Master thesis investigates reproductive barriers in diploid-polyploid complex of Vicia cracca. Complex with basic chromosome number x=7 consists of diploid (2x=14), tetraploid (4x=28) and rare triploid (3x=21) cytotype. I studied prereproductive barriers between diploid and tetraploid cytotype: phenology of flowering, pollinators' behavior (preference of spieces of pollinators to cytotypes, sequence of visited cytotypes), variables, that could explain pollinators' behavior (amount of nectar as the main reward, size and amount of pollen grains as a potentional reward). To find out how strong the triploid block is I analyzed ploidy of seeds and seedlings from mixed-ploidy population. The habitat isolation showed up to be the strongest reproductive barrier. Pollinator's behavior meaningfully contributes to isolation, phenology of flowering contributes only minimally. Index expressing rate of prereproductive barriers is 0,956. Pollinator Bombus pascuorum visited on one locality preferably tetraploid plants and Andrena sp. preferred diploid plants. Even though tetraploid plants produce more nectar, no other analysis showed pollinators' preference to tetraploid plants. I prepared one squash of diploid V. cracca using method of in situ hybridization. This method needs to be optimilized for the studied taxon.

The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

A esquistossomose é uma doença causada por trematódeos do gênero Schistosoma. Dentre os genes identificados no transcriptoma do parasita, membros da família gênica SmVAL (Schistosoma mansoni Venom Allergen-Like) foram apontados como candidatos vacinais. SmVALs foram identificadas em secreções de cercárias e esquistossômulos cultivados in vitro, os transcritos SmVAL4 e 24 foram localizados nas glândulas acetabulares de germ ball e a proteína nativa SmVAL4 foi identificada em extrato de cercárias, indicando funções durante a penetração da pele. Já os transcritos SmVAL13 e 14 foram localizados na glândula esofágica anterior de vermes adultos, sugerindo papéis no processo de alimentação sanguínea. A imunização com as proteínas rSmVAL4, 6, 7, 13, 14 e 18 coadministradas não protegeu camundongos contra o desafio experimental, porém, observou-se uma diminuição do número de fêmeas e do número de ovos no grupo imunizado. A investigação de funções para as proteínas secretadas mostrou que a rSmVAL18 interage com plasminogênio in vitro favorecendo a invasão do hospedeiro. / Schistosomiasis is a disease caused by trematodes of the genus Schistosoma. Among the genes identified in the parasite transcriptome, members of SmVAL (Schistosoma mansoni Venom Allergen-Like) gene family were proposed as vaccine candidates. SmVALs were identified in cercariae and schistosomule secretions in vitro, the SmVAL4 and 24 transcripts were located to the germ ball acetabular glands and SmVAL4 native protein was identified in cercariae extract, indicating functions in skin penetration. On the other hand, SmVAL13 and 14 transcripts were located to the anterior esophageal gland of adult worms, suggesting roles in the blood feeding processes. Immunization with rSmVAL4, 6, 7, 13, 14 and 18 proteins co-administered did not protected mice against experimental challenge, however, there was a decrease in the number of females and the number of eggs in the immunized group. The investigation of functions for secreted proteins showed that rSmVAL18 interacts with plasminogen in vitro thus favoring the host invasion.

Fluorescenční in situ hybridizace (FISH) je široce používaná metoda pro detekci určité sekvence DNA na chromozomech. Cílem práce je porovnání tří různých chemických sloučenin (formamidu, ethylenkarbonátu a sodných kationtů) používaných ve směsích pro in situ hybridizaci. Složení těchto směsí ovlivňuje renaturaci DNA a je důležité porovnat jejich fyzikální vlastnosti. Práce je rozdělena do dvou hlavních částí. První část se zabývá otázkou termodynamických parametrů používaných pro experimenty FISH, jako je teplota tání, entalpie přechodu DNA ze dvoušroubovice na vlákno nám dává přehled o energii potřebné k tomto přechodu a interakcích mezi bázemi a každou složkou směsi. Kromě toho hodnoty entropie určují poř uvnitř směsi - systém DNA. Druhá část porovnává intenzitu fluorescenčního signálu při optimalizovaných teplotách tání sondy použité pro in situ hybridizaci. Jako sonda byla použita sub-telomerní repetice X43.1, která je umístěna na Y chromozomu rostlinného modelového organismu Silene latifolia. Směs obsahující formamid má nejlepší výkon při delším postupu hybridizace, zatímco ethylenkarbonát poskytuje vyšší intenzitu signálu, a proto je vhodnější pro rychlé FISH protokoly.

Supernumerary marker chromosomes (sSMCs) are a relatively rare cytogenetic phenomenon. Their laboratory examination is often difficult, and the clinical interpretation is even more challenging. The main reason is that most sSMC carriers have no clinical manifestations. The chromosome origin and exact range of the aberration are very important, as well as the fact that sSMCs are often found in mosaics that can strongly influence both the phenotype and the interpretation of result. Prenatal sSMC finding is one of the most challenging situations in both clinical and laboratory genetics. This work deals with the investigation process of sSMC carriers using molecular cytogenetic techniques, especially fluorescence in situ hybridization (FISH). We investigated a total of 67 families collected both prospectively and retrospectively, and we found 70 unique sSMCs in a total of 74 individuals. Six cases were familial and in three cases two sSMCs were found in one individual. According to the initial karyotype finding, the cases were divided into two groups, sSMCs supernumerary to a normal karyotype (group A) and sSMCT s supernumerary to the Turner karyotype (group B). The chromosomal origin was successfully determined in 88,6 % sSMCs. In group A the most common findings were sSMCs derived from chromosome 15,...

Asexual modes of reproduction are usually based on the principle of copying (cloning) DNA from the female and passing it on to the offspring. For most asexually reproducing vertebrates the progeny develop from an unreduced and often unfertilised egg. This is driven by the mechanisms of parthenogenetic and gynogenetic reproduction. While in the former the clonal germ cell develops spontaneously and separately, in the latter a sexual partner is needed to activate the cleavage of the ovum, although without the fusion of the sperm and egg. Therefore in both cases there is no fertilization and the clonal progeny consist solely of daughters, hence the majority of previous studies have only focused on asexual female lineages. However, on rare occasions asexual clonal males can arise when the right fertilization occurs. Whilst these offspring are usually infertile, fertile diploid asexual males have been discovered in just three genera of hybrid origin in vertebrates. One of these unique cases is the European water frog complex of the genus Pelophylax, whose distribution includes the Czech Republic. In areas around the upper Odra River populations of hybrid males were recently discovered who form stable all-male lineages, similar to those formed by asexual females. The results of this study show that males produce...

Mammalian oocyte is a highly differentiated cell which gives rise to an embryo after fertilization. Importantly, fully-grown oocytes become transcriptionally inactive at the end of the growth phase. During following stages of development, i. e. meiotic maturation of the oocyte and early embryonic development, only transcripts previously synthesized and stored are used. The tight correlation between mRNA distribution and subsequent protein localization and function provides a mechanism of spatial and temporal regulation of gene expression used by various cell types. However, not much is known about mRNA localization and translation in the mammalian oocyte and early embryo. The aim of my thesis was to determine the localization of transcripts and components of translational machinery in the mammalian oocyte and embryo and to uncover the mechanisms of spatiotemporal regulation of translation as a prerequisite for correct oocyte and embryo development. We have shown that nuclei of both mouse and human oocytes contain RNA molecules and RNA binding proteins. Following the nuclear envelope breakdown (NEBD), translational hot-spots occur in the area surrounding the nuclear region. We suppose that mRNAs previously retained in the nucleus are released to the cytoplasm during NEBD and their subsequent...

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

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