A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (220 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10^13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in selection of non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Esta pesquisa investiga o design e as práticas de governança urbana. O quadro teórico é composto de duas partes. Na primeira, examinamos as dimensões cognitivas, comunicativas, culturais, econômicas e sócio políticas do espaço contemporâneo em um contexto de ampla disseminação das Tecnologias da Informação e da Comunicação (TICs). Na segunda, discutimos as políticas públicas para os serviços urbanos com enfoque na cooperação considerando suas dimensões normativas, estratégicas e operacionais. Na pesquisa empírica, estudamos as redes e interfaces públicas utilizadas pelo poder executivo do município de Ribeirão Preto. Utilizando análise de grafos, categorias da Social Construction of Technology (SCOT) e da Actor-Network Theory (ANT), analisamos em que medida a incorporação das TICs amplia as oportunidades de participação e controle social da gestão do espaço e dos serviços urbanos. A pesquisa é concluída com contribuições para o design de redes e interfaces na constituição de uma forma de governança urbana conectada. / In this study we investigate design and urban governance practices. The theoretical framework has two parts. In the first one, we examines the cognitive, communicative, cultural, economic and socio-political dimensions of space in a context of widespread dissemination of Information and Communication Technologies (ICTs). In the second one, we discusses policy formulation in public urban services focusing on cooperation under its regulatory, strategic and operational dimensions. In the empirical research, we examine the networks and public interfaces used by government agencies of Ribeirão Preto municipality. Graph analysis, elements of social construction of technology and actor-network theory were used to evaluate how ICTs can increase the opportunities for participation and social control of space and urban services management. We conclude the study with contributions to the networks and interfaces design aiming to develop a form of connected urban governance.

La protéine Mlh1 eucaryote est un acteur central de la voie de réparation des mésappariements (MMR). Chez la levure, Mlh1 forme un hétérodimère via sa région C-terminale avec les endonucléases Pms1 et Mlh3. La région C-terminale de Mlh1 est également en interactions avec l’exonucléase Exo1 du MMR et deux protéines Ntg2 et Sgs1 qui sont impliquées dans d’autres voies de réparation. Dans un premier temps, nous avons identifié et caractérisé le site d’interaction de Mlh1 avec les protéines Exo1, Ntg2 et Sgs1, qui utilisent un même motif de 5 acides aminés, (R/K)SK(Y/F)F appelé motif MIP pour Mlh1 Interacting Protein. Nous avons montré que ces 3 protéines interagissent en un même site, appelé site S2. Nous avons identifié 10 positions de Mlh1 impliquées dans le site S2 et caractérisé par microcalorimétrie, une affinité micromolaire entre des peptides contenant le motif MIP et la région C-terminale de Mlh1. Nous avons montré que les protéines EXO1 et BLM humaines qui possèdent également un motif MIP, interagissent spécifiquement avec MLH1 humain par ce motif. Dans un second temps, nous avons résolu la structure cristallographique à 2.6Å de la région C-terminale de l’hétérodimère Mlh1*Pms1. Le site d’hétérodimèrisation présente une surface d’interaction supérieure à celle observée dans les homodimères de MutL bactériens. La structure résolue confirme le rôle des 10 acides aminés de Mlh1 identifiés lors de la caractérisation du site S2. La structure du site endonucléase de Pms1 révèle la présence de deux atomes de zinc chelatés par 5 acides aminés de Pms1 et le dernier acide aminé de la protéine Mlh1, la cystéine C769. Cette première structure d’une région C-terminale d’un complexe Mlh1*Pms1 eucaryote permet d’analyser la position des nombreux mutants ponctuels de MLH1 humain associés à des cancers du côlon HNPCC. / Eucaryotic Mlh1 is a core component of mismatch repair pathway (MMR). In yeast organisms, Mlh1 forms heterodimer with its C-terminal region with endonucleases Pms1 and Mlh3. The C-terminal region of Mlh1 is also involved in interactions with MMR exonuclease Exo1 and two proteins, Ntg2 and Sgs1, which are involved in other DNA repair pathways. First, we identified and charaterised the interaction site between Mlh1 and proteins Exo1, Ntg2, and Sgs1, that share the same motif of 5 amino acids, (R/K)SK(Y/F)F, named MIP box for Mlh1 Interacting Protein. We showed that these 3 proteins bind to the same site, named site S2. 10 positions of Mlh1 important for interactions on site S2 were identified and a micromolar affinity was measured by calorimetry between the C-terminal region of Mlh1 and peptides containing a MIP box. We showed that human EXO1 and BLM specifically with human MLH1 through their MIP box. Secondly, we solved the X-ray structure of the C-terminal region of Mlh1*Pms1 heterodimer at 2.6Å. The structure shows that the surface buried upon heterodimerisation is higher in eucaryotes than in MutL homodimers. The structure confirms the overall structure of the site S2 predicted in the first part of this study. The endonuclease site of Pms1 presents in the crystal two zinc atoms that are bound by five Pms1 residues and the last residue of Mlh1 chain, cystein C769. This structure represents the first image of the C-terminal region of an eucaryote Mlh1*Pms1 heterodimer. It allows localizing the positions of human MLH1 mutants associated with colon cancers named HNPCC.

<p>Now a day, one of the most attractive research topics in the area of Intelligent Traffic Control is </p><p>Inter-vehicle communication (V2V communication). In V2V communication, a vehicle can </p><p>communicate to its neighbouring vehicles even in the absence of a central Base Station. The </p><p>concept of this direct communication is to send vehicle safety messages one-to-one or one-to- </p><p>many vehicles via wireless connection. Such messages are usually short in length and have very </p><p>short lifetime in which they must reach the destination. The Inter-vehicle communication system </p><p>is an ad-hoc network with high mobility and changing number of nodes, where mobile nodes </p><p>dynamically create temporary sensor networks and transferring messages from one network to </p><p>others by using multiple hops due to limitation of short range. </p><p> </p><p>The goal of the project is to investigate some basic research questions in order to organize such </p><p>sensor networks and at the same time highlight the appropriate routing protocol that support </p><p>mobile ad hoc networks in an efficient and reliable manner. </p><p> </p><p>In our investigation, we have answered the technical issues in order to construct a V2V </p><p>communication system. We have also studied some mobile ad hoc network routing protocols in </p><p>detail and then selected the DSR (Dynamic Source Routing) for our V2V communication and </p><p>then simulated it according to our system requirements. We are quite satisfied by the result of </p><p>DSR, but at the same time much more work is required to come up with an absolute application </p><p>for the end user.</p>

Now a day, one of the most attractive research topics in the area of Intelligent Traffic Control is Inter-vehicle communication (V2V communication). In V2V communication, a vehicle can communicate to its neighbouring vehicles even in the absence of a central Base Station. The concept of this direct communication is to send vehicle safety messages one-to-one or one-to- many vehicles via wireless connection. Such messages are usually short in length and have very short lifetime in which they must reach the destination. The Inter-vehicle communication system is an ad-hoc network with high mobility and changing number of nodes, where mobile nodes dynamically create temporary sensor networks and transferring messages from one network to others by using multiple hops due to limitation of short range. The goal of the project is to investigate some basic research questions in order to organize such sensor networks and at the same time highlight the appropriate routing protocol that support mobile ad hoc networks in an efficient and reliable manner. In our investigation, we have answered the technical issues in order to construct a V2V communication system. We have also studied some mobile ad hoc network routing protocols in detail and then selected the DSR (Dynamic Source Routing) for our V2V communication and then simulated it according to our system requirements. We are quite satisfied by the result of DSR, but at the same time much more work is required to come up with an absolute application for the end user.

La coacervation complexe, une séparation de phase associative principalement induite par des interactions électrostatiques, entre la ß-lactoglobuline (BLG, protéine animale) et la gomme d'Acacia (AG, polysaccharide végétal) a été étudiée dans ce travail. La plus grande difficulté pour comprendre la coacervation complexe au niveau moléculaire entre BLG et AG révèle être la polymolécularité élevée d'AG. A partir de là, la motivation principale de cette thèse était de comprendre et contrôler les interactions entre la BLG et les fractions moléculaires d'AG, FI (~88% d'AG) et FII (~10% d'AG) en utilisant la titration calorimétrique isotherme, la diffusion statique et dynamique de lumière, la mobilité électrophorétique, la Granulo- Polarimétrie et la microscopie optique. Une énergie d'interaction plus forte, une stoechiométrie d'association plus faible et ainsi une complexation favorable ont étés montrées entre la BLG et FII en relation avec l'accessibilité et la densité de charges plus élevées de FII. Les résultats majeurs de cette étude ont ainsi montré des rôles différents des fractions de l'AG dans la coacervation complexe avec la BLG.

Quantifying and characterizing isolated tumor cells (ITCs) is of interest in surgical pathology and cytology for its potential to provide data for cancer staging, classification, and treatment. Although the independent prognostic significance of circulating ITCs has not been proven, their presence is gaining clinical relevance as an indicator. However, researchers have not established an optimal method for detecting ITCs. Consequently, this Ph.D. dissertation is concerned with the development and evaluation of dielectric spectroscopy as a low-cost method for cell characterization and quantification. In support of this goal, ultra-wideband (UWB), microwave pulse generator circuits, coaxial transmission line fixtures, permittivity extraction algorithms, and dielectric spectroscopy measurement systems were developed for evaluating the capacity to quantify B16-F10 tumor cells in suspension. First, this research addressed challenges in developing tunable UWB circuits for pulse generation. In time-domain dielectric spectroscopy, a tunable UWB pulse generator facilitates exploration of microscopic dielectric mechanisms, which contribute to dispersion characteristics. Conventional approaches to tunable pulse generator design have resulted in complex circuit topologies and unsymmetrical waveform morphologies. In this research, a new design approach for low-complexity, tunable, sub-nanosecond and UWB pulse generator was developed. This approach was applied to the development of a novel generator that produces symmetrical waveforms (patent pending 60/597,746). Next, this research addressed problems with transmission-reflection (T/R) measurement of cell suspensions. In T/R measurement, coaxial transmission line fixtures have historically required an elaborate sample holder for containing liquids, resulting in high cost and complexity. Furthermore, the algorithms used to extract T/R dielectric properties have suffered from myriad problems including local minima and half-wavelength resonance. In this dissertation, a simple coaxial transmission line fixture for holding liquids by dispensing with the air-core assumption inherent in previous designs was developed (patent pending 60/916,042). In addition, a genetic algorithm was applied towards extracting dielectric properties from measurement data to circumvent problems of local minima and half wavelength resonance. Finally, in this research the capacity for using dielectric properties to quantify isolated B16-F10 tumor cells in McCoy's liquid medium was investigated. In so doing, the utility of the Maxwell-Wagner mixture formula for cell quantification was demonstrated by measuring distinct dielectric properties for differing volumes of cell suspensions using frequency- and time-domain dielectric spectroscopy.

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

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.