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.

L'objectif de cette thèse était de déterminer dans quelle mesure l'équilibre en apports énergétiques et azotés dans la ration de ruminants en croissance (agneaux) pouvait avoir un impact sur l'efficacité d'utilisation de l'azote et en particulier des acides aminés (AA) chez ces animaux. Ce travail s'est particulièrement intéressé à l'utilisation des AA au niveau de l'aire splanchnique (tube digestif et foie) qui est utilisatrice importante d'AA et entre en compétition avec les muscles pour l'utilisation de ces AA. Les résultats montrent qu'une baisse des apports azotés seuls dans la ration relativement aux recommandations alimentaires ne se traduit pas nécessairement par une baisse de croissance, ce qui implique donc une meilleure efficacité d'utilisation de l'azote alimentaire. Ceci s'explique en partie par une diminution du prélèvement hépatique des AA (diminution du catabolisme des AA) et une meilleure efficacité d'utilisation des AA au niveau hépatique. A l'inverse, dans le cas d'une baisse des apports alimentaires en énergie seule dans le régime, la baisse de croissance et de rétention azotée est immédiate, associée à un catabolisme des AA et une uréogenèse intense chez les animaux. Ainsi, les recommandations en terme d'apports azotés chez les ruminants en croissance mériteraient d'être réévalués chez les ruminants afin de maximiser l'efficacité d'utilisation de l'azote dans la ration et limiter les rejets azotés dans l'environnement. A l'inverse, les recommandations énergétiques ne doivent pas être diminuées sous peine de pénaliser la croissance et l'accrétion protéique des animaux.

Magistrantūros studijų baigiamajame darbe pateikiama 2011–2012 metais Aleksandro Stulginskio universiteto Agronomijos fakulteto Agrobiotechnologijos laboratorijoje tirtų veiksnių, lemiančių sėjamosios kanapės dediferenciacijos indukciją hipokotilių ir skilčialapių kultūroje, tyrimų rezultatai. Darbo objektas – 10 dienų eksplantai auginti MS terpėje su skirtingais augimo reguliatorių kiekiais. Darbo metodai: Sėjamosios kanapės sėklos plautos po tekančiu vandeniu 1 val., 15 min. sterilintos 0,2 % kalio permanganato tirpale, 2 min. laikytos 70 % etanolio vandeniniame tirpale, 9 min. 10 % natrio hipochlorite ir 3 kartus po 5 min. mirkytos distiliuotame vandenyje. Sterilios sėklos daigintos ant MS maitinamosios terpės be augimo reguliatorių, papildytoje 10 g l-1 sacharozės ir 8 g l-1 Difco-Bacto agaru. MS terpėje su skirtingais augimo reguliatorių kiekiais auginta po 60 eksplantų. Kas keturias savaites eksplantai perkelti į šviežią tos pačios sudėties maitinamąją terpę. Vertintas kaliaus susidarymo dažnis (%), šaknų formavimosi dažnis (%), šaknų kiekis iš eksplanto (vnt.) bei ūglių formavimosi dažnis (%). Darbo rezultatai. Įvertinus sėjamosios kanapės kaliaus indukcijos tyrimų rezultatus nustatyta, kad sėjamosios kanapės izoliuoti hipokotilio audiniai nedideliu dažniu geba formuoti kalių ir terpėje be augimo reguliatorių. Naudojant vienos grupės (auksiną arba citokininą) augimo reguliatorių didžiausias kaliaus formavimosi dažnis gautas terpėje, papildytoje 0,5 mg l-¹ α... [toliau žr. visą tekstą] / The master work presents the results of factors affecting Cannabis sativa L. dedifferentiation process in vitro. Research was investigated at the Laboratory of Agrobiotechnology, Faculty of Agronomy of Aleksandras Stulginskis University in 2011 - 2012. Investigation in vitro was carried out with Cannabis sativa L. hypocotyls and cotyledons explants. Object of the work – 10 days explants grown in MS medium with different growth regulators concentrations. Method of the work – Cannabis sativa L. seeds were washed under running water for 1 h and surface sterilized with 0.2 % potassium permanganate for 15 min. sterilized and for 2 min. in 70 % ethanol, then sterilized in 10 % sodium hypochlorite for 9 min. and rinsed 3 times for 5 min. with sterile distilled water. Sterile seeds sprouted on MS nutrient medium without growth regulators, supplemented with 10 g l-1 sucrose and 8 g l-1 Difco Bacto-agar. MS medium with different growth regulators concentrations were grown 60 explants. Every four weeks, explants were transferred to the same composition fresh medium. The callus formation frequency (%), root formation frequency (%), root number of the explant (units) and the frequency of shoot formation (%) were evaluated. The results of the work – Cannabis sativa L. hypocotyls tissues with low frequency can form callus and without growth regulators in the medium. Using a single group of growth regulators (auxin or cytokinin) highest callus formation frequency obtained in the medium... [to full text]

Given that chronic hyperglycemia generates toxic methylglyoxal, the detoxifying effect of glyoxalase-1 (Glo1) on chronic hyperglycemia induced explant-derived cardiac stem cell (EDC) dysfunction was investigated. Wildtype (WT) and Glo1 over-expressing (Glo1TG) mice with or without streptozotocin treatment were studied. Hyperglycemia reduced overall culture yields while increasing the reactive dicarbonyl content within WT mice. These intrinsic cell changes reduced the angiogenic potential and nanoparticle production by hyperglycemic EDCs while promoting cell senescence. Compared to transplant of normoglycemic WT EDCs, hyperglycemic EDCs reduced myocardial function following infarction by inhibiting angiogenesis and endogenous repair mechanisms. In contrast, EDCs from hyperglycemic Glo1TG mice decreased reactive dicarbonyl content and restored culture yields. Intramyocardial injection of hyperglycemic Glo1TG EDCs also boosted myocardial function and reduced scarring. These findings demonstrate that, while chronic hyperglycemia decreases the regenerative performance of EDCs, over-expression of Glo1 reduces dicarbonyl stress and rescues the adverse effects of hyperglycemia on EDCs.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I–III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, β1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

Staphylococcus aureus est une bactérie responsable de nombreuses infections. Divers facteurs de virulence sont décrits comme ayant un rôle aggravant dans l’infection staphylococcique. La leucocidine de Panton-Valentine (LPV) en est un. Elle interagit par l’intermédiaire du récepteur de C5a (C5aR) avec les leucocytes et les cellules neuronales dans différents tissus, mais son action au niveau rétinien est méconnue. Nous avons recherché des cibles rétiniennes cellulaires de l’intoxination à la LPV et étudié ses conséquences cellulaires et inflammatoires précoces dans les tissus rétiniens. AINSI, deux modèles de lapins ont été créés : l'injection intravitréenne in vivo et les explants rétiniens ex vivo. Dans les deux modèles, les cellules ganglionnaires étaient les principales cibles cellulaires rétiniennes de la LPV et le seul type de neurones rétiniennes qui exprimait C5aR. Les cellules de Müller comme la microglie étaient activées. L’explant rétinien était facilement manipulé, ils peuvent servir à la recherche de la LPV sur la rétine. La LPV seule pourrait induire une inflammation rétinienne après avoir ciblé spécifiquement les cellules neuronales. / Staphylococcus aureus is responsible for many infections. It secretes various virulence factors aggravating the staphylococcal infections. Panton-Valentine leucocidin (PVL) is a virulent leukotoxin from S. aureus and presents active effects towards leukocytes and neuronal cells via the C5a receptor (C5aR). The effects of PVL on retina is little known. We explored PVL retinal cell target and early retinal inflammation and tried to find the processes of bacterial toxins aggravating bacterial endophthalmitis. We employed two different rabbit models to study the PVL effects on retina: intravitreal injection in vivo and retinal explant ex vivo. In the two models, retinal ganglion cells were the only retinal neurons which express C5aR and the major cell targets of PVL in retina. PVL induced retinal Müller and microglial cell activation. The retinal explants were easily manipulated and showed obvious cellular targets of PVL and glial cell activations, they can contribute to research the effects of PVL on retina in future. PVL alone without S. aureus could induce great retinal inflammation after targeting specifically retinal neurons.

This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.

Linseed flax studies in vitro were carried out at the Laboratory of Genetic – Biotechnological, Department of Crop Science and Animal Husbandry of Lithuanian University of Agriculture in 2002-2005, photomorphogenetic processes were investigated at the Laboratory of Plant Physiology of the Lithuanian Institute of Horticulture in 2004-2005. Optimal conditions for the dedifferentiation of linseed flax stem segments and hypocotyls cells and those of secondary differentiation in vitro were ascertained. Light technology of a solid body was used for investigation and an optimal combination of light parameters has been selected, promoting photomorphogenesis in vitro. Factors determining androgenesis and gynogenesis processes in vitro were evaluated and potential androgenic and gynogenic genotypes for the development of initial selection material were selected.

L’adiponectine (ApN) est une adipokine de 30 KDa produite abondamment par le tissu adipeux qui a été décrite pour la première fois en 1995. Dès lors qu’une corrélation inverse entre les taux circulants d’ApN et l’adiposité a été mise en évidence, de très nombreuses études ont été initiées pour chercher à établir un lien avec les pathologies liées au syndrome métabolique. Ainsi, chez les patients obèses et diabétiques, une perte de poids induite par un traitement médicamenteux ou un régime hypocalorique s’accompagne d’une augmentation des taux circulants d’ApN et d’une amélioration des paramètres biochimiques. Il ressort de ces études que des taux élevés d’ApN sont corrélés à une amélioration de la sensibilité à l’insuline, des paramètres lipidiques et une diminution du risque cardiovasculaire. Cependant, les mécanismes d’action conduisant à l’amélioration du métabolisme lipidique et glucidique restent encore mal connus. Au cours de cette étude, nous avons, dans un premier temps, cherché à déterminer les conséquences d’une variation de la masse grasse sur la production de l’ApN chez la souris. Pour cela, différentes stratégies nutritionnelles et pharmacologiques, conduisant à une fonte ou à une augmentation de la masse grasse, ont utilisées. Les résultats obtenus suggèrent que la production d’ApN n’est pas dépendante de la taille des adipocytes mais plutôt de leur état inflammatoire. Dans un second temps, grâce à un modèle d’explants de foie en culture, nous avons cherché à déterminer les effets directs de l’ApN sur le métabolisme lipidique du foie. Une première approche a consisté à tester les effets à long terme c’est à dire susceptibles de correspondre à des modifications d’expression génique et de synthèse protéique en incubant les explants pendant 21h dans un milieu de culture contenant de l’ApN en présence ou non d’insuline. Les résultats suggèrent que l’ApN accentue les effets de l’insuline, ce qui se traduit par une meilleure utilisation du glucose avec en contre partie une stimulation de la lipogenèse et une réduction de la ß-oxydation en présence d’insuline. De plus, l’ApN apparaît exercer un effet sur les voies de catabolisme des HDL. Par une seconde approche, nous avons testé les effets à court terme de l’ApN sur les capacités β-oxydatives en traitant les explants pendant 45 min. Dans ces conditions plus appropriées pour mettre en évidence l’activation des voies de régulation rapides, la présence d’ApN entraine une stimulation de la ß-oxydation qui s’accompagne d’une augmentation de la p- AMPK. En conclusion, ces résultats suggèrent que l’ApN exerce des effets directs sur le métabolisme hépatique du glucose compatible avec l’effet insulino-sensibilisateur observé in vivo. En revanche, les résultats ne permettent pas, à ce stade, d’établir une relation entre une induction par l’ApN de l’activité ß-oxydative des acides gras dans le foie et ces effets bénéfiques sur les paramètres lipidiques. / Adiponectin (ApN), a 30-kDa adipokine abundantly produced by adipose tissue has been described for the first time in 1995. Early findings demonstrating serum adiponectin levels are inversely correlated with obesity initiated intense investigation of the relationship between adiponectin and all symptoms of the metabolic syndrome. Of these studies, strong evidence suggests that a weight loss induced by medication or hypocaloric diet led to increased circulating ApN levels and improvement of biochemical parameters. Thus, high levels of ApN have been correlated with an improvement of insulin sensitivity and lipid parameters reducing cardiovascular risk. However mechanisms involved in the improvement of glucose and lipid metabolism are not fully understood. In this study, we wanted to determine whether variations of plasma ApN levels are associated with alterations of liver lipid metabolism in mice. For this, we used different nutritional and pharmacological strategies to induce fat mass variations and measured corresponding ApN production and lipid parameters. First, results suggested that production of ApN is not dependent on the size of adipocytes, but rather to their inflammatory state. The effect of ApN of liver metabolism was further studied using an in vitro model of liver explants in culture. A first approach consists of testing the long-term effects of ApN on liver incubating the slices for 21h in the presence or not of insulin. Biochemical and molecular data suggested that ApN accentuates the effects of insulin, resulting in improvement of glucose utilization associated with a stimulation of lipogenesis and a reduction in β-oxidation in the presence of insulin. In addition, ApN appears to affect HDL catabolism pathways. In a second approach, we tested the short-term effects of ApN on β-oxidative capacity treating the slices for 45 min. Under these conditions more appropriated to highlight the activation of fast regulatory pathways, ApN induced a stimulation of ß-oxidation which was accompanied by an increase in p-AMPK. In conclusion, data suggested that ApN has direct effects on hepatic glucose metabolism consistent with the insulin-sensitizer effect demonstrated in vivo. However, at this stage, these results did not give evidence of a relationship between induction of liver fatty acid β- oxidation by ApN and its beneficial effects on lipid parameters.