Oxisteróis são derivados oxigenados do colesterol que podem ser formados por autoxidação ou por atividade de enzimas específicas. Eles apresentam inúmeras atividades biológicas, incluindo inibição da proliferação celular e citotoxicidade. Dentre os oxisteróis, 7-cetocolesterol (7-KC), que difere do colesterol por apresentar um grupamento cetônico no carbono 7, é conhecido por induzir morte celular por diferentes vias. Neste projeto de pesquisa pretendemos avaliar o uso de uma emulsão lipídica contendo oxisterol (OXLE) como veículo para entrega de 7-KC a células tumorais. Os efeitos tóxicos de OXLE foram avaliados nas linhagem celulares RPMI 8226 (mieloma múltiplo) e B16F10 (melanoma). As células foram tratadas com: a) uma emulsão conhecida por atuar funcionalmente como a LDL (LDE); b) OXLE (75 uM até 225 uM de 7-KC); c) 7-KC (100 uM). As distribuições das células nas diferentes fases do ciclo de duplicação celulares foram analisadas por citometria de fluxo com iodeto de propídio. Todos os tratamentos, nestas concentrações, levaram a acúmulo das células de mieloma na fase proliferativa do ciclo celular. Para analisar a resposta apoptótica, nos utilizamos o fluorocromo JC-1, que mede o potencial transmembranar mitocondrial. Ambas a linhagens celulares, quando tratadas com OXLE, apresentaram hiperpolarização do potencial transmembranar associado com progressivo decrescimento do mesmo. Os resultados mostram que os grupos OXLE e 7-KC induziram a uma morte massiva das células de mieloma. OXLE induziu um efeito citostático nas células de melanoma, caracterizadas por alterações de morfologia, com um aumento na polimerização das fibras de actina, hiperpolarização seguida de perda do potencial transmitocondrial (apoptose associada à despolarização) , presença de vacúolos autofágicos como figuras de mielina (morte celular programada do tipo II - Autofagia); e impossibilitando as células de fazer a citocinese, determinado pelo aparecimento de uma população de células poliplóides. Nossos resultados indicam que esta nova emulsão contendo um oxisterol em sua formulação não é apenas um veículo para moléculas hidrofóbicas, mas pode atuar como agente cistotático/citotóxico. A morte mediada por OXLE possui características de apoptose e autofagia. Estes resultados são promissores e o possível use de OXLE in vivo necessita de futuras investigações. / Oxysterols are oxygenated derivatives of cholesterol that may be formed by autoxidation or by action of specific enzymes. They exhibit a number of biologic activities including inhibition of cellular proliferation and cytotoxicity. Among oxysterols, 7-ketocholesterol (7-KC), that differs form cholesterol by a functional ketone group at C7, is known to induce cell death by different ways. Herein we evaluated the use of an oxysterol-containing lipid emulsion(OXLE) as a vehicle to deliver 7-KC to tumor cells. OXLE was evaluated regarding its toxicity on RPMI 8226 (multiple myeloma) and B16F10 (melanoma) cell lines. Cells were treated with: a) an emulsion known to act functionally as LDL (LDE); b) OXLE (75 uM to 225uM of 7KC); c) 7KC (100uM). The distribution of cells in the different phases of cell cycle was analyzed by flow cytometry with propidium iodide. All treatments, according to the concentrations, led the myeloma cells to an arrest in the proliferative phases of the cell cycle. To analyze the apoptotic response, we then used a fluorochrome (JC-1), which measures the mitochondrial transmembrane potential. Both cell lines when treated with OXLE had a hyperpolarization of the transmembrane potential that decrease progressively. The results showed that exposure to either OXLE or 7-KC induced a massive death of myeloma cells. OXLE had an cytostatic effect on melanoma cells characterized by morphologic alterations as increased polymerization of actin fibers; hyperpolarization followed by a dissipation of the mitochondrial transmembrane potential (apoptosis-associated depolarization); presence of IX autophagic vacuoles as myelin figures (programmed cell death type II - Autophagy); and impaired cytokinesis, as determined by the emergence of a polyploid population. Our results indicated that this new oxysterol-containing emulsion is not only a carrier for hydrophobic molecules, but it can act itself as a cytostatic/cytotoxic agent. Cell death mediated by OXLE have both apoptosis and autophagy characteristics. These results are promising and the possible use of OxLE in vivo warrants further investigation

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-05T15:13:46Z No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 1892834 bytes, checksum: bffd5bff4b38c8f75f7aca75cbd5160b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T13:40:45Z (GMT) No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 1892834 bytes, checksum: bffd5bff4b38c8f75f7aca75cbd5160b (MD5) / Made available in DSpace on 2017-05-17T13:40:45Z (GMT). No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 1892834 bytes, checksum: bffd5bff4b38c8f75f7aca75cbd5160b (MD5) Previous issue date: 2013-02-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O gênero Pfaffia (Amaranthaceae) compreende 33 espécies distribuídas na Região Neotropical. Graças a algumas substâncias químicas encontradas no gênero que se assemelham àquelas encontradas no gênero Panax (ginseng coreano), a espécie Pfaffia glomerata é conhecida “ginseng brasileiro ”. Tal planta é utilizada como tônico, afrodisíaco, rejuvenescedor, anti-fadiga, antiestresse e contra distúrbios gástricos. Suas propriedades medicinais são atribuídas a compostos encontrados principalmente em suas raízes tuberosas, sendo a β-ecdisona a mais visada pela indústria farmacêutica e cosmética. Apesar de sua importância econômica e medicinal, são poucos os estudos que investigam aracterísticas genéticas da espécie. Neste trabalho foram feitas análises citométricas e cromossômicas de fáfias diploide e poliploide induzida. A poliploidização se deu por duplicação cromossômica in vitro com uso de agentes antimitóticos (colchicina e orizalina) em diferentes concentrações e tempo de exposição. A quantidade de DNA encontrada foi de 2,41 pg (2n=34) e 4,79 pg (2n=68). O genoma de P. glomerata diploide e tetraploide foi analisado por técnicas de coloração convencional e coloração diferencial dos cromossomos por bandamento com CMA3/DA/DAPI e FISH para DNAr. Embora os cromossomos da espécie sejam pequenos, foi possível identificar suas constrições primárias e encontrar um par de cromossomos com satélite no representante diploide e dois pares no poliploide. Foram montados cariogramas e idiogramas, sendo que as fórmulas cariotípicas foram: 16M + 1SM para o genoma 2n=34 e 32M + 2SM para o genoma 2n=68. Os núcleos interfásicos mostraram-se reticulados. Através do bandeamento com CMA3/DA/DAPI observou-se que todos os cromossomos possuem bandas centroméricas DAPI+, indicando uma região heterocromática ao redor dos centrômeros. Foi localizada uma banda CMA+ no braço curto de um dos cromossomos, sendo registrada diferença na extensão da banda entre os homólogos. O emprego da FISH com as sequências de DNAr 45S e 5S, gerou para cada tipo de sonda, dois sinais em um par de homólogos para o genoma diploide e quatro sinais no genoma poliploide. Os cromossomos homólogos apresentaram heteromorfismo para as regiões de DNAr invetigadas. O teor de β-ecdisona presente no extrato metanólico das raízes de fáfia com quatro meses de cultivo, foi de 0,32% para a planta diploide e 0,48% para a poliploide sugerindo um aumento de 50% na produção do composto no poliploide quando comparado com seu diploide. A estrutura do grão de pólen, determinada por acetólise Erdtman, mostrou-se estável entre acessos diploides da espécie. Este é o primeiro relato da quantidade de DNA no gênero Pfaffia. Também são inéditos os dados obtidos por FISH com sondas para DNAr 45S e 5S para a P. glomerata diploide e poliploide. Os resultados da quantificação do teor de β-ecdisona sugerem a duplicação cromossômica como alternativa para incremento do valor econômico da espécie. / The genus Pfaffia (Amaranthaceae) comprises 33 species distributed in the neotropics. Thanks to some chemicals found in the genus that resemble those found in the genus Panax (Korean ginseng), the species P. glomerata is known as "Brazilian ginseng". P. glomerata is commonly used as tonic, phrodisiac, rejuvenator, anti-fatigue, anti-stress and against gastric disorders. Its medicinal properties are attributed to the compounds mainly found in their tuber roots, being the β-ecdysone the most explored by the pharmaceutical and cosmetic industries. Despite its economical and medicinal importance, few studies have been done to investigate the genetic aspects of the species. In this work, cytometric and chromosomal analyzes were made with diploid and induced polyploid of P. glomerata. The polyploidization occurred by in vitro chromosome doubling, using antimitotic agents (colchicine and oryzalin) at different concentrations and exposure times. Flow cytometry analysis showed 2.41 pg (2n = 34) and 4.79 pg (2n = 68) of DNA amounts. Both P. glomerata diploid and tetraploid were analyzed by conventional staining techniques and chromosomal differential staining by CMA3/DA/DAPI banding and rDNA FISH. In spite of the small sizes of chromosomes, it was possible to identify their primary constrictions and a pair of satellites chromosomes in the diploid and two pairs in the polyploid. Karyograms and idiograms were assembled. Karyotypic formulas were 16M + 1SM considering the 2n = 34 genome and 32M + 2SM considering 2n = 68. The interphase nuclei seem to reticulated. The fluorochrome CMA3/DA/DAPI banding revealed all chromosomes with centromeric DAPI+ bands indicating a heterochromatic region around the centromeres. A CMA+ band, with different extension between the homologous was observed on the short arm of one chromosome pair. The FISH technique with rDNA 45S and 5S sequences revealed two signals to each probe for the diploid genome and four signals in the polyploid genome. Homologous chromosomes are heteromorphic when considering the rDNA regions here investigated. The content of β ecdysone present in the methanol extract of the roots of P. glomerata with four months of cultivation was 0.32% for the diploid and 0.48% for the tetraploid suggesting an increase of 50% on the tetraploid comparing with the diploid. Pollen structure was determined by Erdtman acetolysis, and no differences were observed between diploid accessions. This is the first report of DNA amount in the genus Pfaffia and data obtained with FISH using 45S and 5S rDNA probes have not been published elsewhere. The β-ecdysone quantification suggests that chromosome duplication is an alternative to increase the economical value of the species.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-10-21T01:01:09Z No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-10-21T13:13:03Z (GMT) No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) / Made available in DSpace on 2017-10-21T13:13:03Z (GMT). No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) Previous issue date: 2017-03-22 / As orquídeas do complexo "Zygopetalum maculatum" (Kunth) Garay são típicas dos campos rupestres e de altitude do leste do Brasil. Atualmente são reconhecidas seis espécies para este grupo que, por compartilharem características morfológicas diagnósticas, são reconhecidas aqui como um complexo de espécies. De forma a contribuir para entender o padrão de variação fenotípica observado entre as populações e com isso tentar esclarecer a formação deste complexo, este trabalho caracterizou do ponto de vista genético ar 22 populações de "Z. maculatum", sendo 21 provenientes do Brasil e uma da Bolívia. As análises compreenderam estimativa da quantidade de DNA nuclear, determinação do número cromossômico, comportamento meiótico e diversidade genética identificada por marcadores do tipo ISSR. O conteúdo médio de DNA revelou três níveis de ploidia no complexo, 7,36 pg, 10,52 pg e 14,09 pg de DNA, sendo estes relacionados a números cromossômicos de 2n=48; 2n=72; 2n=96, respectivamente. Tal variação ocorreu entre e dentro das populações. Dados cariotípicos (morfometria, DNAr 5S e 45S) evidenciaram similaridade entre os três níveis de ploidia. A meiose mostrou-se irregular, com ocorrência de atraso e perda cromossômica, pontes, “cromossomos aderentes”, micronúcleos e assincronia em todos os níveis de ploidia. O dendrograma baseado em marcadores ISSR revelou tendência de agrupamento de indivíduos com o mesmo nível de ploidia e originados das mesmas regiões geográficas. Os resultados sugerem que as populações possuem baixos índices de diversidade genética entre indivíduos e que a maior diversidade encontra-se entre as populações. De forma geral, os resultados indicam que eventos de poliploidização estejam relacionados à diversificação e especiação de “Z. maculatum”, sendo um processo importante para explicar as variações morfológicas observadas dentro do complexo. / The orchids of the "Zygopetalum maculatum" (Kunth) Garay complex are typical of the “campos rupestres” of the east side of Brazil. Currently, six species are recognized for this group which, due to their morphological similarity, is recognized here as a species complex. In order to understand the phenotypic pattern observed among the populations and contribute to clarify the formation of this complex, this study characterized 22 populations of “Z. maculatum”, being 21 populations from Brazil and one population from Bolivia. The study included DNA amount estimation, determination of the chromosome number, meiotic behavior and genetic diversity estimation. The average of DNA content revealed three 2C values, 7.36 pg, 10.52 pg and 14.09 pg of DNA, which were associated to 2n = 48; 2n = 72 and 2n = 96 chromosomes, respectively. The variation was observed within and among populations. Karyotypic data (morphometry, 5S and 45S rDNA) showed similarity among all ploidy levels. Meiosis behavior was not regular being observed chromosome delay and loss, bridges, micronuclei, chromosomal stickiness and asynchrony. The dendrogram based on ISSR revealed similarity regarding the ploidy level and the geographical distribution of the individuals. The data also suggested low genetic diversity within populations being most of the diversity observed among populations. In general, the results indicate that polyploidization events are probably related to the Zygopetalum diversification and speciation, being an important process to explain the morphological variations observed within the complex.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-19T18:37:15Z No. of bitstreams: 1 viníciussacramentopacheco.pdf: 1524403 bytes, checksum: da3bc47331a9b9d21aceffe2209ed60f (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Nome do autor não pode ser salvo com acento on 2018-03-19T19:36:45Z (GMT) / Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-19T19:41:33Z No. of bitstreams: 1 viníciussacramentopacheco.pdf: 1524403 bytes, checksum: da3bc47331a9b9d21aceffe2209ed60f (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Nome do autor tem que ser salvo sem acento on 2018-03-19T19:42:52Z (GMT) / Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-19T20:19:33Z No. of bitstreams: 1 viniciussacramentopacheco.pdf: 1524403 bytes, checksum: da3bc47331a9b9d21aceffe2209ed60f (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-03-21T13:00:16Z (GMT) No. of bitstreams: 1 viniciussacramentopacheco.pdf: 1524403 bytes, checksum: da3bc47331a9b9d21aceffe2209ed60f (MD5) / Made available in DSpace on 2018-03-21T13:00:16Z (GMT). No. of bitstreams: 1 viniciussacramentopacheco.pdf: 1524403 bytes, checksum: da3bc47331a9b9d21aceffe2209ed60f (MD5) Previous issue date: 2017-08-04 / Lippia alba (Mill.) N.E. Brown (Verbenaceae) é uma das espécies medicinais mais amplamente utilizadas no Brasil. Nas últimas décadas, grande número de trabalhos vem investigando e comprovando os efeitos farmacológicos dos componentes encontradas no óleo essencial dessa planta. Estudos relacionados à genômica demonstram a ocorrência de poliploidia natural na espécie, condição que pode interferir na composição e produção dessas substâncias. Em contrapartida, fatores ambientais, como a irradiância e disponibilidade hídrica, também são importantes modeladores da biossíntese dessas moléculas, podendo interagir com o fator genético. Diante do exposto, investigou-se os efeitos de níveis contrastantes de irradiância, a 100% (pleno sol) e 30%( à sombra), durante as épocas seca e chuvosa de cultivo, sobre as trocas gasosas e seus posteriores efeitos sobre o crescimento e a produção de óleo essencial em acessos de L. alba que possuíam três níveis de ploidia distintos (Di-; Tetra- e Hexaploide). Para a maioria dos parâmetros de trocas gasosas, as plantas cultivadas à sombra apresentaram valores menores quando comparados às plantas cultivadas a pleno sol, independentemente da época de cultivo. Em ambos os níveis de irradiância, o acesso tetraploide apresentou os menores valores para a fotossíntese liquida, bruta, fotorrespiração, condutância estomática e taxa de transporte de elétrons para carboxilação e oxigenação. Acessos cultivados à sombra apresentaram respostas tipicamente associadas a essa condição de luminosidade, com maior área foliar especifica e unitária, alocação preferencial de biomassa em parte aérea e maior conteúdo de clorofilas, bem como valores menores em biomassa de raiz, massa total e taxa assimilatória liquida. Em comparação aos cultivados a pleno sol, todos os acessos cultivados à sombra apresentaram conteúdo de óleo essencial , em média, 40% menor. Quando cultivados a pleno sol o acesso diploide apresentou o maior conteúdo de óleo essencial, que foi em média 150% maior, que o observados para os acessos tetra- e hexaploide. À sombra, o conteúdo de óleo essencial do acesso diploide foi 120% maior que os demais. Entretanto, nessa condição, este acesso apresentou a menor biomassa total. Os resultados encontrados permitem concluir que o acesso diploide, independentemente da irradiância em que foi cultivado, apresento o maior rendimento da produção de óleo essencial, sugerindo maior atividade das vias do metabolismo secundário, reduzindo o acúmulo de biomassa sob baixa irradiância. / Lippia alba (Mill.) N.E. Brown (Verbenaceae) is one of the most widely used medicinal species in Brazil. In the last decades, a great number of works have been investigating and proving the pharmacological effects of the components found in the essential oil of this plant. Studies related to genomics demonstrate the occurrence of natural polyploidy in the species, a condition that may interfere in the composition and production of these substances. In contrast, environmental factors, such as irradiance and water availability, are also important modellers of the biosynthesis of these molecules, and may interact with the genetic factor. Considering the above, the effects of contrasting levels of irradiance, at 100% (full sun) and 30% (in the shade), during the dry and rainy seasons of cultivation, on the gas exchanges and their subsequent effects on growth were investigated and the production of essential oil in L. alba accesses that had three distinct levels of ploidy (Di-, Tetra- and Hexaploid). For most gaseous exchange parameters, shade-grown plants presented lower values when compared to plants grown in full sun, regardless of the growing season. In both levels of irradiance, tetraploid access presented the lowest values for liquid photosynthesis, crude, photorespiration, stomatal conductance and electron transport rate for carboxylation and oxygenation. Shade - grown accesses presented responses typically associated to this luminosity condition, with a greater specific and unitary leaf area, preferential allocation of biomass in aerial part and higher content of chlorophylls, as well as lower values in root biomass, total mass and net assimilation rate. Compared to those grown in the full sun, all accesses cultivated in the shade had an essential oil content, on average, 40% lower. When cultivated in full sun the diploid access had the highest essential oil content, which was on average 150% higher than that observed for the tetra- and hexaploid accessions. In the shade, the essential oil content of the diploid access was 120% greater than others accessions. However, in this condition, this access presented the lowest total biomass. The results show that diploid access, irrespective of the irradiance in which it was cultivated, presents the highest yield of essential oil production, suggesting higher activity of the secondary metabolism pathways, reducing the accumulation of biomass under low irradiance.

Myriophyllum L. (watermilfoil) belongs to one of the most invasive genus of the Northern Hemisphere. The three most agressive species, one of which is native to Europe (M. spicatum), are widespread throughout the majority of United states and two of them (M. aquaticum and M. heterophyllum) are becoming invasive in several Europaean countries as well. Therefore, Europaean populations of both native and invasive species, represents ideal place where to study processes which account for watermilfoils' invasivness. In this study, flow cytometry, morphometric analyses and germination experiments were employed to evaluate cytotype and morphological variability of Myriophyllum along with its potential to hybridize and propagate generatively. Five ploidy levels were detected in Europe, however no ploidy variability has been found within any species except for M. sibiricum (hexaploids and nonaploids) and one population of M. aquaticum (hexaploids and oktoploids). Flow cytometry proved to be useful tool for purposes of distinguishing species of different ploidy level and their potential hybrids. Nevertheless, the key species - M. sibiricum and M. spicatum - among which probably even more aggressive hybrids were recently identified, have similar hexaploid level. Thus flow cytometry cannot facilitate...

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ecological clines often result in gradients of disease pressure in natural plant communities, imposing a gradient of selection on disease resistance genes. We describe the diversity of a resistance gene homolog in natural populations of the dominant tallgrass prairie grass, Andropogon gerardii, across a precipitation gradient ranging from 47.63 cm/year in western Kansas to 104.7 cm/year in central Missouri. Since moisture facilitates infection by foliar bacterial pathogens, plants along this precipitation gradient will tend to experience heavier bacterial disease pressure to the east. In maize, the gene Rxo1 confers resistance to the pathogenic bacterium Burkholderia andropogonis. Rxo1 homologs have been identified in A. gerardii and B. andropogonis is known to infect natural populations of A. gerardii. The spatial genetic structure of A. gerardii was assessed from central Missouri to western Kansas by genotyping with AFLP markers. Samples were also genotyped for Rxo1 homologs by amplifying an 810 base pair region of the leucine-rich repeat and digesting with restriction enzymes. We compared Rxo1 homolog diversity to AFLP diversity across different spatial scales. Genetic dissimilarity based on AFLP markers was lower than would have occurred by chance at distances up to 30 m, and different prairies were more dissimilar than would have occurred by chance, but there was not a longitudinal trend in within-prairie dissimilarity as measured by AFLP markers. Dissimilarity of the Rxo1 homologs was higher in the east suggesting the presence of diversifying selection in the more disease-conducive eastern environments.

Les légumineuses peuvent interagir avec les bactéries du sol de la famille des Rhizobiaceae. Cette interaction aboutit à la formation d'un organe spécialisé appelé nodosité. Au sein des cellules symbiotiques des nodosités, les rhizobia sont capables de fixer l'azote atmosphérique et de la convertir en ammoniac, qui est une source d'azote assimilable par les plantes. Chez la Légumineuse Medicago truncatula, les cellules symbiotiques produisent une large famille de peptides riches en cystéines appelées (NCRs) spécifiquement exprimés dans les nodosités. Ces NCRs induisent la différenciation des bactéroïdes qui se traduit par un allongement cellulaire couplé à une forte endoréplication du génome (les bactéroïdes deviennent polyploïdes) contribuant ainsi à une augmentation importante de la taille des cellules, ainsi qu’une perméabilité membranaire accrue et une perte de toute capacité reproductrice. Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens, acteurs clés de l’immunité innée. L'analyse de l'expression de 334 gènes NCR dans 267 différentes conditions expérimentales en utilisant la base de données MtGEA (Medicago truncatula Gene Expression Atlas) a révélé que l'ensemble des gènes NCR testés (sauf quatre) n'est exprimé que dans les nodosités, ils ne sont pas exprimés dans d’autres organes de la plante, ni lors d’une infection par des agents pathogènes. De plus l’expression des NCRs n’est induite en réponse à aucune interaction biotique ou abiotique testée ou à des facteurs Nod. Les gènes NCR sont activés en vagues successives au cours de l’organogenèse nodulaire et ce profil temporel est en corrélation avec une localisation spatiale spécifique de leurs transcrit de la zone apicale à la partie proximale de nodosités. En outre, nous avons montré que les NCRs ne sont pas induites pendant la sénescence des nodules. Ces analyses expérimentales ensemble avec des calculs d’entropie de Shannon, une métric pour la spécificité d’expression, montrent que les gènes NCR sont parmi les gènes les plus fortement et le plus spécifiquement exprimés chez M. truncatula. Ainsi, l'expression des NCRs est soumise à une régulation extrêmement stricte et ils sont activés exclusivement pendant l’organogenèse et au cours du développement nodulaire dans les cellules symbiotiques polyploïdes. Cette analyse a suggéré l'implication de la régulation épigénétique des gènes NCR. La formation des cellules symbiotiques s'exerce par une endoreplication et est associée à une reprogrammation transcriptionnelle. En utilisant le tri par cytométrie en flux des noyaux, en fonction de leur contenu en ADN, nous avons montré que les vagues transcriptionnelles sont en correlation avec les niveaux croissants de ploïdie et resultent des modifications épigénétiques durant les cycles d’endoréplication. Nous avons étudié la méthylation de l'ADN génomique et l'accessibilité à la chromatine, ainsi que la présence des marqueurs répresseurs (H3K27me3) ou activateurs transcriptionnels (H3K9ac) sur des gènes spécifiques des nodosités. La méthylation différentielle de l'ADN n'a été trouvée que dans un petit sous-ensemble de gènes symbiotiques spécifiques aux nodosités. Néanmoins, plus que la moitié des gènes NCR était différentiellement méthyles. D'autre part, l'expression des gènes était corrélée avec la décondensation de la chromatine (ouverture), un enrichissement du marqueur H3K9ac et une diminution du marqueur H3K27me3. Nos résultats suggèrent que l’endoréplication, pendant la différenciation cellulaire dans les nodosités, fasse partie des mécanismes qui lèvent l’inactivation transcriptionnelle des gènes spécifiques des nodosités, ceci résultant de modifications des codes épigénétiques au niveau de la chromatine. / Legume plants are able to interact with soil bacteria from the Rhizobiaceae family. This interaction leads to the development of a specialized organ called root nodule. Inside the symbiotic nodule cells, rhizobia are capable to fix atmospheric nitrogen and convert it to ammonia, which is a usable nitrogen source for the plant. In the legume Medicago truncatula the symbiotic cells produce high amounts of Nodule-Specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes. The expression analysis of 334 NCR genes in 267 different experimental conditions using the Medicago truncatula Gene Expression Atlas (MtGEA) revealed that all the NCR genes except five are exclusively expressed in nodules. No NCR expression is induced in any other plant organ or in response to biotic, abiotic stress tested or to Nod factors. The NCR genes are activated in consecutive waves during nodule organogenesis, which correlated with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. Moreover, we showed that NCRs are not induced during nodule senescence. According to their Shannon entropy, a metric for tissue specificity, NCR genes are among the most specifically and highest expressed genes in M. truncatula. Thus, NCR gene expression is subject to an extreme tight regulation since they are only activated during nodule organogenesis in the polyploid symbiotic cells. This analysis suggested the involvement of epigenetic regulation of the NCR genes. The formation of the symbiotic cells is driven by endoreduplication and is associated with transcriptional reprogramming. Using sorted nodule nuclei according to their DNA content, we demonstrated that the transcriptional waves correlate with growing ploidy levels and investigated how the epigenome changes during endoreduplication cycles. We studied genome-wide DNA methylation and chromatin accessibility as well as the presence of repressive H3K27me3 and activating H3K9ac histone tail modifications on selected genes. Differential DNA methylation was found only in a small subset of symbiotic nodule-specific genes, including over half of the NCR genes, while in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes’ active or repressed state. On the other hand, expression of these genes correlated with ploidy-dependent opening of the chromatin and in a subset of tested genes with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in differentiation of symbiotic cells.

Nuclear DNA content (genome size) is one of the basic characteristics of living organisms. In the Angiosperms, the range of genome size is 2,300-fold, which raises questions about the causes and consequences of this tremendous variation. This thesis deals with genome size in plants from the level of intraspecific homoploid variation, through intraspecific ploidy variation, to interspecies comparisons. On various study systems we investigated the dynamics and ranges of genome size variation, tried to reveal possible associations between genome size and selected biological traits, and assessed the extent to which differences in genome size are manifested at the ecological and evolutionary level. As a means of estimating genome size we applied flow cytometry (FCM). In Taraxacum stenocephalum we conducted a detailed study of its enormous genome size variation. We carried out crossings of parents with various genome sizes and compared these parental genome sizes with those of F1 offspring. We also attempted to reveal the association of genome size with various growth traits. In Galium valdepilosum and Arabidopsis arenosa we carried out an extensive flow-cytometric ploidy level screening and compared the distribution and ecological preferences of detected cytotypes. We studied the Andean genus...

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.

The allocation of resources to male or female progeny, or to male or female reproductive function more generally, is one of the most important life history decisions a sexually reproducing individual must ever make. Sex determination is thus a fundamental process, yet the mechanisms which control it are surprisingly diverse. In this thesis, I examine sex determination in the plant species Mercurialis annua L. (Euphorbiaceae). I assess the mechanism of sex determination operating in dioecious and androdioecious populations of M. annua and also investigate the conservation and evolution of sex-determining mechanisms across the annual mercury clade, the lineages of which display exceptional variation in sexual system. First, using crosses, I establish that sex in dioecious M. annua is controlled by a single-locus genetic mechanism, consistent with recent work that identified a single male-linked DNA marker in the species. My search for new sex-linked genes revealed none, however, suggesting that M. annua possesses at most a small non-recombining region around sex-determining loci. Why many dioecious plants lack heteromorphic sex chromosomes is still poorly understood and I consider explanations for this. I extend my investigation by comparing genetic diversity between loci that differ in their linkage to the sex-determining locus. I find a single male-linked marker to possess significantly lower diversity than autosomal loci, but no difference in the diversity of partially sex-linked and non-sex-linked genes. I also assess the conservation of a sex-linked marker among annual mercury lineages and conduct crosses between lineages to examine the conservation of sex determination. My findings indicate a conserved mechanism of single-locus genetic sex determination and I consider the role polyploidisation and hybridisation have played in sexual system evolution and the modification of sex-determining mechanisms in the clade. Finally, I assess the presence of environmental sex determination in androdioecious M. annua, concluding that although male frequency is not influenced by growing density, a degree of sexual lability exists in the lineage.