Chez Escherichia coli la terminaison de la transcription peut intervenir selon deux mécanismes distincts : tout d'abord la terminaison intrinsèque qui correspond à une séquence ADN, codant pour une structure en tige boucle riche en GC suivie d'une répétition d'uridine sur l'ARN, induisant le relargage de l'ARN polymérase. Le second mécanisme est gouverné par un facteur de terminaison nommé Rho qui gouverne environ 50 % des événements de terminaison chez E. coli. Au cours de ma thèse, je me suis intéressé à ce deuxième mécanisme, et plus particulièrement à sa régulation in vivo (antiterminaison). Rho, sous la forme d'un anneau héxamèrique, se fixe à l'ARN naissant au niveau d'un site d'entrée (également appelé RUT), puis utilise son activité ATPase pour longer l'ARN et dissocier le complexe ternaire d'élongation stoppé au niveau d'un site de pause. Les terminateurs Rho-dépendants sont assez mal définis et peu d'entre eux on été analysés en détail. Le terminateur du tR1 du phage lambda (λ) est le terminateur Rho-dépendant le plus étudié à la fois in vitro et in vivo. La terminaison Rho-dépendante au niveau de ce terminateur est gouvernée principalement par les séquences localisées en 5', codant deux régions du transcript nommées RUTA et RUTB. Ces deux régions sont séparées par le motif ARN BOXB qui n'est pas indispensable à l'action de Rho mais sert, dans le mécanisme d'antiterminaison, de site de fixation pour la protéine N du paghe λ. Grâce à un système minimal d'étude in vivo, nous avons montré que le motif BOXB possède une fonction double dans les mécanismes de terminaiso/antiterminaison au niveau de λtR1 régulant l'expression temporale du génome du phage λ. Sous la forme d'une tige boucle hautement structurée, BOXB agit comme un lien permettant de placer RUTA et RUTB l'un à côté de l'autre pour une interaction optimale avec Rho et une terminaison efficace. A l'inverse, la fixation de la protéine N sur BOXB induit l'antiterminaison au niveau de λtR1 en empêchant l'accès de Rho à l'ARN. Ce double rôle a été démontré in vivo en substituant au couple N/BOXB la « coat protein » du phage MS2 et son motif cible en tige boucle. En complément de ce travail, j'ai utilisé cette faculté d'un complexe ribonucléoprotéïque de bloquer la terminaison Rho-dépendante, pour développer une nouvelle approche d'étude in vivo, des complexe ARN-protéine.

L'épigénétique est un composant essentiel du fonctionnement des génomes eucaryotes. Les divers phénomènes épigénétiques modifient l'état chromatinien et participent à la plasticité du génome, mais aussi au maintien de son identité fonctionnelle à travers les générations cellulaires. Parmi ces processus, la méthylation de l'ADN joue un rôle fondamental dans la régulation de l'expression des gènes.Chez les mammifères, la méthylation de l'ADN est associée à la répression transcriptionnelle, et elle remplit au moins trois fonctions essentielles. Premièrement, elle permet de réprimer les séquences répétées afin de préserver l'intégrité du génome. Deuxièmement, la méthylation contrôle l'expression des gènes soumis à l'empreinte parentale, qui sont des régulateurs cruciaux du développement et de la vie adulte. Enfin, la méthylation permet de réprimer certains gènes tissu-spécifiques dans les organes où ils doivent être silencieux. En plus de ces rôles physiologiques, la méthylation est liée au cancer. En effet, des patrons de méthylation anormaux sont fréquemment observés dans les cellules tumorales, et ces anomalies participent à la transformation cellulaire par plusieurs mécanismes.La méthylation exerce ces effets par l'intermédiaire de protéines dédiées, qui reconnaissent spécifiquement l'ADN méthylé et contrôlent la transcription en modulant la chromatine. Trois familles de protéines liant l'ADN méthylé sont connues chez les mammifères, et elles totalisent entre elles neuf membres. De nombreux arguments suggèrent que cette liste est encore incomplète, et que des protéines humaines liant l'ADN méthylé restent à découvrir. Dans cette optique, nous avons opté pour deux types d'approches distinctes, une approche basée sur la littérature et une approche génétique. L'étude des protéines candidates ne nous a pas permis d'identifier de nouvelles protéines liant l'ADN méthylé et l'approche génétique par phage display a révélé deux protéines intéressantes, CHD3 et HMGB1 qui doivent désormais être validées par des approches in vivo et in vitro.Par ailleurs, nous avons entrepris l'étude de la régulation des éléments répétés par la protéine Zbtb4 chez la souris. Les expériences préliminaires indiquent une possible régulation des satellites mineurs par Zbtb4. Le rôle de cette régulation sera, par la suite, approfondi.

L'endoribonucléase de restriction RegB est une enzyme produite par le bactériophage T4. Elle est impliquée dans la transition phase précoce-phase moyenne durant le cycle lytique du virus. RegB coupe avec une spécificité quasi absolue la séquence GGAG impliquée notamment dans l'initiation de la traduction chez la bactérie Escherichia coli. Nous avons caractérisé dans cette thèse de façon précise la toxicité de RegB dans la bactérie et nous avons proposé des outils pour contourner cette toxicité tels la manipulation du nombre de copies du vecteur d'expression ou l'atténuation de l'efficacité du site d'initiation de la traduction. Nous avons, par ailleurs, proposé une application de RegB pour la construction d'un vecteur de clonage à sélection positive et à expression duale dans les systèmes procaryotes et eucaryotes. L'étude par RMN du 31P de la cinétique de clivage d'un ARN par RegB a permis de définir RegB comme une "transphosphorylase libérant un phosphodiester 2', 3'-cyclique". Des études de mutagenèses dirigées et aléatoires combinées à l'évolution du gène regB dans un virus apparenté au phage T4 (le virus RB49) ont mis en évidence le rôle des résidus Glutamate 19, Histidine 48, Arginine 52 et Histidine 68 dans l'activité de RegB. Le mutant RegB H48A a été choisi pour construire un modèle structural du site actif de RegB. L'attribution séquentielle de cette protéine par RMN hétéronucléaire 1H/15N/13C a été entreprise avec succès.

<p>It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.</p><p>The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma. </p>

<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

<p>Bacteria of the genus <i>Bartonella</i> inhabit the red blood cells of many mammals, including humans, and are transmitted by blood-sucking arthropod vectors. Different species of <i>Bartonella</i> are associated with different mammalian host species, to which they have adapted and normally do not cause any symptoms. Incidental infection of other hosts is however often followed by various disease symptoms, and several <i>Bartonella</i> species are considered as emerging human pathogens.</p><p>In this work, I have studied the genomic diversity within and between different <i>Bartonella</i> species, with focus on the feline-associated human pathogen <i>B. henselae</i> and its close relatives, the similarly feline-associated <i>B. koehlerae</i> and the trench-fever agent <i>B. quintana</i> which is restricted to humans.</p><p>In <i>B. henselae</i>, the overall variability in sequence and genome content was modest and well correlated, suggesting low levels of intra-species recombination in the core genome. The variably present genes were located in the prophage and the genomic islands, which are also absent from <i>B. quintana</i> and <i>B. koehlerae</i>, indicating multiple independent excision events. In contrast, diversity of genome structures was immense and probably associated with rearrangements between the repeated genomic islands located around the terminus of replication, possibly to avoid the host’s immune system. In both <i>B. henselae</i> and the mouse-associated species <i>B. grahamii</i> a large portion of the chromosome was manifold amplified in long-time cultures and packaged into phage particles, allowing for different recombination rates for different chromosomal regions.</p><p>In B<i>. quintana</i>, diversity was studied by sequencing non-coding spacers. The low variability might be due to the recent emergence of this species. Surprisingly, also this species displayed high variability in genome structures, despite its lack of repeated sequences.</p><p>The results indicate that genome rearrangements and gain or loss of mobile elements are major mechanisms of evolution in <i>Bartonella</i>.</p>

The food safety and human diagnostics markets are in need of faster working, reliable, sensitive, specific, low cost bioassays and biosensors for bacterial detection. This thesis reports the use of P22 bacteriophage tailspike proteins (TSP) immobilized on silanized silicon surfaces, roughened at a nano-scale, for specific capture and detection of Salmonella. Towards developing TSP biosensors, TSP immobilization characteristics were studied, and methods to improve bacterial capture were explored. Atomic force microscopy was used to count TSP immobilized on gold thin-films. Surface density counts are dependent on the immobilization scheme used. TSP immobilized on flat silicon (Si), silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde, showed half the bacterial capture of gold thin-films. To improve bacterial capture, roughened mountain-shaped ridge-covered silicon (MSRCS) surfaces were coated with TSP and tested. Measurements of their bacterial surface density show that such MSRCS surfaces can produce bacterial capture close to or better than TSP-coated gold thin-films. / Biomedical Engineering

Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40% of TSP for Pal and 20% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics. / Lytische Enzyme aus Bakteriophagen bieten Eigenschaften, die sie zu vielversprechenden Medikamenten im Einsatz gegen bakterielle Krankheiten machen. Obwohl sie speziell beim Einsatz gegen bakterielle Infektionen, welche durch Antibiotika resistente Erreger hervorgerufen werden, eine maßgebende Rolle spielen könnten, waren bisher die hohen Produktionskosten ein Hindernis für die medizinische Anwendung. Ein kostengünstiges und einfach zu handhabendes System, wie beispielsweise Chloroplasten in Pflanzen, würde diese lytischen Enzyme zu einer effizienten Alternative zu herkömmlichen Antibiotika machen. In dieser Arbeit wird erstmals die erfolgreiche Produktion von lytischen Enzymen in Tabak-Chloroplasten vorgestellt, welche mit einem Fremdproteingehalt von mehr als 70% des gesamtlöslichen Proteins der Pflanze eine Menge beschreibt, die bisher mit diesem Verfahren noch nicht erreicht wurde. Alle in Chloroplasten hergestellten lytischen Enzyme zeigten hohe spezifische bakteriolytische Aktivität gegen die gewählten Humanpathogene und waren innerhalb von Minuten in der Lage diese Bakterien abzutöten. Zur Herstellung von zwei lytischen Enzymen wurde in dieser Arbeit ein spezieller Shuttle-Vektor entworfen, der die Expression von toxischen Genen innerhalb von E. coli Zellen im Zuge der DNA Replikation vermeidet, jedoch die Herstellung einer ungehinderten Expression der toxischen Gene in den Chloroplasten nach Beseitigung des Selektionsmarkers erlaubte. Ein Vergleich zwischen einem herkömmlich verwendeten Transformationsvektor und dem Shuttle-Vektor mittels eines Reportergens zeigte, dass das neu entwickelte System bis zu 4 mal mehr Protein produzierte. Diese Ergebnisse zeigen das Potential von Chloroplasten als kostengünstige und leicht zu handhabende Produktionsplattform für lytische Enzyme, welche als neue Generation von Antibiotika attraktive Alternativen zu herkömmlichen Therapien bieten.

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting. In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines. In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.