Le moteur flagellaire bactérien (BFM) est un complexe moléculaire qui permet aux bactéries de nager dans un milieu liquide. La rotation du moteur est générée à l’interface entre deux éléments clés: les protéines formant le stator (MotA and MoB) et l’anneau C “switching complex” à la base du rotor. Les stators sont des modules du moteur structurellement et fonctionnellement différentiables du reste du moteur, et leurs association et dissociation dynamique autour du rotor contrôle la génération du couple. Quand une protéine fluorescente (PF) est fusionnée à MotB, le moteur est en état de marche mais une réduction générale de la mobilité de la cellule a été observée. La raison précise d’une telle réduction de mobilité n’a pas été étudiée.Le but de cette étude est de comprendre comment la fusion PF de la protéine du stator modifie la génération du couple et le sens de rotation du moteur. C’est particulièrement important car le tag FP se trouve à l’interface entre le stator et le rotor, là où le couple et le changement du sens de rotation sont produits. Trois différentes PFs (eGFP, YPet, Dendra2) ont été fusionnées à la protéine MotB. Malgré la haute similarité de leurs structures, notre analyse a montré que les trois stators fusionnés génèrent des couples différents. Les stators marqués avec YPet produisent un couple moyen similaire au WT (stators sans tag PF), alors que les stators marqués avec eGFP et Dendra2 produisent respectivement 70% et 40% du couple moyen du WT. De plus, les moteurs utilisant les stators fusionnés ont montré des capacités de changement de sens de rotation réduites. Lors d’un changement de sens de rotation, la valeur absolue de la vitesse des moteurs WT ne change pas. Cette “symétrie” de vitesse lors du changement n’apparaît pas avec les moteurs à stators fusionnés et le changement peut être accompagné d’une importante diminution (~30%) de la vitesse absolue.En observant par microcopie TIRF avec détection de molécules uniques, des stators marqués dans un moteur en état de marche, les signaux de fluorescence sont détectés à la membrane comme prévu pour ces protéines, montrant une population de stators diffusant dans celle-ci. Les clusters fluorescents étaient visibles au centre des cellules en rotation, attachés au couvre-glace par une seule flagelle, confirmant que le tag de fluorescence peut être visualisé dans des moteurs en état de marche. Dans un second projet développé dans le laboratoire Bertus Beaumont à TU Delft, en prenant le BFM en tant que système modèle d’évolution expérimentale, sa modularité et son « évolubilité » ont été explorés pour apprendre les détails au niveau moléculaire de l’évolution de ce type de machine. Les stators de E.coli ont été échangés par un set de 21 stators étrangers homologues. L’expérience a révélé que les protéines du stator peuvent être échangées entre espèces de bactéries distantes et certains stators non compatibles peuvent être modifiés positivement par un procédé d’évolution pour devenir fonctionnels. Au cours de cette évolution, les bactéries ont accumulé des mutations avantageuses dans leurs gènes MotA et MotB étrangers, tout particulièrement dans leur domaine fonctionnel. Des mutations identiques dans des stators différents ont été observées, indiquant que l’évolution peut se reproduire. L’analyse fonctionnelle au niveau d’un seul moteur a révélé que ces mutations avantageuses amélioraient la génération du couple et/ou la capacité du moteur à changer de sens. Les investigations détaillées du génotype et du phénotype du BFM modifié par évolution apportés par cette étude, pourraient donner une idée sur la façon dont des machines moléculaires comme le BFM ont évolué, et les effets fonctionnels des mutations bénéfiques qui facilitent l'intégration fonctionnelle. / The bacterial flagellar motor (BFM) is the macromolecular complex which allows bacteria to swim in liquid media. Located at the base of the flagellum, anchored in the cell membrane, this remarkably small (~45nm) yet powerful rotary motor rotates each flagellum of the cell switching between counterclockwise (CCW) and clockwise (CW) direction. The motor rotation is generated at the interface between the two key components of the motor: the stator protein complexes (each composed of 4 MotA and 2 MotB proteins) and the C- ring protein complex at the base of the rotor. The stator complexes are structurally and functionally discernible modules of the motor, and their dynamical association and dissociation around the rotor controls the generation of torque.The first project of this study aims to investigate how the FP tag on the stator protein modifies the torque generation and switching of the motor. This is particularly important because the fluorescent protein tag lies at the interface between stator and rotor, where torque and switching are produced. Three different FPs (eGFP, YPet, Dendra2) were fused to MotB. Interestingly, despite the high similarity of their structures, our analysis revealed that the three fusion stators generate different torque. Furthermore, in the presence of fusion stators, the motor showed significantly impaired switching abilities. When switching direction of the rotation, the absolute value of the speed of WT motors does not change, whereas this symmetry of speed upon switching is not observed in the fusion stator motors, and switching can be accompanied with a significant (~30%) decrease in absolute speed. Both the impaired torque generation and the switching ability were improved by introducing a rigid linker between the stator and the FP tag. Taken together, this study provides a further insight into the dynamics of the stator and rotor interaction at its interface.When the cells carrying the fluorescently labeled stators were observed in a custom made TIRF-fluorescence microscope with single molecule capability, the fluorescence signals were detected as concentrated clusters in the membrane as expected for these membrane proteins around the motors, together with a population of stators diffusing in the membrane. Fluorescent clusters were visible at the center of rotating cells tethered to the glass slide by a single flagellum, confirming that the fluorescent tags can be visualized in functioning motors.In a second project developed in Bertus Beaumont lab at TU Delft, taking BFM as an experimental evolutionary model system, its modularity and evolvability have been explored to learn the molecular details of the evolution of molecular machines. The stators of E.coli have been exchanged by a set of 21 homologue foreign stators. The experiments revealed that the stator proteins can be exchanged between distant bacteria species, and some of the non-compatible stators can be positively modified by evolution to become functional. Those evolved strains accumulated beneficial mutations in their foreign motA and motB genes, especially on their functional domains. Identical mutations in different stators were common, indicating that evolution is repeatable. The functional investigation at the single motor level revealed that those beneficial mutations improved the torque generation and/or the switching ability of the motor. The detailed genotype and phenotype investigations of the evolutionary modified BFM may bring an insight into how molecular machines such as BFM have evolved as well as the functional effects of the beneficial mutations that facilitate functional integration.

No description available.

Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on thrombocyte function in zebrafish could enhance fundamental knowledge on hemostasis and may provide future target candidates for therapies.

Orientadores: Marcelo Brocchi, Gustavo Bueno Gregoracci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:27:26Z (GMT). No. of bitstreams: 1 SartiSprogis_AdrianeCristina_M.pdf: 1852459 bytes, checksum: 397af9ee41d085346189ca76eee83f96 (MD5) Previous issue date: 2014 / Resumo: A salmonelose é uma zoonose que representa um sério problema de saúde pública mundial, devido: à sua alta prevalência, à dificuldade de seu controle, ao seu caráter endêmico, à morbidade e à mortalidade. O conhecimento da ocorrência das diferentes sorovariedades de S. enterica em diferentes regiões e países pode ajudar no rastreamento e reconhecimento de patógenos emergentes, e assim implementar políticas de tratamento e prevenção. A grande maioria das sorovariedades expressa dois tipos diferentes de antígenos flagelares codificados pelos genes fliC (fase 1) e fljB (fase 2), sendo assim denominadas bifásicas. Contudo, algumas sorovariedades expressam apenas uma das fases, e são denominadas monofásicas. É possível que a variação de fase flagelar em S. enterica esteja associada a uma função de escape do sistema imunológico, por aumentar o repertório de antígenos expressos pela célula bacteriana, evitando temporariamente a resposta imune celular. Assim sendo, S. enterica bifásicas possuiriam uma vantagem seletiva sobre as monofásicas, porém isso não é totalmente verificado nos estudos epidemiológicos, pois no Estado de São Paulo, S. enterica I,4,[5],12:i:-, (monofásica) é uma das mais comumente associadas aos casos de diarreia e/ou infecções sistêmicas em pacientes humanos. De fato, a partir da década de 1990 houve um aumento significativo da S. enterica I,4,[5],12:i:- em muitos países. Sequências de profagos são muito comuns em S. enterica, sendo de conhecimento que esses fagos codificam vários fatores que contribuem para patogenicidade, diversidade genética e/ou características que aumentam o fitness. Coculturas experimentais de linhagens de S. enterica podem induzir espontaneamente profagos, que matam bactérias sensíveis, e assim a indução espontânea de fagos em uma população lisogênica acentua a competitividade entre populações. Neste estudo foram analisadas culturas puras de S. enterica Enteritidis (bifásica) adicionadas de fagos líticos induzidos de S. enterica I,4,[5],12:i:-, bem como coculturas entre as duas sorovariedades citadas, nas quais foram observadas induções espontâneas de fagos associados à alta densidade populacional e alterações das taxas de crescimento em ambos os estudos, corroborando a hipótese de que S. enterica monofásica pode alterar a dinâmica populacional a seu favor, pela liberação de fagos líticos à outra sorovariedade, interferindo no crescimento populacional de S. enterica Enteritidis, e que o sucesso evolutivo de S. enterica I,4,[5],12.:i:- pode estar associado a fagos líticos atuando como um regulador na ecologia bacteriana. Esses dados podem mudar nosso conhecimento sobre a interação bactéria-fago de uma simples relação parasita-hospedeiro para uma coevolução de duas vias entre seus genomas / Abstract: Salmonellosis is a zoonosis that is a serious public health problem worldwide, due to its high prevalence, difficulty controlling, their endemicity, morbidity and mortality. The knowledge of the occurrence of different serovars of S. enterica in different regions and countries can help in tracking and recognition of emerging pathogens and thus implement policies for treatment and prevention. The majority of serovars express two different types of flagellar antigens encoded by genes: fliC (phase 1) and fljB (phase 2), so called biphasic. However, some serovars express only one of the phases and are termed monophasic. It is possible that flagellar phase variation of S. enterica is associated with an escape function of the immune system to increase the repertoire of antigens expressed by the bacterial cell temporarily preventing cellular immune response. Thus, S. enterica biphasic would have a selective advantage over monophasic, but this is not fully verified in epidemiologic studies, because in the State of São Paulo, S. enterica I,4,[5],12:i:-, (monophase) is the one most commonly associated with cases of diarrhea and/or systemic infections in human patients, in fact, from the 1990s there was a significant increase of S. enterica I,4,[5],12:i:- in many countries. Prophages sequences are very common in S. enterica, with the knowledge that these phages encode several factors that contribute to pathogenicity, genetic diversity and/or characteristics that increase fitness. Cocultures experimental strains of S. enterica prophages can induce spontaneous, killing susceptible bacteria, and thus the spontaneous induction in a population of lysogenic phage enhances the competitiveness between populations. This study analyzed pure cultures of S. enterica Enteritidis ( biphasic ) added lytic phage induced from S. enterica I,4,[5],12:i:-, as well as cocultures between the two serovars cited where inductions were observed spontaneous phage associated with high population density and changes in growth rates in both studies, supporting the hypothesis that S. enterica monophase can alter the population dynamics to their advantage by releasing lytic phage to another serovar, interfering with the population growth of S. enterica Enteritidis and the evolutionary success of S. enterica I,4,[5],12:i:- may be associated with lytic phages acting as a regulator in bacterial ecology. These data may change our understanding of bacteria- phage from a simple parasite-host coevolution for a two-way between their genomes / Mestrado / Clinica Medica / Mestra em Clínica Médica

Im Fokus dieser Arbeit stand die Analyse von Faktoren, die den Zusammenbau des Flagellenapparates von Legionella pneumophila (Lp) regulieren. Mit einem kombinierten Replikations-/ Überlebensversuchs mit Lp Corby oder Lp Paris und ihren zugehörigen Regulationsmutanten wurde eine verminderte Fitness für dfliA und erstmals für drpoN, dfleQ defiziente Stämme nachgewiesen. Zur Validierung von Microarray-Daten aus Lp Paris wurden wachstumsphasenabhängige Transkriptions- und Translationsanalysen mit Lp Corby Wildtyp und drpoN, dfleQ, dfliA und dflaA defizienten Stämmen durchgeführt. Es wurde gezeigt, dass die basale Expression von fliA in den späteren Phasen unabhängig von RpoN und FleQ stattfindet. In dieser Arbeit konnte erstmals der Transkriptionsstartpunkt des Hauptregulators FliA bestimmt werden. Es zeigte sich eine putative RpoD (S70) Bindungsstelle. Ein Modell zur Regulation der fliA Expression wurde weiterentwickelt. Demnach kommt es in der exponentiellen Phase durch das Zusammenwirken von RpoD und DksA, aber unabhängig von FleQ, zur basalen fliA Promotoraktivität. Durch den Übergang in die transmissive Phase und direkte oder indirekte Interaktion mit FleQ sowie dem Alarmon ppGpp scheint es zu einem Austausch des Sigmafaktors S70 gegen SS und zu einer Aktivierung der fliA Expression zu kommen. Elektronenmikroskopische Studien zeigten, dass drpoN und dfleQ defiziente Mutanten wahrscheinlich aufgrund des fehlenden Basalkörpers nicht flagelliert sind. Mutanten für dfliA, dflaA und dfliD hatten ebenfalls keine Flagelle, zeigten aber eine ungewöhnliche, gerade Hook Struktur, die den Zusammenbau des Basalkörpers demonstriert. Weiterhin wurden durch in silico Studien 15 Legionella Spezies in Bezug auf das Flagellensystem und ein putatives Chemotaxissystem untersucht. So konnte L. oakridgensis als erste Art ohne beide Systeme sequenziert werden. Andererseits konnten mit LLAP12, L. bozemanii, L. gormanii und L. lytica Stämme beschrieben werden, die beide Systeme tragen. / This work focused on the analysis of factors contributing to the regulation of the flagellum self-assembly of Legionella pneumophila (Lp). With a combined replication/survival assay with Lp Corby or Lp Paris and their corresponding regulatory mutants a reduced fitness could be verified for dfliA and for the first time for drpoN, dfleQ deficient strains. For validation of microarray-data for Lp Paris with strain Lp Corby a growth phase dependent analysis of transcription and translation rates was done with wild-type and the drpoN, dfleQ, dfliA and dflaA deficient strains. A regulation of basal fliA expression independently from RpoN and FleQ was shown in the later growth phases. Furthermore the transcriptional start site of fliA could be shown for the first time. A RpoD (S70) binding site could be identified. According to a further developed model for the regulation of the fliA expression RpoD and DksA lead to a basal fliA promotor activity, independently from FleQ. Most likely, during transition to stationary phase, direct or indirect interaction with FleQ and the alarmone ppGpp results in the exchange of the sigma factor S70 and the binding of RpoS. This leads to the activation of fliA expression. Electron microscopic studies revealed that drpoN and dfleQ deficient mutants are not flagellated caused by the missing basal body. Mutants of dfliA, dflaA and dfliD were also aflagellated, but there was a uncommon straight hook structure visible which demonstrates a filament-independent assembly of the basal body. Furthermore, in silico analysis was done with 15 Legionella species with regard to the flagellum regulation system and a putative chemotaxis system. Analysis revealed that the strain L. oakridgensis is the first strain lacking both systems. On the other hand the strains LLAP12, L. bozemanii, L. gormanii and L. lytica could be characterized as strains carrying both systems.