Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms. / Master of Science

Made available in DSpace on 2016-04-28T20:18:25Z (GMT). No. of bitstreams: 1 ELISE MIYUKI YAMASAKI.pdf: 7116323 bytes, checksum: 0698deb473a21808a4e19ade2a6072b4 (MD5) Previous issue date: 2010-01-21 / The epidemic and clinic-pathological aspects of paratuberculois in a dairy cattle herd in the Rio Claro county, southern Rio de Janeiro region, are described. In the years 2006-2009, eight adult cows presented chronic-intermittent diarrhea, chronic weight loss and normal appetite. At necropsy, the subserosal lymphatics were proeminent and dilated, mesenteric nodes were enlarged, and intestinal mucosa was corrugated, thickened and of microgranular aspect. Especially, in duodenum, was observed polipoids lesions in mucosa surface. Histopathology revealed, from the duodenum to the rectum, severe and diffuse granulomatous inflammation of the lamina propria and submucosa, broadened and distorced villi, marked dilatation of the lymphatic vessels in their apex, lymphangioectasia and granulomatous lymphangitis in the submucosa. Ziehl-Neelsen stain revealed variable amounts of acid-fast bacilli in macrophages, Langhan s giant cells and freely in the musosa and submucosa of the small intestine and colon, and in lymphnodes. Lesions in the lamina propria, particularly in the jejun and ileum, in some animals, were severe hypertrophy; polipoids lesions observed in duodenum mucosa was markedly muscularis mucosa hipertrophy , intestinals and duodenal glands hiperplasia. Mycobacterium avium subsp. paratuberulosis was isolated by bacterial cultivation of samples from feces, intestinal mucosa and milk, and identified through IS900 PCR. From 298 cows, older than three years, the percentage of reactive animals was 40%, in indirect ELISA test. The diagnosis of paratuberculosis was based on clinic-epidemiological data, sorology, bacterial isolation and IS900 PCR. After the adoption of control measures, as slaughter of sick cows and selective slaughter of soropositive animals, was observed reduction of clinical cases in the herd, from six cases to one case per year, in three years of study. / Descreve-se os aspectos epidemiol?gicos e cl?nico-patol?gicos da paratuberculose em um rebanho bovino leiteiro no munic?pio de Rio Claro, regi?o Sul-Fluminense, Rio de Janeiro. No per?odo de 2006 a 2009, oito vacas adultas da ra?a Girolanda apresentaram diarreia cr?nico-intermitente, perda de peso progressivo e apetite normal. ? necropsia observou-se vasos linf?ticos subserosos proeminentes, linfonodos mesent?ricos aumentados de volume, ?midos ao corte, serosa do intestino com aspecto anelado e cerebr?ide, mucosa espessada, pregueada e com aspecto microgranular. Em especial, no duodeno havia les?es polip?ides na supef?cie da mucosa. ? microscopia, desde o duodeno at? o intestino grosso, havia acentuada inflama??o granulomatosa difusa, marcada dilata??o dos vasos linf?ticos no ?pice das vilosidades, linfangiectasia e linfangite granulomatosa na submucosa, muscular e serosa, altera??es tamb?m vistas nos linfonodos mesent?ricos. A colora??o de Ziehl-Neelsen revelou, vari?vel quantidade de bacilos ?lcool-?cido resistentes no interior de macr?fagos, c?lulas gigantes de Langhans e livres na mucosa e submucosa do intestino delgado e grosso e em linfonodos mesent?ricos. A l?mina pr?pria da mucosa, principalmente do jejuno e ?leo de alguns animais, apresentava acentuada hipertrofia; as les?es polip?ides correspondiam ? marcada hipertrofia da muscular da mucosa, hiperplasia de gl?ndulas duodenais e intestinais. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano a partir de amostras de fezes, raspado de mucosa intestinal e leite e identificado pela t?cnica de PCR IS900. Atrav?s da avalia??o sorol?gica semestral, foram analisadas 298 vacas, a partir de tr?s anos de idade, e observou-se cerca de 40% de animais reagentes ao teste ELISA indireto no per?odo estudado. O diagn?stico da paratuberculose foi baseado nos dados cl?nicopatol?gicos, sorologia, isolamento e identifica??o do agente em amostras de fezes, raspado de mucosa e leite, atrav?s do cultivo bacteriano e PCR IS900. Ap?s implementa??o de medidas de controle, tais como elimina??o de animais doentes, abate seletivo dos soropositivos, separa??o dos bezerros ao nascer com utiliza??o de banco de colostro, observou-se diminui??o da ocorr?ncia de casos cl?nicos no rebanho, de seis casos por ano para cerca de um caso por ano, em tr?s anos de estudo.

Chez les espèces fruitières, la dormance est une période clé qui influencera la floraison et la fructification. La dormance est finement régulée par de nombreux facteurs génétiques et environnementaux. Elle est composée de deux phases principales, l’endodormance (ou dormance « vraie ») et l’écodormance (ou dormance « environnementale »). L’endodormance est caractérisée par une incapacité totale des bourgeons à débourrer, même dans des conditions normalement propices à la croissance. C’est le froid hivernal qui permet la sortie de l’endodormance, nécessaire à la floraison puis la fructification. Les besoins en froid, quantité nécessaire de froid pour accéder à la levée de l’endodormance, sont génétiquement déterminés, et sont relativement élevés chez la plupart des variétés de cerisier doux (Prunus avium L.) comparativement à d’autres espèces. Dans un contexte de réchauffement climatique, les besoins en froid pourraient ne plus être satisfaits et conduire à des pertes économiques.. L’objectif de cette thèse est donc d’approfondir les connaissances sur les voies impliquées dans la levée d’endordormance. L’étude porte sur un panel de variétés contrastées pour leurs besoins en froid, depuis des variétés très précoces comme ‘Cristobalina’ jusqu’à des variétés très tardives comme ‘Fertard’. Des approches métabolomiques, moléculaires et d’apport exogène de molécules ont été utilisées. Les résultats montrent que les voies 1) hormonales, 2) du stress oxydatif et 3) des sucres, ont un rôle différent. La voie des sucres donne un aperçu de l’activité métabolique ainsi que des marqueurs des différentes phases de la dormance. La voie hormonale est un système permettant de contrôler la croissance sans lien causal sur la dormance. La voie du stress oxydatif indique que les variétés précoces sont dans un état globalement plus oxydé que les variétés tardives lors de cette période. Les données de RNA-seq permettent l’identification de gènes ayant un rôle majeur dans la dormance et de confirmer les hypothèses des mécanismes impliqués. Ces résultats seront déterminants pour la création de variétés adaptées aux conditions climatiques futures. / Among fruit tree species, dormancy is a key period determining the success of flowering and fruit producing. Dormancy is tightly regulated by numerous genetic and environmental factors. It can be divided in two main phases: endodormancy (“true dormancy”) and ecodormancy (“environmental dormancy”). Endodormancy is characterized by a total incapacity of buds to burst, even in conditions proper to growth. Winter chilling enables the end of endodormancy, necessary for flowering and fruit production. Chilling requirements, necessary amount of chill for endodormancy release, are genetically fixed and relatively elevated in sweet cherry (Prunus avium L.) varieties compared to other species. In a climate change context, chilling requirements may not be entirely fulfilled and lead to drastic economic losses. The objective of this work is to increase the knowledge on the pathways involved in endodormancy release. The study relies on a panel of varieties contrasted for their chilling requirements, varying from a very low chill cultivar ‘Cristobalina’ to a high chill cultivar ‘Fertard’. Metabolomics and molecular studies, and exogenous molecules supply were used for dormancy deciphering. Hormonal pathway, oxidative stress and sugar pathway seem to have different roles. Sugar pathway enables to have an overview of the metabolic activity, and may be used as molecular markers to identify dormancy phases. Hormonal pathway has a role on growth control but no causal link with dormancy release was assessed. Major results concern the role of oxidative stress, with low chill cultivars in a more oxidized status than high chill cultivars. Moreover, RNAseq data enable the identification of key genes controlling dormancy and confirms the hypothesis on mechanisms involved. These results will be crucial for breeding new verities well adapted to future climatic conditions.

Chez les espèces fruitières, la floraison est un évènement majeur qui influencera fortement la fructification. Ce processus, finement régulé par de nombreux facteurs génétiques et environnementaux, est encore peu connu. Chez le cerisier doux (Prunus avium), les fleurs ne s’épanouissent qu’après avoir satisfait des besoins en froid et en chaud. Les effets du changement climatique sur la floraison sont déjà notables et pourraient induire d’importantes pertes économiques. La compréhension des déterminants génétiques et moléculaires impliqués dans la floraison permettra l’amélioration des programmes de sélection variétale visant l’obtention d’arbres adaptés aux futures conditions climatiques. L’objectif de cette thèse est d’accroître les connaissances sur ces déterminants et d’identifier les gènes contrôlant la floraison chez le cerisier. En étudiant les deux familles intra spécifiques ‘Regina’ × ‘Lapins’ et ‘Regina’ × ‘Garnet’, la détection de nombreux quantitative trait loci (QTL) sur l’ensemble des groupes de liaisons (GL) a permis de confirmer la forte implication des besoins en froid dans la floraison ainsi que la complexité de ces caractères. Un QTL à effet majeur a été localisé sur le GL4. Dans les régions couvertes par les QTL contrôlant la date de floraison, une centaine de gènes candidats (GC) pour ce caractère a été identifiée. Un sous ensemble de ces GC a ensuite été étudié pour leur expression au cours du développement des bourgeons par PCR quantitative (qPCR). A terme, ces travaux serviront de bases pour l’identification et la sélection de gènes qui permettront l’obtention de génotypes adaptés aux futures conditions climatiques. / In fruit species, the flowering is a major event which strongly influences fructification. This process tiny controlled by many genetic and environmental factors is still little known. In sweet cherry (Prunus avium), flowers open out only after having satisfied chill and heat requirements. The effects of climate change on the flowering are already notable and could induce important economic losses. Identification of genetic and molecular determinants involved in the flowering will allow the improvement of varietal selection programs to obtain trees adapted to future climate conditions. Objective of this thesis is to increase the knowledge of these determinants and identify genes involved in flowering in sweet cherry. By studying two intraspecific progenies ‘Regina’ × ‘Lapins’ and ‘Regina’ × ‘Garnet’, detection of many quantitative trait loci (QTL) on all linkage groups (LG) has enabled us to confirm the strong involvement of chill requirements in the flowering as well as the complexity of these traits. QTL with major effect was localized on the LG4. In regions covered by all the QTLs controlling flowering date, a hundred candidate genes (CG) for this trait was identified. A subset of these GC was then studied for their expression during development of buds by quantitative PCR (qPCR). In the long term, this work will serve as a basis for the identification and selection of genes that allow obtaining genotypes adapted to future climate conditions.

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 5 uppsatser.

Lic.-avh. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 2 uppsatser.