Дисертації: "Microscopic pathogens"

1

Pascault, Alice. "Investigating Candida albicans epithelial infection using a high-throughput microscopy-based assay." Electronic Thesis or Diss., Sorbonne université, 2023. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2023SORUS277.pdf.

Повний текст джерела

Анотація:

Les infections fongiques représentent un problème émergeant de santé publique dans les pays développés. C. albicans est une levure dimorphique, commensale des muqueuses orales, génitales et intestinales d’une majorité de la population saine. Elle peut cependant conduire à des infections locales, comme les candidoses orales et vaginales voire à des infections systémiques chez les patients immunodéprimés. Si les interactions entre C. albicans et le système immunitaire de l’hôte sont désormais de mieux en mieux connues, l’invasion des épithélia qui est pourtant à l’origine des processus d’infection, demeure mal comprise. Quatre étapes ont été décrites : l’adhésion de la levure aux cellules de l’hôte, suivi par la filamentation qui permet d’obtenir une hyphe capable d’invasion et parfois de dommage infligé aux les cellules hôtes pouvant aboutir à une translocation dans les tissus profonds. Plusieurs facteurs ont été décrits, tant du côté de l’hôte que du pathogène, comme jouant un rôle dans l’invasion des epithelia, incluant les adhésines et invasines fongiques, la toxine candidalysine et l’E-cadherin de la cellule hôte. Une étude récente de notre équipe basée sur une technique de microscopie live à l’échelle cellulaire a permis de déterminer que l’invasion précoce des cellules épithéliales HeLa et Caco-2 peut faire intervenir deux modes d’interaction et donc deux modes de vie fongiques distincts : (1) l’invasion conduisant au dommage cellulaire, au cours de laquelle la membrane plasmique de la cellule hôte est rompue, fréquemment suivie de la mort de la cellule hôte (2) l’invasion au travers de tunnels trans-cellulaires (CaTCT) au cours de laquelle les hyphes envahissent les cellules hôtes entourés de tunnels de membrane cellulaire, sans dommage. Ces tunnels peuvent être multicellulaires et sont constitués de couches membranaires successives. Les mécanismes moléculaires conduisant à la formation de ces tunnels tant du point de vue fongique que du point de vue de l’hôte ne sont pas connus. L’objectif de ma thèse était d’identifier et de caractériser les facteurs d’hôte et du pathogène impliqués dans l’infection des cellules épithéliales Caco-2, qui ne fait intervenir que l’invasion au travers de CaTCT jusqu’à 9 heures d’infection. A cette fin j’ai développé un outil expérimental de microscopie permettant de quantifier de façon objective, universelle (i.e. pouvant s’appliquer à de nombreux modèles fongiques et de cellules hôtes) et à haut débit l’adhésion, la filamentation l’invasion et le dommage cellulaire au cours d’une seule expérience d’infection in vitro. Cet outil a ensuite été utilisé afin d’étudier plusieurs aspects dede la formation des CaTCT : (1) le rôle de la protéine fongique Als3 au cours de l’adhésion et de l’invasion (2) l’origine cellulaire des membranes impliquées dans les CaTCT (3) le rôle de la toxine peptidique candidalysine et des aspartyl-protéases (4) le métabolisme fongique au cours de l’invasion et notamment du métabolisme du glycogène (5) l’immunité de l’hôte au niveau des muqueuses au travers de la secrétion d’IgA. Le protocole mis en place m’a également permis de screener des souches cliniques commensales et issues d’infections systémiques afin de rechercher de nouveaux facteurs de virulence fongiques. Pour conclure, la nouvelle méthode expérimentale développée au cours de ce projet a permis quelques avancées dans la compréhension du processus énigmatique des CaTCT, et pourra servir à de futures études de l’infection de epithelia par C. albicans mais aussi à celles d’autres pathogènes
Fungal infections are an emerging threat to human health in developed countries. Candida albicans is a dimorphic yeast which colonizes the oral, genital and intestinal mucosa as part of the commensal flora of most of the healthy population. However, it can also lead to local infections such as oral and vaginal thrush and in susceptible patients to severe systemic infections. While much effort has been made in deciphering the interplay between C. albicans and the host at the immunological level, infection begins with invasion of the host epithelium, a process that is only partially understood. At the onset of infection, C. albicans transforms from a yeast to a filamentous hyphal form that can invade and damage epithelial cells, sometimes followed by translocation deeper into host tissues. Several fungal and host molecular factors have been shown to regulate epithelial invasion, including fungal adhesins, invasins and secreted factors such as the fungal toxin candidalysin, as well as host factors such as E-cadherin, which plays a role in C. albicans endocytic uptake. Recent work from our lab based on single cell, live imaging of early invasion into HeLa and Caco-2 cell lines revealed that two invasive lifestyles involving distinct host cellular niches can be exploited by the fungus: (1) Damaging invasion, in which host membranes are breached, leading most often to host cell death; (2) C. albicans trans-cellular tunnelling (CaTCT), in which hyphae extend within host membrane-derived transcellular tunnels without host damage. During CaTCT, hyphae can traverse through several host cells in sequence, leading to the formation of multi-layered tunnel structures. Currently, the molecular factors and cellular mechanisms regulating CaTCT from both the fungal and host sides remain almost entirely undescribed. The objective of my thesis project was to identify and characterize molecular factors and cellular processes regulating early Caco-2 infection by C. albicans, which occurs exclusively via CaTCT for up to 9 hours post-infection. For this purpose, I developed a novel quantitative, high-throughput and universal (i.e. applicable to a wide variety of fungal and host models) experimental imaging assay that uses an automated non-biased approach to provide single cell readouts pertaining to adhesion, hyphal formation, invasion and host damage in a single experiment. I then applied this assay to study several distinct aspects of CaTCT: (1) the function of the fungal Als3 protein in C. albicans adhesion and invasion; (2) the reservoir of host membranes implicated in trans-cellular tunnel formation and extension; (3) the function of the fungal toxin candidalysin and fungal secreted aspartyl proteases (Saps); (4) nutrient uptake and glycogen metabolism ; (5) the role of host secreted IgA in immune defence at the epithelial surface. In order to identify new potential virulence factors, I also employed the assay to screen for differences in epithelial infection between C. albicans clinical strains isolated from commensal and invasive origins. Overall, my work has provided several new insights into the mechanism of CaTCT, which act to further enhance our knowledge of this enigmatic process. Furthermore, the experimental assay developed in this project has important potential applications for future targeted studies and screens relating to C. albicans epithelial infection, as well as infection by other fungal pathogens

Стилі APA, Harvard, Vancouver, ISO та ін.

2

Galtier, Eloïse. "Etude du dégradosome à ARN de la bactérie pathogène Helicobacter pylori." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC002.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

3

Lin, Xiaonan. "Chemical and Cellular Defenses against Foreign Pathogens." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10354.

Повний текст джерела

Анотація:

Bacterial and viral infections affect billions of people each year. While bacterial infections are treated by the use of antibiotics, viral infections are eradicated by the immune system. This thesis comprises two parts. Part I presents the reconstitution of enzymes required for the synthesis of the minimal pharmacophore of moenomycin A (MmA), a molecule with antibacterial activity. Part II details single-particle electron microscopy studies of MDA5 alone and in complex with double-stranded RNA (dsRNA). MmA is a natural product antibiotic from Streptomyces ghanaensis that possesses remarkable potency against clinically relevant Gram-positive bacteria. MmA exerts its antibacterial activity by binding directly to peptidoglycan glycosyltransferases, enzymes involved in the synthesis of the glycan strands of peptidoglycan. The genes responsible for MmA biosynthesis have been identified, and a complete biosynthetic pathway has been proposed. Part I of this thesis describes the reconstitution of enzymes required for the synthesis of two trisaccharide scaffolds of MmA that retain antibacterial activity. It also describes the synthesis of unnatural phosphoglycerate lipid acceptors and UDP-amino sugars that can be used to probe the substrate tolerances of key glycosyltransferases in MmA biosynthesis. This work lays the foundation for the synthesis of unnatural MmA analogs that may possess better pharmacokinetic properties than the parent molecule. MDA5 is a helicase that detects viral dsRNA in the cytoplasm of vertebrate cells. Upon dsRNA recognition, MDA5 initiates a series of signal transduction events that activate the innate immune response. Part II of this thesis presents the structures of apo MDA5 protein and the MDA5-dsRNA complex obtained by using single-particle electron microscopy. Two-dimensional averages of apo MDA5 revealed that the protein is very flexible and can adopt multiple conformations. This finding suggests that MDA5 does not adopt an autorepressed conformation in the absence of viral dsRNA. When MDA5 is incubated with dsRNA, the protein assembles onto the dsRNA to form a linear oligomer. Two-dimensional averages and a three-dimensional reconstruction reveal the complex to consist of seven to eight stacked discs per strand of 112 base pair dsRNA. This work lays the foundation for further structural studies aimed at elucidating the mechanism by which MDA5 is activated.
Chemistry and Chemical Biology

Стилі APA, Harvard, Vancouver, ISO та ін.

4

Ijaz, Usman. "Molecular Mapping and Microscopic analysis of Faba Bean- Uromyces viciae-fabae Host-Pathogen Interaction." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/18416.

Повний текст джерела

Анотація:

This investigation covered characterisation of various faba bean rust isolates by identifying a differential set in the host; identification of molecular markers linked with rust resistance genes Uvf-2 in Doza#12034 and Uvf-3 in Ac1655 and their validation across diverse backgrounds; and elucidation of the host-pathogen interactions of Uromyces viciae-fabae with faba bean, field pea (Pisum sativum), lentil (Lens culinaris), chickpea (Cicer arietinum), lupin (Lupinus albus and Lupinus angustifolius) and mungbean (Vigna radiata). The differential pathogenicity in the interaction of Vicia faba × Uromyces viciae-fabae led to the identification of nine faba bean rust pathotypes, characterised by the set of twelve genotypes (regarded as differential), and named 0-10, 0-46, 40-31, 40-55, 24-40, 63-53, 63-49, 55-63 and 63-63. This information will help faba bean breeders to carefully deploy rust resistance gene(s) which can effectively insight resistance against pathotypes of targeted environment. Genetic analysis, using pathotype 24-40, revealed monogenic inheritance of rust resistance in faba bean genotypes Doza#12034 and Ac1655, respectively. After genotyping Fiord/Doza#12034 and Fiord/Ac1655 RILs, two closely linked KASP markers KASP_Vf_0703 and KASP_Ac×F165 were mapped on chromosome III and V with Uvf-2 and Uvf-3, respectively and validated successfully in a set of local/exotic faba bean genotypes. These closely linked markers will allow breeders to implement markers assisted selection for both resistance genes. The histopathology of Australian U. viciae-fabae revealed a host range: both faba bean and field pea were competent hosts showing varietal differences to pathogen responses, with differential expression in resistance; lentil showed complete hypersensitive resistance by expressing cell death; and chickpea, lupin and mungbean appeared as non-hosts.

Стилі APA, Harvard, Vancouver, ISO та ін.

5

Alves, Eduardo. "Xylella fastidiosa adesão e colonização em vasos do xilema de laranjeira doce, cafeeiro, ameixeira, fumo e espécies de cigarrinhas vetoras e formação de biofilme sobre película de poliestireno." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-09052003-141508/.

Повний текст джерела

Анотація:

X. fastidiosa é uma bactéria fitopatogênica limitada ao xilema, que tem afetado um grande número de plantas no Brasil e no mundo. Muitos trabalhos já foram realizados sobre esta bactéria, mas pouco se conhece a respeito da adesão, colonização e expressão dos sintomas. Os objetivos deste trabalho foram: a) através do uso da microscopia eletrônica e de luz, determinar e relacionar o número de vasos colonizados de citros, cafeeiro e ameixeira com a sintomatologia em folhas; b) estudar a adesão, migração radial e colonização dos vasos do xilema do pecíolo de folhas de citros pela bactéria; c) estudar algumas variáveis experimentais que afetam a expressão dos sintomas em fumo; d) verificar os sítios de ligação da bactéria em cigarrinhas vetores; e) estudar a adesão e a formação do biofilme por X. fastidiosa em superfície de poliestireno, como uma nova metodologia. Os resultados mostraram em ameixeira e cafeeiro uma relação entre o número de vasos colonizados e a expressão de sintomas necróticos, relação esta que não pode ser observada para citros, o qual apresentava um número de vasos colonizados do pecíolo bem menor que o das outras duas espécies. No estudo da bactéria nos vaso do xilema de citros foi possível verificar as diversas fases do processo de colonização do xilema, bem como a capacidade da bactéria em degradar a parede celular primária da pontuação e migrar para os vasos adjacentes. Neste estudo foi também possível verificar respostas da planta à bactérias caracterizadas pela produção de cristais no lúmen dos vasos do xilema e o acúmulo de goma e hiperplasia de células nas folhas. No estudo com variedades de fumo verificou-se que a cultivar Havana apresentou expressão de sintomas mais intensa que as variedades TNN e RP1 e que o aparecimento dos mesmos não foi influenciado pelo volume de inóculo e pelo local de inoculação, mas sim pela adubação com sulfato de amônio, a qual retardou o aparecimento dos sintomas e reverteu os sintomas inicias em folhas após a aplicação. Em cigarrinhas, células bacterianas com morfologia similar as de X. fastidiosa, foram visualizadas aderidas pela parte lateral na câmara do cibário (sulco longitudinal, parede lateral e membrana do diafragma) de Acrogonia citrina, e Oncometopia facialis, no canal do apodeme de Dilobopterus costalimai e pela parte polar no precibário de O. facialis. Finalmente, no estudo da adesão de X. fastidiosa a película de poliestireno, os resultados revelaram as várias fases da formação do biofilme, aspectos da sua arquitetura, e indicaram que a técnica é uma ferramenta adequada para o estudo da formação do biofilme e também da morfologia das bactérias. Os resultados são discutidos em termos de modelos de adesão e colonização, da bactéria e importância para o conhecimento dos mecanismos de patogenicidade da bactéria em plantas e transmissão pelos vetores.
X. fastidiosa is a xylem-limited bacterium that has been affecting a high number of plants in Brazil and in the world. A lot of researches were already accomplished on this bacterium, but little is known regarding the adhesion, colonization and expression of the symptoms in plants. The objectives of this work were: a) through the use of electron microscopy and of light microscopy determine and to correlate the number of xylem colonized vessels of petiole of sweet orange, coffee and plum with chlorosis and leaf scorching in leaves; b) study the adhesion, radial migration and colonization of the vessels of the petiole xylem of sweet orange by the bacterium; c) study some experimental variables that affect the expression of symptoms in tobacco; d) verify the retention sites of the bacterium in sharpshooters; d) study the adhesion and biofilm formation by X. fastidiosa on polystyrene surface. The results showed a relationship between the number of colonized vessels in plum and coffee and the expression of necrotic symptoms. However, that relationship was not observed for sweet orange, which presented a number of colonized vessels smaller than the other two species. In the study of the bacterium in the xylem vessels of sweet orange it was possible to verify the several phases of the colonization process of the xylem as well as the ability of the bacterium to degrade the primary cell wall of the pit and migrate to adjacent vessels. It was also possible to verify responses of the plant to the bacterium characterized by the production of crystals in the lumen of the xylem vessels and gum accumulation and hyperplasia in the leaf cells. Regarding the tobacco varieties it was verified that the expression of symptoms is more intense in the cultivar Havana than in the cultivars TNN and RP1. It was also seen that symptoms expression was not influenced by the inoculum volume or the inoculation place, but it was altered by fertilization with ammonium sulfate, which delayed the beginning of the symptoms and reverted the symptoms in leaves after the application. In sharpshooters, bacterial cells exhibiting morphology similar to X. fastidiosa were visualized attached to the lateral side in the cibarium camera (longitudinal, lateral wall and membrane of the diaphragm) of Acrogonia citrina, and Oncometopia facialis, in the apodemal channel of Dilobopterus costalimai, and in the polar part in the pre-cibarium of O. facialis. Finally, in the study of the adhesion of X. fastidiosa on polystyrene surface, the results revealed the several phases of biofilm formation; aspects of its architecture, and it also indicated that the technique is an appropriate tool to study of the formation of biofilms and also of the bacterial morphology. The results are discussed regarding adhesion models, colonization, and distribution of the bacterium in the plant and the importance of knowing the pathogenicity mechanisms of X. fastidiosa and its transmission by the insect vectors.

Стилі APA, Harvard, Vancouver, ISO та ін.

6

Morán, Cruz Gabriela. "Luminescent surfaces to fight or detect bacteria." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS214/document.

Повний текст джерела

Анотація:

Le 20ème siècle a vu le recul des maladies infectieuses grâce aux antibiotiques. Cependant leur importante utilisation a rendu certaines bactéries, comme Staphylococcus aureus ou Pseudomonas aeruginosa (multi)résistantes. Un des moyens de lutte est de réduire la consommation d’antibiotiques ou de cibler ceux qui seront actif sur une souche identifiée. Nous souhaitons développer des surfaces et des dispositifs sensibles pour la détection précoce, rapide de bactéries pathogènes dans des fluides. Cela permettra de limiter la contamination et donc l’usage de médicaments. Ce projet regroupe 3 partenaires qui travaillent en synergie en mettant à profit leur expertise en physico-chimie, chimie de synthèse et microbiologie. Des nano-objets fluorescents, biocompatibles, et sensibles à la croissance bactérienne seront immobilisés sur des surfaces de verre. Ils seront rendus sélectifs de bactéries pathogènes par des traitements post-synthétiques. Il s’agit in fine de mettre au point un dispositif de détection miniaturisé et de tester la résistance aux antibiotiques des pathogènes détectés
Infectious diseases have recessed during the 20th century thanks to antibiotics. However, some bacterial strains like Staphylococcus aureus or Pseudomonas aeruginosa have become (multi)resistant to antibiotic treatments because of overuse. One way to combat this is to reduce consumption of drugs or to better target those that will eliminate a given strain. We wish to develop sensitive surfaces and devices for the early and rapid detection of pathogenic bacteria in fluids. They will help limit contaminations and the use of drugs. The project gathers 3 partners working in synergy because they combine expertise in physical-chemistry, synthetic chemistry and microbiology. Fluorescent nanoobjects that are biocompatible and sensitive to bacterial growth will be immobilized on glass surfaces. They will be selective for pathogenic bacteria by post-synthetic modifications. The final goal is to build miniaturized sensitive devices that can detect pathogens and further test their resistance to antibiotics

Стилі APA, Harvard, Vancouver, ISO та ін.

7

Logan, Savannah. "Imaging Vibrio Cholerae Invasion and Developing New Tools for 3D Microscopy of Live Animals." Thesis, University of Oregon, 2019. http://hdl.handle.net/1794/24524.

Повний текст джерела

Анотація:

All animals harbor microorganisms that interact with each other and with their hosts. These microorganisms play important roles in health, disease, and defense against pathogens. The microbial communities in the intestine are particularly important in preventing colonization by pathogens; however, this defense mechanism and the means by which pathogens overcome it remain largely unknown. Moreover, while the composition of animal-associated microbial communities has been studied in great depth, the spatial and temporal dynamics of these communities has only recently begun to be explored. Here, we use a transparent model organism, larval zebrafish, to study how a human pathogen, Vibrio cholerae, invades intestinal communities. We pay particular attention to a bacterial competition mechanism, the type VI secrection system (T6SS), in this process. In vivo 3D fluorescence imaging and differential contrast imaging of transparent host tissue allow us to establish that V. cholerae can use the T6SS to modulate the intestinal mechanics of its host to displace established bacterial communities, and we demonstrate that one part of the T6SS apparatus, the actin crosslinking domain, is responsible for this function. Next, we develop an automated high-throughput light sheet fluorescence microscope to allow rapid imaging of bacterial communities and host cells in live larval zebrafish. Light sheet fluorescence microscopy (LSFM) has been limited in the past by low throughput and tedious sample preparation, and our new microscope features an integrated fluidic circuit and automated positioning and imaging to address these issues and allow faster collection of larger datasets, which will considerably expand the use of LSFM in the life sciences. This microscope could also be used for future experiments related to bacterial communities and the immune system. The overarching theme of the work in this dissertation is the use and development of advanced imaging techniques to make new biological discoveries, and the conclusions of this work point the way toward understanding pathogenic invasion, maximizing the use of LSFM in the life sciences, and gaining a better grasp of host-associated bacterial community dynamics. This dissertation includes previously published and unpublished co-authored material.

Стилі APA, Harvard, Vancouver, ISO та ін.

8

Lee, Miin-Huey. "Microscopic, physiological and molecular studies of pathogenesis in Monilinia fructicola, the brown rot pathogen for stone fruits /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

9

Lavik, John-Paul. "Intravital Microscopy of Borrelia burgdorferi: Delineation of Dissemination Kinetics and Persistence Within Murine Skin." University of Toledo Health Science Campus / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=mco1340104556.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

10

Theodoropoulos, Christina. "Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro." Thesis, Queensland University of Technology, 2003. https://eprints.qut.edu.au/37160/6/37160_Digitised%20Thesis.pdf.

Повний текст джерела

Анотація:

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Стилі APA, Harvard, Vancouver, ISO та ін.

11

PRATES, RENATO A. "Avaliacao dos efeitos da terapia fotodinamica antimicrobiana sobre leveduras patogenicas." reponame:Repositório Institucional do IPEN, 2010. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9536.

Повний текст джерела

Анотація:

Made available in DSpace on 2014-10-09T12:27:45Z (GMT). No. of bitstreams: 0
Made available in DSpace on 2014-10-09T14:05:04Z (GMT). No. of bitstreams: 0
Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Стилі APA, Harvard, Vancouver, ISO та ін.

12

Götz, Ralph [Verfasser], Markus [Gutachter] Sauer, Michael [Gutachter] Hudecek, and Sören [Gutachter] Doose. "Super-resolution microscopy of plasma membrane receptors and intracellular pathogens / Ralph Götz ; Gutachter: Markus Sauer, Michael Hudecek, Sören Doose." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1213659752/34.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

13

Vieira, Flávia Campos Freitas [UNESP]. "Anatomia e ultraestrutura do processo de infecção de Xanthomonas causadoras de doenças em citros." Universidade Estadual Paulista (UNESP), 2018. http://hdl.handle.net/11449/154415.

Повний текст джерела

Анотація:

Submitted by FLÁVIA CAMPOS FREITAS VIEIRA (flavia.camposfv@yahoo.com.br) on 2018-06-28T14:09:18Z No. of bitstreams: 1 Tese_Flávia_Vieira_2018.pdf: 8871588 bytes, checksum: e1ee1b7f2e2e7170f04d44c1a1e76d6a (MD5)
Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-06-29T18:42:12Z (GMT) No. of bitstreams: 1 vieira_fcf_dr_jabo.pdf: 8871588 bytes, checksum: e1ee1b7f2e2e7170f04d44c1a1e76d6a (MD5)
Made available in DSpace on 2018-06-29T18:42:12Z (GMT). No. of bitstreams: 1 vieira_fcf_dr_jabo.pdf: 8871588 bytes, checksum: e1ee1b7f2e2e7170f04d44c1a1e76d6a (MD5) Previous issue date: 2018-06-04
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Os citros são um grupo de espécies vegetais, com alto potencial produtivo, porém, acometido por várias doenças. Do gênero de bactérias Xanthomonas, três espécies são patogênicas a citros: Xanthomonas citri subsp. citri, agente causal do cancro cítrico, uma das doenças de maior preocupação da citricultura; X. fuscans subsp. aurantifolii tipos B e C, que causam a cancrose; e X. alfalfae subsp. citrumelonis, responsável por causar a mancha bacteriana dos citros. Diante disso, o objetivo desse trabalho foi a análise e descrição, em nível anatômico e ultraestrutural, dos processos de infecção de bactérias do gênero Xanthomonas, causadoras de doenças em citros, a correlação do fenótipo da interação XfaC-citros com o transcriptoma e a análise dos fatores de virulência das estirpes estudadas. O processo de infecção das estirpes Xac306, Xac636, Xac828, Xacm1510, XfaC1632 e XfaC1630 em lima ácida ‘Galego’ e/ou laranja doce ‘Hamlin’, foi avaliado em diferentes períodos de infecção, pelas técnicas de microscopia de luz e eletrônicas de varredura e transmissão. As seguintes características relacionadas a virulência das estirpes foram avaliadas: formação in vitro de biofilme, produção de goma, motilidade “swimming” e auto agregação celular. Os resultados mostraram que Xac306 é a estirpe mais agressiva dentre as demais, visto pelos sintomas e pela intensidade das alterações anatômicas no tecido de ‘Hamlin’ e ‘Galego’. O processo de infecção em lima ácida ‘Galego’ foi semelhante em todas as estirpes de Xanthomonas estudadas, pois induziram hiperplasia e hipertrofia, porém, em diferentes intensidades, sendo que as alterações provocadas pelas estirpes Xac828 e Xacm1510 não resultaram no rompimento da epiderme; todas as estirpes produziram goma, envolvendo as bactérias e favorecendo a colonização no ambiente intercelular. A microscopia de luz permitiu identificar alterações na anatomia de lima ácida ‘Galego’ infectada e a diferença de susceptibilidade entre as variedades de citros inoculadas com Xac306 e estirpes de XfaC. As microscopias eletrônicas de varredura e transmissão permitiram a identificação de aspectos em comum às estirpes, independente da variedade cítrica. A HR induzida por estirpes de XfaC foi caracterizada pela microscopia eletrônica de transmissão. Com a análise de microscopia, não foi possível estabelecer uma relação direta entre os testes de formação in vitro de biofilme, produção de goma e motilidade “swimming” das estirpes. A análise dos dados de transcriptoma revelou que a resposta de defesa de laranja ‘Hamlin’ contra XfaC foi uma resposta rápida e eficiente que levou a indução de HR, que em última instância, conteve a infecção pela morte celular programada no local da infecção. Esse trabalho ampliou o conhecimento da área sobre o comportamento de diferentes estirpes de Xanthomonas causadoras de doenças em citros, fornecendo informações sobre sua interação com o hospedeiro suscetível e a resposta de defesa de XfaC em interação incompatível.
Citrus are a diverse group of plant species, with high yield potential, but affected by several diseases. The bacteria genus Xanthomonas has three species pathogenic to citrus: Xanthomonas citri subsp. citri, causal agent of citrus canker, one of the most important diseases of citrus crop; X. fuscans subsp. aurantifolii types B and C, causing citrus cancrosis; and X. alfalfae subsp. citrumelonis, which causes citrus bacterial spot. Therefore, the goal of this work was to analyze and describe, at the anatomical and ultrastructural level the infectious processes of Xanthomonas causing diseases in citrus, the correlation of the XfaC-citrus interaction phenotype with the transcriptome and the comparative analysis of virulence related factors, in order to elucidate morphophysiological traits of host-pathogen interaction. The infection process of Xac306, Xac636, Xac828, Xacm1510, XfaC1632 and XfaC1630 strains in 'Galego' Mexican lime and 'Hamlin' sweet orange were evaluated at different points of infection by light microscopy and scanning and transmission electron microscopy. The following virulence related characteristics of the strains were evaluated: in vitro biofilm formation, gum production, swimming motility and cellular auto aggregation. The results showed that Xac306 is the most aggressive strain among the others, due to the intensity of the symptoms and anatomical alterations in 'Hamlin' and 'Galego' tissue. The infection process in the susceptible host ('Galego' Mexican lime) was similar for all Xanthomonas strains studied, since they induced hyperplasia and hypertrophy, however in different intensities, and the alterations caused by Xac828 and Xacm1510 strains did not result in epidermis rupture; all strains produce gum, involving bacteria and assisting colonization in the intercellular environment. Light microscopy was able to identify anatomical alterations in 'Galego' Mexican lime infected tissue and the susceptibility differences between citrus varieties inoculated with Xac306 and XfaC strains. The scanning and transmission electron microscopies was able to identify common traits to the strains, regardless of the citrus variety. The HR induced by XfaC strains was characterized by transmission electron microscopy. We could not stablish a direct relation between the in vitro biofilm formation, gum production and "swimming" motility of the strains with the microscopy analysis. The transcriptome analysis revealed that 'Hamlin' defense response against XfaC is a quick and effective response that led to an HR induction, which ultimately contained the infection, by programmed cell death at the site of infection. This work expanded our knowledge about the behavior of different Xanthomonas strains causing citrus diseases, providing information on its interaction with the susceptible host and the defense response of XfaC in an incompatible interaction.

Стилі APA, Harvard, Vancouver, ISO та ін.

14

Liu, Yang Li Men`gshi. "Study of antimicrobial activity and mechanism of zinc oxide nanoparticles against foodborne pathogens." Diss., Columbia, Mo. : University of Missouri-Columbia, 2009. http://hdl.handle.net/10355/6718.

Повний текст джерела

Анотація:

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 23, 2010). Thesis advisor: Dr. Mengshi Lin. Includes bibliographical references.

Стилі APA, Harvard, Vancouver, ISO та ін.

15

Dafalla, Elgasim Abdalla. "Contribution à l'étude des jaunisses à mycoplasmes au Soudan : inventaire partiel dans la région centrale : étude de la phyllodie de la fèverole : premiers essais de caractérisation sérologique des agents pathogènes." Paris 11, 1987. http://www.theses.fr/1987PA112309.

Повний текст джерела

Анотація:

Un inventaire des maladies susceptibles d'être liées à la présence de MLO (Mycoplasma-like-Organisms) a été entrepris au SOUDAN. Chez cinq d'entre elles : la maladie des petites feuilles d'Eucalyptus microtheca, la phyllodie de Catharanthus roseus, la phyllodie de Crotalaria Saltiana, les feuilles blanches de Cynodon dactylon et la virescence de Zinnia elegans, la présence de ces microorganismes a été décrite. L'importance économique et écologique de ces maladies résulte du rôle joué par ces espèces végétales au Soudan. L'Eucalyptus est utilisé pour limiter l'extension du désert, C. Roseus pour l'extraction de substances médicinales Crotalaria sp, Cynodon dactylon interviennent dans l'équilibre des écosystèmes. La détection rapide de l'infection en microscopie photonique à fluorescence (M. P. ), l'étude en microscopie électronique par transmission (MET) de l'ultrastructure de l'agent pathogène sur coupe ultrafine et de sa morphologie tridimensionnelle sur coupes épaisses ont été réalisées. Différents aspects de la Phyllodie de la féverole ont été étudiés. L'incidence des variétés et l'effet de la température sur l'évolution des symptômes sont décrits. La variété française Ascott est beaucoup moins affectée par la maladie que les variétés soudanaises les plus couramment utilisées : BF 2/2, Hudeiba et Selaim. La transmission expérimentale de l'infection à quatre espèces de légumineuses : Phaseolus vulgaris, Medicago saliva, Lablab vulgaris et Cajanus cajanus par greffage et à un hôte différentiel C. Roseus par Cuscuta subinclusa a été obtenue. Le rôle de C. Saltiania comme réservoir de la maladie a été mis en évidence. L'étude en MET sur coupes ultrafines et sur coupes épaisses a confirmé la présence de MLO. La concentration importante de ces microorganismes chez la féverole et la structure anatomique des tubes criblés de cette plante ont facilité la mise au point d'une méthode de détection rapide en M. P. Des extraits semi-purifiés de : Vicia faba et C. Roseus infectés par la phyllodie de la féverole et de C. Roseus et Nicotiana tabacum atteints du Stolbur des solanacées ainsi que des plantes saines correspondantes ont été utilisés pour immuniser des souris. L'immunofluorescence indirecte (IF) a été comparée à l'autofluorescence observée en lumière bleue et à la fluorescence secondaire de l'ADN observée en lumière U. V. Les résultats obtenus avec les anticorps polyclonaux ainsi préparés ont montré une amélioration de la spécificité de l'immunofluorescence comparativement aux deux autres modes d'observation. Elle a permis la distinction des deux maladies. Ces études ont été effectuées sur coupes de matériel frais et de matériel fixé, inclus dans la paraffine.

Стилі APA, Harvard, Vancouver, ISO та ін.

16

Adonne, Moulin-Traffort Joëlle. "Etude cytologique ultrastructurale de l'action de quelques substances antifongiques sur les champignons pathogènes." Aix-Marseille 2, 1990. http://www.theses.fr/1990AIX22994.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

17

Emerson, Ray Jenkins. "A nanoscale investigation of pathogenic microbial adhesion in biomaterial systems." Link to electronic dissertation, 2006. http://www.wpi.edu/Pubs/ETD/Available/etd-042706-075421/.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

18

Wall, Michael L. "The Starch Granule Surface: Technological and Biological Implications of Puroindoline and Host-pathogen Interactions." Thesis, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19731.

Повний текст джерела

Анотація:

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identiﬁed the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

Стилі APA, Harvard, Vancouver, ISO та ін.

19

Ansari, Aiysha R. "Comparison of Visual vs. Microscopic Methods to Detect Blood Splatter from an Intravascular Catheter with Engineered Sharps Injury Protection (ESIP)." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/3951.

Повний текст джерела

Анотація:

Intravascular devices with engineered sharps injury protection (ESIP) are designed to reduce sharps injuries, but have not been investigated for blood splatter potential. In this laboratory-based experiment, which did not use human subjects, 100 intravenous catheters of the same type with a retraction mechanism, were tested for blood splatter. Once blood was obtained from a simulated brachial vein containing mock venous blood, the devices were placed in a testing chamber and scientific filters labeled A, B & C were used to capture blood splatter after activation. The blood splatter was examined visually and microscopically, and the filters were weighed pre- and post-activation on an analytical scale. The research questions in this study were: 1) do retractable intravenous devices produce blood splatter, and 2) does blood splatter frequency differ between visual methods vs. microscopy? The differences in filter mass, visual inspection, and microscopic analysis for presence of blood on filters were the units of analysis. Descriptive statistics, paired t-tests to determine pre and post activation filter weights and kappa statistics to assess degree of agreement between methods were used to analyze the data. For filters B and C, the proportions with blood detected by the naked eye were 12 and 13% respectively. However, for filter A, both visual and microscopic methods detected blood splatter on 70% and 71% of the time respectively. In addition, a statistically significant difference was observed in the mean mass of filter A between pre- and post-activation confirmed by the naked eye (t= - 0.0013, p= 0.01400) and confirmed microscopically (t= - 0.00014, p=0.0092). Substantial agreement between methods was observed for filter A (kappa=0.78; 95% CI: 0.64-0.92), filter B (kappa= 0.73; 95% CI: 0.51-0.95) and filter C (kappa= 0.75; 95% CI: 0.55-0.96). However, in 7 instances (7%), blood was detected by microscopy but not by the naked eye on filters A (5 %), B (1%), and C (1%), respectively. Also, in 6 instances (6%), blood was detected by the naked eye but now by microscopy on filter B (3%), and filter C (3%). Consequently, there is potential for a total of 13 % blood splatter. The findings indicate potential for bloodborne pathogen exposure with use of a specific retractable intravascular catheter. The finding that blood splatter was detected by microscopy in 7% of the instances has important occupational health implications. Healthcare workers (HCWs) may not be able to detect this blood splatter when it occurs and may not report a splash to mucous membranes or non-intact skin. This study therefore reinforces the need for HCWs to wear personal protective equipment, such as masks, face shields, goggles, when using intravascular catheters with retractable mechanisms. It is recommended that the research protocol used in this study be replicated by other investigators and tested on all brands of retractable intravascular devices.

Стилі APA, Harvard, Vancouver, ISO та ін.

20

Franke, JanaLynn. "Identification of the Infection Route of a Fusarium Seed Pathogen into Non-Dormant Bromus tectorum Seeds." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/4318.

Повний текст джерела

Анотація:

The genus Fusarium has a wide host range and causes many different forms of plant disease. These include seed rot and seedling blight diseases of cultivated plants. The Fusarium-caused diseases of wild plants are less well-known. In this study we examined Fusarium sp. n-caused disease development on non-dormant seeds of the important rangeland weed Bromus tectorum as part of broader studies of the phenomenon of stand failure or ‘die-off’ in this annual grass. We previously isolated an undescribed species in the Fusarium tricinctum species complex from die-off soils and showed that it is pathogenic on seeds. It can cause high mortality of non-dormant B. tectorum seeds, especially under conditions of water stress, but rarely attacks dormant seeds. In this study, we used scanning electron microscopy (SEM) to investigate the mode of attack used by this pathogen. Non-dormant B. tectorum seeds (i.e., florets containing caryopses) were inoculated with isolate Skull C1 macroconidia. Seeds were then exposed to water stress conditions (-1.5MPa) for 7 d, then transferred to free water. Time lapse SEM photographs of healthy vs. infected seeds revealed that hyphae under water stress conditions grew toward and culminated their attack at the abscission layer of the floret attachment scar. A prominent infection cushion, apparent macroscopically as a white tuft of mycelium at the radicle end of the seed, developed within 48 hours after inoculation. Seeds which lacked an infection cushion completed germination upon transfer to free water, whereas seeds with an infection cushion were almost always killed. In addition, hyphae on seeds that did not initiate germination lacked directional growth and did not develop the infection cushion. This strongly suggests that the fungal attack is triggered by seed exudates released through the floret attachment scar at the initiation of germination. Images of cross-sections of infected seeds showed that the fungal hyphae first penetrated the caryposis wall, then entered the embryo, and later ramified throughout the endosperm, completely destroying the seed.

Стилі APA, Harvard, Vancouver, ISO та ін.

21

Abou, al Fadil Taissir Dechamp-Guillaume Grégory. "Déterminisme de la tolérance du tournesol a Phoma Macdonaldii au collet et sur racines approches génétique et histologiques /." Toulouse : INP Toulouse, 2006. http://ethesis.inp-toulouse.fr/archive/00000300.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

22

Koyyappurath, Sayuj. "Étude histo-pathologique et moléculaire de la résistance des vanilliers (Vanilla spp., Orchidaceae) à Fusarium oxysporum f.sp. radicis-vanillae, agent de la pourriture des racines et des tiges." Thesis, La Réunion, 2015. http://www.theses.fr/2015LARE0008/document.

Повний текст джерела

Анотація:

La production de vanille (Vanilla planifolia, Orchidaceae) est limitée par la pourriture des racines et des tiges (PRT) provoquée par des champignons telluriques du genre Fusarium contre lesquels la lutte chimique ou prophylactique est inefficace. Ce travail décrit finement l'interaction entre les vanilliers et les Fusarium dans le but de développer la lutte génétique. L'analyse moléculaire (gène EF1α et IGS) et la détermination du pouvoir pathogène de 365 souches de Fusarium isolées de vanilliers à La Réunion et à Madagascar ont démontré que les souches pathogènes sur vanillier appartiennent presque toutes au complexe d'espèce F. oxysporum et ont une origine polyphylétique. Les observations en microscopie à champ large et multi-photons démontrent que la colonisation des racines débute au niveau des poils absorbants puis gagne le cortex, mais que le système vasculaire est épargné. Cela conduit à renommer l'agent pathogène en F. oxysporum f. sp. radicis-vanillae (Forv). Dans les interactions incompatibles (souche pathogène et vanillier résistant), l'épaississement et la lignification des parois de l'hypoderme, constitutive et induite par le champignon, joue un rôle significatif dans le blocage de l'invasion mycélienne. Le rôle important des composés phénoliques dans la résistance des vanilliers a été confirmé par les analyses d'expression différentielle de transcrits obtenus de novo par RNA-seq Illumina. Enfin, un test robuste de détermination in-vitro de la résistance à Forv a été validé et de nouvelles sources de résistance génétique à la PRT ont été identifiées. Nos résultats ouvrent des perspectives prometteuses pour la sélection de variétés améliorées de vanilliers
Vanilla is a high value cash crop that is continuously demanded by the agri-food and cosmetics industries for its incomparable flavor. Most of vanilla comes from the cured fruits of V. planifolia G. Jackson, a hemi-epiphytic climbing orchid cultivated in the humid tropics. In all the countries were it is cultivated, the vanilla vines suffer from a root and stem rot (RSR) caused by the soil borne fungus Fusarium oxysporum which dramatically reduces plant production and the durability of plantations. No efficient control method is currently available for this disease. Sources of genetic resistance to RSR exist in few vanilla relatives, but so far no commercial resistant variety has been produced. The purpose of this thesis was to better describe the diversity and histopathology of the causal agent of RSR and to evaluate the potential sources of genetic resistance that could be used in breeding programs. In a first step, a collection of 377 single-spored Fusarium isolates recovered from rotten roots and stems during surveys conducted in 52 vanilla plots from Reunion Island, Madagascar and French Polynesia were characterised. Representative subsets of isolates were genotyped using the Elongation Factor 1α and Intergenic Spacer gene sequences. Their pathogenicity was assayed by root dip inoculation on the susceptible V. planifolia accession pla0001. Results showed that F. oxysporum was the principal species responsible for the disease in the field, although a few F. solani isolates showing slight pathogenicity were also isolated. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Remarkably, their pathogenicity varied in gradient between non- pathogenic (about 42% of isolates) to highly pathogenic (14%). In a second step, 254 vanilla accessions comprising 18 species and six types of hybrids were assessed for resistance to RSR in the field (natural inoculum) and in the lab (in-vitro plants inoculated with Fo072). The strong resistance to RSR of all V. pompona accessions and hybrids of V. planifolia X V. pompona or V. phaeantha, were confirmed, and novel sources of resistance to RSR were added including, V. bahiana, V. costariciensis and V. crenulata. Most of the V. planifolia accessions, V. ×tahitensis and V. odorata were susceptible to RSR. However, three inbreeds of V. planifolia showed a high level of resistance to Forv. To our knowledge this is the first report of resistance to RSR in V. planifolia accessions. For the 26 accessions evaluated in both conditions, a strong correlation was observed between long term (9 years) evaluation in the field and ratings on in-vitro plants at 15dpi. Thirdly, we monitored by wide field and multiphoton microscopy the root infection process and the responses of one susceptible accession (V. planifolia pla0001) and two resistant accessions (V. planifolia pla0020 and V. pompona pom0018) to challenge inoculation with the severe isolate Fo072. In the compatible interaction (Fo072 – pla0001) invasion started from penetration of hyphae emitted from germinated conidia in the hairy region of root rapidly colonizing the cortex but never expanded to the vascular bundles up to the 9th dpi. It was therefore suggested to prefix the forma specialis name of the causal agent of RSR with radicis to point out its non-vascular pathogenicity in vanilla. In the two incompatible interactions, the important role played by hypodermis cells for impeding the invasion of the cortex by Fo072 was demonstrated by specific staining and spectral analysis of lignin precursors. Both constitutive and pathogen induced defense mechanism were described in pla0020 and pom0018. The mechanisms included the deposition of lignin in the hypodermal cell wall, entrapment of hyphae in specific hypodemal cells and polyphenolics secretion in intercellular spaces. Further, a de novo transcriptome analysis was experimentedon 8 pooled samples

Стилі APA, Harvard, Vancouver, ISO та ін.

23

Formosa, Cécile. "Comprehension of the mechanisms of action of antimicrobial molecules using nanobiotechnologies." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2424/.

Повний текст джерела

Анотація:

Mon travail de thèse consiste à utiliser les techniques de Microscopie à Force Atomique (AFM) pour étudier les microorganisms pathogènes, et leurs interactions avec des antimicrobiens. Ces dernières décennies, la résistance microbienne a augmenté de façon dramatique. Les bactéries et levures pathogènes sont à l'origine d'infections mettant en jeu la vie de patients. Il y a donc deux urgences : la première est de trouver de nouveaux antimicrobiens; pour cela il faut acquérir des données fondamentales sur la paroi des microorganismes, afin d'identifier des cibles. La deuxième urgence est donc de développer des nouvelles techniques pour explorer la paroi des. Durant cette thèse nous avons donc utilisé l'AFM, adapté aux conditions biologiques. Un avantage de l'AFM est la possibilité de travailler en liquide, ce qui nous a permis d'imager l'élongation de cellules de P. Aeruginosa traitées avec un antibiotique, ainsi que la disparition de la capsule de K. Pneumoniae traitée avec de la colistine. L'AFM est aussi une machine de force qui enregistre des courbes de force permettant d'accéder aux propriétés nanomécaniques et d'adhésion des cellules. Nous avons ainsi observé les modifications des propriétés adhésives de la levure C. Albicans traité avec de la caspofongine. Enfin il est possible de fonctionnaliser des pointes AFM avec des biomolécules ; cette stratégie nous a permis de localiser des protéines spécifiques à la surface de levures et cellules animales, et d'étudier la paroi de P. Aeruginosa traité par un antibactérien innovant, le Cx1. Pour conclure, cette thèse a permis d'adresser spécifiquement la contribution de la biophysique en microbiologie clinique
My PhD work consists in using Atomic Force Microscopy (AFM) techniques to study pathogenic microorganisms, and to probe their interactions with antimicrobials. During the last three decades, microbial resistance has dramatically increased and spread around the world. Pathogenic bacteria and yeasts are the cause of life-threatening infections in some patients. There are therefore two emergencies; the first one is to find new antimicrobial molecules; for that, it is mandatory to get further knowledge on the microbial cell wall. Therefore the second emergency is to develop new techniques to explore microbial surfaces. During my PhD, we took advantage of a technology coming from physics, and adapted to biological conditions, AFM. An advantage of AFM is the possibility to work in liquid on living cells, which allowed us to image the elongation of cells of P. Aeruginosa treated by ticarcillin, and the removal of capsular polysaccharides from K. Pneumoniae upon treatment with colistin. AFM is also a force machine, able to record force distance curves that give access to nanomechanical and adhesive properties of cells. We could observe the modifications of the adhesive properties of the yeast C. Albicans, treated by caspofungin. Finally it is possible to functionalize AFM tips with biomolecules; we used this strategy to localize specific proteins at the surface of living yeasts and mammalian cells, and to study the cell wall of P. Aeruginosa treated by an innovative antibacterial, Cx1. In conclusion, during my PhD, we especially addressed the contribution of biophysics in clinical microbiology

Стилі APA, Harvard, Vancouver, ISO та ін.

24

Månsson, Lisa. "Visualizing the dynamic interplay between the host and bacterial pathogen : a real-time study of renal infection /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-218-7/.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

25

Gatto, Mariele. "Novel Pathogenic Pathways and Therapeutic Implications in Lupus Nephritis: The Emerging Role of PTX3-Related Immunity." Doctoral thesis, Università degli studi di Padova, 2020. http://hdl.handle.net/11577/3426251.

Повний текст джерела

Анотація:

Background. Abnormalities affecting regulatory molecules and pentraxin (PTX) 3 have been suggested to contribute to development of lupus glomerulonephritis (LN). Characterization of novel pathogenic pathways could pave the way to targeted therapeutic approaches. Aims. To investigate the role and relevance of PTX3/anti-PTX3 related immunity in systemic lupus erythematosus (SLE) patients and in lupus murine models and to explore the in vivo effects of restoration of SERPINB3 levels. Methods. The overall project comprised four experimental phases. Three out of four experiments were carried out on murine models of SLE, either New Zealand Black/White (NZB/W F1) or Mrl/lpr mice, while one experiment was conducted in humans. In the first experiment, intraperitoneal administration of recombinant SERPINB3 (7.5 μg/0.1mL or 15 μg/0.1mL) or placebo (PBS 0.1 ml) was carried out in 40 NZB/W F1 mice divided into four groups of 10 mice each. Group 1 and 2 were treated before (preventive approach and group 3 and 4 after (therapeutic approach) development of proteinuria ≥100mg/dl. Two additional groups included 20 MRL/lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). The second experiment involved 30 NZB/W F1 mice which underwent subcutaneous immunization with PTX3+alum (n=10), PBS+alum (n=10) or PBS alone (n=10) three times three weeks apart, starting before development of proteinuria. For both experiments, time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Subanalysis regarding anti-PTX3 antibody levels and function were performed in the second experiment. The last experiment on mice involved 22 NZB/W F1 female littermates divided into two groups of 10 mice each and treated with the same approach described in the second experiment. Ten mice (5 from each group) were sacrificed at week 22 and the other 10 at 29 weeks. Histological and ultrastructural lesions were compared using optical microscopy, immunoelectron microscopy (IEM), immunofluorescence (IF) and confocal microscopy. Data on humans were retrieved enrolling 38 SLE patients (12 with biopsy-proven LN and 26 without LN) and 22 matched healthy donors (HD). Characterization and comparison of circulating levels of PTX3 specific (PTX3+) B cells between patients and controls was performed by flow-cytometry. The differences between groups for nonparametric continuous variables were analyzed using Mann-Whitney U test or Kruskal-Wallis’s ANOVA test when appropriate; proteinuria-free survival rate (proteinuria <300 mg/dl) and survival rates were evaluated by Kaplan-Meyer method using Mantel-Cox test for comparison. Chi-squared test was used for histological comparison. A p value<0.05 was considered statistically significant. Results. Experiments on mice showed positive results in terms of clinical effects deriving from restoration of SERPINB3 levels or immunization with PTX3. SERPINB3-administered mice displayed a milder LN, with lower and delayed occurrence of nephritogenic antibodies and milder proteinuria at several timepoints, as well as a prolonged survival versus PBS groups. Immunization with PTX3 evoked a vaccine-like response with occurrence of anti-PTX3 antibodies only in immunized mice and delayed and decreased levels of nephritogenic antibodies and proteinuria, resulting in a significantly longer disease-free and overall survival. Among mice sacrificed at given timepoints, notable differences were observed at IEM, IIF and confocal microscopy. PTX3-immunized mice displayed less or no electron-dense deposits (EDD) along the glomerular basement membrane and the mesangium, and remarkably decreased glomerular deposition of IgG, C1q and PTX3 compared to PBS-treated mice. Moreover, PTX3 was found inside the EDD at IEM and was shown to co-localized with nuclear material. LN patients displayed significantly lower levels of circulating PTX3+ B cells in comparison to SLE and HD, showing a persistent decrease among naïve and memory PTX3+ specific subsets. Conclusions. Clinical improvement of a lupus-like disease following restoration of SERPINB3 levels and immunization with PTX3 in lupus murine models suggests that very early abnormalities affecting molecules involved in tissue homeostasis, apoptosis and removal of apoptotic debris may induce further development of SLE and SLE-specific manifestations within an unpredictable lag time. PTX3 more strikingly emerged as a novel antigen in LN progression and PTX3/anti-PTX3 immunity appear to function as an early level of regulation which may fail in patients developing LN. Consistently, acquisition of a targeted anti-PTX3 immunity could hinder the progression from the preclinical to the clinical stages of disease. Altogether, these findings may add a piece of knowledge on the mechanisms supporting LN development and progression, and suggest that a targeted modulation of the native immunity could improve renal manifestations in selected patients. In the short term, dosage of serum anti-PTX3 antibodies may become a handful tool to help in stratifying lupus patients according to the risk of developing LN.

Стилі APA, Harvard, Vancouver, ISO та ін.

26

Kunz, Tobias C. [Verfasser], Vera [Gutachter] Kozjak-Pavlovic, Georg [Gutachter] Nagel, Thomas [Gutachter] Rudel, and Markus [Gutachter] Sauer. "Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens / Tobias C. Kunz ; Gutachter: Vera Kozjak-Pavlovic, Georg Nagel, Thomas Rudel, Markus Sauer." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1227189931/34.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

27

Ramirez-Suero, Montserrat. "Etude de l'interaction de Medicago truncatula avec Fusarium oxysporum et du rôle de l'acide salicylique dans les interactions de la plante avec différents agents pathogènes et symbiotiques." Thesis, Toulouse, INPT, 2009. http://www.theses.fr/2009INPT015A/document.

Повний текст джерела

Анотація:

Des études de l'interaction de la plante modèle des légumineuses Medicago truncatula avec des microorganismes pathogènes et symbiotiques ont été réalisées. Tout d'abord un pathosystème fongique a été caractérisé: M. truncatula en interaction avec Fusarium oxysporum spp., champignon responsable des fusarioses, soit du flétrissement vasculaire ou de la pourriture racinaire chez de nombreuses plantes cultivées. Deux lignées de M. truncatula: A17 et F83005.5 ont été identifiées comme sensible et tolérante respectivement à F. oxysporum f.sp. medicaginis, la forma specialis attribuée à la luzerne. De plus 9 souches de F. oxysporum isolées de différentes plantes hôtes et une souche non pathogène du sol ont été testées dans des expériences d'inoculation avec ces deux lignées. Elles ont toutes été capables d'induire les symptômes de la maladie chez M. truncatula. A l'aide d'une souche de F. oxysporum f.sp. medicaginis exprimant le gène rapporteur GFP, les étapes de colonisation de la racine par le champignon ont été caractérisées. Les observations en microscopie à fluorescence et microscopie confocale des racines d'A17 et F83005.5 ont indiqué un patron de colonisation inhabituel et ont montré que la tolérance de F83005.5 n'était pas corrélée à un mécanisme d'exclusion du cylindre central. Cependant, des différences dans l'expression de gènes de défense ont été détectées entre les deux lignées. Dans la deuxième partie, le rôle de l'acide salicylique a été étudié. Les résultats d'expériences avec l'acide salicylique exogène ont indiqué que le prétraitement de racines avec ce composé pouvait conférer une protection aux plantes vis-à-vis de F. oxysporum f.sp. medicaginis et la bactérie phytopathogène Ralstonia solanacearum. Avec l'objectif d'étudier le rôle de l'acide salicylique endogène dans les interactions avec les microorganismes, la transformation génétique de M. truncatula avec le gène NahG codant le salicylate hydroxylase a été entreprise. Cette enzyme dégrade l'acide salicylique en catechol. Seulement la lignée 2HA a pu être transformée et régénérée en plantes transgéniques. Ces plantes 2HA-NahG ont été inoculées avec des microorganismes pathogènes (R. solanacearum, Verticillium albo-atrum, F. oxysporum f. sp. medicaginis Colletotrichum trifolii et C. higginsianum) ainsi qu'avec le champignon endomycorhizien Glomus intraradices. Les limitations expérimentales n'ont pas permis de conclure définitivement, mais indiquent qu'il est possible que la signalisation par l'acide salicylique ne soit pas impliquée dans les défenses de M. truncatula face à ces microorganismes pathogènes et symbiotiques
A study on the interactions of the plant model legume Medicago truncatula (M.t.) with pathogens and symbiotic microorganisms was undertaken. First, a fungal pathosystem was characterized: M. truncatula in interaction with Fusarium oxysporum spp., the causal agent of Fusariosis, of Fusarium wilt and of root rot in many crop plants. Two M. truncatula lines, A17 and F83005.5, were identified as susceptible and tolerant respectively to F. oxysporum f.sp. medicaginis, the forma specialis related to alfalfa. Besides, 9 strains of F. oxysporum isolated from different host plants and a non-pathogenic soil-borne strain were tested in inoculation experiments with both lines. All the strains were able to trigger disease symptoms in M. truncatula. Using the F. oxysporum f.sp. medicaginis strain transformed with the GFP reporter gene, the stages of the root colonization by the fungi were characterized. Fluorescence and confocal microscopy observations on A17 and F83005.5 roots showed an unusual pattern of colonization and showed that the F83005.5 tolerance was not related to an exclusion mechanism in the central cylinder. However, differences on defence gene expression were detected in both lines. In the second part, the role of salicylic acid was studied. Results of experiments with exogenous salicylic acid indicated that prior treatment of roots with this compound may confer a protection towards F. oxysporum f. sp. medicaginis and the phytopathogenic bacterium Ralstonia solanacearum. With the goal to study the role of endogenous salicylic acid, the genetic transformation of M. truncatula with the NahG gene was initiated. This gene codes for a salicylate hydroxylase which degrades salicylic acid to catechol. Only the highly embryogenic 2HA line could be transformed and regenerated into transgenic plants. These 2HA plants were inoculated with pathogenic microorganisms (Ralstonia solanacearum, Verticillium albo-atrum, Fusarium oxysporum f.sp. medicaginis Colletotrichum trifolii and C. higginsianum) as well as the mycorrhiza fungus Glomus intraradices. Experimental limitations did not allow us to conclude definitely, but it seems possible that the salicylic acid signaling way may not be implicated in the defence of M. truncatula against these pathogenic and symbiotic microorganisms

Стилі APA, Harvard, Vancouver, ISO та ін.

28

Stévenin, Virginie. "Epithelial cell invasion and intracellular trafficking triggered by Salmonella Typhimurium." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC267.

Повний текст джерела

Анотація:

La Salmonellose est une maladie gastro-intestinale d’origine alimentaire contractée par plusieurs dizaines de millions de personnes chaque année. Bien que généralement bénigne, cette maladie engendre encore aujourd’hui des milliers de morts par an, en particulier chez les jeunes enfants et les personnes immunodéprimées. Au cours de cette thèse, nous avons disséqué les stratégies mises en place par la bactérie Salmonella lors de l’infection de cellules épithéliales. Salmonella est dotée de flagelles qui lui permettent de se déplacer à la surface des cellules de l’intestin et de cibler certaines cellules hôtes à infecter. Les critères de sélection de la bactérie étaient jusqu’à présent méconnus. Nous avons combiné une approche in vitro avec des outils de modélisation, afin de prédire mathématiquement les chances pour une cellule épithéliale d’être infectée ou non. Nous avons ainsi démontré que les salmonelles sont capables de sélectionner certaines cellules à infecter en fonction de leur microenvironnement local. Qui plus est, l’invasion d’une cellule hôte par Salmonella augmente les chances de cette cellule d’être infectée par d’autres salmonelles, révélant un mécanisme de coopération au long-terme entre salmonelles lors d’une infection. Pour pénétrer au sein de sa cellule hôte, Salmonella induit la formation d’expansions membranaires et s’engouffre dans une vacuole intracellulaire appelée SCV pour “Salmonella-Containing Vacuole”. Les SCVs peuvent soit se développer en une niche vacuolaire réplicative, soit se rompre, entraînant la libération de Salmonella dans le cytosol. La nature précise du compartiment d’entrée de Salmonella est demeurée longtemps débattue. En combinant des techniques de microscopie optique et électronique, nous avons révélé que Salmonella entre dans les cellules épithéliales dans une vacuole “étroite”, entourée de larges macropinosomes formés au site d’infection. La formation de ces macropinosomes dépendant de l’infection bactérienne, nous avons renommé ces compartiments “IAMs” pour “Infection-Associated Macropinosomes”. Quelques minutes après leur formation, les IAMs fusionnent avec la vacuole de Salmonella, permettant un rapide élargissement de la vacuole bactérienne. L’absence de fusion entraine quant à elle, la rupture de la vacuole bactérienne et la libération de Salmonella dans le cytosol. Ainsi, l’interaction entre les IAMs et la vacuole de Salmonella détermine les conditions de vie intracellulaire de la bactérie. Afin d’identifier les facteurs hôtes impliqués dans ce processus, nous avons développé une méthode d’isolation spécifique des IAMs et identifié les protéines qui les constituent. En particulier, nous avons révélé la présence de plusieurs protéines SNAREs pouvant permettre la fusion entre les IAMs et la vacuole bactérienne. Cette fusion est suivie d’une perte de membrane de la SCV via la formation de tubules membranaires. Nos résultats suggèrent que le mécanisme de rupture dépend de l’équilibre entre le gain et la perte de membrane apportés réciproquement par la fusion avec les IAMs et la formation de tubules.Ensemble, ces travaux nous ont permis d’élucider de nouvelles étapes clefs du mécanisme d’infection intracellulaire de Salmonella
Salmonellosis is a foodborne gastroenteritic disease causing tenths of millions of cases per year. While the disease is commonly benign, it still leads today to hundreds of thousands of deaths per year, especially in young children and immunodepressed people. Along this Ph.D., we dissected the molecular and cellular strategies of Salmonella during its infection of epithelial cells. Thanks to its flagella, Salmonella can move on the surface of intestinal cells and target specific host cells for infection. The selection criteria of the bacteria have been mostly unknown so far. We set up a pipeline of automatic acquisition and image analysis combined with mathematic modeling to forecast the probability of a given epithelial cell to be infected or not. We demonstrated that Salmonella can target specific host cells depending on features of their local environment, such as the localcell density. Besides, we found that the infection of an epithelial cell by Salmonella directly increases the vulnerability of the same cell to be reinfected, demonstrating the existence of a mechanism of long-term cooperation between bacteria.To penetrate targeted cells, Salmonella induces membrane ruffles leading to its engulfment into a membrane-bound compartment. This compartment can maturateand form a replicative vacuolar niche called the “Salmonella-Containing Vacuole”(SCV). Alternatively, the bacteria can rupture its vacuole leading to Salmonella release into the cytosol. A discrepancy existed in the field regarding the nature of theearly Salmonella-containing compartment. Coupling live fluorescence microscopy with correlative light electron microscopy, we revealed that Salmonella enters inepithelial cells in a tight compartment distinct from the surrounding macropinosomes formed at the infection site. As the bacterial infection induces the formation of those macropinosomes, we termed them “Infection-Associated Macropinosomes” (IAMs). Few minutes post-infection these endomembrane compartments can fuse with the SCV leading to its enlargement. Besides, the absence of fusion results in SCV destabilization and vacuolar escape. Thus, the interaction between IAMs and SCV determine the lifestyle of Salmonella.To identify the host factors driving SCV-IAM interactions, we developed a highlyspecific fractionation method to isolate IAMs. After proteome analysis by massspectrometry, we revealed the recruitment of specific SNAREs at the IAMs that couldallow SCV-IAMs fusion. Concomitantly to SCV-IAM fusions, we also monitored theloss of portions of the SCV membrane through the formation of membrane tubules.Our results suggest that the rupture mechanism depends on the equilibrium between the gain and the loss of membrane at the SCV via IAM fusions and tubular extraction, respectively. Together, our work provides a new comprehensive model of the early steps of Salmonella invasion of epithelial cells

Стилі APA, Harvard, Vancouver, ISO та ін.

29

Hahn, Sabine. "Virologische Untersuchungen an Stieleichen (Quercus robur L.) zum verursachenden Pathogen der pfropfübertragbaren chlorotischen Ringflecken." Doctoral thesis, Humboldt-Universität zu Berlin, Landwirtschaftlich-Gärtnerische Fakultät, 2006. http://dx.doi.org/10.18452/15457.

Повний текст джерела

Анотація:

Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen.
Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.

Стилі APA, Harvard, Vancouver, ISO та ін.

30

Le, May Nicolas. "Mécanismes de pathogenèse de la protéine non structurale NSs du virus de la Fièvre de la Vallée du Rift." Paris 7, 2005. http://www.theses.fr/2005PA077205.

Повний текст джерела

Анотація:

Le virus de la fièvre de la Vallée du Rift (RVFV) est un arbovirus appartenant à la famille des Bunyaviridae et au genre Phlebovirus. Il est transmis par les moustiques et infecte l'homme et les ruminants. De graves épidémies/épizooties ont eu lieu en Afrique et le virus circule maintenant en Arabie Saoudite ainsi qu'au Yémen. Le génome viral est composé de trois segments d'ARN simple brin: les segments L. M sont de polarité négative et le segment S utilise une stratégie ambisens. Bien que le cycle viral ait heu dans le cytoplasme, la protéine NSs (256 acides aminés, 31 kDa), codée par le segment S, a une localisation nucléaire où elle forme des filaments. NSs est un facteur de pathogenèse du virus qui inhibe la synthèse des ARNm du gène codant pour l'IFN bêta sans perturber pour autant l'enhanceosome (NF-KB, IRF3 et ATF2/cjun). Pour comprendre les mécanismes par lesquels NSs inhibe la réponse anti-virale, nous avons recherché ses partenaires cellulaires. Nous avons montré que l'infection par le VFVR a pour conséquences : i) une rapide diminution de la synthèse des ARN cellulaires parallèle à la décroissance de la concentration du facteur de transcription TFIIH, ii) l'inhibition du recrutement de CBP et de l'acétylation des histones au niveau du promoteur IFNp et iii) la protéolyse de STAT1. Par les techniques du double hybride, d'immunoprécipitations de protéines ou de la chromatine et en microscopie confocale, nous avons démontré que chacun des événements est lié aux interactions de la protéine NSs avec respectivement - la sous unité p44 du TFIIH, la sous unité SAP30 de certains co-répresseurs dont Sin3 et N-coR et la protéine Socs 1 présente dans une E3 ligase : ces différents partenaires de NSs sont présents dans les filaments nucléaires. NSs en interagissant avec p44 empêche la néo-formation du TFIIH. NSs, via SAP30 et son association avec le facteur de transcription YY1, stabilise des co-répresseurs responsables de la déacétylation des histones et empêche l'interaction entre CBP et YY1 au niveau du promoteur IFNp. Enfin NSs provoque l'accumulation de Socs 1 qui recrute un complexe E3 ligase CBCSo' responsable de la dégradation de STAT1 inhibant l'induction par l'IFNy. Ainsi la protéine multifonctionnelle NSs inhibe par trois mécanismes indépendants la réponse anti-virale de l'hôte
The Rift Valley fever virus is a phlebovirus of the Bunyaviridae family transmitted by mosquitoes and affecting cattle, sheep, goats and humans. It causes many dramatic epidémies and epizootics in Africa and recently it was introduced in Yemen and in Saudi Arabia with a high mortality rate. The viral genome is composed of three segments of RNA: the L and M segments are of negative polarity and encode respectively for the RNA polymerase RNA dependent and the precursor of envelope glycoproteins. The S segment utilises an ambisense strategy and codes for the nucleoprotein N and the non structural protein NSs. Although the viral cycle is cytoplasmic, the NSs protein (256 amino acids, 31 kDa) is nuclear and forms filament. Moreover, it was shown that NSs is the major pathogenicity factor, inhibiting IFN beta messenger RNA synthesis but do not disturb the formation of the enhanceosome (NF-KB, IRF3 and ATF2/cjun). We found that infection by RVFV leads to i) a rapid and drastic suppression of host cellular RNA synthesis that parallels a decrease of the TFIIH transcription factor concentration, ii) an inhibition of CBP recruitment and histones acetylation on IFNp promoter and iii) STAT1 proteolysis. Using yeast two hybrid System, immunoprecipitations, Chips and confocal microscopy, we further demonstrated that each event is linked to the association of the nonstructural viral NSs protein with respectively the TFIIH subunit p44, co-repressors subunit SAP30 and Socs 1 in the nuclear filaments. NSs prevents the assembly of newly synthesized TFIIH subunits. NSs, through the interaction between SAP30 and YY1 transcription factor, stabilizes co-repressors like N-coR or Sin3 responsible of histones deacetylation on IFNp promoter and preventing the association between CBP and YY1. Finally NSs provokes Socs 1 accumulation and, through a Socs 1 containing-E3 ligase complex, it degrades STAT1 and inhibes induction by IFNy. These observations shed light on the mechanisms utilized by RVFV to evade the host response

Стилі APA, Harvard, Vancouver, ISO та ін.

31

Berger, Pascale. "Les composés phénoliques foliaires de l'hévéa et leur implication dans la résistance à "Colletotrichum gloeosporioides" Penz. Et "Microcyclus ulei" Henn." Montpellier 2, 1992. http://www.theses.fr/1992MON20062.

Повний текст джерела

Анотація:

Des etudes statistiques ont permis de correler la resistance des jeunes feuilles d'hevea a colletotrichum gloeosporioides et microcyclus ulei a l'abondance de certains flavanes et flavonols. La localisation epidermique des flavonols semble bien adaptee a leur eventuelle implication dans la defense contre les pathogenes. La resistance des feuilles agees de tous les clones pourrait etre due a des flavanes. La resistance de certains clones vis-a-vis de m. Ulei pourrait etre en rapport avec la fongitoxicite de leurs extraits foliaires aqueux et avec des phenols presents dans ces extraits. L'inoculation par c. Gloeosporioides induit apparemment de plus fortes productions de scopoletine et accumulations de flavanes et de derives hydroxycinnamiques chez un clone resistant que chez un clone sensible. L'extension de l'infection s'accompagne d'un appauvrissement en phenols solubles vraisemblablement par fixation aux parois et/ou oxydation

Стилі APA, Harvard, Vancouver, ISO та ін.

32

Noronha, Marissônia de Araujo. "Escala diagramática para avaliação da mancha preta em folhas de citros e efeito da temperatura e da duração do molhamento na pré-penetração de conídios de Guignardia citricarpa Kiely [Phyllosticta citricarpa (McAlp.) Van der Aa]." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-21032003-133754/.

Повний текст джерела

Анотація:

A mancha preta dos citros causada pelo fungo Guignardia citricarpa Kiely [Phyllosticta citricarpa (McAlp.) Van der Aa], possui duas formas de infecção, conídios e ascósporos. Informações a respeito da importância dos conídios na epidemiologia da doença são escassas ou controversas. Visando uma maior compreensão sobre o patossistema citros-G. citricarpa (P. citricarpa), os objetivos desta dissertação foram: elaborar e validar uma escala diagramática para avaliação da severidade da mancha preta em folhas de citros; verificar o efeito da temperatura e da duração do período de molhamento na formação de apressórios formados a partir de conídios; observar por meio de microscopia eletrônica de varredura a germinação de conídios e a formação de apressórios sobre folhas destacadas de limão 'Siciliano' submetidas a diferentes temperaturas e períodos de molhamento. A escala diagramática com níveis de severidade de 1; 3; 6; 12; e 24% de área foliar lesionada foi validada por dois grupos de avaliadores, com e sem experiência na quantificação de doenças. Comparada com a avaliação sem escala, o uso da escala proporcionou melhor precisão e acurácia tanto para avaliadores experientes como inexperientes, quando considerada a estimativa média dos mesmos. Na maioria dos casos, os desvios entre estimativas e severidade atual da doença foram mais evidentes para os níveis de severidade entre 5 e 15%. A reprodutibilidade das avaliações resultou em valores de R 2 mais uniformes para a maioria dos avaliadores experientes. Diferenças consideráveis de precisão foram observadas entre avaliadores inexperientes. O efeito da temperatura (10 o C 40 o C) e da duração do molhamento (4 48 h) na formação de apressórios formados a partir de conídios de G. citricarpa (P. citricarpa) foi avaliado sob condições "in vitro" e sobre a superfície de folhas de limão 'Siciliano'. A formação de apressórios ocorreu em todas as temperaturas a partir de 12 horas de molhamento, sendo os extremos de temperatura (10 o C e 40 o C) menos favoráveis à formação de apressórios. A temperatura mínima para formação de apressórios, estimada pela função beta generalizada foi de 3 o C e a máxima de 48,4 o C, ambas para 48 horas de molhamento. A formação de apressórios foi consideravelmente favorecida pela duração do período de molhamento, com o máximo de apressórios formados a 24 horas de molhamento, para a maioria das temperaturas. O período de molhamento constituído de 48 horas foi essencial para que os esporos submetidos a temperaturas de 10 o C e 40 o C, formassem apressórios. A superfície de resposta obtida pela multiplicação das funções beta generalizada e monomolecular apresentou um ajuste satisfatório para os dados observados na estimativa da porcentagem relativa de apressórios formados (R 2 =0,75). As amostras observadas em microscopia eletrônica de varredura possibilitaram a aquisição de imagens de conídios e apressórios sobre a superfície de folhas de limão 'Siciliano' em todas as combinações de temperatura e molhamento avaliadas.
Citrus black spot caused by Guignardia citricarpa Kiely [Phyllosticta citricarpa (McAlp.) van der Aa] presents two infection forms, conidia and ascospores. Information regarding the importance of the conidia in the epidemiology of the disease is scarce and controversial. Seeking a better understanding on the pathosystem citrus-G. citricarpa (P. citricarpa), the objectives of this dissertation were: elaborate and validate a diagrammatic scale for assessments of the citrus black spot; verify the effect of the temperature and of the wetness duration in the appressorium formation; observe through scanning electron microscopy the germination and formation of appressorium on outstanding lemon 'Siciliano' leaves submitted to different temperatures and wetness duration. The diagrammatic scale with severity levels of 1; 3; 6; 12; and 24% of diseased leaf area was validated by two groups of raters, with experience and without experience in the quantification of diseases. The scale provided better precision and accuracy for both experienced and inexperienced raters, considering the estimates average of them. In the majority of cases, the bias between estimated and actual disease severity were more evident for disease severity levels between 5 and 15%. The reproducibility of assessments resulted in R 2 with more uniforms values for the majority of the experienced raters, considerable differences of precision were observed among inexperienced raters. The effect of the temperature (10 o C - 40 o C) and of the wetness duration (4 48 h) in the germination of conidia and appressoria formation of G. citricarpa (P. citricarpa), was assessed "in vitro" and on the surface of lemon 'Siciliano' leaves. The appressoria formation occurred in all the temperatures starting from 12 hours of wetness. The extreme temperatures (10 o C and 40 o C) were less favorable to the apressorium formation. The minimum temperature for appressorium formation, estimated by generalized beta function was of 3 o C and the maximum of 48,4 o C, both for 48 hours of wetness. The appressorium formation was favored considerably by the wetness duration period, with the maximum of apressoria formed at 24 hours of wetness, for majority of the temperatures. The wetness duration period constituted of 48 hours was essential so that the spores submitted to temperatures of 10 o C and 40 o C, formed appressorium. The response surface obtained by the multiplication of the generalized beta and monomolecular functions provided a close fit to observed data in the estimate of the relative percentage of formed appressorium (R 2 =0,75). The samples observed in scanning electron microscopy made possible the acquisition of images of conidia and appressoria on the surface of lemon 'Siciliano' leaves in all the temperature combinations and wetness evaluated.

Стилі APA, Harvard, Vancouver, ISO та ін.

33

Abou, al Fadil Taissir. "Déterminisme de la tolerance du tournesol a Phoma Macdonaldii au collet et sur racines : approches génétique et histologiques." Toulouse, INPT, 2006. http://ethesis.inp-toulouse.fr/archive/00000300/.

Повний текст джерела

Анотація:

La maladie des taches noires du tournesol, ou Phoma du tournesol, est la deuxième maladie plus importante après le Mildiou en France. Elle provoque des pertes de rendement qui peuvent atteindre jusqu'à 70%. Les études réalisées à ce jour portent essentiellement sur la contamination du tournesol par Phoma macdonaldii au niveau de la tige. Cependant, aucun travail n'a été réalisé sur les attaques de Phoma au niveau des racines et du collet. Nos travaux ont porté sur : mise au point d'un test de contamination racinaire et au collet à la transformation de souches de Phoma macdonaldii avec une protéine verte fluorescente (GFP) ; étude de l'interaction entre 4 génotypes de tournesol et 10 souches monospores de Phoma macdonaldii sur tige, collet et racine ; variabilité génétique de la résistance partielle à la maladie des tache noires ; contrôle génétique de la résistance partielle du tournesol au Phoma macdonaldii ; identification de QTLs impliqués dans la résistance du tournesol au Phoma.

Стилі APA, Harvard, Vancouver, ISO та ін.

34

Ganash, Magdah. "X-ray crystallographic and electron microscopy studies on members of the ClyA/Nhe family of the pore-forming toxins avian pathogenic E. coli cytolysin A and B. cereus non-hemolytic enterotoxin." Thesis, University of Sheffield, 2012. http://etheses.whiterose.ac.uk/3830/.

Повний текст джерела

Анотація:

Escherichia coli cytolysin A (ClyA, also known as hemolysin E, HlyE) is a 34 kDa cytolytic α-helical pore-forming toxin. The crystal structure of soluble monomeric E. coli K-12 ClyA was previously solved at high resolution and this showed that ClyA had a novel structure that had not previously been seen in the data bank of proteins. Avian pathogenic E. coli (APEC), strain JM4660 ClyA is 75% sequence identical to E. coli K-12 ClyA and has many significant similarities. However, two significant differences between them are that JM4660 ClyA pores are more homogeneous when observed by electron microscopy (EM), and JM4660 ClyA is more thermostable than E. coli K-12 ClyA. Consequently, JM4660 ClyA could be a good model system to investigate ClyA membrane interaction. The expression and purification of JM4660 ClyA was successful. Crystals were grown of the pore form, but so far diffract to about 7 Å resolution. Bacillus cereus Nhe is a complex, pore-forming toxin consisting of three related proteins: NheA (43 kDa), NheB (39 kDa), and NheC (40 kDa). It is able to lyse mammalian cells from several organisms including humans in both Agar and liquid media and all three components of Nhe are necessary for toxicity. NheA has only 22% identity with NheB and NheC and its binding is the final stage of pore formation. Nhe proteins have sequence identity to B. cereus Hbl proteins. A previous structure solution of HblB revealed a structural resemblance to E. coli ClyA. To date, no crystal structure of any Nhe protein has been available in either the soluble form or in the pore form. Therefore, the project aimed to resolve the crystal structures of Nhe proteins in their water-soluble forms. The expression and purification of NheA was very successful. NheA was crystallized using PEG3350 as a precipitant by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 308 Å, b = 58 Å, c = 172.4 Å, α = γ = 90° and β =110° and were estimated to contain 8 protein molecules per asymmetric unit. The three-dimensional crystal structure was solved at 2.05 Å resolution using Multi-wavelength anomalous dispersion (MAD) data sets. NheA is a rod-shopped structure. The main body is formed by a bundle of four helices, each at least 70 Å long and near the end of C terminal there is a extra fifth helix known as αG. NheA has two subdomains: the tail domain and the head domain that contains amphipathic α/β hairpin. The structure of NheA reveals strong structure similarity to ClyA and HblB, which further suggests that Nhe, Hbl and ClyA belong to the some novel family of toxins. In addition, B. cereus NheB expressed in B. subtilis strain JH642, was purified and crystallized. This thesis also presents the initial electron microscopy studies involving NheA and NheB pore formation.

Стилі APA, Harvard, Vancouver, ISO та ін.

35

Verdier, Valérie. "Contribution à l'étude de la variabilité de Xanthomonas campestris pv. Manihotis (Arthaud Berthet et Bondar) Starr. Agent causal de la bactériose vasculaire du manioc (Manihot esculenta Crantz)." Paris 11, 1988. http://www.theses.fr/1988PA112100.

Повний текст джерела

Анотація:

Quelques étapes du processus infectieux de Xanthomonas campestris pv. Manihotis sont étudiées sur vitroplants, ainsi que sur cals issus de tiges et sur feuilles excisées. Le comportement de deux souches, non agressive et agressive de Xanthomonas campestris pv. Manihotis est évalué sur cinq variétés de manioc présentant différents niveaux de résistance à la bactériose au champ. Nous avons pu montrer, à l'appui d'analyses statistiques, que quelle que soit la technique mise au point sur vitroplant, cal ou feuille isolée, la dynamique de l'implantation de la bactérie est significativement différente pour les deux souches. L'association vitroplant-souche agressive conduit à l'expression de symptômes semblables à ceux observés après infection au champ. Sur les cinq variétés inoculées, seule la variété 30555 présente un comportement différent des autres. Par contre, sur toutes les variétés inoculées avec la souche non agressive, l'absence de symptôme est corrélée à la non colonisation interne des différents organes du vitroplant. La colonisation épiphylle est contrôlée en microscopie électronique de balayage. L'observation des sites d'adhésion et de multiplication des bactéries d'une part, et des réactions des tissus d'autre part, confirme la divergence de comportement des deux souches. La seconde partie de l'étude porte sur la mise en évidence de plasmides dans 54 souches de Xanthomonas campestris pv. Manihotis différant par leur origine géographique et leurs caractéristiques biochimiques, lysogéniques et de pouvoir pathogène. Les résultats permettent d'établir une corrélation entre la nature des plasmides et les caractéristiques lysogéniques d'une part et le pouvoir pathogène d'autre part. La discussion porte sur :- les possibilités d'utilisation des cultures in vitro pour :1 -étudier les relations hôte-bactérie et dans le cas présent, les étapes de pollution,. Adhésion et pénétration,2 - évaluer rapidement la résistance variétale. - les corrélations établies entre le pouvoir pathogène, la présence et la nature des plasmides dans les souches de Xanthomonas campestris pv. Manihotis
Several stages of the infection process of Xanthomonas campestris pv. Manihotis , were studied using in vitro plantlets as well as stem callus tissue and sectionned leaves. Two strains of Xanthomonas campestris pv. Manihotis, agressive and non-agressive, were compared on 5 varieties of cassava presenting different levels of resistance under field conditions. Statistical analysis revealed that bacterial implantation differed significantly according to which of the two strains was used, whichever mode!applied, in vitro plantlets, callus tissue or sectionned leaves. The association agressive strain in vitro plantlet results in symptoms similar to those observe upon infection under field conditions. Only one variety out of the 5 tested, variety 30555, presented a different type of reaction to the agressive strain. However, the absence of symptoms with the non-agressive strain was correlated with the failure to colonize internai organs for the 5 varieties tested in vitro. S. E. M. Observations were used for further observation of the adhesion and growth patterns of the bacterial strains as well as plant tissue reaction. In the second part of this study, the existence of plasmids was revealed in a collection of 54 strains of Xanthomonas campestris pv. Manihotis differing by their geographical origin, biochemical caracteristics and pathogenicity. Plasmid type was correlated with lysogenic caracteristics a s well as with pathogenicity. The use of in vitro methods for : 1 - Studying the host-bacteria interaction and, in the present case pollution, adhesion and penetration stages 2 - Rapid evaluation of varieta! resistance. The correlations established between the presence and nature of plasmids in strains of Xanthomonas campestris pv. Manihotis and pathogenicity are considered

Стилі APA, Harvard, Vancouver, ISO та ін.

36

Bergeau, Dorian. "Contribution à l'études des systèmes de sécrétion chez la bactérie Pseudomonas fluorescens." Rouen, 2015. http://www.theses.fr/2015ROUES026.

Повний текст джерела

Анотація:

Les systèmes de sécrétion de type III (SST3) sont utilisés par certaines bactéries pathogènes, afin d’injecter des effecteurs au travers des membranes cellulaires eucaryotes, entraînant la colonisation de l’hôte et la paralysie de ses défenses. Récemment, des gènes SST3 ont été détectés chez Pseudomonas fluorescens, une rhizobactérie saprophyte retrouvée inopinément en milieu clinique. La diversité, la phylogénie et la virulence associée aux gènes SST3 ont été étudiés chez des P. Fluorescens issus de milieux hospitaliers ou de plantes. Un 1er groupe réunit les isolats d’infections sanguines, proches des espèces P. Putida et P. Mosselii. Ces bactéries portent des gènes SST3 de la famille Ysc présentant 99% d’homologie à ceux du pathogène opportuniste, P. Aeruginosa. Un 2d groupe intègre les autres isolats hospitaliers (expectoration, abcès) aux côtés des souches environnementales. Ces bactéries sont psychrotrophes et présentent une diversité phylogénétique plus vaste. Les membres sont porteurs de gènes SST3 appartenant à la famille Hrp1, trouvés chez le phytopathogène P. Syringae. Une étude des composants externes de P. Fluorescens modèles issus de ces travaux a été réalisée par microscopie électronique en transmission. Elle révèle les 1ères images d’un SST3 chez P. Fluorescens. Il s’agit d’un pilus Hrp1 d’environ 1,5 μm de long, dont la production est induite par des sucres végétaux et fongiques. Ces SST3 seraient impliqués dans l’induction des systèmes de défense des plantes. Notre étude révèle aussi la présence de faisceaux dendritiques imposants et de vésicules, possiblement impliqués dans l’adhésion, la nutrition et/ou la communication des colonies microbiennes
Type III secretion systems (T3SS) are used by some pathogenic bacteria to inject effectors through the eukaryotic cell membranes. These structures allow the colonization of the host cell and the paralysis of its defenses. Recently, T3SS genes were detected in Pseudomonas fluorescens saprophytic rhizobacterium surprisingly found in clinical environment. The T3SS genes diversity, phylogeny and virulence were investigated in P. Fluorescens found in hospital or plant environments. A cluster integrates isolates of blood infections, close to P. Putida and P. Mosselii species. These bacteria harbor T3SS genes belonging to Ysc family and share 99% of homology with T3SS of the opportunistic pathogen P. Aeruginosa. The second cluster includes other hospital isolates (respiratory tract or abscess) close to environmental P. Fluorescens. These bacteria are psychrotrophs and have a broader phylogenetic diversity. Their T3SS genes belong to the Hrp1 family, usually found in the plant pathogen P. Syringae. A study of external components of some P. Fluorescens models was performed by transmission electron microscopy. It reveals the first images of a P. Fluorescens T3SS. This T3SS is a Hrp1 pilus of around 1. 5 μm long whose production is induced by plant and fungal sugars. It could be involved in the induction of plant defense systems. Our study also reveals the presence of dendritic fibril bundles and vesicles, possibly involved in adhesion, nutrition and /or communication at the scale of the microbial colony

Стилі APA, Harvard, Vancouver, ISO та ін.

37

Glavier, Marie. "Études structurales par cryo-microscopie électronique d’un système d’efflux multi-drogues bactérien, impliqué dans la résistance aux antibiotiques." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0239/document.

Повний текст джерела

Анотація:

L'apparition croissante de bactéries pathogènes multi-résistantes à la plupart des antibiotiques disponibles apparaît comme un problème mondial de santé publique. Malheureusement, un usage excessif à la fois en médecine humaine et animale a conduit à l’apparition de souches multi-résistantes à la plupart des antibiotiques disponibles sur le marché. Il est donc urgent de mieux comprendre les mécanismes mis en place par les bactéries pour résister aux antibiotiques afin de trouver des solutions pour combattre les souches multi-résistances.Dans ce contexte, le projet de la thèse vise à mieux comprendre les bases moléculaires de l’efflux actif de drogues chez Pseudomonas aeruginosa, qui est un des plus importants mécanismes utilisés par la bactérie pour lutter contre l’action de plusieurs antibiotiques. Les systèmes d’efflux forment des complexes protéiques situés dans la paroi de la bactérie et expulsent de manière active les antibiotiques avant même qu’ils aient pu atteindre leur cible intracellulaire, les rendant ainsi inactif.L’étude structurale se focalise sur le système RND (Resistance-Nodulation and cell Division) MexA-MexB-OprM qui est constitutivement exprimé chez la bactérie sauvage et est surexprimé chez les souches résistantes. Ce complexe tripartite est composé d'un transporteur inséré dans la membrane interne, d'une protéine canal insérée dans la membrane externe et d’une protéine adaptatrice périplasmique qui relie les deux autres protéines pour former un conduit étanche traversant le périplasme. En l’absence de la connaissance de la structure du complexe tripartite, l’objectif de la thèse a été de développer une stratégie originale pour reconstituer in vitro le complexe entier dans un environnement lipidique à partir des trois composants natifs produits séparément.L’assemblage du complexe tripartite est réalisé en mélangeant MexB et OprM en Nanodisque avec MexA mimant les deux bicouches lipidiques. La structure de ce complexe tripartite a été obtenu en combinant la cryo microscopie électronique et à l’approche dite ‘des particules isolées’. La structure tridimensionnelle du complexe calculée à une résolution inférieure à 4 Å a permis de construire un modèle atomique du complexe tripartite assemblé entre deux Nanodisques.Le complexe tripartite est composé d’un trimère d’OprM, d’un trimère de MexB et d’un hexamère de MexA entourant MexB et en interaction avec OprM. Ces données ont permis de résoudre la structure complète de MexA dans le complexe dont la partie N-terminale jusqu’alors inconnue car trop flexible et décrivent pour la première fois l’ancrage de MexA dans une membrane lipidique. Les changements conformationnels sont observés sur OprM et MexB lorsqu’ils sont engagés dans le complexe avec l’ouverture de l’extrémité périplasmique d’OprM et le basculement d’une boucle de MexB permettant d’établir un contact supplémentaire avec MexA.Pour replacer cette structure tripartite dans le cycle d’efflux de l’antibiotique, celle-ci décrit un état qui s’apparente probablement à un état au repos, sachant qu’aucun ligand spécifique n’a été ajouté au cours de l’assemblage. De plus, le complexe forme un canal ouvert à son extrémité extracellulaire, fournissant le conduit pour évacuer les drogues transportées par MexB qui utilise la force protomotrice comme source d’énergie.Ce travail ouvre la perspective à des études structurales d’autres états conformationnels du système d’efflux en condition « énergisé » pour compléter la compréhension du mécanisme du cycle d’efflux. Par ailleurs, la connaissance de cette première structure du complexe natif tripartite constitue le premier pas vers le développement de molécules capables de bloquer l’assemblage du complexe à des fins thérapeutiques. En effet, de telles molécules inhiberaient l’efflux actif et restauraient l’efficacité perdue des antibiotiques actuels
The increasing appearance of multi-drug-resistant pathogenic bacteria to most available antibiotics is emerging as a global public health problem. Unfortunately, excessive use in both human and animal medicine has led to the emergence of multi-drug-resistant strains for most antibiotics available on the market. It is therefore urgent to better understand the underlying mechanisms by which bacteria resist to antibiotics to combat multi-resistance strains. In this context, this work aims at better understanding the molecular basis of active drug efflux in Pseudomonas aeruginosa, which is one of the most important mechanisms used by the bacterium to resist to several antibiotics. Efflux systems form protein complexes in the bacterial wall and actively expel antibiotics even before they reach their intracellular target, rendering them inactive. The structural study focuses on the MexA-MexB-OprM RND (Resistance-Nodulation and cell Division) system that is constitutively expressed in wild-type bacteria and is over-expressed in resistant strains. This tripartite complex is composed of a transporter inserted into the inner membrane, a channel protein inserted in the outer membrane and a periplasmic adapter protein that connects the other two proteins to form a sealed conduit through the periplasm. In the absence of knowledge of the structure of the tripartite complex, the aim of the thesis was to develop an original strategy to reconstitute the whole complex in vitro in a lipid environment from the three native components produced separately.The assembly of the tripartite complex is made by mixing MexA with MexB and OprM in Nanodisc mimicking the two lipid bilayers. The structure of this tripartite complex was obtained by combining cryo electron microscopy and the so-called 'isolated particles' approach. The three-dimensional structure of the complex, calculated at a resolution of less than 4 Å, was used to build an atomic model of the tripartite complex assembled between two Nanodiscs. The tripartite complex is composed of an OprM trimer, a MexB trimer and a MexA hexamer surrounding MexB and interacting with OprM. We solve the complete structure of MexA whose N-terminal part hitherto unknown because of a high flexibility and describe for the first time the anchoring of MexA in a lipid membrane. The conformational changes are observed on OprM and MexB when they are assembled in the complex with the opening of the periplasmic end of OprM and the spatial re-orientation of a MexB loop to establish additional contact with MexA.To integrate this tripartite structure into the antibiotic efflux cycle, it describes a state that is probably a resting state, knowing that no specific ligand was added during assembly. In addition, the complex forms an open channel at its extracellular end, providing the conduit to evacuate the drugs carried by MexB that uses the proton motive force as a source of energy. This work opens new perspective for structural studies of other conformational states of the efflux system in "energized" conditions to fulfill our understanding of the efflux cycle mechanism. Moreover, the knowledge of this first tripartite native complex structure constitutes the first step towards the development of molecules capable of blocking the assembly of the complex for therapeutic uses. Indeed, such molecules would inhibit active efflux and restore the lost efficiency of current antibiotics

Стилі APA, Harvard, Vancouver, ISO та ін.

38

Rey, Camille. "Cytosolic bacterial subversions of mucosal immunity : a study of microfold (M) cell and enterocyte infections by S. flexneri and L. monocytogenes." Thesis, Sorbonne Paris Cité, 2018. https://theses.md.univ-paris-diderot.fr/Rey_camille_1_va_20180321.pdf.

Повний текст джерела

Анотація:

Les pathogènes bactériens cytosoliques S. flexneri et L. monocytogenes échappent à l'immunité extracellulaire de la muqueuse en induisant leur entrée et leur mode de vie intracellulaire dans l'épithélium intestinal. Dans leur cellule hôte, ils peuvent rapidement s’échapper de leur vacuole d'internalisation, envahir le cytosol et éviter l’élimination par la dégradation cellulaire en se propageant directement de cellule à cellule.Afin d’initier l’invasion intestinale, ces deux pathogènes ciblent les cellules M préleveusesd'antigènes qui recouvrent les sites d'induction immunitaire. Toutefois le mode de vie de ces pathogènes dans les cellules M, le mécanisme de propagation de l'infection à partir de ce pointd’entrée vers les entérocytes voisins ainsi que le mécanisme d'évasion de l'induction de l'immunité adaptative sont très peu caractérisés. Dans cette étude, nous présentons un nouveau modèle physiologique d'infection apicale par S. flexneri de cellules M humaines in vitro, qui récapitule les étapes précoces de l'invasion de l’épithelium intestinal par le pathogène. Nous montrons qu'une population de S. flexneri est rapidement transcytosée, en 15 minutes, à travers les cellules M. Nous amenons une nouvelle approche microscopie en temps réel de l'infection des cellules M, qui révèle qu'une deuxième sous-population de bactéries induit son entrée plus tardivement dans les cellules M, accompagnée de projections membranaires apicales, suivie par une rupture vacuolaire et l’initiation de la réplication cytosoliique des bactéries dans les cellules M. Nous découvrons que S.flexneri a également la capacité de se propager des cellules M aux cellules voisines par la motilité liéeà l'actine, qui constitue la voie principale de propagation basolatérale de l'infection. En étendant notre étude à L. monocytogenes, nous observons qu’à la différence de S. flexneri, cette bactérie détourne le processus de transcytose à travers les cellules M en utilisant le facteur de virulence ActA. Cependant, nous notons que L. monocytogenes se propage dans l'épithélium exclusivement par la motilité liée à l'actine, de façon similaire à S. flexneri. Nous proposons que la subversion de la voie de transcytose au travers des cellules M et l'évitement des tissus immunitaires sous-jacents sont des caractéristiques partagées par les pathogènes cytosoliques, leur permettant d'échapper à l'induction de l'immunité adaptative.Par ailleurs, nous présentons une approche de tri basée sur la fluorescence d’entérocytes individuels aux stades successifs de l'infection par S. flexneri, combinée avec une analyse transcriptomique parPCR quantitative en multiplex. Cette méthode révèle la production de réponses distinctes chez les entérocytes hôtes en fonction de la localisation subcellulaire du pathogène. Nous observons la production d'une réponse bystander forte, impliquant de multiples voies de signalisation corrélées chez les enterocytes non infectés. De plus nous détectons la production de profils de réponses distincts chez l’hôte en fonction de la localisation vacuolaire ou cytosolique de la bactérie chez les entérocytes infectés. Nous montrons que le facteur de virulence OspF contribue à atténuer les réponses des entérocytes infectés et à perturber des voies de signalisation autrement corrélées chez l’hôte.En conclusion, nos études exposent de nouvelles stratégies de subversion immunitaire liées aux modes de vie intracellulaires de bactéries entériques cytosoliques, soulignant l'importance des cellules M dans la propagation bactérienne initiale et le détournement de l'immunité adaptative, ainsi que l'organisation et la perturbation des réponses immunitaires innées chez les entérocytes au cours de l’infection
Cytosolic bacterial pathogens S. flexneri and L. monocytogenes subvert extracellular mucosal immunity by inducing their uptake and intracellular lifestyle in the intestinal epithelium. Within the host, they are able to rapidly escape their internalization vacuole, invade the cytosol and escape cellular degradation by spreading from cell-to-cell. Antigen sampling M cells overlying immune induction sites are targeted by these pathogens to initiate intestinal invasion. However, the intracellular lifestyle of these pathogens within M cells, the mechanism of spread of the infection toneigh boring enterocytes from this entry point and the mechanism of S. flexneri evasion of adaptive immunity is poorly characterized. We present a novel physiologic model of apical S. flexneri infection of human in vitro M cells which recapitulates the early steps of epithelial invasion. We show that a subset of S. flexneri is rapidly transcytosed, within 15 minutes, through M cells. We establish a newtime-lapse imaging approach of M cell infections, which reveals that another subset of bacteriainduces apical ruffling upon entry, vacuolar rupture and replicates within the M cells at later timepoints. Remarkably, these bacteria are able to spread from M cells to neighboring cells by actinbased-motility, which we show constitutes the main route of basolateral spreading of the infection.As we extend our study to L. monocytogenes, we observe that unlike S. flexneri, the bacterium diverts M cell transcytosis via the virulence factor ActA. However, we discover that L. monocytogenes spreads within the epithelium exclusively by actin-based motility, similar to S. flexneri. We propose that subversion of M cell transcytosis and avoidance of underlying immune tissues are features shared by cytosolic pathogens, allowing their escape from induction of adaptive immunity.In addition, we submit a pipeline of fluorescence-based single cell sorting of enterocytes atsuccessive stages of infection combined with transcriptional analysis by multiplex qPCR. This methodreveals the production of distinct responses in host enterocytes according to subcellular pathogen localizations. We observe the production of a strong bystander response involving multiplecorrelated host pathways in non-infected enterocytes. Moreover, we detect the output of distinct host response patterns according to vacuolar or cytosolic bacterial localizations in infectedenterocytes. We further show that the virulence effector OspF contributes to dampen infected host responses and disrupt otherwise correlated host signaling pathways. To conclude, our studies expose new immune subversion strategies linked to the intracellular life styles of cytosolic enteric bacteria, highlighting the importance of M cells in initial bacterial dissemination and diversion of adaptive immunity, and the organization and disruption of innate immune responses provoked in enterocytes during infection

Стилі APA, Harvard, Vancouver, ISO та ін.

39

Imori, Priscilla Fernanda Martins. "Caracterização do potencial patogênico de linhagens de Yersinia enterocolitica-like." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-02052016-104428/.

Повний текст джерела

Анотація:

Dentre as 18 espécies do gênero Yersinia, as espécies Y. enterocolitica, Y. pseudotuberculosis e Y. pestis foram extensivamente caracterizadas em diversos aspectos como ecologia, epidemiologia e mecanismos de patogenicidade. Sete das 15 espécies restantes (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii e Y. rohdei), usualmente conhecidas como Y. enterocolitica-like, até o momento, não tiveram seu potencial patogênico caracterizado e são, geralmente, consideradas não-patogênicas. Entretanto, dados da literatura sugerem que algumas dessas espécies possam causar doença. Esses dados estimularam o surgimento de questões sobre os mecanismos pelos quais as espécies de Y. enterocolitica-like possam interagir com as células do hospedeiros e causar doenças. Esse projeto teve como principal objetivo caracterizar o potencial patogênico de linhagens de Y. enterocolitica-like, especificamente das espécies Y. frederiksenii, Y. kristensenii e Y. intermedia. No presente trabalho, o potencial patogênico de 118 linhagens de Y. enterocolitica-like (50 Y. frederiksenii, 55 Y. intermedia e 13 Y. kristensenii) foi avaliado pela pesquisa da presença dos genes relacionados à virulência ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB e virF por PCR. Além disso, a habilidade de algumas linhagens de Yersinia de aderir e invadir células Caco-2 e HEp-2 após diferentes períodos de incubação, bem como, de sobreviver no interior de macrófagos humanos U937 foi testada. Aspectos morfológicos da adesão bacteriana foram visualizados por microscopia eletrônica. Finalmente, a presença de possíveis novos mecanismos de virulência foi avaliada a partir do sequenciamento de RNA de uma linhagem de Y. enterocolitica-like. As linhagens estudadas apresentaram os seguintes genes: Y. frederiksenii, fepA (44%), fes (44%) e ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) e tccC (35%); e Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) e hreP (54%). De modo geral, as linhagens de Y. enterocolitica-like tiveram a habilidade de aderir e invadir células Caco-2 e HEp-2 inferior à da linhagem altamente patogênica Y. enterocolitica 8081. Contudo, Y. kristensenii FCF 410 e Y. frederiksenii FCF 461 apresentaram elevado potencial de invasão a células Caco-2 após cinco dias de pré-incubação, os quais foram 45 e 7,2 vezes maiores do que o controle Y. enterocolitica 8081, respectivamente, porém, o gene ail não foi detectado nessas linhagens. O ensaio de sobrevivência em macrófagos humanos U937 ii mostrou que as linhagens de Y. frederiksenii FCF 461 (40,0%) e Y. frederiksenii FCF 379 (24,6%) tiveram porcentagens de sobrevivência superior à de Y. enterocolitica 8081 (13,4%). Todavia, linhagens de Y. intermedia e Y. kristensenii apresentaram uma capacidade reduzida de sobreviver em macrófagos. A microscopia eletrônica de varredura mostrou as bactérias em contato com a filipódia celular. As bactérias foram distribuídas tanto individualmente quanto em pequenos aglomerados. Portanto, podemos concluir que a presença dos genes relacionados à virulência encontrados nas Y. enterocolitica-like estudadas indicou o possível potencial patogênico de algumas dessas linhagens. Os ensaios de adesão e invasão a células de mamíferos sugerem que a patogenicidade de Y. kristensenii e Y. frederiksenii possa ser linhagem-dependente. O ensaio de sobrevivência em macrófagos humanos U937 evidenciou o potencial patogênico de algumas linhagens de Y. frederiksenii. Em conjunto, os resultados obtidos sugerem a existência de mecanismos de virulência alternativos aos mecanismos clássicos descritos para Y. enterocolitica patogênica. Contudo, a presença de possíveis novos mecanismos de virulência não pode ser verificada, uma vez que a plataforma 454 GS Junior (Roche) não se mostrou adequada para a realização de sequenciamento de RNA de amostras provenientes de interações com células devido à baixa cobertura obtida.
Among the 18 species of the Yersinia genus, Y. enterocolitica, Y. pseudotuberculosis and Y. pestis were extensively characterized in different subjects as ecology, epidemiology and pathogenicity mechanisms. Seven among the remaining 15 species (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii and Y. rohdei), often called Y. enterocolitica-like have not their pathogenic potential characterized and are usually considered to be nonpathogenic. However, literature data suggest that some of these species can cause diseases. These data stimulate the upsurge of questions about the mechanisms of which Y. enterocolitica-like species may interact with host cells and cause diseases. The main objective of this preject was to characterize the pathogenic potential of Y. enterocolitica-like strains, specifically of the species Y. frederiksenii, Y. kristensenii and Y. intermedia. This work evaluated the pathogenic potential of 118 Y. enterocolitica-like strains (50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii) searching for the presence of the virulence-related genes ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB and virF by PCR. Besides, Yersinia strains ability of adhesion and invasion to Caco-2 and HEp-2 cells after different pre-incubation periods, and its survival within human macrophages U937 were tested. Morphologic aspects of bacterial adhesion were observed by scanning electronic microscopy. Finally, the presence of new possible virulence mechanisms was evaluated through RNA sequencing of one Y. enterocolitica-like strain. The studied strains showed the following genes: Y. frederiksenii, fepA (44%), fes (44%) and ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) and tccC (35%); and Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) and hreP (54%). Usually Y. enterocolitica-like strains presented less ability of adhere and invade Caco-2 and HEp-2 cells than the highly pathogenic strain Y. enterocolitica 8081. On the other hand, Y. kristensenii FCF 410 and Y. frederiksenii FCF 461 showed high potential of invasion in Caco-2 cells after 5 days of pre-incubation, which were 45 and 7.2 times higher than the control Y. enterocolitica 8081 respectively, but ail gene was not found in these strains. Survival assay in human macrophages U937 showed that Y. frederiksenii FCF 461 (40.0%) and Y. frederiksenii FCF 379 (24.6%) strains presented survival percentages higher than Y. enterocolitica 8081 (13.4%). However, Y. intermedia and Y. iv kristensenii strains showed a reduced capability of surviving in macrophages. Scanning electron microscopy showed bacteria at the surface in contact with the cellular filopodia. The bacteria were distributed either individually or in small clumps. Therefore, it may be concluded that the presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Mammal cells adhesion and invasion assays suggest that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent. Human macrophages U937 surviving assay highlighted the pathogenic potential of some Y. frederiksenii strains. Together, the results suggest the existence of alternative virulence mechanisms other than the classical mechanisms described for pathogenic Y. enterocolitica. However, we could not verify the presence of possible new virulence mechanisms because 454 GS Junior (Roche) platform was not suitable for RNA sequencing of strains from cells interaction due its low coverage obtained.

Стилі APA, Harvard, Vancouver, ISO та ін.

40

Crisanti-Lassiaz, Patricia. "Effets de rétrovirus aviaires sur la différenciation de la neurorétine d'embryons d'oiseaux en culture cellulaire." Paris 7, 1985. http://www.theses.fr/1985PA077021.

Повний текст джерела

Анотація:

Le travail présenté dans cette thèse à pour but d'étudier les interactions : virus oncogènes et différenciation; et en particulier, l'effet du virus de Sarcome de Rous sur différents marqueurs de la différenciation des cellules de neurorétines d'embryon de caille ou de poulet in vitro. Cette thèse est composée de trois chapitres : une introduction générale expose les relations connues entre c-onc et la différenciation cellulaire. Le premier chapitre est consacré aux propriétés du virus de Sarcome de Rous et des autres virus oncogènes utilisés pour ce travail. Le chapitre II présente le système biologique : la Neurorétine et sa différenciation normale in vitro. Les effets des différents rétrovirus sur la différenciation de la NR, font l'objet du dernier chapitre.

Стилі APA, Harvard, Vancouver, ISO та ін.

41

(9828155), Top Pun. "Computer visualisation for microscopic discernment and counting of plant-parasitic nematodes." Thesis, 2024. https://figshare.com/articles/thesis/Computer_visualisation_for_microscopic_discernment_and_counting_of_plant-parasitic_nematodes/29147576.

Повний текст джерела

Анотація:

Plant-parasitic nematodes are invasive pathogens that cause severe damage to crops. To minimise the infestation of plant-parasitic nematodes, appropriate identification techniques and accurate population estimation of plant-parasitic nematodes are necessary to employ optimal control strategies. Conventionally, nematodes are identified using a manual microscope, and their morphological features are investigated to discern nematode species and count their population in the sample. However, this process is laborious and time consuming with the increasing number of samples. Although several nematode identification techniques have been proposed such as morphological, and molecular methods, these methods require complicated high-cost equipment, nematologists, or microbiologists, and bioinformatic skills. This study investigated image processing methods using state-of-the-art computer vision and deep learning (YOLO, you look only once) models to detect and quantify plant-parasitic nematode juveniles and eggs. The novel computer vision algorithm implemented graph network-based skeleton analysis to identify unique features of nematode juveniles in microscopic images. A new feature was revealed from the middle and average width difference of nematode eggs using the medial axis transform. The extreme point method was implemented to detect the accurate length of nematode eggs. Subsequently, this study explored the novel application of mosaic augmentation to detect and count plant-parasitic nematodes using state-of-the-art YOLO models. The YOLOv5-608 model attained the highest accuracy in the detection and counting of nematodes, whereas YOLOv7-512 performed well in detecting overlapped nematodes. The deep learning-based decision support tool was developed to automate the detection and enumeration of plant-parasitic nematode, calculate its population in the microscopic images, and suggest suitable nematode management strategies.

Стилі APA, Harvard, Vancouver, ISO та ін.

42

Götz, Ralph. "Super-resolution microscopy of plasma membrane receptors and intracellular pathogens." Doctoral thesis, 2020. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-207165.

Повний текст джерела

Анотація:

Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the “Receptome”, the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in naïve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi
Menschen neigen schon immer dazu, vor allem das zu glauben, was sie mit eigenen Augen sehen können, weswegen mikroskopische Methoden seit ihrer Erfindung im 17. Jahrhundert schon immer sehr beliebt waren. Mit der Einführung der hochauflösenden Mikroskopie konnte das Auflösungslimit von ~200 - 250 nm durchbrochen werden, was genauere Einblicke in biologische Proben ermöglichte. Insbesondere die Einzelmolekül-Lokalisations-Mikroskopie Methode dSTORM bietet hierbei die Möglichkeit der quantitativen Bildgebung. Sie nutzt das wiederholte Schalten organischer Farbstoffe in Anwesenheit von Thiolen, was eine Auflösung von bis zu 20 nm möglich macht. Eine weitere kürzlich entwickelte hochauflösende Mikroskopiemethode ist die Expansionsmikroskopie (ExM), in welcher die Probe isotrop vier- bis sogar zwanzigfach vergrößert wird, womit sich auch die Auflösung um diesen Faktor vergrößert. Um dies zu ermöglichen, wird die Probe in ein Hydrogel eingebettet, mittels einer unspezifischen Proteinase homogenisiert und in destilliertem Wasser expandiert. Innerhalb dieser Arbeit wurden beide Methoden genutzt, um sowohl die Verteilung von Plasmamembran Rezeptoren als auch unterschiedliche bakterielle und pilzliche Pathogene zu beleuchten Im ersten Teil dieser Arbeit wurde dSTORM genutzt, um das „Rezeptom“, die Gesamtheit aller Membranrezeptoren, sowohl von Jurkat T-Zellen als auch von primären Patientenzellen zu entschlüsseln. In dieser Arbeit konnten die Rezeptoren CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 und CD105 erfolgreich visualisiert und quantifiziert werden, welche Dichten von 0,8 Cluster pro µm² im Falle von CD20 und 81,4 Cluster pro µm² für den stark exprimierten Rezeptor CD45 in aktivierten primären T-Zellen auf der basalen Membran aufwiesen. Hierbei konnten wir für einen Großteil der Rezeptoren eine homogene Verteilung nachweisen, wohingegen nur wenige andere Rezeptoren Cluster zeigten. Für CD3 konnten sowohl in Jurkat T-Zellen als auch in aktivierten primären Zellen Cluster detektiert werden, was auf deren Aktivierung hinweist, wohingegen CD3 in naiven Zellen homogen verteilt war. Im Weiteren wurde dSTORM im Rahmen von drei klinischen Fragestellungen angewandt, in welche die Rezeptoren CD38, BCMA und CD20 involviert waren, die in Immuntherapien mit monoklonalen Antikörpern oder auch CAR T-Zellen adressiert werden. In den beiden erstgenannten Projekten wurde dSTORM genutzt, um die Erhöhung der Rezeptoren-Expression nach Zugabe verschiedener Medikamente sowohl in der Zelllinie MM1.S als auch in primären Zellen von Patienten mit multiplen Myelomen zu bestimmen. Durch das CD20-Projekt hingegen wurde die überlegene Sensitivität von dSTORM gegenüber der Durchflusszytometrie unter Beweis gestellt. Hier konnte verglichen mit der Durchflusszytometrie eine deutlich höhere CD20-positive Fraktion in Patientenzellen detektiert werden, welche nach Behandlung mit CD20 CAR T-Zellen eliminiert wurde. Hierauf folgte hochauflösende Bildgebung ganzer Zellen sowohl mit LLS-3D dSTORM als auch 10x ExM, um Interaktionen mit der Glasoberfläche ausschließen zu können. Bei 10x ExM wurde eine Signalamplifikation mittels Biotin und Streptavidin ATTO 643 benötigt, wonach sogar einzelne Antikörper, welche gegen den Heterodimer CD11a gerichtet waren, an einem herkömmlichen konfokalen Mikroskop detektiert werden konnten. Obwohl dies aufgrund der Prozesse von Gelierung, Verdau und Expansion während des ExM-Protokolls vermutlich nicht quantitativ ist, konnten sogar mutmaßliche Dimere des Rezeptors CD2 mit 10x ExM-SIM visualisiert werden, welche ähnlich in dSTORM Experimenten auftraten. Im zweiten Teil dieser Arbeit wurde die Expansionsmikroskopie für bakterielle und pilzliche Pathogene eingesetzt. ExM ermöglichte nicht nur eine isotrope vierfache Expansion von Chlamydia trachomatis, sondern auch die Unterscheidung der beiden Entwicklungsformen, der Retikulär- und Elementarkörperchen, aufgrund der Größe der einzelnen Chlamydien. Anschließend wurde ein neues α-NH2-ω-N3-C6-Ceramid eingeführt, was eine effiziente Fixierung und zum ersten Mal die Nutzung von Lipiden in 4x und 10x ExM ermöglichte, was wir Sphingolipid ExM nannten. Diese Verbindung wurde genutzt, um die Ceramid-Aufnahme und den -Einbau in die Zellmembran von Chlamydia trachomatis und Simkania negevensis zu untersuchen. Im Falle von Chlamydia trachomatis wurde die hohe Auflösung von 10x ExM mit SIM kombiniert, was die Visualisierung beider bakterieller Membranen in einem Abstand von ~30 nm ermöglichte. Hiernach wurde ExM bei den drei unterschiedlichen Pilzen Ustilago maydis, Fusarium oxysporum und Aspergillus fumigatus nach enzymatischen Verdau der pilzlichen Zellwand angewandt. Im Falle von Ustilago maydis Sporidien konnte der Verdau sowohl an lebenden Zellen, was in Protoplasten resultierte, als auch an fixierten Zellen verwendet werden, was die Morphologie erhielt. Mittels dieses neuen Protokolls konnten sowohl Immunfärbungen als auch fluoreszierende Proteine der drei genannten Pilze expandiert werden

Стилі APA, Harvard, Vancouver, ISO та ін.

43

Kunz, Tobias C. "Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens." Doctoral thesis, 2021. https://doi.org/10.25972/OPUS-22333.

Повний текст джерела

Анотація:

The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm
Aufgrund der Beugungseigenschaften des Lichtes wurde bereits 1873 durch Ernst Abbe für die Lichtmikroskopie eine theoretische Auflösungsgrenze von 200-250 nm definiert. Durch die Einführung verschiedener hochauflösender Mikroskopiemethoden, wie beispielsweise SIM-Mikroskopie (structured illumination microscopy), STED-Mikroskopie (stimulated emission depletion) und (d)STORM-Mikroskopie ((direct) stochastic optical reconstruction microscopy), konnte im letzten Jahrzehnt jedoch die Auflösung auf unter 100 nm verbessert werden. Allerdings benötigen solche Hochauflösungstechniken sowohl spezialisierte und kostenintensive Geräte als auch Expertenwissen zur Vermeidung von Artefakten, sodass diese nur in wenigen Laboren angewendet werden können. Ein alternativer Ansatz, die sogenannte Expansionsmikroskopie, wurde kürzlich von der Arbeitsgruppe um Ed Boyden etabliert. Hierbei wird eine Probe mit einem quellfähigen Gel vernetzt, welches daraufhin isotrop expandiert wird, sodass auch an konventionellen konfokalen Mikroskopen Hochauflösung ermöglicht wird. Seit ihrer Einführung im Jahre 2015 hat sich die Expansionsmikroskopie schnell entwickelt und bietet Protokolle für 4-fache, 10-fache oder sogar 20-fache Expansion von Proteinen als auch RNA in Zellen oder sogar komplexen Geweben. Mitochondrien besitzen zwei Membranen und sind für die Zelle von großer Bedeutung, da sie eine Vielzahl wichtiger Aufgaben übernehmen - von der ATP-Produktion durch die oxidative Phosphorylierung über die Produktion vieler wichtiger Metabolite bis hin zur Regulation zellulärer Signalwege. Die innere Mitochondrienmembran ist stark gefaltet und bildet Einstülpungen, die sogenannten Cristae, in welchen die oxidative Phosphorylierung und somit die Energieumwandlung und ATP-Synthese stattfindet. Morphologische Veränderungen der Cristae können sowohl beim Altern von Zellen, als auch bei verschiedenen Infektionen beobachtet werden und können darüber hinaus auch im Rahmen diverser Erkrankungen, wie beispielsweise Krebs, Diabetes oder neurodegenerativen Erkrankungen auftreten. Die Visualisierung der Cristae durch Fluoreszenzmikroskopie ist herausfordernd, da der Abstand zwischen einzelnen Cristae oftmals unter 100 nm beträgt. In der vorliegenden Arbeit wird gezeigt, dass die Expression der mitochondrialen Kreatinkinase gekoppelt an das Fluoreszenzprotein GFP (MtCK-GFP) als Cristaemarker genutzt werden kann. In Kombination mit vierfacher Expansion ermöglicht unser Marker die Untersuchung morphologischer Veränderungen von Cristae, sowie die Lokalisierung mitochondrialer Proteine relativ zu den Cristae. Darüber hinaus wird im Rahmen dieser Arbeit die Anwendbarkeit der Expansionsmikroskopie für mehrere bakterielle Pathogene, und zwar Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae und Staphylococcus aureus, gezeigt. Hierbei verdeutlichen wir wichtige Aspekte für den vollständigen Verdau unterschiedlicher bakterieller Zellwände und somit isotropen Expansion. Die Expansion der intrazellulären Pathogene C. trachomatis und S. negevensis ermöglichte es uns an konventionellen konfokalen Mikroskopen zwischen den zwei verschiedenen Entwicklungsstadien, der katabolisch aktiven Retikulärkörperchen (RBs) und der infektiösen Elementarkörperchen (EBs), zu unterscheiden. Außerdem konnte die Möglichkeit der präzisen Lokalisierung chlamydialer Proteine wie CPAF und Cdu1 innerhalb und außerhalb der chlamydialen Inklusion gezeigt werden und Bakterien, in diesem Fall S. aureus, in LAMP1 und LC3-II Vesikeln visualisiert werden. Mit der Einführung des unnatürlichen α-NH2-ω-N3-C6-Ceramides, präsentieren wir zudem ein erstes Konzept für die Expansion von Lipiden, welches möglicherweise auch für deutlich unzugänglichere Molekülklassen wie beispielsweise Kohlehydrate geeignet ist. Die effiziente Akkumulierung unseres funktionalisierten α-NH2-ω-N3-C6-Ceramides in Zellen sowie Bakterien ermöglicht in Kombination mit zehnfacher Expansion die Untersuchung der Interaktion von Proteinen mit der Zellmembran, Membranen von Organellen und Bakterien mit einer räumlichen Auflösung von 10-20 nm. Ceramid ist das zentrale Molekül des Sphingolipidstoffwechsels, ein wichtiger Baustein zellulärer Membrane und reguliert viele essentielle Prozesse wie die Zelldifferenzierung, die Proliferation als auch die Apoptose. Viele Studien berichten von der Bedeutung der Sphingolipide während der Infektion verschiedener Pathogene. So wurde beispielsweise zuvor berichtet, dass Ceramide aktiv zu Chlamydien transportiert und in deren Membranen eingebaut werden. Hierbei verblieb allerdings die Frage, ob Ceramide in der äußeren oder inneren bakteriellen Membran lokalisiert sind. Die Expansion unseres α-NH2-ω-N3-C6-Ceramides ermöglichte es uns Ceramide in der inneren und äußeren Membran von C. trachomatis zu visualisieren und den Abstand zwischen beiden Membranen auf 27.6 ± 7.7 nm zu bestimmen

Стилі APA, Harvard, Vancouver, ISO та ін.

44

Zaraee, Negin. "Interferometric imaging for pathogenic bacteria identification and antibiotic susceptibility testing." Thesis, 2021. https://hdl.handle.net/2144/42614.

Повний текст джерела

Анотація:

Pathogenic bacterial infections are a serious threat to public health, claiming millions of lives every year. In order to contain the spread of infectious diseases sensitive and timely diagnosis of pathogenic bacteria is of significant importance. The rapid detection of low abundance analytes is still challenging in the most common bacteria detection techniques including, culture and colony counting, Enzyme-linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). Conventional bacteria detection techniques suffer from limitations such as low sensitivity, cost, long procedural time and requiring complex lab equipment. Thus, there is a critical need for rapid, sensitive and low-cost bacterial detection platform in various applications ranging from water and food safety to medical diagnosis. The quest to overcome these limitations have sparked significant interest in innovative biosensor development, with considerable emphasis on optical techniques. Among optical biosensors, label-free methods are highly desirable over label-based alternatives for eliminating the additional cost and sample processing required for labeling. Also, techniques for whole-cell bacteria detection are preferred to detection of pathogenic molecular components detection due to the requirement for extracting and isolating the desired bacterial components such as nucleic acids or proteins. Overall, label-free whole-cell detection of pathogenic bacteria has a significant advantage of simplicity in sample preparation that translates to time and cost reduction. An additional benefit of detecting whole-cell bacteria without labels, thus in their natural environment, is the ability of monitoring the growth and replication of individual pathogens with a potential application in antimicrobial susceptibility determination. Despite the significant advantages of antibiotics as one of our most powerful tools for fighting infections, their extensive misuse and overuse over the years, have resulted in the emergence of antibiotic resistant bacteria as the global health crisis of our time. The current gold-standard technique for antibiotic susceptibility testing (AST) used in clinics, is culture-based disk diffusion assays. The time-consuming diagnosis method of the common clinical susceptibility testing, which is an inherent limitation of culture-based techniques, have necessitated the need for an alternative AST analysis platform. A clinical diagnosis test that could perform rapid pathogenic bacteria identification and determine its susceptibility to a panel of selected antibiotics, would greatly reduce the hospital stay time for patients with bacterial infection, therefore decreasing mortality and morbidity rate. In addition, it will have a great economic impact on the global healthcare system by advising optimal antibiotic use and maintaining the value of existing drugs. In this dissertation, we describe the design and development of a rapid, sensitive, and multiplexed biosensor platform that can both identify pathogenic bacteria and perform image-based AST on a single reader instrument. The simple and low-cost design of our biosensing platform makes it a perfect candidate as a point-of-care (POC) diagnostic tool in clinical setting. The biosensor presented in this dissertation is based on interferometric enhancement of the visibility of individual biological particles, such as viruses and bacteria, afforded by Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS), previously developed in our group. The integration of SP-IRIS with microfluidic flow cells provides kinetic measurements capability, by enabling in-liquid imaging of the sensor surface in real-time, therefore making it a promising diagnostic platform. Here, we build upon the SP-IRIS platform and utilize it for pathogenic bacteria identification and image-based AST analysis. To validate our biosensor's functionality, we demonstrate E. coli detection and characterization in end-point and real-time measurement modality through particle detection and tracking analysis of the acquired images from sensor surface. In addition, we perform rapid image-based AST analysis for E. coli bacteria against two antibiotics, ampicillin and gentamicin, by monitoring single cell morphological variations and tracking their growth rate under various antibiotic challenges.

Стилі APA, Harvard, Vancouver, ISO та ін.

45

ZENG, ZHEN, and 曾珍. "CHARACTERISTICS OF XANTHOMONAS CAMPESTRIS PV. MAGIFERAEIDICAE AND THE MICROSCOPY OF THIS PATHOGEN IN AND ON MANGO LEAVES." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/64455491452193392670.

Повний текст джерела

Стилі APA, Harvard, Vancouver, ISO та ін.

46

Roth, Karina Adriana. "Proliferation of Pathogenic Biofilms within Sealer-root Dentin Interfaces is Affected by Sealer Type and Aging Period." Thesis, 2011. http://hdl.handle.net/1807/31413.

Повний текст джерела

Анотація:

Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP). Methods: Standardized human root specimens were filled with the test materials and were aged for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each incubation period was assessed using fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation periods.

Стилі APA, Harvard, Vancouver, ISO та ін.

Дисертації з теми "Microscopic pathogens"

Оформте джерело за APA, MLA, Chicago, Harvard та іншими стилями