La présente étude visait à développer un protocole de fixation et d'échantillonnage pour le poumon équin suivant les directives publiées sur l’utilisation d’une approche stéréologique de type « design-based ». Les poumons gauches de chevaux contrôles et atteints du souffle ont été fixés avec du formaldéhyde 10% pendant 48h à une pression constante de 25-30 cm d’H2O. Les poumons ont été sectionnés en 20-21 tranches d’une épaisseur d'environ 2,5 cm chacune; de 10-11 tranches ont été sélectionnées de façon aléatoire et systématique pour la mesure du volume de référence avec la méthode de Cavalieri. Un protocole d’échantillonnage systématique, aléatoire et uniforme utilisant le principe du « smooth fractionator » et un poinçon à biopsie de 17 mm ont été utilisés pour échantillonner une fraction représentative de chaque poumon. Les méthodes d’échantillonnage de sections verticales, uniformes et aléatoires (VUR) et d’échantillonnage isotropique, uniforme et aléatoire (IUR) ont toutes deux été effectuées pour comparer le nombre de voies respiratoires en coupe perpendiculaire obtenues à partir de chaque méthode. L'architecture globale et la qualité des tissus fixés ont également été évaluées. Des spécimens pulmonaires équins ont été échantillonnés avec succès selon un protocole visant à produire des données morphométriques valides. Les tissus ont été fixés avec un minimum d'artéfacts et contenaient une quantité suffisante de voies respiratoires en coupe perpendiculaire dans les deux types d’échantillons. En conclusion, un protocole de fixation et d'échantillonnage adapté au poumon équin permettant l'utilisation d'une approche stéréologique de type « design-based » a été élaboré pour l’étude du remodelage des voies respiratoires. / The present study aimed at developing a fixation and sampling protocol for the horse lung in agreement with recent published guidelines for a design-based stereology approach. The left lungs from control and from heaves-affected horses were fixed in 10% formaldehyde for 48hr at a controlled constant pressure of 25-30 cm H2O. Lungs were cut into 20-21 slices of a thickness of approximately 2.5cm each; 10-11 slices were then randomly and systematically selected for the measurement of the reference volume using the Cavalieri method. A systematic, uniform and random sampling (SURS) protocol using a 17 mm punch biopsy and the smooth fractionator principle was used to select a representative fraction of each lung. The vertical uniform random (VUR) and isotropic uniform random (IUR) sampling methods were both performed to compare the number of perpendicular airways obtained with each method. The general architecture and the quality of the fixed tissues were also evaluated. Equine lung tissues were successfully sampled with a protocol designed to yield accurate morphometric data. The tissues were fixed with minimal artifacts and contained an adequate amount of perpendicular airways in both VUR and IUR sections. In conclusion, we developed a fixation and sampling protocol adapted to the equine lung allowing the use of a design-based stereology approach to study airway remodeling.

KUBÍNOVÁ, Zuzana. The effect of elevated CO2 concentration and irradiation on quantitative parameters of mesophyll cells of Norway spruce. Prague, 2010. 74 p. Master's degree thesis. Faculty of Science, Charles University in Prague. Abstract The aim of the present thesis was to choose and adjust a suitable methodology for counting particles in 3D space, which would be suitable for unbiased estimation of chloroplast number in needle mesophyll cells. The disector method was used for the first time to determine the number of chloroplasts. This method enables unbiased estimation of chloroplast number in needle volume from optical sections captured from fresh free-hand sections by confocal microscope. The sections did not need any pre-processing. Another aim was to compare selected photosynthetic and anatomical characteristics of sun and shade Norway spruce needles, which were grown under different CO2 concentration. The trees were grown for eight years in ambient (during the experiment increasing from 357 up to 370 µmol CO2 ∙ mol-1 ) CO2 concentration or elevated (700 µmol ∙ mol-1 ) CO2 concentration in special glass domes on an experimental research site of the Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic at Bílý Kříž in Moravskoslezské Beskydy mountains. The sun needles...

6 Abstract Temperate forests serve as long term carbon storage and are affected by increasing carbon dioxide (CO2) concentration in the atmosphere. Norway spruce (Picea abies (L.) Karst.) is the most abundant conifer in the forests of the Czech Republic, therefore I studied the response of its photosynthetic aparatus to elevated CO2 concentration. The aim of my thesis was to analyze the impact of CO2 concentration on chloroplast ultrastructure in both shaded and exposed needles, focusing on the volume density of starch in the median cross-sections of mesophyll cell chloroplasts. The next aim of the study was to test whether the chloroplasts of the first subepidermal layer of mesophyll are representative for the whole needle with respect to starch volume density. The study was performed on eleven years-old Norway spruce trees that had been exposed to ambient or elevated concentration of CO2 for six years; the experiment had been carried out at the Bílý Kříž experimental station in the Beskids Mountains in cultivation chambers with automatically adjustable windows. First year needles of trees grown under abient (382-395ppm) or elevated (700 ppm) CO2 concentration were collected in October 2011. The needles were used to prepare ultrathin sections and the images of median chloroplast cross-sections were...