1 Abstract: The diploma thesis focuses on study of sex chromosomes evolution and repetitive organized genes of chosen mygalomorph and araneomorph spiders. Spiders are characterized by complexicity of sex chromosome systems, their karyotypes contain multiple sex chromosomes X. Besides multiple X chromosomes they also contain a pair or two pairs of nondiferentiated sex chromosomes X and Y. The used methods include methods of classical cytogenetics (preparation of chromosome slides, C-banding) and methods of molecular cytogenetics (fluorescent in situ hybridization and comparative genome hybridization). Complex sex systems were discovered in the studied Theraphosidae spiders. In Theraphosidae spiders Atropothele socotrana and Poecilotheria vittata neo-sex chromosomes were found. Analysis of molecular differentiation of sex chromosomes suggests low differentiation of Y chromosome in neo-sex chromosomes and pair of nondifferentiated sex chromosomes XY. In haplogyne spider Kukulcania aff. hibernalis (X1X2Y), the Y chromosome was significantly differentiated, male specific signal covered the whole chromosome. Detection of 18S rDNA showed that karyotypes of majority of analysed Theraphosidae spiders and haplogyne spiders contain low number (1 or 2) of nucleolar organizing regions localized terminally, which...

Cytogenetics of mesothelid spiders is largely unkown. The presented diploma thesis is focused on the karyotype evolution of these spiders. As it is the most basal group of spiders, the analysis of its cytogenetics can bring important data about ancestral spider karyotype. In the framework of my thesis, I analysed diploid chromosome numbers, chromosome morphology, meiotic division, sex chromosomes and the pattern of selected molecular markers that were detected by fluorescence in situ hybridization. According to my results, mesothelid spiders have a high number of chromosomes and the prevalence of monoarmed chromosomes. Unlike other spiders, mesothelids have little differentiated sex chromosomes. Key words: evolution, spider, chromosome, karyotype, fluorescence in situ hybridization, nucleolar organiser region, sex chromosomes

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

Abstract : Current prenatal diagnosis depends on invasive procedures and is thus offered only to high-risk pregnancies. Development of non-invasive prenatal diagnosis (NIPD) would change the risk-benefit ratio and make it likely that more women would benefit from prenatal testing. Scientists have documented the presence of rare fetal cells in maternal blood and envisioned targeting them with specific markers and their use in NIPD. Considering their extremely low frequency in maternal blood, fetal cells have been difficult to retrieve and use in clinical practice. Therefore, there is a pressing need for systematic sequential studies to evaluate their feasibility in NIPD. Generally, detection of rare cells within a large cell population carries great potentialities for the prospects of cancer management and NIPD. Manual scanning is very cumbersome and time-consuming Therefore; the first part of our project was, dedicated to the optimization of an effective strategy to evaluate retrieval of rare cells. We have developed a way of distributing a controlled number of target cells among hundreds of thousands of other cells on microscope slides. This strategy allows the precise evaluation of the retrieval of rare events and the comparizon of the efficacy of different techniques and enrichment approaches by knowing the definite number and locations of target cells on the slides. Furthermore, it allows the evaluation of hybridization of missed events. We have also developed a robust custom-made detection algorithm for rare cells using the MetaSystems automated platform and have used this strategy in the validation of manual and automatic scanning of 60 slides with a pre-defined number of rare male cells among a pure population of female cells using XY-FISH. Consequently, we tested the developed classifier for the detection of real fetal cells from maternal blood in both normal and aneuploid pregnancies with Down syndrome. We further evaluated the number of fetal cells with different methods of enrichments in the first and second trimesters. The data collected confirmed the early presence of fetal cells in all of the pregnancies tested and their frequencies were higher in cases of aneuploidies. Fetal cells are in a state of dynamic change throughout the pregnancy. Higher numbers of these cells can be obtained by optimizing the harvest time and methods of enrichment. We found that automatic scanning is more sensitive and reliable than manual detection. Furthermore, it alleviates the burden of scanning large numbers of cells and thus is more suitable for clinical application. We also demonstrated the feasibility of using rare cells in NIPD. Five microdissected amniotic fetal cells from 26 cases of normal and aneuploid pregnancies were quite enough to provide accurate NIPD through using whole genome amplification coupled with QF-PCR. Our findings laid the ground for the use of rare fetal cells in maternal blood for NIPD. // Résumé : Le diagnostic prénatal résulte encore aujourd’hui de procédures invasives, qui présentent des risques pour la grossesse. Le développement du diagnostic prénatal non-invasif (DPNI) changerait le rapport risque : bénéfice, rendant le diagnostic prénatal plus intéressant pour les femmes enceintes. Plusieurs chercheurs ont montré la présence de cellules fœtales dans le sang maternel et des travaux ont été entrepris afin de les cibler et de les utiliser éventuellement en DPNI. Toutefois, la faible concentration des cellules fœtales dans le sang maternel réduit les possibilités d’isolement ainsi que celles de leur utilisation en clinique. Un autre aspect technique du DPNI, le balayage manuel, est très laborieux, surtout en terme de temps technique. Il y a donc un besoin certain pour des études approfondies afin d’évaluer et d’améliorer la faisabilité du DPNI. La détection d’évènements rares dans une grande population cellulaire offre un potentiel pour le diagnostic en oncologie mais aussi en diagnostic prénatal. Dans cette thèse, la première étude était dédiée à l’optimisation d’une stratégie pour détecter les cellules rares. Nous avons développé une méthode d’étalement sur lame d’un nombre précis de cellules cibles parmi des centaines de milliers de cellules. Cette stratégie a permis d’évaluer le taux de détection d’évènements rares et de comparer l’efficacité des techniques d’enrichissement en connaissant le nombre exact et la localisation de cellules cibles sur les lames. De plus, il a été possible d’évaluer les problèmes d’hybridation des évènements manqués. Nous avons, par la suite, développé un algorithme robuste pour la détection de cellules rares en utilisant la plateforme de microscopie automatisée MetaSystems et utilisé cette approche dans la validation des balayages manuel et automatique d’un nombre précis de cellules mâles parmi une large population de cellules femelles marquées avec la technique FISH. Nous avons testé ce classificateur avec des échantillons de sang de femmes enceintes de grossesses normales et aneuploïdes et évalué la fréquence de cellules fœtales isolées par différentes méthodes d’enrichissement au cours des premier et second trimestres de grossesse. Les données accumulées ont confirmé la présence de cellules fœtales chez toutes les grossesses et leur fréquence plus élevée dans les grossesses aneuploïdes. Le nombre de cellules fœtales est dynamique tout au long de la grossesse. De plus, un nombre plus élevé de cellules fœtales peut être obtenu en optimisant le moment du prélèvement et les méthodes d’enrichissement. De plus, le balayage automatique s’est avéré plus sensible et constant que le balayage manuel, ce qui permet de balayer un grand nombre de cellules et devient plus approprié pour une application clinique. Nous avons aussi montré la faisabilité d’utiliser des cellules fœtales dans le cadre du DPNI. Cinq cellules amniotiques microdisséquées, provenant de grossesses normales et aneuploïdes, ont suffi pour poser un diagnostic prénatal par une combinaison de l’amplification du génome complet et de la technique QF-PCR (réaction quantitative en fluorescence d’amplification entraînée par une polymérase) permettant la détection d’anomalies chromosomiques. Nos résultats ouvrent la voie à l’utilisation de cellules fœtales dans le sang maternel pour le DPNI.

Thèse réalisée en co-tutelle à l'Université de Montréal et de Paris Sud / Ce travail de thèse traite de la construction des masculinités par des athlètes investis dans une carrière sportive de haut niveau. Notre enquête s’appuie sur des observations ethnographiques durant les entraînements et sur des entretiens (n=48) réalisés avec des athlètes (n=38) et entraineurs (n=10) dans un Pôle Espoir de handball (n=8), de rugby (n=13) et dans un Pôle France de boxe française (n=13), de patinage artistique dans la catégorie individuelle (n=11), ainsi que dans trois clubs au Québec (n=3). Un premier apport de cette thèse réside dans la mise en relation entre deux cadres théoriques utilisés respectivement en sociologie du sport de langue anglaise et française. Nous avons analysé nos données en utilisant les concepts — d’ordre de genre, de régime de genre et de masculinité hégémonique — développés par Raewyn Connell et ceux de — champ, d’habitus et de capital — développés par Pierre Bourdieu. Nous avons proposé de considérer que le champ sportif est régi par une forme de masculinité hégémonique. Cette dernière se définirait comme une configuration idéale d’appartenances, de dispositions et de capitaux, propice à assurer la re-productivité des agents dans le jeu social. Nous avons vu que cette forme était malléable. Il existe une pluralité de masculinités hégémoniques en relation avec le régime de genre spécifique à chaque discipline et plus précisément avec l’ordre de genre local ancré géographiquement et institutionnellement. Un deuxième apport de cette thèse réside dans l’étude du processus d’hybridation des masculinités en relation avec la multiplication des exigences institutionnelles. En rupture avec une conception essentialiste de la masculinité hégémonique, nous relevons que cette dernière se caractérise par la pluralité de ces facettes et par sa plasticité. Les athlètes les plus conformes à la forme hégémonique dans leur discipline développent une capacité à entrer dans des registres dispositionnels variés, voire contrastés. Ils sont toujours plus aptes au combat, mais plus fins dans les usages de la violence et de leur corps, qu’ils apprennent à gérer comme un capital (esthétique, hygiénique). Par ailleurs, ils mettent à distance l’expression de sexisme et d’homophobie, ce qui ne permet pas pour autant de remettre en question l’inégale valeur des sexes, des genres et des sexualités dans le champ sportif / This thesis examines the construction of masculinities by athletes invested in a high-level sporting career. Our investigation is based on ethnographic observations during sports training and interviews (n = 48) conducted with athletes (n = 38) and coaches (n = 10) in a Pole Espoir of handball (n = 8) of rugby (n = 13) and in a Pole France of french boxing (n = 13), in two Poles France of ice skating in the individual category (n = 11), and in three clubs at Quebec (n = 3). The first contribution of this thesis lies in the linkage between the two theoretical frameworks used in Sport Sociology respectively in English and French language. We analyzed our data using the concepts - gender order, gender regime and hegemonic masculinity - developed by Raewyn Connell and those - field, habitus and capital - developed by Pierre Bourdieu. We proposed to consider that the sports field is governed by a form of hegemonic masculinity. This form would be defined as an ideal configuration of affiliations, dispositions and capital favourable to promote the re-productivity of agents in the social game. We have seen that this form was malleable. There is a plurality of hegemonic masculinities in relation to specific gender regime of each discipline and more precisely with the local gender order which is geographically and institutionally anchored. The second contribution of this thesis lies in the study of the process of masculinities hybridization in relation to the increase of institutional requirements. Contrary to an essentialist conception of hegemonic masculinity, we note that this latter is characterized by the plurality of its facets and its plasticity. The athletes, who are the most conform to the hegemonic form in their discipline, develop a capacity to enter into a variety or mixed dispositional uses. They are always more "fit for fighting", but more subtle in the uses of violence and their bodies, that they learn to manage as a capital (aesthetic, hygienic). In addition, they put away the expression of sexism and homophobia, which still does not really allow reconsidering the unequal value of sexes, genders and sexualities in sports field.

L’organisation cellulaire repose sur une distribution organisée des macromolécules dans la cellule. Deux ARNm, cen et ik2, montrent une colocalisation parfaite aux centrosomes. Ces deux gènes font partie du même locus sur le chromosome 2L de Drosophila melanogaster et leur région 3’ non traduite (3’UTR) se chevauchent. Dans le mutant Cen, le transport de Ik2 est perturbé, mais dans le mutant Ik2, la localisation de cen n’est aucunement affectée. Ces résultats suggèrent que cen est le régulateur principal de la co-localisation de cen et ik2 aux centrosomes et que cette co-localisation se produit par un mécanisme impliquant la région complémentaire au niveau du 3’UTR des deux transcrits. La localisation de cen au niveau des centrosomes dans les cellules épithéliales de l’embryon est conservée dans différentes espèces de Drosophile : D. melanogater, D. simulans, D. virilis et D. mojavensis. Cependant, la localisation de ik2 n’est pas conservée dans D. virilis et D. mojavensis, deux espèces dont les gènes cen et ik2 sont dissociés dans le génome. Ces résultats suggèrent que la proximité de Cen et Ik2 dans le génome est importante afin d’avoir un événement de co-localisation de ces deux transcrits. J’ai généré différentes lignées de mouches transgéniques dans lesquelles un transgène contenant la séquence GFP fusionnée à différentes partie de Cen (partie codante, 3’UTR, Cod+3’UTR) qui sont sous le contrôle du promoteur UAS et qui sont gal4 inductibles. La région codante de l’ARNm cen était suffisante pour avoir un ciblage précis du transcrit aux centrosomes. / Messenger RNA (mRNA) localization plays a key role in establishing cellular architecture and function. The centrocortin (cen) and IkB Kinase-like 2 (ik2) mRNAs are co-localized to centrosomes in embryonic epithelial cells. Interestingly, both of these genes are organized in a head-to-head configuration in the genome, with their 3’ untranslated regions (3’UTRs) overlapping on opposite DNA strands. Here we show that gene positioning of cen and ik2 is important for the co-localization of these transcripts during Drosophila embryogenesis. The localization of cen is conserved within the Drosophila phylogeny and ik2 cannot localize when it is separated from the cen locus. Also, loss of function mutants of cen show a complete loss of ik2 localization, proposing that cen is the main driver of the co-localization. Structure-function analysis revealed that the coding region of cen is necessary for its centrosomal targeting, suggesting that a cis-regulatory motif that drives its localization is located in the coding region. This study reveals for the first time the importance of gene positioning for RNA localization. We suggest a model where cen mRNA is the main driver of centrosomal localization, which may occur through post-transcriptional interaction/annealing of these mRNAs via their 3’UTRs.

Abstract This MSc. thesis deals with karyological, genetic and phenotypic variation of selected taxa from the genus Dianthus L. in the Czech Republic and adjacent countries. The evolutionary history of the genus has been shaped by several microevolutionary processes, including interspecific hybridization, genome duplication, and edaphic speciation. These processes led to the origin of a number of phenotypically similar taxa, which are often restricted to a narrow geographic area. One subendemic and three endemic taxa occur in the Czech Republic. These (sub)species were used as model groups to gain insight into microevolutionary processes in small populations and the postglacial development of the genus in Central Europe. The thesis consists of three parts, each addressing different evolutionary phenomenon: Dianthus arenarius subsp. bohemicus is a critically endangered endemic psammophyte currently known from a single population in Central Bohemia. The site is also inhabited by widespread D. carthusianorum. Interspecific hybridization has been suspected on the basis of morphological characters, but this has never been confirmed by any other technique. I exploited differences in the number of chromosomes between both species and, with the aid of DAPI flow cytometry, estimated relative DNA contents of...

This study investigates the hybridization of two species of cattails, Typha latifolia (Common Cattail) and T. angustifolia (Narrow-leaved Cattail) in the Czech Republic. The aim of this study was to determine, how often T. latifolia and T. angustifolia hybridize, whether hybridization is allowed by overlapping flowering time of these species and whether it is possible these species controlled cross in a culture. For detection of hybrid individuals were used microsatellite DNA markers, which allow to detect hybridization events and differentiate hybrids from the parental species. Molecular analysis revealed that hybridization of T. latifolia and T. angustifolia occurs in the Czech Republic, but it is not frequent. Of the 267 analyzed individuals, 130 individuals were pure species T. latifolia, 108 individuals pure species T. angustifolia and 29 individuals were hybrids. Of the hybrids, 23 were advanced hybrids, 5 were backcrosses and only one individual was F1 hybrid. Flowering time of T. latifolia and T. angustifolia overlaps, which allows hybridization, and flowering time to not act as a prezygotic reproductive isolation barrier and gametes T. latifolia and T. angustifolia can blend together. In controlled crosses the female spikes T. latifolia and T. angustifolia created seeds, but these were...

On the 4th of October 1562, the Polish princess Catherine Jagiellon married the Swedish prince and duke of Finland Johan Vasa. Leaving Poland, Catherine Jagiellon was equipped with a very rich dowry and followed by an entourage of nearly 50 people. The objective of this study is to investigate the objects and people surrounding the newly wedded 16th century princess and asses if the document mirrors the complex cultural interactions of the early modern world. The analysis of the inventory is carried out using two theoretical approaches: material culture and culture transfer. The study is constructed in two parts. The first part focuses on the analysis of the sections of the inventory following the order of the document. If possible, the objects are mapped, their history is traced through the sources, their appearance and function are discussed. The examination of the members of the court is also carried out. With the deepened analysis of the inventory as a basis, the second part of the study is dedicated to the search of culture hybridization markers in described artefacts, people, practices as well as the language of the document itself. With the result of this investigation, the author is able to pinpoint the complex international cultural processes that were occurring in an early modern world.

1 Phylogenetic Studies in the Polyploid Genus Curcuma L. SUMMARY Curcuma is genetically one of the most complex genera within the tropical family Zingiberaceae, with hybridization and polyploidization being the major forces in its evolution. In this thesis, I have focused mainly on the genetic background of Curcuma species variation, relationships and overall genome structure, as a key to solve long standing taxonomic problems. Results of my molecular studies on the genus Curcuma performed since 2007 represent an extension of ongoing taxonomic and nomenclatural work started by Jana Leong- Škorničková in 2000. The first part of the thesis consists of a broad, general introduction to the subject to reflect the current state of knowledge, formulate the major problems to be confronted within the genus, and summarise the major results of the studies presented in the second part of the thesis. As the main obstacles in studying Curcuma are consequences of its reticulate evolution, it is also outlines the importance of understanding the genetic background and species relationships using molecular markers. Common molecular methods used for assessing phylogenetic relationships on the intraspecific and infrageneric levels - AFLP and sequencing of selected markers from cpDNA, nrDNA and nDNA - are described, with the...