Zugl.: Leipzig, Univ., Diss., 2008

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met lit.opg. en samenvattingen in het Nederlands en Russisch.

Universiẗat, Diss.--Kaiserslautern, 1997.

Proefschrift Maastricht. / Lit.opg. - Samenvatting in het Engels.

Proefschrift Maastricht. / Lit.opg. - Met samenvatting in het Nederlands.

The aim of this work is to propose reliable method for distinguishing between diploid (2n) and tetraploid (4n) taxa of the genus Betula L., based on leaf measurements. These measurements are realized by common tools, such as ruler or protractor. Main advantage of these methods is that they can be applied even in the field. The further aim of work is to describe and evaluate genetic variability within tetraploid taxa, whose taxonomic relationships are still unclear and under discussions. For statistical analysis, 97 individuals from 6 localities within Šumava region were selected and 4 leaves from every individual were analysed. In total, 20 parameters were measured on every leaf (16 quantitative and 4 qualitative). Each individual was also analysed by flow cytometry method to determine real genome size (ploidy). Differences in selected parameters between individuals with different ploidy were statistically tested. Significant results were observed in 12 of 16 quantitative and 2 of 4 qualitative parameters. For real ploidy prediction, the classification function was designed, whose reliability was 96 % for the primary data. The reliability of classification function was verified on samples from three different regions within the Czech Republic and compared with functions suggested by other authors. Function designed in this work was able to correctly determine real ploidy in 89 %, which is the most of all compared functions. Genetic variability, especially within tetraploid taxa is mentioned in relation to other author's previous studies. Distinguishing between B. pubescens and B. carpatica was not possible using selected genetic markers. Genetic conformity of these taxa is also mentioned in publications of foreign authors, as contrasted to Czech literature, which generally distinguishes B. pubescens and B. carpatica as separated species.

Many studies describe the beneficial effect of probiotic bacteria of the gastrointestinal tract and their immunomodulatory effects. The aim of this diploma thesis was to evaluate the effect of probiotics, which are commonly used in human and animal nutrition, on the viability of the cells of the immune system. For the experiment were selected blood monocytes, which were isolated from porcine and human blood by dextran sedimentation. Monocyte population was co-cultivated in vitro with probiotic strains Bifidobacterium bifidum, Lactobacillus rhamnosus and Enterococcus faecium for 2 and 4 hours. The percentage of apoptosis and necrosis of monocytes was analysed by flow cytometry. The results showed statistically significant differences in the proportion of apoptotic and necrotic of porcine and human monocytes. It is obvious that the selected strains of probiotics have immunomodulatory effect on immune cells. Enterococcus faecium and Lactobacillus rhamnosus induce cell death of porcine and human monocytes during short-term cultivation in vitro.

Assessment of semen quality (sperm viability) is an essential for the efficient honeybee semen storage. The motility of spermatozoa is not the ultimate parameter of sperm viability. Instead, the viability of spermatozoa is assessed by testing their structural or functional plasma membrane integrity. These assays are based primarily on the technique of microscopy evaluation of either fluorescent DNA dyes or hypo-osmotic "tail-coiling" reaction of spermatozoa. However, the technique of microscopy evaluation is time-consuming, subjective and often is not accurate. On the other hand, the technique of flow cytometry is rapid, objective and precise. Nowadays, this technique is affordable for many institutions, as the price of "benchtop" flow cytometer is relatively low. To best of our knowledge, there is currently no flow cytometry assay available for evaluation of functional integrity of honeybee spermatozoa. Here we report the establishing of novel flow cytometry assay for simultaneous evaluation of structural and functional plasma membrane integrity. DRAQ7, the new fluorescent DNA dye was used to test the structural integrity of plasma membranes. The hypo-osmotic "tail-coiling" reaction was used for flow cytometric assessment of functional integrity of spermatozoa plasma membranes. The combination of both is a valuable tool for rapid and precise evaluation of sperm viability in honey bees.

Les levures non conventionnelles, dont Yarrowia lipolytica, sont devenues desusines cellulaires attractives pour la production de protéines recombinantes. Lasélection de promoteurs régulés impliqués dans le métabolisme de substratshydrophobes est d'un grand intérêt pour une telle application. Dans ce cadre,l’objectif de ce projet est de mieux comprendre la régulation des promoteurs desgènes POX2 et LIP2 dans le but d’améliorer la production de protéinesrecombinantes chez Y. lipolytica.D’un point de vue biotechnologique, l'analyse à l’échelle cellulaire est devenueune approche répandue pour l’analyse et l’optimisation des bioprocédés. Ainsi,l'objectif de la première partie de ce projet vise le développement d’une méthoded’analyse en ligne des paramètres de culture Y. lipolytica dans un milieu contenantde l’acide oléique. Cette technique consiste au couplage d’un bioréacteur à uncytomètre en flux via une interface d’échantillonnage automatique. Cetteméthodologie a conduit principalement à une analyse rapide de la croissancecellulaire, de l'accumulation des lipides, du dimorphisme ainsi qu’à l'analyse duniveau d’induction des promoteurs chez Y. lipolytica.Les systèmes d’expression basés sur les promoteurs LIP2 et POX2 sont difficilesà manipuler, principalement en raison de l’utilisation de substrats insolubles dansl’eau (acide oléique) comme inducteur. Dans ce travail, il a été clairement démontréque pLIP2 est le promoteur de choix à utiliser pour développer un procédé deproduction de protéines recombinantes par culture de Y. lipolytica dans un milieucomplexe. De plus, l’utilisation d’un mélange acide oléique-glucose 60/40 (w/w) aconduit à une amélioration du niveau d’induction du promoteur LIP2 par un facteurde 10 par rapport à utilisation l'acide oléique. De plus, l’analyse à l’échelle cellulairemontre que ces deux substrats sont co-consommés par les cellules.Enfin, la dernière partie de cette thèse a eu pour but d'étudier la régulation dupromoteur LIP2 par rapport à la transition dimorphique. Nos résultats ont clairement montré que le changement morphologique chez Y. lipolytica n'a pas d'impact sur larégulation de pLIP2.L’ensemble de ces études a conduit à une meilleure compréhension de laphysiologie de Y. lipolytica, soulignant son potentiel avéré de production desprotéines recombinantes. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished