Tie1 é um receptor tirosina quinase expresso em células endoteliais importante em angiogênese, formação de vasos sanguíneos a partir de vasos pré-existentes. Este receptor pertence a uma família pequena composta por apenas dois membros (Tie1 e Tie2) para os quais angiopoietinas foram identificadas como ligantes de Tie2. No entanto, Tie1 continua a ser um receptor órfão, sem ligantes identificados até o momento. Sendo assim, é difícil compreender completamente as propriedades biológicas de Tie1 e seus mecanismos moleculares em angiogênese sem um ligante identificado. Entretanto, como sugerido através de estudos de deleção gênica, este receptor é uma molécula essencial na angiogênese, apresentando um papel importante no desenvolvimento da vascularização da retina e desenvolvimento de tumores. O nosso objetivo foi estudar a participação do domínio extracelular de Tie1 na neovascularização e, no processo, identificar possíveis ligantes para este receptor. Através da tecnologia de phage display, identificamos um peptídeo específico e seletivo para Tie1, sugerindo a existência de um sítio de ligação único neste receptor. Mostramos que este peptídeo é capaz de inibir a proliferação de células endoteliais induzida por Ang1, um ligante bem caracterizado de Tie2 que também modula a atividade de Tie1. Além disso, também mostramos que este peptídeo inibe a angiogênese in vivo num modelo animal bastante relevante para estudo de doenças humanas, o modelo da retinopatia induzida por oxigênio. Uma vez que este peptídeo liga-se a um sítio único e seletivo para Tie1, hipotetizamos que o mesmo poderia mimetizar possíveis ligantes naturais deste receptor. Para identificá-los, proteínas com mimetopo cruzado com este peptídeo foram identificadas em extrato proteico de diferentes linhagens celulares. Tais proteínas são possíveis candidatos a interação com Tie1. Em resumo, demonstramos que o domínio extracelular de Tie1 é importante para a angiogênese patológica e identificamos proteínas como possíveis ligantes deste receptor, o que poderá contribuir para um melhor entendimento da participação de Tie1 na formação de vasos. O peptídeo aqui identificado poderá ser ainda uma ferramenta útil para o desenvolvimento de novas terapias anti-angiogênicas com importantes aplicações à saúde humana. / Tie1 is a tyrosine kinase receptor expressed by endothelial cells and important in angiogenesis, the formation of new blood vessels from pre-existing ones. This receptor belongs to a small family of receptors composed of two members only (Tie1 and Tie2) to which angiopoietins have been identified as ligands for Tie2. On the other hand, Tie1 is still an orphan receptor with no ligand identified to date. Thus, it is difficult to assess Tie1 mechanism of action in neovascularization without a known ligand. Nevertheless, gene deletion studies have shown that Tie1 is essential in angiogenesis, and plays an important role in retinal and tumoral vascularization. The aim of our study was to evaluate the participation of Tie1 extracellular domain in angiogenesis, and in the process, to identify putative ligands for this receptor. Utilizing phage display, we have identified and characterized a Tie1 specific and selective ligand peptide, which suggests the existence of a binding site unique to this receptor and not shared by other family members. We show that this peptide prevents endothelial cells proliferation, induced by angiopoetin-1, a ligand for Tie2 but which also modulates Tie1 activity. Using a well-accepted mouse model for human diseases, the oxygen induced retinopathy model, we show that this peptide inhibits angiogenesis in vivo. Since this peptide maps to a unique binding site in Tie1, we hypothesized that it might mimic a natural ligand for this receptor. To identify them, proteins with cross reactive epitopes with an anti-peptide sera were identified by proteomic approaches. These proteins are thus possible ligands for Tie1. In summary, we have shown that Tie1 extracellular domain is important in angiogenesis and we have identified putative ligand for this receptor, which might contribute to a better understanding of the molecular mechanisms associated with Tie1 in blood vessel formation. The peptide here characterized may also be an important tool for the development of novel anti-angiogenesis therapeutic approaches for disesase with an angiogenic component.

Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas. / Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro / In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.

Le diabète sucré est un problème majeur de santé publique. L’une de ses principales complications est l’ulcère du pied (UPD) à l’origine d’infections et de nombreuses amputations. Ces plaies sont majoritairement polymicrobiennes. La principale bactérie isolée est Staphylococcus aureus (SA) qui pose d’importants problèmes en thérapie courante. Bactérie commensale notamment des fosses nasales, elle est un vrai pathogène au niveau des UPD. La compréhension du passage du commensalisme à la pathogénicité reste peu connue. Or récemment une souche colonisante de SA présentant un faible potentiel de virulence, et isolée sur un UPD a été identifiée. Cette souche présente un phage (ROSA-like) inséré dans son génome dont la stabilité a été démontrée. Ce phage est responsable de la faible virulence de cet isolat. Ce travail de doctorat a permis : i) de mettre en évidence l’implication du phage ROSA-like dans la dérégulation des mécanismes d’acquisition du fer, mécanismes essentiels pour la survie du SA ainsi que dans le métabolisme global de cette souche. Ces résultats ont confirmé le rôle clé du phage dans la baisse de la virulence du SA colonisant ; ii) à partir d’un modèle in vitro d’infections chroniques mimant les conditions rencontrées dans les UPD, d’étudier le comportement de souches cliniques de SA (colonisantes/infectantes) en fonction des différentes conditions environnementales : hyperglycémie, anaérobie, antibiotiques (linézolide et vancomycine). Les résultats ont montré qu’une exposition prolongée (plus de 24 semaines) à ces conditions de stress notamment aux antibiotiques, réduisait la virulence des souches de SA ; iii) d’évaluer la virulence des SA en présence d’autres bactéries qui l’entourent, élément essentiel du polymicrobisme de l’UPD. Ce travail s’est particulièrement intéressé aux interactions existant entre SA et Helcococcus kunzii, bactérie fréquemment isolée en association avec le SA dans les UPD. Il a montré pour la première fois une baisse de la pathogénicité des souches de SA par une bactérie commensale non virulente. / Diabetes mellitus is a global problem. One of the main complications is diabetic foot ulcer (DFU) which can evolve towards infection and induces lower limb amputations. Those wounds are mostly polymicrobial and Staphylococcus aureus (SA), the most prevalent pathogen isolated in this situation causes numerous therapeutic problems. This bacterium, known to be a commensal organism present notably in nasal cavity, is a real pathogen in DFU. However the comprehension between the commensalism and the pathogenicity of SA remains still unknown. Recently, a colonizing SA strain with very low virulence potential was discovered in DFU. This strain possesses a phage named Rosa-like responsible of its low virulence.This work helped us: i) to highlight the involvement of the Rosa-like phage in the disregulation of iron uptake mechanisms, the main source for SA survival and its global metabolism. The obtained results confirm the key role of the phage in the decrease of the colonizing SA strain virulence; ii) to study the behaviour of clinical SA strains (colonizing/infecting) using an in vitro chronic infection model miming different environmental conditions encountered in DFU: hyperglycemia, anaerobic condition, antibiotics (linezolid and vancomycin). Results showed that prolonged exposure (up to 24 weeks) to those stress conditions notably antibiotics reduced SA strains virulence; iii) to evaluate SA virulence in presence of other bacteria present in DFU. Particularly, in this work a focus was performed in the interaction between SA and Helcococcus kunzii, gram positive bacteria frequently isolated with SA in DFU. It was shown for the first time a decrease of SA pathogenicity by a commensal non virulent bacterium.

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. / Epigenetic phenomena are key contributors to the function of eukaryotic genomes. These processes act on chromatin, and they are used to render the genome dynamic, but also stable throughout successive rounds of cell division. Among epigenetic processes, DNA methylation is especially well known for its role in the regulation of gene expression.In mammals, DNA methylation is strongly correlated with transcriptional repression, and fulfills at least three essential roles. First, it maintains repeated sequences transcriptionally silenced, thus ensuring the stability of the genome. Second, it is responsible for the proper regulation of parentally imprinted genes, which are crucial regulators of embryonic development and adult life. Finally, DNA methylation ensures that some tissue-specific genes are kept inactive in the organs in which they should be repressed. Besides these roles in the physiology of normal cells, DNA methylation has strong links to cancer. Indeed the pattern of DNA methylation on the genome is frequently altered in cancer cells, and these anomalies contribute to transformation by several mechanisms.DNA methylation does not control transcription directly, but instead acts via a set of dedicated proteins that specifically recognize methylated DNA and repress transcription by acting at the chromatin level. At present, three families of such proteins, totalling 9 members altogether, are known in humans. However, several lines of evidence suggest that the list is not exhaustive, and that other human proteins that bind methylated DNA remain to be found. This was the goal of the current project.To this end, we opted for two distinct types approaches, an approach based on literature and a genetic approach. The study of candidate proteins does not allow us to identify new methylated DNA binding proteins and the genetic approach by phage display revealed two proteins of interest, HMGB1 and CHD3 that must now be validated by in vivo and in vitro approaches.Furthermore, we studied the regulation of DNA repeats by Zbtb4 in mice. Preliminary results show a regulation of minor satellites by Zbtb4. The role of this regulation will be analyse further in the future.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

Les matériaux inorganiques ont des propriétés uniques à l'échelle nanométrique. Ces propriétés ont généré beaucoup d'intérêt pour fabriquer des nouveaux matériaux utilisant des nano-objets comme unité de construction. Nous avons suivi une approche biomimétique pour la fabrication de dispositifs à base de nanoparticules afin d'améliorer les méthodes actuelles de fabrication top-down et bottom-up. Certaines protéines naturelles se lient en effet spécifiquement à des matériaux inorganiques, et déclenchent notamment la croissance de cristaux inorganiques. Une première étape dans cette approche biomimétique est de comprendre comment des protéines se lient spécifiquement à des nanomatériaux inorganiques. Nous avons exploré ce mécanisme de reconnaissance en sélectionnant des anticorps (les protéines de notre système immunitaire spécialisées dans les interactions avec de nombreuses cibles) contre des matériaux inorganiques par la méthode combinatoire biotechnologique appelée "phage display". Cette technique permet d'obtenir la séquence génétique codante des anticorps sélectionnés se liant à leur cible à partir d'une banque aléatoire d'anticorps. L'analyse statistique des séquences des anticorps sélectionnés fournit de nouvelles informations sur les interactions protéines/matériaux inorganiques. Notre principale conclusion est l'identification de l'acide aminé arginine en tant que contributeur majeur dans les interactions protéine/or. L'ingénierie génétique des anticorps permet de fonctionnaliser ces nouvelles sondes de matériaux inorganiques en vue de leur utilisation pour des applications dans le domaine des nanomatériaux. Les anticorps recombinants sélectionnés et leurs dérivés fonctionnalisés peuvent être exprimés par sécrétion à l'aide d'un hôte eucaryote (Dictyostelium discoideum) mis au point au cours de cette thèse. / Inorganic materials have unique properties at the nanometer scale. These properties have generated a lot of interest among researchers to fabricate novel materials using nano objects as building units. In this PhD thesis, we have attempted to mimick nature in the fabrication of nanoparticle based devices in order to improve upon current top-down and bottom-up nanomaterial fabrication methods. Proteins can specifically bind inorganic materials and trigger crystal growth and thus are considered as the main building units for a biomimetic approach of fabrication. The first step towards mimicking nature is to explore how proteins bind specifically to nanomaterials. We have explored this recognition mechanism by selecting antibodies (the protein binders of our immune system) against inorganic nanomaterials using the combinatorial biotechnology method of phage display. This technique provides us with the genetic sequence of selected antibodies from a random antibody library exposed against a target. Statistical analysis of selected antibody sequences provides new information on proteins/inorganics interactions. Our main finding in this regard is the identification of the amino acid arginine as a major contributor to protein/gold interactions. Additional functionality to these new binders of inorganic materials is obtained by antibody engineering, allowing for their value added use in nanomaterial science applications. Selected recombinant antibodies and their engineered derivatives along with other recombinant protein can be expressed and secreted using a eukaryotic expression platform (Dictyostelium discoideum) developed during this thesis.

The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe. The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria. Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed. Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host. Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results. Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

The long, contractile tails of myophages are the conduit for phage DNA transfer into the bacterial host cell and the most important part of the myophage tail is the baseplate; a complex structure, distal to the phage head. To better understand the structure and function of myophage baseplates, a component of the phage P2 baseplate, gpW was studied. This protein is widely conserved among myophages and is essential for the formation of infectious phage particles. Bioinformatic work confirmed that gpW homologues are found in almost all myophages and in many prophages. Moreover, gpW was shown to be a structural component of the virion; and, using electron microscopy, it was found to be at the top of the P2 baseplate. It was also found that some single residue substitutions can completely disrupt gpW function. Finally, evidence is presented that at least eight different proteins may be required to form intermediate P2 baseplate structures while other proteins may be necessary for the formation of stable baseplate complexes.

The long, contractile tails of myophages are the conduit for phage DNA transfer into the bacterial host cell and the most important part of the myophage tail is the baseplate; a complex structure, distal to the phage head. To better understand the structure and function of myophage baseplates, a component of the phage P2 baseplate, gpW was studied. This protein is widely conserved among myophages and is essential for the formation of infectious phage particles. Bioinformatic work confirmed that gpW homologues are found in almost all myophages and in many prophages. Moreover, gpW was shown to be a structural component of the virion; and, using electron microscopy, it was found to be at the top of the P2 baseplate. It was also found that some single residue substitutions can completely disrupt gpW function. Finally, evidence is presented that at least eight different proteins may be required to form intermediate P2 baseplate structures while other proteins may be necessary for the formation of stable baseplate complexes.