Shigella flexneri is a bacterial pathogen which causes dysentery by invading the epithelial cells of the colon. In order to survive and replicate inside the host, S. flexneri requires many genes present on both its chromosome and the large virulence plasmid it carries. This study examines which genes are required for infection of cultured epithelial cells in order to understand which processes are used by S. flexneri during the infection process. This analysis pinpointed genes involved in metabolism, LPS synthesis, protein homeostasis and virulence effector proteins. The role of Elongation factor P (EF-P) in S. flexneri virulence is also investigated in this study. EF-P is a bacterial translation factor that is post-translationally modified with a [Beta]-lysine by the action of PoxA. Here it is shown that both EF-P and PoxA are necessary for virulence of S. flexneri. Loss of either EF-P or PoxA leads to an impaired ability of S. flexneri to invade epithelial cells. Proteomic analysis of efp and poxA deletion mutants revealed decreased levels of several virulence effector proteins, as well as proteins for the biosynthesis of the siderophore aerobactin. Virulence proteins were affected due to decreased levels of the master virulence regulator VirF. Reduction in VirF transcription is likely due to decreased levels of CpxA, which activates virF through the response regulator CpxR. The role of CpxAR in reduced synthesis of VirF and its downstream effectors was confirmed by showing increased invasion when a mutation resulting in constitutively vii activated CpxR was introduced into the efp mutant. Thus, modified EF-P is one of the chromosomal factor necessary for the virulence of this bacterial pathogen. / text

This report is the result of a project in the course Independent Projekt in Molecular Biotechnology at Uppsala University during the spring of 2014. The foremost purpose of the course is to give students the opportunity to carry through exstensive work in a project environment. This project was formed based on a comission from the biotechnology company SweTree Technologies, and the goal has been to compose a summary of the different techniques and methods that exist in the field of mass propagation of trees through the method of somatic embryogenesis. The project group has obtained information about the area mainly throgh reading patents, trying to find key components and bottlenecks in other companies’ somatic embryogenesis technologies. This paper is divided into different sections, containing the patents of the automation of different steps in the process. This is to make it easier for readers to find information about the area they are interested in, as well as to illustrate the main parts of the process as percieved by the project group. Currently, there are several automated solutions for almost every step in the process, some of which are already in use. All the information obtained shows that the cost and labour has decreased with the development of this technology. While there is still room for significant devolopment in order to produce a complete automated process, there is no doubt that this method is becoming an ever more important asset in the area of forestry. Our hope is that this report may be a useful tool for companies or laymen to geta grasp of the field of automated mass production of trees.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Techn. University, Diss., 2005--München.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Orientador: Wanderley Dias da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T11:08:23Z (GMT). No. of bitstreams: 1 Angelini_Michelle_D.pdf: 4094056 bytes, checksum: b80e9ba086ee8f30b823ce5b5718f3c7 (MD5) Previous issue date: 2009 / Resumo: As bactérias do gênero Shigella spp. são responsáveis por infecções intestinais endêmicas e epidêmicas denominadas shigueloses. As shigueloses, transmitidas por via fecal-oral, principalmente através das mãos e água contaminadas, são responsáveis por 10% dos casos de diarréia entre crianças até cinco anos de idade em países em desenvolvimento. Estima-se que, anualmente, 1,1 milhões de pessoas morram de infecções causadas por Shigella. No Brasil, estudos revelam que as shigueloses estão entre as principais causas de diarréia. O presente trabalho teve por objetivo analisar a análise clonal comparativa de 119 linhagens de Shigella spp., entre elas 57 S. flexneri e 62 S. sonnei, isoladas de diferentes casos de shiguelose ocorridos nas regiões metropolitanas de Campinas e Ribeirão Preto, Estado de São Paulo, pelos métodos de PCR da seqüência consenso intergênica repetitiva enterobacteriana (ERIC-PCR), de PCR de elementos extragênicos palindrômicos repetitivos (REP-PCR), análise do padrão de macro-restrição em gel de eltroforese com campo pulsado e análise da presença de integrons. A maior parte dos casos de shiguelose estudados aconteceram no verão, estação de maior precipitação de chuvas. Do total de casos, 57,9% das infecções causadas por S. sonnei e 64,4% das causadas por S. flexneri afetaram crianças com menos de 5 anos de idade. Pela análise de ERIC e REP-PCR foram caracterizados grupos de identidade entre as linhagens isoladas em ambas regiões. Pela análise de PFGE caracterizaram-se os genótipos circulantes nas regiões. Não foi possível determinar uma correlação entre a presença dos integrons, o perfil de resistência a antimicrobianos e os resultados obtidos pelos métodos utilizados neste trabalho nas linhagens de S. sonnei pesquisadas. / Abstract: The bacteria of the genus Shigella spp. are responsible for endemic and epidemic intestinal infections called shigellosis. The shigellosis, transmitted by fecal-oral route, mainly through the hands and contaminated water, are responsible for 10% of cases of diarrhea among children under five years of age in developing countries. It is estimated that annually 1.1 million people die from infections caused by Shigella. In Brazil, studies show that shigellosis are among the main causes of diarrhea. The present study was carried out to examine the clonal variation of 119 strains of Shigella spp., including 57 S. flexneri and 62 S. sonnei, isolated from different cases of shigellosis occurred in the metropolitan regions of Campinas and Ribeirão Preto, São Paulo state, by ERIC-PCR, REP-PCR, and PFGE methods and by integrons profile analysis. Most of shigellosis cases happened in summer, season of highest precipitation of rain. Of the total cases, 57.9% of infections caused by S. sonnei and 64.4% of infections caused by S. flexneri affected children under 5 years of age. By analysis of ERIC and REP-PCR groups were characterized for identity between the strains isolated in both regions. By the analysis of PFGE circulating genotypes were characterized in the regions. It was not possible to determine a correlation among the presence of integrons, the profile of resistance to antibiotics and the results obtained by the methods used in this work in strains of S. sonnei investigated. / Doutorado / Doutor em Genetica e Biologia Molecular

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Fresh produce has been implicated in several foodborne outbreaks. A primary site of microbial contamination for produce occurs on the surface during production and handling. An approach to reduce contamination is to sample the surface of produce. This study used different detergent agents at 22&#176;C and 40&#176;C to determine their efficacy for recovery of pathogenic bacteria, from surfaces of several produce types and examined survival of organisms in detergents over time. Strawberries, tomatoes and green leaf lettuce were dip inoculated in a 6-6.5 LOG CFU/ml cocktail of nalidixic acid resistant organisms. After drying, produce were rinsed with either 0.1 % sodium lauryl sulfate (SLS), 0.1% Tween 80, or water at different temperatures. Rinse solutions were plated onto Tryptic Soy agar supplemented with 50-ppm nalidixic acid (TSAN). About 4 LOG CFU/ml of Salmonella, and 3-LOG CFU/ml Shigella were recovered, with slightly lower recovery from tomatoes. Inoculated strawberries rinsed with SLS, displayed minimal recovery at ~1.5-LOG CFU/ml at 22&#176;C, and <1-LOG CFU/ml at 40&#176;C. When whole strawberries treated with SLS were analyzed, few Salmonella were recovered. Lack of recovery of Salmonella rinsed with SLS, suggests SLS may be inactivating Salmonella, especially at elevated temperatures. Detergent solutions were inoculated with 3-LOG CFU/ml cocktail and incubated for up to 32 hours at 22&#176;C, and 40&#176;C. Aliquots were plated onto TSAN at varying times. All solutions at 40&#176;C allowed Shigella to grow. SLS gave initial drops in Salmonella populations followed by slight recovery. SLS may cause an initial injury of Salmonella. While organisms were able to survive in detergents, the application of detergents to produce was no more effective in recovery of organisms from produce than water. / Master of Science

Shigella est l'agent causal de la shigellose, une maladie à transmission oro-fécale, et responsable d'une grande partie des cas diarrhéiques dans les pays en voie de développement. L'infection par Shigella résulte en la colonisation et l'inflammation de la muqueuse colique. Sa virulence est liée à son Système de Sécrétion de Type 3 (SST3) qui agit comme une "seringue" moléculaire pour l'injection des protéines directement dans le cytoplasme de la cellule hôte, via un pore de translocation, provoquant la subversion de sa physiologie et l'internalisation de la bactérie. Le SST3 est composé d'un bulbe cytoplasmique, d'un corps basal et d'une aiguille extracellulaire assemblée suite à la polymérisation hélicoïdale des sous-unités MxiH, formant ainsi un canal qui permet le transit des substrats du SST3. L'extrémité de l'aiguille comprend les translocateurs IpaBCD qui constituent le complexe d’extrémité. IpaD module l'insertion membranaire des protéines hydrophobes IpaB et IpaC, qui forment le pore de translocation, et prévient, avec IpaB, la sécrétion prématurée des effecteurs avant le contact cellulaire. La sécrétion est également contrôlée à la base du SST3 par la protéine cytoplasmique MxiC qui perçoit le contact cellulaire par un signal transmis à travers l'aiguille. Ces deux dispositifs de contrôle participent à l'établissement d'une hiérarchie de sécrétion entre translocateurs, effecteurs précoces et tardifs. <p>Lors de cette étude, nous avons essayé de mieux comprendre le fonctionnement du SST3 en ciblant la protéine IpaD. Des variants obtenus par délétions de 10 acides aminés (Schiavolin, 2013) et par mutations ponctuelles (Meghraoui, 2014) d’IpaD ont été générés pour caractériser leurs effets sur l'exposition à la surface des translocateurs, le contrôle de sécrétion, la formation du pore de translocation, et enfin l'invasion cellulaire. Nos résultats ont permis d’identifier trois phénotypes correspondant à i) une sécrétion contrôlée, similaire à la souche sauvage, ii) une sécrétion constitutive de tous les substrats, et iii) un phénotype de sécrétion intermédiaire. Les variants par délétions nous ont permis de comprendre l'importance de la localisation d'IpaD et IpaB à la surface pour le fonctionnement du SST3. Les variants par mutations ont révélé l'indépendance entre le contrôle de sécrétion et l’invasion cellulaire, ainsi qu’une corrélation entre la sécrétion prématurée des translocateurs/effecteurs précoces et l'augmentation de la virulence in vitro. Nous avons aussi étudié les partenaires d'interaction d'IpaD (Meghraoui, in prep) et MxiC (Cherradi, 2013) respectivement à l'extrémité de l'aiguille et à la base du SST3. L'interaction d'IpaD avec elle-même et avec MxiH a pu être montrée uniquement après délétion du domaine auto-chaperon d’IpaD. Ce domaine semble essentiel au maintien d'IpaB à l'extrémité de l'aiguille, au contrôle de sécrétion, à l'insertion du translocon, mais pas à l'invasion cellulaire. D'autre part, nous avons démontré que l'interaction entre MxiC et le composant de la tige interne MxiI participe au contrôle interne de la sécrétion. En conclusion, nos travaux contribuent à une meilleure compréhension du lien entre différents composants et fonctions du SST3 et de l'implication d'IpaD dans la régulation allostérique de la sécrétion. Ces résultats dépassent le cadre de Shigella puisque les composants étudiés dans le cadre de cette thèse sont conservés chez d’autres bactéries utilisant le SST3 comme dispositif principal de virulence./<p><p>Shigella is the causative agent of shigellosis, an oro-fecally transmitted disease, among major causes of diarrhoea in developing countries. Infection by Shigella results in the colonisation and inflammation of colonic mucosa. The virulence of this bacterium is related to a Type 3 Secretion System (T3SS) that acts as a molecular syringe to inject proteins directly into host cell cytoplasm, through a translocation pore, leading to subversion of cell physiology and bacterial internalisation. The T3S apparatus (T3SA) is composed of a cytoplasmic bulb, a basal body and an extracellular needle. The needle is assembled through the helical polymerisation of MxiH subunits that form a channel allowing the passage of T3S substrates and topped by the translocators IpaBCD (needle tip complex). IpaD is a hydrophilic protein that modulates the membrane insertion of the hydrophobic IpaB and IpaC (translocation pore) and prevents, along with IpaB, the leakage of proteins before cell contact. Secretion is also controlled at the base of the T3SA by cytoplasmic MxiC that senses cell contact through the transmission of a signal along the needle. These two control devices are involved in the establishment of a secretion hierarchy between translocators, early and late effectors.<p>In this study, we aimed to better understand the function of the T3SS by targeting the tip protein IpaD. Ten amino-acid deletion (Schiavolin, 2013) and point mutation (Meghraoui, 2014) variants of IpaD were generated to characterise their effects on translocators exposure, secretion control, pore formation and cell invasion. Three secretion phenotypes were identified and correspond to wild-type like secretion control, constitutive secretion and an intermediate secretion phenotype. Deletion variants allowed us to understand the requirement of IpaB and IpaD surface exposure for the T3SS functions. Mutation variants highlighted the uncoupling between secretion control and cell entry and the correlation between the premature secretion of translocators/early effectors and the enhanced in vitro virulence. We also studied interaction partners of IpaD (Meghraoui, in prep) and MxiC (Cherradi, 2013) at the needle tip and T3SA base, respectively. The bindings of IpaD to itself and to the needle subunit MxiH were only possible after deletion of the self-chaperoning domain. This domain was essential for the correct maintenance of IpaB at the needle tip, the secretion control, the insertion of the translocon, but not for cell entry. Besides, we showed that the interaction of MxiC with inner rod component MxiI participates in the internal control of secretion. In conclusion, these observations facilitated our understanding of the links between the different components and functions of the T3SS and the involvement of IpaD in the allosteric regulation of secretion. Our work is relevant beyond the Shigella field as genes studied here are conserved among several pathogenic bacteria using T3SS as a virulence weapon. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les bactéries du genre Shigella sont responsables de la dysenterie bacillaire, ou shigellose, chez l'être humain, causant plus de 125 millions d'épisodes et 14 000 morts par an. Cette infection est caractérisée par l'inflammation et la destruction de la muqueuse intestinale. La bactérie utilise un système de sécrétion de type III (SST3) pour manipuler la physiologie des cellules épithéliales intestinales et du système immunitaire favorisant l'invasion de la muqueuse et enrayant la mise en place d'une réponse adaptative efficace. Le SST3 peut être comparé à une seringue moléculaire traversant la paroi bactérienne sous la forme d'anneaux membranaires, contenant une tige interne (MxiI), et d’une aiguille extracellulaire (MxiH). L'assemblage de cette dernière se termine par la mise en place d'un complexe d'extrémité formé par plusieurs copies des protéines IpaD et IpaB. Le SST3 prend en charge différentes classes de substrats à sa base via un complexe protéique comprenant l'ATPase Spa47. Les translocateurs (IpaB et IpaC) sont les premiers substrats à être sécrétés. Ceux-ci sont stockés dans le cytoplasme en complexe avec leur chaperon IpgC et sont recrutés à l'extrémité de l'aiguille lors du contact avec la membrane de la cellule hôte pour y former un pore à l'aide de la protéine IpaD. Ce pore permet l'injection des autres substrats du SST3 (effecteurs) qui vont interférer avec les voies de signalisation cellulaire. Il existe deux classes d'effecteurs, les effecteurs précoces (dont OspD1) stockés au préalable dans le cytoplasme et sécrétés suite au contact cellulaire. Ce contact active l’expression des effecteurs tardifs via un couplage assuré par deux complexes, OspD1-MxiE et translocateurs-IpgC. La sécrétion d’OspD1 et des translocateurs libère leurs partenaires qui agissent comme activateurs transcriptionnels. La régulation de la sécrétion dépend de plusieurs acteurs situés dans les différentes parties du SST3. Le complexe d'extrémité et la protéine MxiC contrôlent la sécrétion aux niveaux extra- et intracellulaires alors que l'aiguille transmettrait le signal de sécrétion entre ces deux complexes. Ce paradigme reste cependant encore peu compris et le mode de fonctionnement du complexe d’extrémité et de la protéine MxiC reste à éclaircir.<p>Nos travaux menés sur la protéine IpaD nous ont permis de mettre en évidence un phénotype de sécrétion intermédiaire. Celui-ci est caractérisé par la sécrétion des translocateurs et des effecteurs précoces, sans toutefois observer de sécrétion d’OspD1 et des effecteurs tardifs, suggérant un mécanisme de discrimination entre OspD1 et les effecteurs précoces. Ce phénotype de sécrétion est similaire à celui induit par l’interaction IpaD-désoxycholate. En effet, les variants d’ipaD restant fonctionnels pour la mise en place du pore provoquent également une augmentation de l’insertion des translocateurs et du pouvoir invasif. Nous avons également identifié la région d’IpaD nécessaire au maintien d’IpaB au niveau du complexe d’extrémité ainsi qu’un rôle de son domaine central dans l’insertion du pore. Nous avons enfin étudié l’effet d’anticorps monoclonaux anti-IpaD. Ces résultats nous ont permis de proposer un modèle de fonctionnement du complexe d’extrémité lors de l’insertion du pore, d’identifier les épitopes conférant une protection in vitro et in vivo ainsi que l’existence d’un polymorphisme qui empêche la liaison de ces anticorps à IpaD provenant d’autres sérotypes.<p>Notre étude sur MxiC a mis en évidence de nouveaux partenaires d’interaction (MxiI et IpgC). Ces résultats montrent que l’interaction MxiC-MxiI est nécessaire pour la régulation de la sécrétion des effecteurs précoces par MxiC. De même, la mutation mxiIQ67A provoque un phénotype similaire à la mutation mxiHK69A, ce qui suggère que le mécanisme de régulation impliquant l’aiguille est similaire pour la tige interne. Enfin, l’interaction renforcée MxiC-Spa47, via IpgC probablement couplée à un translocateur, apporte des pistes quant au rôle de MxiC dans la sécrétion des translocateurs.<p>Les rôles identifiés pour les différents régulateurs de la sécrétion ouvrent de nouvelles pistes pour la compréhension du fonctionnement du SST3. Leurs modes de fonctionnement restent cependant encore flous et nécessitent des études complémentaires.<p><p><p>Shigella are responsible for bacillary dysentery, or shigellosis, in human beings causing over 125 million episodes and 14 000 deaths per year. This infection is characterized by inflammation and destruction of the intestinal mucosa. The bacteria use a type III secretion system (T3SS) to manipulate the physiology of intestinal epithelial cells and the immune system favoring the invasion of the mucosa and halting the development of an efficient adaptive response. The T3SS can be compared to a molecular syringe that extends from the bacterial cell wall which contains an internal rod (MxiI), and an extracellular needle (MxiH). The assembly of the latter ends with assembly of a tip complex formed by multiple copies of IpaB and IpaD proteins. The T3SS recruits different classes of substrates at its base via a complex comprising the Spa47 ATPase. The translocators (IpaB and IpaC) are the first substrates to be secreted. They are stored in the cytoplasm in complex with their chaperone (IpgC) and are recruited at the needle tip upon contact with host cell membrane to form a pore via IpaD. This pore allows the injection of other substrates of the T3SS (effectors), which will interfere with the cellular signaling pathways. There are two classes of effectors, early effector (including OspD1) stored in the cytoplasm and secreted upon cell contact. This contact activates the expression of late effectors genes through a complex formed by MxiE (blocked by OspD1) and IpgC. Both proteins are released through OspD1 and translocators secretion. Secretion regulation depends on several actors located at different parts of the T3SS. The tip complex and the gatekeeper MxiC regulate secretion at the T3SS tip and base, the needle subunits transmitting a secretion signal between these two complexes. This paradigm, however, is still poorly understood and the operating mode of the tip complex and MxiC remains unclear.<p><p>Our work on IpaD protein allowed us to identify an intermediate secretion phenotype which is characterized by the secretion of translocators and early effector, but no secretion of OspD1 and late effectors, suggesting a discriminating mechanism between early effectors and OspD1. This secretion phenotype is similar to that induced by deoxycholate-IpaD interaction. Indeed, IpaD point mutants responsible for this phenotype cause an increase in the pore insertion and cell invasion. We also identified the region of IpaD necessary to maintain IpaB at the needle tip as well as a role of IpaD central domain in the pore insertion. We finally studied the effect of anti-IpaD monoclonal antibodies. These results allowed us to propose a working model of the tip complex end upon pore insertion, identify epitopes conferring protection in vitro and in vivo as well as the existence of a polymorphism that prevents the binding of these antibodies to IpaD from other serotypes.<p><p>Our MxiC study showed new interaction partners (MxiI and IpgC). These results showed that the MxiC-MxiI interaction is necessary for the regulation of early effectors secretion of by MxiC. Moreover, a mxiIQ67A mutation causes a phenotype similar to the mutation mxiHK69A, suggesting that the regulatory mechanism involving the needle is shared by the inner rod. Finally, the enhanced interaction MxiC-Spa47 through IpgC, probably in complex with a translocator, provides clues for the role of MxiC in translocators secretion.<p><p>The roles identified for the various regulators of secretion open up new avenues for understanding how the T3SS functions. Their ways of working are however still unclear and require further study.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished