Canada is a global leader in flax production, but flax acreage in Canada remains limited since flax is not well adapted to the northern Prairies. Therefore, breeding early-flowering and early maturing flax cultivars that are adapted to the climate of the northern Prairies is one of the major strategies to expand flax acreage in Canada. The objective of this project is to understand flowering time in flax and generate early flowering genotypes that are adapted to the continental climate of the Canadian Prairies. This project examined photoperiod sensitivity in five Canadian flax cultivars (CDC Sorrel, CDC Bethune, Flanders, Prairie Thunder and Royal) and three M9 genotypes derived from 5-azacytidine (5-azaC) treatment (RE1, RE2 and RE3). To investigate how each cultivar or genotype responds to photoperiod changes, a reciprocal transfer experiment between long day and short day conditions was conducted. All cultivars and genotypes were photoperiod sensitive. However, the level of sensitivity and length of the sensitive phase varied among cultivars and genotypes. The five cultivars were more sensitive to photoperiod changes compared with the three mutant genotypes, while RE2, which was the earliest flowering genotype, was the least sensitive genotype. This project, in addition, examined the expression pattern of ELF4 (EARLY FLOWERING 4), a specific flowering-related gene. This experiment was conducted with three Canadian flax cultivars (CDC Sorrel, CDC Bethune and Royal) and one 5-azaC mutant genotype (RE2). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as a reference gene in RT-qPCR. Results of RT-qPCR demonstrated that CDC Sorrel and CDC Bethune had a similar expression pattern, while Royal and RE2 had a similar expression pattern. This project also sought to generate early-flowering genotypes by treating CDC Sorrel with 5-azaC as well as to introgress the early-flowering trait from RE genotypes into CDC Sorrel via hybridization. Mutant populations (M2, M3, bulk M3) and hybrid populations (F2, F3, and bulk F3) were grown and evaluated for time to flowering, maturity and height under latitude (53° N) field conditions in 2012 and 2013. 5-azaC treatment did not induce significant differences in flowering or maturity in the CDC Sorrel background. However, the early flowering trait was successfully introgressed into CDC Sorrel background since selected progeny lines flowered significantly earlier than the later flowering CDC Sorrel parental line.

Spray application of 5­azacytidine on established plant seedlings was tested for its demethylating efficiency, as it represents a novel method for plant experimental demethylation with a potentially lower negative impact on plant development compared to the traditional application of the demethytaling agent through germination of seeds in its solution. Further, the 5­azacytidine spray application was used in practice to erase the epigenetic memory in offspring of Taraxacum brevicorniculatum plants from different competitive conditions. The impact of parental competition on the juvenile phenotype was estimated by measuring growth related traits, while the experimental demethylation allowed for evaluating the significance of DNA methylation marks in bioticaly induced transgenerational effects in T. brevirorniculatum.

Die heterogene Biologie der diffusen großzelligen B-Zell-Lymphome (DLBCL) und die daraus resultierenden Unterschiede in Therapieansprechen und Prognose stellen ein wesentliches Problem in der aktuellen Therapie von DLBCL dar. Anomalien im kanonischen Wnt-Signalweg und seinen natürlichen Antagonisten wie secreted frizzled-related proteins (SFRPs) sind für verschiedene solide und hämatologische Malignome als wichtige Bestandteile der Tumorgenese und Tumorprogression identifiziert worden. Vorarbeiten unserer Arbeitsgruppe lieferten Hinweise für die Bedeutung von Wnt3a und SFRP4 in der Regulation von DLBCL. Im Rahmen dieser Arbeit wurde untersucht, welche Rolle dem Protein SFRP4 in der Wachstumsregulation von diffusen großzelligen B-Zell-Lymphomen zukommt. Die Synthese von SFRP4 wurde in den als Modellzelllinien genutzten humanen DLBCL-Zelllinien nachgewiesen. Unter Verwendung von rekombinantem humanen SFRP4 sowie einer mittels lentiviraler shRNA erzeugten Expressionsreduktion von SFRP4 konnte gezeigt werden, dass SFRP4 zu einer Expansionshemmung sowie zu einer reduzierten Klonogenität bei DLBCL-Zellen führt. Als Ursache für diese Effekte konnte die Inhibition des kanonischen Wnt-Signalwegs durch SFRP4 identifiziert werden. SFRP4 wurde hinsichtlich seiner Bedeutung für die Ausprägung der Side Population (SP), einer Tumorzellsubpopulation mit repopulativem Potenzial, in DLBCL-Zellen untersucht. Dabei wurde sowohl durch exogene Zugabe als auch durch Reduktion von SFRP4 auf Transkriptebene die Bedeutsamkeit von SFRP4 für die SP-Regulation gezeigt. Untersuchungen zur differentiellen SFRP4-Expression sowie SFRP4-Promotormethylierung in Side Population versus non-Side Population wiesen epigenetische Mechanismen in der Regulation des SP-Phänotyps nach. Das DNA-demethylierende Medikament 5-Azacytidine reduzierte in DLBCL-Zelllinien Expansion sowie Klonogenität. Darüber hinaus beeinflusste 5-Azacytidine den kanonischen Wnt-Signalweg und den SFRP4-Gehalt der DLBCL-Zellen. Die Bedeutung von Exosomen als interzelluläre Kommunikatoren, die Wnt-Proteine transportieren, wurde unter besonderer Berücksichtigung von SFRP4 evaluiert. Sowohl SFRP4 als auch Wnt3a waren in der Exosomen- und der Mikrovesikelfraktion von DLBCL-Zellen nachweisbar. Die Exosomen waren in der Lage, den kanonischen Wnt-Signalweg an den Zielzellen zu aktivieren und Einfluss auf die Side Population zu nehmen. Insgesamt wurde für SFRP4 erstmalig eine hemmende Wirkung auf das Wachstum von DLBCL-Zellen demonstriert. Weiterführende Studien können adressieren, inwiefern die aufgezeigten Mechanismen der Wachstumsmodulation von DLBCL-Zellen durch SFRP4 in therapeutischen Anwendungen genutzt werden können.

Adipose tissue is not homogenous as individual fat depots display regional variation in their physiological properties. It follows that body fat distribution is increasingly being recognised as a major determinant of metabolic disease risk. At the cellular level, depot-specific properties are exhibited by adipocyte precursors during in vitro culture and persist for many generations, suggesting these cells retain an ‘intrinsic memory’ of their anatomical origin which is epigenetic in nature. A primary aim was to identify depot-specific genes whose expression may be regulated by DNA methylation in adipose precursors. Using two paired preadipocyte cell lines derived from human subcutaneous abdominal and gluteal adipose tissue - to represent upper and lower body fat with their opposing associations with cardiovascular disease and diabetes respectively - depot-specific gene expression and DNA methylation profiles were detected. Furthermore, the expression of certain genes in preadipocytes was found to change in response to treatment with the DNA demethylating agent 5-azacytidine, which suggests DNA methylation may regulate depot-specific gene expression. A secondary aim was to investigate whether glucocorticoids – which are important determinants of body fat distribution – exert their effects through DNA methylation. The synthetic glucocorticoid dexamethasone was found to modulate the expression of some of the differentially expressed genes in preadipocytes, with this effect possibly being mediated by DNA methylation. It has been postulated that depot-specific phenotypes in adipose tissue may arise from developmental differences. Several genes were found to be expressed in a depot-specific fashion during a differentiation time course, suggesting regional variation in adipogenesis may contribute to the generation of depot-specific phenotypes. Overall, the data presented suggests regional variation within subcutaneous white adipose tissue exists and supports the notion that DNA methylation patterns can, in part, determine adipose tissue heterogeneity.

Tumor microenvironment is an area, where the local immunosuppressive effects dominate and prevents the immune system to perform its physiological functions. The cells infiltrating the microenvironment have an important function among many cell types since they produce a large quantity of factors suppressing the immune response. In our work, we monitored the immune changes in the microenvironment during tumor growth and chemotherapy. For these purposes, we utilized the methods for analysis of the proportion and phenotype of the distinct populations of immunocytes and for analysis of the total level of expressions of selected genes associated with immunosuppression or with distinct populations of immunocytes. The aim of our work was to discover, using two types of mouse tumors (TRAMP-C2 and TC-1/A9), how 5-azacytidine (5AC), a cytostatic drug with epigenetic activity, affects the proportion of leukocytes infiltrating the tumor microenvironment and, further, whether these changes are accompanied by decreased expression of immunosuppressing genes. In addition, we have also focused on the changes of relative expression of genes encoding markers of lymphoid lines and, on other immunoregulating genes, encoding IL-6, IL-10, IL-12, IL-4 and IFNγ cytokines, in the microenvironment of these tumors....

Orientador: Eliana Pereira de Araujo / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T05:27:47Z (GMT). No. of bitstreams: 1 Gomes_FabianadeSouza_M.pdf: 1469055 bytes, checksum: 01ebfd4043c839e80e30bebeeb9bef95 (MD5) Previous issue date: 2013 / Resumo: O desenvolvimento de métodos que tem por objetivo acelerar e melhorar a qualidade do processo de cicatrização de feridas tem impacto positivo na condução de distúrbios de cicatrização associados a inúmeras condições médicas. Neste estudo, avaliamos os efeitos moleculares, celulares e clínicos da aplicação tópica de 5-azacitidina na cicatrização de feridas em ratos. De acordo com estudos pregressos, a 5-azacitidina reduz a expressão de folistatina, que é um regulador negativo das ativinas. Estas, por sua vez, promovem o crescimento de células em diferentes tecidos, incluindo a pele. Ratos Wistar machos com oito semanas de vida foram submetidos a um ferimento cutâneo com punch de oito milímetros na região dorsal. A seguir os ratos foram aleatoriamente separados em grupo controle (veículo) ou submetidos à aplicação tópica de 5-azacitidina (10 mM), uma vez por dia por até 12 dias, iniciando-se no terceiro dia após a lesão. A documentação fotográfica e coleta de amostras ocorreram nos dias 5, 9 e 15. O emprego desta droga resultou em aceleração da cicatrização da ferida, (99,7±7,0% versus 71,2±2,8% no dia 15, p <0,01). Este resultado clínico foi acompanhado pela redução de aproximadamente três vezes na expressão protéica de folistatina. O exame histológico da pele revelou re-epitelização eficiente com aumento da expressão de queratinócitos e aumento significativo na expressão do gene de TGF-? além da diminuição significativa de citocinas, tais como TNF-? e IL-10. Analisamos também a proliferação celular na lesão de pele através do método de incorporação de BrdU. O número de células positivas para BrdU aumentou significativamente quando comparado ao controle. No entanto, quando folistatina exógena foi aplicada na pele em paralelo ao tratamento tópico de 5-azacitidina a maioria dos benefícios do medicamento foi perdida. Assim, 5-azacitidina atua, pelo menos em parte, através da via folistatina/ativina para melhorar a cicatrização de feridas em ratos. Este trabalho pertence à linha de pesquisa Processo de Cuidar em Saúde e Enfermagem / Abstract: The development of new methods aimed at improving wound healing may have an impact on the outcomes of a number of medical conditions. Here we evaluate the molecular and clinical effects of topical 5-azacytidine, a compound used in myelodysplasia, on the wound healing in rats. According to previous studies, 5-Azacytidine decreases the expression of follistatin 1, which is a negative regulator of activins. Activins, in turn, promote cell growth in different tissues, including the skin. Eight-week old male Wistar rats were submitted to an 8 mm punchwound in the dorsal region. After three days, rats were randomly assigned to either control or topical application of a solution containing 5-azacytidine (10mM), once a day. Photo documentation and collection of samples occurred at days 5, 9 and 15. Overall, 5-azacytidine resulted on a significant acceleration of complete wound healing (99.7% ±0.7.0 vs. 71.2%±2.8 on days 15; n=10; p<0,01). This was accompanied by an up to 3-fold reduction in follistatin expression. Histological examination of the skin revealed efficient reepithelization with increase in gene expression of TGF-? and keratinocytes markers, involucrin and citokeratin, besides the significant decrease of cytokines such as TNF-? and IL-10. In addition, we analyzed cell proliferation in injured skin employing the BrdU incorporation method. The treatment with 5-azacytidine led to a progressive increase of BrdU positive cells. Finally when recombinant follistatin was employed in the skin in parallel to topical 5-azacytidine most of the benefits of the drug were lost. Thus, 5-azacytidine acts, at least in part, through the follistatin/activin pathway to improve wound healing in rats. This study belongs to online research process Caring in Nursing and Health / Mestrado / Enfermagem e Trabalho / Mestra em Ciências da Saúde

No description available.

Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...

No description available.

The myelodysplastic syndrome (MDS) is a group of hematopoietic clonal disorders resulting in the inefficient production of myeloid lineage blood cells, with the prevalence of patients older than 65 years. One of the possible treatment options for MDS is 5- azacytidine and 5-aza-2'-deoxycytidine therapy. These compounds have been shown to cause the induction of cell-cycle arrest, cell differentiation and/or apoptosis. The in vitro experiments with 5-aza-2'-deoxycytidine indicated that this compound causes the premature cellular senescence, a state of the irreversible cell-cycle arrest. We have asked, whether 5-azacytidine, as a molecule with similar structure, is capable of causing the same effect. This treatment strategy could be beneficial in case that the negative pro- inflammatory effect of senescent cells on their surroundings can be nullified. In this thesis we have shown that 5-azacytidine induces DNA damage response, which is described as a fundamental event for the onset of the cell senescence. We tested 5- azacytidine treated HeLa cells for several markers of the cell senescence - the increase of the β-galactosidase activity, the PML and PML nuclear bodies and the formation of persistent DNA damage signaling lesions - albeit all these markers were positive, it was the very low increase in...