billes et par un SAFB modifié, les iBeads®.Ces deux tests montraient de bonnes fiabilité et concordance, mais le traitement acide pouvait parfoisêtre mis en défaut alors que les iBeads® auraient une sensibilité légèrement inférieure aux SAFBclassiques. Deuxièmement, nous avons déchiffré l’interférence liée au complément : les IgG anti-HLA de forte MFIsont capables d’activer le complément à la surface des billes, conduisant à une accumulation desproduits de dégradation du C4 et du C3, capables de réduire la détection des IgG anti-HLA. Nousavons également démontré que les IgM anti-HLA étaient capables d’interférer avec la détection desIgG à travers une compétition pour l’épitope, un encombrement stérique et une activation ducomplément. Troisièmement, nous avons montré que la détection des DSA avec les SAFB dans les éluats debiopsies de poumons transplantés, preuve formelle que ces DSA interagissent avec le greffon,constituait un facteur de risque de perte du greffon. Nous avons également développé un système decapture en résonance plasmonique de surface permettant de déterminer la concentration et l’affinitédes anticorps anti-HLA, ce qui pourrait permettre d’étudier la façon dont les DSA interagissent avec legreffon. / IgG HLA antibodies are a cause of graft loss in organ transplantation. The single antigen flow beadsassays (SAFB) are the most precise and sensitive assays to identify donor specific HLA antibodies(DSA) in recipient’s sera. Their semi-quantitative readout, the mean fluorescence intensity (MFI), is notperfectly associated with graft outcomes, which could be due to several factors.Firstly, we showed that class I SAFB frequently detects denatured class I HLA antibodies which areunable to bind cell surface and then are clinically irrelevant, while they actually impact the access to atransplant. Their identification was performed through SAFB acid-treatment and a modified SAFBassay, the iBeads®. They had a high reliability and a good concordance, but the acid-treatment assaycan be put at fault in a few cases whereas iBeads® appeared slightly less sensitive than classicalSAFB. Secondly, we deciphered the complement interference phenomenon: high MFI level IgG HLAantibodies activate the complement cascade at bead surface, leading to the deposition of C4 and C3degradation products which are able to reduce IgG HLA antibodies detection. We also demonstratedthat IgM HLA antibodies interfere with IgG detection through competition for the epitope, allosterichindrance and complement activation. Thirdly, we demonstrated that the detection of DSA with SAFB in lung biopsy eluates, proving that theDSA interact with the graft, was a risk factor for graft loss. We further developed a capture system insurface plasmon resonance allowing the concentration and affinity of HLA antibodies to bedetermined, which could allow the way that the DSA interact with the graft to be studied.

The first part of the thesis describes the synthesis and application of functional magnetic polymer beads. The traditional suspension polymerization approach was used to synthesize polystyrene-iron oxide (Fe 3 O 4 ) based magnetic beads. The beads were coupled to different surface functional groups. The Fe 3 O 4 particles were encapsulated into a polystyrene shell. The surface functional groups were generated by graft-polymerization with functional monomers. The average size of the beads was in the range of 100-500 μm. Chemical tests showed that the beads were stable in strong acid, strong base and polar solvent. The beads had a fast response to an external magnetic field. A self-emulsion-polymerization approach was developed to synthesize smaller magnetic beads with the - OH groups on the surface. A modified approach based on traditional suspension-polymerization was developed to synthesize acid-durable beads with more Fe 3 O 4 encapsulated inside the beads. A novel emulsion-suspension polymerization method was successfully developed to synthesize much smaller magnetic beads ( A new peptide synthesis approach was developed using functional magnetic beads as the resin for solid phase synthesis. In this application, synthesized magnetic beads were further modified by a two-step reaction. The amino group was anchored onto the surface of these beads, followed by coupling with the Rink amide linker. The resulting beads were used as the resin to synthesize several model peptides. The peptides were successfully synthesized, and the sequences were confirmed by mass spectrometry analysis. The yields of the peptides were comparable to those obtained from commercial Rink amide resin. The second part of the thesis describes the synthesis and mass spectrometry analysis of two series of model peptides. One series has the linear (non-cyclic) structure, A n K, KA n , P n K, and AcA n K. The other series contains cyclic peptides, c-Ac-DAKAK and c-Ac-DADapAK. All peptides were synthesized using solid phase peptide synthesis. The relative proton affinities of the model peptides were measured using the collision induced dissociation experiments using a triple quadrupole mass spectrometer. It was found that the effective proton affinity of a cyclic peptide was significantly reduced compared to a linear analogue. The reduced proton affinity implies an increased lipophilicity of the peptide.

<p>Abstract</p><p>Terrace III in the Birka Garrison. An analysis of function based on artifact quantification and find preparation. This paper deals with Terrace III in the Garrison of Birka, Uppland, Sweden. The main purpose is to investigate the function of Terrace III. The analysis consists of two parts. One is to analyse the stratigraphy including layers, constructions and finds. Beads and coins are selected for a discussion of dating. The other part consists of a comparison between the find material from the Hall building, the Smithy and Terrace III. Beads are discussed separately. The results are not definite due to the fact that the terrace is not completely excavated. The finds indicate that Terrace III could have been used as storage house or a dwelling house. The pottery could be taken as evidence for a storage house (and possibly the amount of rivets and nails). The presence of personal finds show that the house might have been used as a dwelling house. A few finds indicate some kind of workshop activity. The finds from Terrace III can be dated to the end of the 10th Century.</p>

Abstract Terrace III in the Birka Garrison. An analysis of function based on artifact quantification and find preparation. This paper deals with Terrace III in the Garrison of Birka, Uppland, Sweden. The main purpose is to investigate the function of Terrace III. The analysis consists of two parts. One is to analyse the stratigraphy including layers, constructions and finds. Beads and coins are selected for a discussion of dating. The other part consists of a comparison between the find material from the Hall building, the Smithy and Terrace III. Beads are discussed separately. The results are not definite due to the fact that the terrace is not completely excavated. The finds indicate that Terrace III could have been used as storage house or a dwelling house. The pottery could be taken as evidence for a storage house (and possibly the amount of rivets and nails). The presence of personal finds show that the house might have been used as a dwelling house. A few finds indicate some kind of workshop activity. The finds from Terrace III can be dated to the end of the 10th Century.

Content removed from thesis due to copyright restrictions: Winger, R.J. and L. Ren (2009). "Solubility of sodium and potassium iodates in saturated salt solutions." Food Chemistry 113: 600-601. / This paper was to improve the production of calcium-induced alginate gels manufactured by a company in Auckland. Problems encountered included yield and syneresis of the beads post-gelation. Essentially the alginate, sugars and other ingredients were dissolved in water at 80ºC. The pH of the solution was adjusted and the alginate beads were extruded into a 5% CaCl2 bath before being drained and dried. The chemical reaction between sodium alginate and calcium ions is dependent upon the solubility and availability of calcium ions. Some calcium salts (e.g., CaCl2, calcium lactate) were readily soluble and fully dissociated in water and resulted in an immediate gelation of the alginate. Dicalcium phosphate (DCP) was sparingly soluble at pH 7 and calcium ions were not released significantly until the pH reached about pH 4.2. Sodium hexametaphosphate (SHMP) is a chelating agent and this was used to soak up small quantities of Ca+2 to ensure no gelation occured while the alginate was being mixed. The optimum quantities of alginate, DCP and SHMP were defined in the laboratory trials. The use of SHMP, maltodextrin, and gums significantly affected the hardness and stickiness of gel beads. It was found that the combination of xanthan and alginate Protanal LF 120 gave the best results in terms of minimal stickiness and maximum yield after drying. Key words: alginate gel beads, syneresis, formula, pH, citric acid, gelation time, SHMP, setting time, yield rate, drying, hardness, stickiness, maltodextrin, xanthan gum, guar gum, stickiness by touching, leakage, apparent viscosity.

Notre génome, véritable mode d'emploi de chaque cellule et organisme, est constamment menacé par de multiples agents endogènes ou exogènes qui endommagent la biomolécule d'ADN. Ces lésions résultantes, de nature diverse, sont notamment impliquées dans les processus de vieillissement cellulaire, de cancérogénèse et de mort cellulaire. Afin de contrer ces effets néfastes, les organismes ont développé différents systèmes de réparation de l'ADN capables de prendre en charge spécifiquement chaque type de dommages. Parmi ces voies métaboliques, la réparation par excision de base (BER) répare chaque jour des dizaines de milliers de dommages, incluant les bases alkylées, oxydées ou désaminées, les sites abasiques ou encore certaines cassures de brin. Dans le présent travail, nous exposons la mise au point d'un nouveau biocapteur pour la détection des activités enzymatiques du BER. L'outil se caractérise par un set de sondes nucléiques autocomplémentaires, fluorescentes ou pro-fluorescentes, immobilisées sur microbilles paramagnétiques. Chaque sonde est modifiée par l'introduction sélective d'une lésion, substrat d'une activité enzymatique ciblée (ADN N-glycosylase, AP-endonucléase). L'activité d'excision/incision de la lésion, conduit à la coupure de la sonde et à la déshybridation de la structure. L'analyse et la quantification du clivage spécifique est réalisée en fluorescence, soit à partir du surnageant par spectrofluorimétrie, soit des billes par cytométrie en flux. Ce dispositif permet la détection multiplexée des activités enzymatiques de protéines purifiées ou au sein d'extraits nucléaires. Egalement, des applications dans le criblage d'inhibiteurs de la réparation de l'ADN sont envisageables dans le cadre de recherches pré-cliniques. L'adaptation de ces tests in vitro à la détection de la réparation de l'ADN in cellulo a fait l'objet de développements préliminaires. / Our genome, which may be considered as the program of each cell and organism, is constantly threatened by multiple endogenous and exogenous agents that damage the DNA biomolecule. These lesions, that show a wide array of structures, are particularly involved in cell aging, carcinogenesis and cell death. To thwart these negative effects, organisms have developed various DNA repair pathways that take in charge the alterations in a specific manner. Among them, the base excision repair (BER) removes every day dozens of thousands of damages, including alkylated, oxidized or deaminated bases, abasic sites or single strand breaks. In this study, we present the development of a new biosensor for the detection of BER enzymatic activities. The tool is characterized by a set of (pro)fluorescent hairpin-shaped DNA probes, immobilized on paramagnetic beads. Each probe is modified by the selective insertion of a lesion, substrate for the targeted repair enzyme (DNA glycosylase, AP-endonuclease). The excision/incision activity of the lesion leads to the cleavage of the probe together with the dehybridization of the structure. The analysis and quantification of the repair process is carried out by direct fluorescence measurements from the supernatant, or by analysis of the functionalized beads by flow cytometry. This device allows the multiplexed enzymatic activities detection of purified proteins or within nuclear extracts. Applications to the screening of DNA repair inhibitors have been successfully initiated. Finally, the adaptation of these in vitro tests to in cellulo detection of DNA repair activities was investigated in preliminary studies.