This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.

Cette thèse se place à l'interface entre la topologie et la combinatoire. On s'intéresse dans un premier temps au problème de réalisation des boules unités duales des normes d'intersections sur les surfaces orientables. On montre aussi un certain lien entre les normes d'intersections et la norme de Thurston sur les 3-variétés.On montre par ailleurs l'existence d'un graphe dit de chirurgie sur l'ensemble des cartes unicellulaires d'une surface orientable. Dans le cas des collections unicellulaires et de cartes cubiques unicellulaires, le graphe de chirurgie s'avère connexe. / This thesis stay in between topology and combinatory. Our first concerned is the problem of realization of dual unit ball of intersection norms on orientable surfaces. We also show a certain relation between intersection norms and Thurston norms on 3-manifolds. On the other part, we show the existence of graph structure on unicellular maps on orientable surface coming from a surgery operation on unicellular maps: a surgery graph. Its happen that surgery graph on unicellular collections and cubic unicellular maps is connected.

The method of directed evolution represents a new approach to generate proteins with new or altered properties. The principle of directed evolution is random mutagenesis of the coding sequence for a protein of our interest followed by selection of generated mutants for the desired property. The aim of this pilot study was to investigate the possibility of utilization of directed evolution for alteration of mouse polyomavirus original tropism and virus retargeting to a model prostate cancer cell line. To generate randomly mutated gene encoding the major capsid protein of mouse polyomavirus, which is responsible for the interaction of the virus with cellular receptor for viral cell entry, error-prone PCR and DNA shuffling methods were used. Production of viruses composed of mutant major capsid protein was ensured by Cre/loxP site-specific recombination. The thesis also dealt with the design and characterization of the system for viral mutant selection. It was found that the prostate cancer cell lines markedly vary in their ability to bind and internalize particles derived from mouse polyomavirus. This knowledge can be used for the preparation of virus-like particles for prostate cancer diagnostics in the future. The study demonstrated that the method of directed evolution can be used for production...

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Le transport des molécules à travers les membranes est un processus cellulaire fondamental. Les transporteurs ABC (pour ATP-binding cassette) sont des protéines transmembranaires médiant le transport actif de substrats variés (peptides, ions, acides aminés, antibiotiques, etc). Le gène ATMI de Saccharomyces cerevisiae code pour un transporteur ABC de 690 acides aminés localisé dans la membrane interne de la mitochondrie. La protéine Atml est une proche homologue de la P-glycoprotéine impliquée dans la résistance aux agents anticancéreux. Il a été démontré qu'Atml est essentielle pour la croissance des cellules, procurant ainsi un phénotype idéal pour l'analyse fonctionnelle de ce transporteur. Nous utilisons une technique, appelée plasmid shuffling', basée sur l'échange de plasmides par contre-sélection d'un plasmide portant le marqueur de sélection URA3 en présence d'acide 5-fluoroorotique (5-F0A) pour étudier Atml. Nous avons construit une souche haploïde de S. cerevisiae dans laquelle la copie chromosomique du gène ATMI est excisée mais qui est viable grâce à la présence d'un plasmide URA3 portant une copie fonctionnelle du gène ATMI. Cette souche a été transformée avec une banque d'allèles mutés d'ATM1 portés par un plasmide LEU2 et criblée pour des mutants thermosensibles (ts) d'Atml après contre-sélection du plasmide URA3 sur 5-F0A. Trois mutants ts d'Atml ont été isolés et les mutations causant le phénotype ts ont été identifiées par séquençage de l'ADN. Des courbes de croissance de ces mutants ont été tracées, démontrant une croissance normale à température permissive mais un arrêt de croissance après quelques divisions à température restrictive. Nous avons produit des anticorps polyclonaux anti-Atml qui nous ont permis d'étudier la protéine de type sauvage et celle des trois mutants ts. Ces études ont révélé l'absence d'Atml seulement dans les mitochondries des mutants ts à température restrictive. D'autre part, des expériences de coimmunoprécipitation ont démontré qu'Atml forme des homodimères et que cette interaction est médiée par la partie C-terminale d'Atml, qui contient le domaine de liaison à l'ATP. Nos expériences ont donc permis d'identifier des acides aminés importants pour la fonction d'Atml ainsi qu'un domaine impliqué dans la structure quaternaire de ce transporteur.

Cryptography is the foundation of modern IT security,it provides algorithms and protocols that can be usedfor secure communications. Cryptographic algorithmsensure properties such as confidentiality and data integrity.Confidentiality can be ensured using encryption algorithms.Encryption algorithms require a secret information called a key.These algorithms are implemented in cryptographic devices.There exist many types of attacks against such cryptosystems,the main goal of these attacks is the extraction of the secret key.Side-channel attacks are among the strongest types of attacksagainst cryptosystems. Side-channel attacks focus on the attacked device, they measure its physicalproperties in order to extract the secret key. Thus, these attacks targetweaknesses in an implementation of an algorithm rather than the abstract algorithm itself.Power analysis is a type of side-channel attacks that can be used to extract a secretkey from a cryptosystem through the analysis of its power consumption whilethe target device executes an encryption algorithm. We can say that the secret information is leaking from the device through itspower consumption. One of the biggest challenges in the domain of side-channel analysisis the evaluation of a device from the perspective of side-channel attacksor in other words the detection of information leakage.A device can be subject to several sources of information leakageand it is actually relatively easy to find just one side-channel attack that works(by exploiting just one source of leakage),however it is very difficult to find all sources of information leakage or to show that there is no information leakage in the givenimplementation of an encryption algorithm. Evaluators use various statistical tests during the analysis of a cryptographic device to checkthat it does not leak the secret key. However, in order to performsuch tests the evaluation lab needs the device to acquire the measurementsand analyse them. Unfortunately, the development process of cryptographicsystems is rather long and has to go through several stages. Thus, an information leakagethat can lead to a side-channel attackcan be discovered by an evaluation lab at the very last stage using the finalproduct. In such case, the whole process has to be restarted in order to fix the issue,this can lead to significant time and budget overheads. The rationale is that developers of cryptographic systems would like to be able to detect issues related to side-channel analysis during the development of the system,preferably on the early stages of its development. However, it is far from beinga trivial task because the end product is not yet available andthe nature of side-channel attacks is such that it exploits the properties ofthe final version of the cryptographic device that is actually available to the end user. The goal of this work is to show how simulators can be used for the detection of issues related to side-channel analysis during the development of cryptosystems.This work lists the advantages of simulators compared to physical experimentsand suggests a classification of simulators for side-channel analysis.This work presents existing simulators that were created for side-channel analysis,more specifically we show that there is a lack of available simulation toolsand that therefore simulators are rarely used in the domain. We present threenew open-source simulators called Silk, Ascold and Savrasca.These simulators are working at different levels of abstraction,they can be used by developers to perform side-channel analysisof the device during different stages of development of a cryptosystem.We show how Silk can be used during the preliminary analysisand development of cryptographic algorithms using simulations based on high level of abstraction source code. We used it to compare S-boxesas well as to compare shuffling countermeasures against side-channel analysis.Then, we present the tool called Ascold that can be used to find side-channel leakagein implementations with masking countermeasure using the analysis of assembly code of the encryption.Finally, we demonstrate how our simulator called Savrasca can be used to find side-channelleakage using simulations based on compiled executable binaries. We use Savrascato analyse masked implementation of a well-known contest on side-channel analysis (the 4th edition of DPA Contest),as a result we demonstrate that the analysed implementation contains a previouslyundiscovered information leakage. Through this work we alsocompared results of our simulated experiments with real experiments comingfrom implementations on microcontrollers and showed that issues found using our simulatorsare also present in the final product. Overall, this work emphasises that simulatorsare very useful for the detection of side-channel leakages in early stages of developmentof cryptographic systems. / Option Informatique du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213. The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies. Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis. The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.

Les attaques par canaux auxiliaires sont apparues dans la deuxième moitié des années 1990. Ces attaques exploitent différentes informations qu’il est possible de collecter lors de l’exécution d’un algorithme sur un appareil cryptographique. Il est ainsi possible, entre autres, de mesurer la consommation d’énergie d’un appareil cryptographique, ou encore d’observer le temps d’exécution d’un certain algorithme sur un appareil. C’est à ces deux sources d’in- formation que nous nous intéressons dans ce travail. Après une présentation des concepts utiles à la lecture du travail et de l’état de l’art des attaques et des contre-mesures du domaine, nous abordons les résultats de nos recherches effectuées lors de ce travail de thèse. Nous présentons d’abord nos contributions aux attaques par mesure de consommation d’énergie :(1) une approche com- binant apprentissage semi-supervisé et attaques par templates pour retrouver le poids de Hamming des différents bytes d’une clé de chiffrement et (2) une approche utilisant des techniques d’apprentissage automatique pour attaquer une implantation protégée d’AES. Ensuite, nous abordons les contre-mesures investiguées durant nos recherches qui se résument (1) en la possibilité de rendre l’ordre des instructions d’AES le plus aléatoire possible en jouant sur la relation de dépendance entre celles-ci ainsi qu’en (2) l’étude de l’application partielle (sur un sous-ensemble de données) de certaines contre-mesures, afin de protéger les données sensibles d’un algorithme. Enfin, nous terminons ce travail par l’emploi de la programmation orientée aspects comme manière d’implanter des contre-mesures pour les attaques temporelles (sur RSA) et pour les attaques par mesures de consommation d’énergie (sur AES). / Doctorat en Sciences / info:eu-repo/semantics/nonPublished