This work assesses the importance of nonlinearity in the stability of flows over compliant and rigid walls, and comprises three main parts. The first part considers inviscid flow with a free surface over a flexible boundary. The dispersion relation is obtained, and the conditions for linear instability investigated. The linear dispersion relation is then used to show that the conditions for nonlinear three-wave resonance are often met. In some circumstances, the resonance may be of 'explosive' sort, involving waves of opposite energy sign; but non-explosive resonant configurations are most common. Next, the wave- amplitude evolution equations for three-wave resonance are derived, firstly by a 'direct' approach, and then via a variational (averaged Lagrangian) method. Results agree with those of Case & Chiu (1977) for capillary-gravity waves, and Craik & Adam (1979), for three-layer fluid flow, on taking the appropriate limits. We also consider a nonlinear model for the flexible boundary. In the second part, stability of Blasius flow over a compliant surface is studied. This extension of rigid-wall work of Craik (1971) and Hendriks (appendix to Usher & Craik 1975) determines the quadratic interaction coefficients of three-wave resonance, and complements the linear analysis of Carpenter & Garrad (1985, 1986) and others. First, the linear eigenvalue spectrum is investigated for various values of the wall parameters. Then, resonant triads are located and the quadratic interaction coefficients determined numerically. By way of introduction some rigid-wall results are also presented, extending those of Hendriks.

The major peculiarities of the language of the Apocalypse related to the usage of the Verb and of the Clause are here explained as due to the influence of the Hebrew and Aramaic languages, not to the author's lack of familiarity with Greek. The approach has been to isolate in each case the usage which is not compatible with Hellenistic Greek syntax, and then to extract from the Septuagint identical constructions. The next step was to determine the type of Hebrew/ Aramaic construction responsible for the anomaly in the Septuagint, and then to apply the same explanation to the identical construction in the Apocalypse. While this method has proven to be fundamentally sound and reliable, it is a strange fact that previous treatments of the Semitic element in the language of the Apocalypse have failed to apply it consistently. Another basic presupposition of this thesis is that the research should not be restricted to a single-Greek manuscript or printed text of the Apocalypse. Instead, an eclectic approach is made, which respects any manuscript evidence supporting the more semitised construction. The study included every significant facet of verbal usage from that of lexicography, Voice, Mood, Tenses of the Finite Verb, case additions to the verb, and the Infinitive and Participle. Included in the section on Clauses were Noun - and Verbal - Clauses, plus the Subordinate Clauses such as Relative, Circumstantial, Conditional, Temporal, Final, and Consecutive. Specific types of Semitic influence were seen to be present in each section listed. In at least some cases the Semitic influence was of such a direct nature that it could be explained only as due to direct translation from a Semitic source. While the evidence points predominantly to Hebrew sources underlying the Apc., the case for Aramaic influence at some points cannot be ruled out without doing an injustice to the facts. The results of this study, based as they are on directly observable Semitic influence on the Greek of the LXX, can be applied to any Jewish translation Greek text, to serve as a syntactical survey of that language. The study also demonstrated that the eclectic approach to the text of the Apocalypse is the only sound one, because no single manuscript or family of manuscripts has preserved a majority of the semitised (and therefore more nearly original) readings.

Le système d’expression le plus utilisé pour la production des protéines membranaires, est le système basé sur l’ARN polymérase T7 (ARNpol T7) (Hattab et al., 2015). L'inconvénient de ce système est néanmoins que la vitesse de transcription de l’ARNpol T7 est dix fois plus rapide que celle de l’enzyme bactérienne. Depuis l’isolement de mutants spontanés, notamment C41 (DE3) et C43 (DE3) (Miroux et Walker, 1996) et l’identification de leurs mutations dans le génome, il apparaît clairement que la toxicité provoquée par la surproduction des protéines membranaires est liée à la quantité trop élevée d’ARNpol T7 dans la cellule (Wagner et al., 2008 ; Kwon et al., 2015). Les protéines membranaires ont besoin d’une vitesse de transcription/traduction plus basse pour se replier correctement dans la membrane de la bactérie. Le premier objectif de ma thèse était d’étendre l’amplitude du promoteur du système T7 sur laquelle est basée l’expression des protéines. Pour cela, nous avons isolé et caractérisé de nouvelles souches bactériennes dans lesquelles le niveau d’ARNpol T7 était efficacement régulé par un mécanisme non transcriptionnel très favorable à l’expression des protéines membranaires (Angius et al., 2016). Le deuxième objectif était de comprendre la prolifération des membranes intracellulaires chez E. coli suite à la surexpression de la protéine AtpF, une sous unité membranaire du complexe de l’ATP synthétase (Arechaga et al., 2000). Pour mieux comprendre les voies métaboliques impliquées dans la biogenèse, la prolifération et l’organisation des membranes, nous avons utilisé une approche de séquençage d’ARN à haut débit à différents temps après induction de la surexpression de la sous-unité AtpF dans la souche C43 (DE3). Ensuite, et en collaboration avec Gerardo Carranza and Ignacio Arechaga (Université de Cantabria, Espagne), nous avons construit et étudié des mutants de C43 (DE3) déficients pour les trois gènes codants pour des enzymes de la biosynthèse des cardiolipides afin d’évaluer leur participation dans la biogénèse des membranes intracellulaires / The most successful expression system used to produce membrane proteins for structural studies is the one based on the T7 RNA polymerase (T7 RNAP) (Hattab et al., 2015). However, the major drawback of this system is the overtranscription of the target gene due to the T7 RNAP transcription activity that is over ten times faster than the E. coli enzyme. Since the isolation of spontaneous mutants, namely C41(DE3) and C43(DE3) (Miroux and Walker, 1996) and the identification of their mutation in the genome, it becomes clear that reducing the amount of the T7 RNAP level removes the toxicity associated with the expression of some membrane proteins (Wagner et al., 2008; Kwon et al., 2015). Also, some membrane proteins require a very low rate of transcription to be correctly folded at the E. coli membrane. The first objective of my PhD was to extend the promoter strength coverage of the T7 based expression system. We used genetic and genomic approaches to isolate and characterize new bacterial strains (Angius et al., 2016) in which the level of T7 RNAP is differently regulated than in existing hosts. A second objective was to understand intracellular membrane proliferation in E. coli. Indeed it has been shown that over-expression of membrane proteins, like overexpression of AtpF of E. coli F1Fo ATP synthase is accompanied by the proliferation of intracellular membranes enriched in cardiolipids (Arechaga et al., 2000). To understand metabolic pathways involved in membrane biogenesis, proliferation and organization, we used a RNA sequencing approach at several time point upon over-expression of the F-ATPase b subunit in C43(DE3) host. On the other hand, in collaboration with Gerardo Carranza and Ignacio Arechaga (University of Cantabria, Spain) we studied C43(DE3) cls mutants, in which the cardiolipids genes A, B and C are deleted, to test how they participate to intracellular membranes structuration

In this work, we seek to expand synthetic in vitro biological systems by using water-in-oil emulsions to provide an environment conducive to directed evolution. We approach this primarily by utilizing a model transcription system, the T7 RNA polymerase and promoter, which is orthogonal to both bacterial and eukaryotic transcription systems and is highly functional in vitro. First, we develop a method to identify functional promoter sequences completely in vitro. This method is tested using the T7 RNA polymerase-promoter model system. We then configure the T7 transcription system as an ‘autogene’ and investigate how this positive feedback circuit (whereby a T7 promoter expresses a T7 RNA polymerase gene) functions across various in vitro platforms, including while compartmentalized. The T7 autogene can be envisioned as a self-replicating system when compartmentalized, and its use for directed evolution is examined. Finally, we look towards future uses for these in vitro systems. One interesting application is to expand the utilization of unnatural base pairs within the context of a synthetic system. We investigate the ability of T7 RNA polymerase to recognize and utilize unnatural base pairs within the T7 promoter, complementing existing work on the utilization of unnatural base pairs for in vitro replication and transcription with an investigation of more complex protein-dependent regulatory function. We envision this work as a foundation for future in vitro synthetic biology efforts. / text

The homodimeric Ocr protein of bacteriophage T7 is a molecular mimic of a bent double-stranded DNA molecule ~24 bp in length. As such, Ocr is a highly effective competitive inhibitor of the bacterial Type I restriction modification (R/M) system. Thus, Ocr facilitates phage infection of the bacterial cell to proceed unhindered by the action of the R/M defense system. The main aim of this work was to understand the basis of the DNA mimicry displayed by Ocr. The surface of the protein is replete with acidic residues, most or all of which mimic the phosphate backbone of DNA. Aspartate and glutamate residues on the surface of Ocr were either mutated or chemically modified in order to investigate their contribution to the tight binding between Ocr and the EcoKI Type I R/M enzyme. Single or double mutations of Ocr had no discernable effect on binding to EcoKI or its methyltransferase component (M.EcoKI). Chemical modification was then used to specifically modify the carboxyl moieties of Ocr, thereby neutralizing the negative charges on the protein surface. Ocr samples modified to varying degrees were analysed to establish the extent of derivatisation prior to extensive biophysical characterisation to assess the impact of these changes in terms of binding to the EcoKI R/M system. The results of this analysis revealed that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by at least ~800-fold. In addition, based on the known 3-D structure of the protein, a set of multiple mutations were introduced into Ocr aimed at eliminating patches of negative charge from the protein surface. Specifically, between 5 and 17 acidic residues were targeted for mutation (Asp and Glu to Asn and Gln, respectively). Analysis of the in vivo activity of the mutant Ocr along with biophysical characterisation of the purified proteins was then performed. Results from these studies identified regions of the Ocr protein that were critical in forming a tight association with the EcoKI R/M system. Furthermore by comparing the relative contribution of different groups of acidic residues to the free energy of binding, the actual mechanism by which Ocr mimics the charge distribution of DNA has been delineated.

Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thema dieser Arbeit ist die evolutive Optimierung der T7-RNA-Polymerase. Zur Stabilisierung technischer oder therapeutischer RNA-Moleküle gegenüber RNAsen wäre es wünschenswert eine RNA-Polymerase zu generieren, welche RNA vollständig aus 2’- modifizierten Nucleotiden synthetisieren kann. Zu diesem Zweck wurde ein kombiniertes Selektions- und Screeningverfahren zur Durchmusterung von Variantenbibliotheken der T7- RNA-Polymerase hinsichtlich des Einbaus von 2’-Methoxy-modifizierten Nucleotiden in RNA entwickelt. Es wurden ein gut handhabbarer, cis-regulierter Expressionsvektor sowie ein Selektionsplasmid erzeugt, die zusammen in E. coli ein in-vivo-Selektionssystem bilden, mit dessen Hilfe man Zellen, welche T7-RNA-Polymerase-Aktivität zeigen anhand ihrer grünen Fluoreszenz identifizieren konnte. Durch error-prone PCR wurden Mutantenbibliotheken generiert, und diese in das Selektionssystem eingesetzt. So konnte die Anzahl der potentiell zu testenden Varianten erheblich gesenkt werden. Zur Bestimmung der T7-RNA-Polymerase- Aktivität mit 2’-Methoxy-modifizierten Nucleotiden wurde ein Fluoreszenz-basierendes Assay etabliert. Dieses Assay, das nicht mit radioaktiv-markierten Nucleotiden arbeitete und keinen gelelektrophoretischen Separationsschritt benötigte, konnte in allen Schritten zur parallelen Bearbeitung von 96 Proben in einem Mikrotiterplatten-Format angepasst werden, so dass es prinzipiell hochdurchsatzfähig war und sich zum Screening umfangreicher Variantenbibliotheken eignete. Die Assay-Reaktion kann dabei auch unkompliziert auf ein Screening von RNA- oder DNA-Polymerase-Bibliotheken hinsichtlich anderer Eigenschaften der Polymerase-Aktivität übertragen werden.

La famille des Flexiviridae a été créée en 2004 et regroupe plusieurs genres viraux affectant particulièrement des espèces ligneuses dont des arbres fruitiers. Grâce à diverses approches plusieurs nouveaux Flexiviridae ont été partiellement caractérisés au cours de ces dernières années. En revanche la position taxonomique précise de certains d’entre eux et leur contribution à des pathologies particulières restent encore incertaines du fait de difficultés inhérentes à l’étude de ces agents. Dans le présent travail, nous avons obtenu les séquences génomiques complètes pour quatre agents proches de l’Apricot latent virus. Ceci a permis de préciser l’organisation génomique de ces virus et d’en déterminer la position taxonomique. Cette étude a également permis de montrer que la partie C-terminale de la capside et la protéine TGBp1 sont soumises à une pression sélective particulièrement forte. Dans un second volet de ce travail, plusieurs approches permettant l’obtention simple et rapide d’ADNc infectieux, sous forme clonée ou non ont été développées. Travaillant sur plusieurs Flexiviridae, dont le virus des taches foliaires chlorotiques du pommier (Apple chlorotic leaf spot virus, ACLSV), nous avons mis au point l’amplification d’ADNc génomiques complets en une seule étape à partir d’extraits d’acides nucléiques totaux obtenus à partir de plantes infectées. Des amplifiats comportant l’ADNc viral sous le contrôle du promoteur 35S du CaMV ou du promoteur de la RNA polymérase du phage T7 ont été obtenus et utilisés pour infecter des plantes directement par biolistique (promoteur 35S) ou pour obtenir des ARN infectieux par transcription in vitro (promoteur T7). Ces données ont mis en évidence des différences importantes dans le comportement de deux hôtes de l’ACLSV, Chenopodium quinoa et Nicotiana occidentalis 37B. Nous avons également utilisé le système de recombinaison homologue de la levure Saccharomyces cerevisiae simplifier le clonage d’ADNc complets amplifiés par PCR ou pour réaliser en une seule étape la construction d’un vecteur navette ternaire levure-E. coli-A. tumefaciens et l’obtention d’un clone ADNc de l’ACLSV inoculable par agroinfiltration. Ces différentes stratégies devraient trouver une large application, en particulier pour tester plus rapidement des hypothèses d’étiologie pour les virus de plantes réputés "difficiles", tels que ceux infectant des hôtes ligneux. / The Flexiviridae family was created in 2004 and contains several viral genera affecting in particular woody hosts, including fruit trees. Using various strategies several new Flexiviridae have been partially characterized in the past few years. However, due to difficulties inherent in studying these agents, the precise taxonomic position of some of them and their contribution to particular diseases are still uncertain. In the present work, the complete genomic sequences of four Prunus-infecting Apricot latent virus (ApLV) like isolates have been determined. This has allowed to determine the genomic organization and the taxonomic position of these viruses. The results obtained also indicate that the C-terminal half of the coat protein and the TGBp1 are the genomic regions under the strongest purifying selection pressure. In the second part of this work, a set of approaches to simplify and streamline the construction of cloned or uncloned infectious full-length viral cDNAs were developed. working with several Flexiviridae and, in particular, with the Apple chlorotic leaf spot virus (ACLSV), we have developed protocols allowing the one-step amplification from total nucleic acids extracts of full-length cDNAs. under the control of the CaMV 35S or phage T7 RNA polymerase promoters. Successful inoculation of plants with these uncloned amplification products was obtained by biolistic bombardment (35S promoter) or using in vitro synthesized RNA transcripts (T7 promoter). Results obtained showed significant differences in the behavior of the two ACLSV hosts, Chenopodium quinoa and Nicotiana occidentalis 37B. We also used the yeast homologous recombination system for the efficient cloning of full-length cDNAs and for the simultaneous one-step construction of a ternary yeast-E. coli-Agrobacterium tumefaciens shuttle vector and generation of an agroinfiltrable infectious ACLSV construct. These various strategies should find broad applications, in particular for the validation of etiological hypotheses in the case of “difficult” plant viruses, such as those infecting woody hosts.

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.