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In the eukaryotic cell nucleus, spatial organization and dynamics of the genome is important in the regulation of gene expression. This thesis describes the use of the fission yeast, Schizosaccharomyces pombe, to study chromatin regulation and dynamics. We used nitrogen starvation to induce transcription of genes in fission yeast cells. In induced genes, nucleosomes get evicted in both the promoter and in the open reading frame (ORF). In the genes with the highest expression more nucleosomes get evicted from the ORF than from the promoter. This indicates that large rearrangements of the chromatin are occurring during a drastic gene induction. Many of the genes that become expressed early after nitrogen starvation are located together in clusters. In a cell where nitrogen is present in the surrounding media the gene clusters locate close to the nuclear periphery. When the nitrogen source is removed from the media, the clusters move to a more internal position. Thus rearrangement of chromatin due to gene induction, described in the first study, is accompanied by subnuclear changes of localization. Another type of regulation is the silencing of genes. We have studied a factor necessary for correct repression of genes located in silent chromatin, in S. pombe. The protein, Clr2, is part of the SHREC complex containing a remodeler (Mit1) and a histone deacetylase (Clr3). By bioinformatic analysis of Clr2 and newly sequenced fungi genomes, three motifs were identified. To gather more information about important parts of the Clr2 protein, deletions were made. When removing from about 20 to 100 amino acids in the middle of the protein, silencing of a reporter gene inserted at the mating-type region, inner repeats of centromere 1 and at the central core of centromere 2, failed. This indicates that Clr2 has an important role in establishing silent chromatin.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Brasil à o segundo maior produtor mundial de soja, destacando-se por sua multiplicidade de uso. Entretanto, perdas na produtividade de seus grÃos em campo sÃo ainda considerÃveis, particularmente oriundas das doenÃas causadas por fungos. Diante desse obstÃculo, emerge a necessidade de busca de molÃculas naturais capazes de inibir o progresso dessas doenÃas, sem causar impactos ambientais. Dentre as molÃculas presentes na soja, com potencial de uso para essa finalidade, destaca-se a toxina da soja (SBTX), uma proteÃna isolada de sementes, composta por duas subunidades (17 e 27 kDa) e ativa contra fungos fitopatogÃnicos. Esse trabalho teve como objetivos verificar a localizaÃÃo tecidual da SBTX em cotilÃdones de sementes maduras, bem como avaliar seu perfil de expressÃo gÃnica ao longo do desenvolvimento da soja e, tambÃm, em resposta ao tratamento com elicitores de defesa vegetal. Sementes de soja foram cultivadas em casa de vegetaÃÃo e, ao longo do desenvolvimento da planta, diferentes tecidos vegetais coletados. Em adiÃÃo, folhas primÃrias da soja foram tratadas com Ãcido salicÃlico (AS) ou inoculadas com esporos do fungo Cercospora kikuchii (CK) e coletadas em diferentes tempos apÃs os tratamentos. Iniciadores foram desenhados com base nas sequÃncias NH2-terminal das subunidades de SBTX e a expressÃo gÃnica foi avaliada pela tÃcnica de RT-PCR quantitativa. A localizaÃÃo de SBTX em sementes foi avaliada por imunohistoquÃmica, usando anti-SBTX. Transcritos dos genes SBTX17 e SBTX27 foram detectados em todos os tecidos vegetais coletados, porÃm seus nÃveis de expressÃo foram diferenciados. NÃveis mais elevados de transcritos para ambas as subunidades da SBTX foram detectados em sementes maduras, cotilÃdones e folhas unifoliadas. Nos cotilÃdones, SBTX foi encontrada na epiderme. InduÃÃo da expressÃo de transcritos da SBTX ocorreu em ambos os tratamentos, porÃm essa resposta se manifestou mais rÃpida (a partir de 6 h) com CK ao invÃs de AS (a partir de 12 h). Praticamente, em todas as anÃlises, transcritos do gene SBTX27 prevaleceram em relaÃÃo Ãqueles do SBTX17. A presenÃa constitutiva e ubÃqua de transcritos dos genes da SBTX ao longo do desenvolvimento da planta, a induÃÃo da expressÃo desses genes por elicitores de resposta de defesa e a localizaÃÃo da toxina na superfÃcie dos cotilÃdones validam o papel de defesa atribuÃdo a SBTX, suscitando a possibilidade de uso dessa proteÃna na produÃÃo de soja resistente ao ataque de fungos de relevÃncia agronÃmica. / Brazil is the second major global soybean producer, whose magnitude is due to its use multiples. However, losses in productivity of soybean grains in the field are still significant, especially those caused by pathogenic fungi. In view of this obstacle, it is important to search natural molecules able to inhibiting the progress of fungal diseases in an environmental friendly practice. Among the soybean molecules which could be used for this purpose, the soybean toxin (SBTX) stands out. SBTX is a protein composed of two subunits (17 and 27 kDa) isolated from seeds with in vitro activity against phytopathogenic fungi. The present study aimed to verify the SBTX tissue localization in soybean seed cotyledons, as well as to evaluate the gene expression profile of two SBTX subunits, both in different stages of plant development and in response to treatment with plant defense elicitors. Soybean seeds were grown in a greenhouse and plant tissues harvested at different days. In addition, soybean primary leaves were treated with salicylic acid (SA) or inoculated with the Cercospora kikuchiii (CK) spores and harvested at different times after the treatments. Based on the N-terminal sequences of the SBTX subunits, primers were designed and their gene expression evaluated by quantitative real-time PCR technique. SBTX tissue localization was performed by immunohistochemistry using anti-SBTX. Transcripts for both SBTX subunits were detected in all plant tissues, predominantly in cotyledons and unifoliate leaves in the early stages of their development, as well as in mature seeds. SBTX was found in the epidermis of the cotyledons. Transcripts were detected for both genes SBTX17 e SBTX27 in all tissues collected, but their expression levels were different. The highest transcript levels for both SBTX subunits were found in mature seeds, cotyledons and unifoliate leaves. In cotyledons, SBTX was found in the epidermis. Leaves treated with elicitors showed induction of the corresponding 17 and 27 kDa subunit transcripts, however this response was earlier in the CK treatment (from 6 h) compared to AS treatment (from 12 h). In almost all analyses, the highest transcript levels were found for the 27 kDa subunit. The ubiquitous and constitutive gene expression during plant development, the induction of gene expression by defense response elicitors and the localization on the surface of cotyledons support the role of SBTX in plant defense and its use to produce fungal-resistant transgenic soybean plants.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kryštof Dobečka Supervisor: PharmDr. Tomáš Smutný, Ph.D. Advisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Experimental approaches for studying hepatic enzyme induction mediated by pregnane X receptor The thesis focuses on hepatic pregnane X receptor (PXR)-mediated induction of biotransformation enzymes. Emphasis is placed on experimental models and methods which are used for the assessment of enzyme induction. In addition to summarizing its well- established role as a xenobiotic-sensing receptor, PXR is also presented as a transcription factor with an important role in endogenous pathways. Furthermore, cell and animal models are evaluated in terms of expression and function of PXR and its target xenobiotic-metabolising enzymes. Primary human hepatocytes in 2D cultures are considered to be the gold standard of in vitro hepatic models. However, 3D technologies are expected to be increasingly used in the future. The use of animal models is limited due to pronounced interspecies differences in PXR activation. Thus, humanized models have been established to overcome these limitations. Next, this thesis comments screening methods for an assessment of interaction between PXR and...