Endometrial adenocarcinoma (EAC) is the most frequently diagnosed gynecological cancer of the female genital tract in the Western world. Research studies in EC is difficult to conduct on human tumor samples due to the complex nature of tumor arousal and genetic heterogeneousness in the human population. Therefore, inbred animal models can be promising tools to use in EC research due to similar histopathology and pathogenesis as humans. Studies performed on MAP2K1 and CCND1 has shown that their altered expression play a crucial role in carcinogenesis. CCND1 has been demonstrated to have oncogenic properties when overexpressed in human neoplasias. The aim of this study is to investigate gene expression levels of MAP2K1 and CCND1 in BDII rat model of endometrial adenocarcinoma cells. Quantitative real-time PCR was used to analyze expression levels of MAP2K1 and CCND1 genes in BDII/Han rat model of endometrial cancer cells using TaqMan approach. The differences in gene expression levels of MAP2K1 and CCND1 between pathologically EAC malignant and nonmalignant cells showed an upregulation of MAP2K1 and CCND1 in EAC malignant cells. The analyzed data presented observable mean differences between MAP2K1 and CCND1 in several endometrial cell lines that were examined. Although no statistical significance was reached, an alteration in gene expression levels in malignant and nonmalignant endometrial cells could be observed. Furthermore, this present study shows observable upregulation of MAP2K1 and CCND1 in endometrial carcinoma cells vs. nonmalignant endometrium cells and encourages further investigation of the role of CCND1 and MAP2K genes in endometrial carcinogenesis.

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Herbal medicines are growing in popularity in the Western world and are becoming more stringently regulated under new EU legislation. Within the arena of herbal medicines, St. John’s Wort (SJW), Hypericum perforatum, is a top ten best seller with clinical evidence to support its use as an anti-depressant. A fundamental requirement of the new legislation is to prove the identity of the plant material in question. This is currently achieved via morphological and chemical methods, neither of which are ideal. A wide range of DNA based methods have been applied to this arena, standardisation is required to realise the potential of DNA based techniques. The DNA barcoding initiative aims to produce sequence data for all plant species, capable of species identification. The proposal is to use these data to design fast and effective DNA based methods of identification. For assay design, the putative barcode region nrITS was selected as a platform. Three assays were designed; • A PCR assay designed to hyper variable sequences within a barcode region. This assay is capable of distinguishing SJW from other closely related species. • A quantitative qPCR assay designed to measure total DNA and specific SJW DNA within a mixed sample. • A multiplex PCR incorporating fluorescently labelled primers, allowing amplicon detection by capillary electrophoresis. This assay identifies four separate Hypericum species, including SJW, with a mixed sample in one reaction. The suitability of the nrITS and three other barcode regions is assessed based on sequence data generated for 32 vouchered samples of different Hypericum species, and a Lithuanian sample set of 22 and 16 H. perforatum and H. maculatum samples respectively. The matK is currently unusable, the rbcL highly conserved, trnH-psbA problematically variable and the nrITS proved to be ideal for assay design.

Tidal freshwater wetlands (TFW) are at significant risk of loss or alteration due to global climate change, and saltwater intrusion from sea level rise is of particular concern for these habitats due to their proximity to coastal areas. A space-for-time model was used to investigate the effects of saltwater intrusion on soil methanogen communities along naturally occurring salinity gradients on the Waccamaw, James, and Hudson Rivers. Amplification of the methyl coenzyme-M reductase (mcrA) functional gene was used in qPCR, reverse transcription qPCR, and T-RFLP to measure the abundance, activity, and community composition of soil methanogens. Both the abundance and activity of methanogens decreased with increasing salinity, and the both total and active methanogen community composition shifted in response to changes in salinity. This research demonstrates that saltwater intrusion will alter carbon cycling in TFWs, potentially altering their ability to sequester carbon and keep pace with rising sea level.

Genome transcription is much more widespread than has been traditionally thought because our view of a "gene" or "transcription unit" has changed dramatically over the past 4 to 5 years with the identification of many different non-coding ribonucleic acids. In the yeast, Saccharomyces cerevisiae, meiosis and sporulation are an important part of the life cycle and IME4 gene expression is required for these processes. IME4 sense transcript levels of expression are influenced by the level of its complementary non-coding antisense strand by mechanisms that are currently unknown. The a1-alpha2 heterodimer binding in the downstream 3' region of IME4 is one component required for repression of IME4 antisense transcription. However, this thesis shows that the general regulatory protein Reb1 is also required in this system. Reb1 involvement is most likely to create a nucleosome-free zone in the promoter region of the IME4 antisense strand therefore contributing to transcription.

Trematode peptidases are important molecules responsible for biocatalysis in many basal biological processes and are crucial in host-parasite interactions. Therefore, these enzymes are intensively studied in order to characterize their biological functions and to use them as potential diagnostic or therapeutic targets. Lately, investigation of transcriptome and secretome revealed, that adult Fascioloides magna (giant liver fluke) expresses and secretes a variety of peptidases. Thus, this thesis focuses on three newly identified enzymes: cathepsin L (FmCL), cathepsin B (FmCB) and cathepsin D (FmCD). In other trematode species, these cathepsins are being linked mainly with the digestion of host blood. We applied quantitative PCR (qPCR) to investigate relative expression levels of the three enzymes among three developmental stages - egg, miracidium and adult. It was revealed that all cathepsins have the highest expression in adult flukes in comparison to eggs and miracidia. We also localized the place of transcription of FmCL, FmCB and FmCD in adult fluke using RNA in situ hybridization. All of the peptidases were detected in gastrodermis, and in addition, they were localized in the reproductive system. The latter surprising finding is suggesting that these enzymes might have multiple functions in adult F....

Cells are the basic units of life. Studying complex tissues and whole organs requires an understanding of cell heterogeneity and responses to stimuli at the single-cell level. Even the cells, which belong to the same cell type, behave differently at a specific moment and contain different amount of mRNA. Quantitative polymerase chain reaction (qPCR) is one the most sensitive methods for the detection of mRNA, however, gene expression profiling in single cells leads to a large amount of missing data due to the fact that the transcript is missing, or is below the level of detection. Therefore, it is necessary to establish a new statistical approach for analysis of single cells. In this thesis the potential of single-cell gene expression profiling using the high throughput instrument Biomark, focusing on data analysis and biological interpretation, is discussed. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at the single-cell level. Single cells are not normalized by reference genes but the number of cells as a normalizer is applied. Missing data are replaced by value, which is equaled one quarter of transcript amount in the cell. Furthermore it is shown how single-cell gene expression data can be viewed and how subpopulations...

Breeding for abiotic stress tolerance is one of main topics in plant breeding. Oilseed rape breeding programs were for a long time focused on morphological and physiological parameters. In this thesis few experiments focused on identification of genes involved in abiotic stress reaction were performed using RT-qPCR (quantitative reverse transcription PCR). Simultaneously SPR (surface plasmon resonance) method were used as modern optical method facilitating very low native protein concentration even in presence of other substances. This method facilitates quantification of concrete proteins by binding them to specific antigen and in oilseed rape research it was not used by now. ERD10 protein was identified by SPR as protein involved in cold stress reaction (or acclimation). The results show ERD10 accumulation in standard conditions affects dynamics of its accumulation change during cold stress. In case we are searching for genotypes great in acclimation ability even during short and warm autumn SPR method should be suitable method for fast, easy and relatively cheap screening of large number of genotypes in breeding collections. Also genes LTI78, RCI2A, NRP1 and two genes for hypothetical proteins were analysed. Their relative expression during cold stress was markedly increased too. Very little is known about these genes and proteins nowadays therefor it will be interesting topic of our oncoming experiment. Relative expression of genes picked according to MALDI-TOF/TOF analysis results was also tested in microspore embryo regenerants stressed by simulated drought. Genes for lactoylglutathione lyase I, phospholipase D 1 and peroxiredoxin antioxidase were tested. In tolerant cultivar was markedly decreased gene expression of peroxiredoxin antioxidase in standard conditions and early stress. These gene will be subject for next research as potential marker for more tolerant genotypes selection.

Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.