Clonally variant gene expression (CVGE) is a common survival strategy used by many pathogens, including the malaria parasite Plasmodium falciparum. Among the genes that show CVGE are several members of the clag and eba families. The active or repressed state of clag3.1, clag3.2 and eba‐140 is transmitted epigenetically and is controlled by the euchromatic or heterochromatic state of bistable chromatin domains, characterised by H3K9ac and H3K9me respectively. Both of these modifications are maintained throughout the asexual cycle to transmit epigenetic memory. We have characterised CVGE on a genome‐wide level and found that it is widespread. Using clag3.2 in proof‐of‐principle experiments, we demonstrate that CVGE for genes other than var can be reproduced in an episomal system and provide preliminary evidence that CVGE is regulated by the spontaneous formation of facultative heterochromatin. The mutually exclusive expression of clag3.1 and clag3.2 is also addressed, including the characterisation of a neighbouring noncoding RNA. / La variació clonal en l'expressió gènica (CVGE) és una estratègia de supervivència comú utilitzada per molts patògens, incloent el paràsit de la malària Plasmodium falciparum. Entre els gens que mostren CVGE hi ha diversos membres de les famílies clag i eba. L'estat actiu o inactiu de clag3.1, clag3.2 i eba‐140 es transmet epigenèticament i és controlat per l'estat eucromatínic o heterocromatínic de dominis de cromatina biestables, caracteritzats respectivament per H3K9ac i H3K9me. Ambdues modificacions es mantenen durant tot el cicle asexual per transmetre la informació epigenètica. També hem caracteritzat la CVGE a nivell de tot el genoma, i hem vist que afecta un gran nombre de gens. Experiments preliminars amb clag3.2 ens demostren que la CVGE de gens diferents que var es pot reproduir en un sistema episomal I proporcionen evidència preliminar de què la CVGE està regulada per la formació espontània d’heterocromatina facultativa. També s’ha estudiat l'expressió mútuament exclusiva de clag3.1 i clag3.2, incloent la caracterització d’un ARN no‐codificant veí.

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Seedling production of canjerana has been limited by difficulty in germination, caused by recalcitrant behavior of their seeds. The objective of this study was to develop micropropagation to auxiliate preserving and multiplication of superior genotypes, to study the plantlet production by micro-cutting and mini-cutting, and to evaluate the genetic diversity of canjerana. In micropropagation, seeds of canjerana were disinfected with 0, 2.5, 5.0, 7.5 and 10% of NaOCl solution to produce aseptic seedlings, which were cultivated on MS and WPM media. Nodal segments were treated with 0 and 2.5 μM of BAP, KIN and TDZ and with 0, 1, 3, 6, 9 and 12 μM of BAP, which were cultivated on WPM media. Micro-cuttings, were cultivated on MS and WPM media with either 0 or 5.0 μM of IBA and NAA. The rooted micro-cuttings were acclimatizated in a humid chamber in a greenhouse. The highest percentage of decontaminated seeds was produced using a solution of 7.5% of NaOCl and immersion times of 10, 20 and 30 minutes. The same concentrations of BAP, KIN and TDZ and increasing concentrations of BAP in the WPM media did not increase shoot number and length. Neither the base medium nor the auxin had a significant effect on the survival of micro-cuttings after 60 days of cultivation, but the addition of 5.0 μM of NAA did increase the percentage of rooting and survival during the acclimatization. Both nodal segments and microstumps of canjerana have a low rate of multiplication. Shoots produced from microstumps may be rooted in WPM or MS medium added with 5.0 μM of NAA. These complete plantlets can be mantained in vitro or acclimatized as a source of stock plants for the microclonal hedge. For production of canjerana plantlets by mini-cutting, different concentrations of indolbutyric acid (IBA) and substrate combinations were evaluated. Mini-cuttings were treated with 2000 mg L-1 of IBA and planted in commercial substrate; coarse sand; carbonized rice husks; and a combination of the three. Apical and nodal minicuttings were treated with 0, 1000, 2000 and 3000 mg L-1 of IBA and planted in a combination of commercial substrate, coarse sand and carbonized rice husks. The productivity of microstumps and mini-cutting rooting were evaluated in three clones of canjerana. The combination of commercial substrate, coarse sand and carbonized rice husks maximized mini-cuttings rooting. Nodal mini-cuttings had higher rooting capability than apical ones. The application of 3000 mg L-1 of IBA improved rooting differentiation and growth of canjerana mini-cuttings. Canjerana clones differ in rooting capability and survival rates. The genetic diversity of canjerana, within and among progenies of three stock plants, was assessed with previously defined species-specific SSR markers. The allele frequency was calculated for each band and the heterozygosity and the polymorphic information content were calculated for each SSR pair of primers, progeny and for the combination of the 32 canjerana genotypes. The results showed high level of genetic diversity, both within and among progenies, making possible that genotypes from different stock plants grouped together. Based upon these results, high level of genetic diversity can be maintained in clones from progenies of selected stock plants. / A produção seminal de mudas de canjerana tem sido limitada pela dificuldade de germinação, ocasionada pelo comportamento recalcitrante das sementes. O objetivo deste trabalho foi desenvolver a micropropagação para auxiliar a conservação e multiplicação de genótipos superiores, estudar a microestaquia e miniestaquia para a produção massal de mudas, e avaliar a diversidade genética da canjerana. Na micropropagação, sementes de canjerana foram desinfetadas com 0; 2,5; 5,0; 7,5 e 10,0% de hipoclorito de sódio para a produção de plantas assépticas e cultivadas em meios MS e WPM. Segmentos nodais das plântulas foram inoculados em meio WPM acrescido de 0 ou 2,5 μM de BAP, KIN ou TDZ, bem como acrescido de 0; 1; 3; 6; 9 e 12 μM de BAP. Microestacas foram cultivados nos meios MS e WPM acrescido de 0 ou 5,0 μM de AIB e ANA. As microestacas enraizadas foram aclimatizadas em câmara úmida e em casa de vegetação. O maior percentual de sementes descontaminadas foi produzido usando uma solução de 7,5% de NaOCl por 10, 20 e 30 minutos. Tanto BAP, KIN e TDZ em iguais concentrações quanto o aumento das concentrações de BAP no meio WPM não aumentaram o número e nem comprimento das brotações. O meio de cultura e a auxina não afetaram a sobrevivência de microestacas, mas a adição de 5,0 μM de ANA aumentou a porcentagem de enraizamento e sobrevivência durante a aclimatização. Segmentos nodais e microcepas tiveram baixa taxa de multiplicação. Microestacas enraizaram em meio WPM ou MS acrescido de 5,0 μM de ANA. As mudas produzidas podem ser mantidas in vitro ou aclimatizadas para serem utilizadas como plantas matrizes do microjardim clonal. Para a produção de mudas de canjerana por miniestaquia foram avaliadas as concentrações de AIB e diferentes substratos. Miniestacas foram tratadas com 2000 mg L-1 de AIB e plantadas em substrato comercial; areia grossa; casca de arroz carbonizada; e a combinação em iguais proporções de substrado comercial, areia grossa e casca de arroz carbonizada. Miniestacas apicais e nodais foram tratados com 0; 1000; 2000 e 3000 mg L-1 de AIB e plantadas em uma combinação de substrato comercial, areia grossa e casca de arroz carbonizada. Além disso, a produtividade de minicepas e o enraizamento de miniestacas foram avaliados em três clones de canjerana. A combinação de substrato comercial, areia grossa e casca de arroz carbonizada maximizaram o enraizamento das miniestacas. Miniestacas nodais tiveram maior capacidade de enraizamento do que as apicais. A aplicação de 3000 mg L-1 de AIB aumentou o enraizamento e o crescimento de miniestacas de canjerana. Clones de canjerana diferem na porcentagem de enraizamento e na sobrevivência das miniestacas. A diversidade genética entre e dentro de progênies de três matrizes de canjerana foi avaliada por microsatélites. A frequência alélica foi calculada para cada banda e a heterozigose e o conteúdo de informação polimórfica foram obtidos para cada par de primers, cada progênie e para a combinação dos 32 genótipos de canjerana. Os resultados indicam a existência de alta variabilidade genética, tanto entre quanto dentro das progênies avaliadas, possibilitando a formação de grupos com genótipos oriundos de diferentes progênies. Assim, alta variabilidade genética pode ser mantida a partir de clones de progênies de matrizes selecionadas.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-30T22:08:29Z No. of bitstreams: 1 DISS_2015_Anne Francis Agostini Santos.pdf: 973075 bytes, checksum: 6e46b2f1d3699849f939c805ae26d7f9 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-05-03T17:40:08Z (GMT) No. of bitstreams: 1 DISS_2015_Anne Francis Agostini Santos.pdf: 973075 bytes, checksum: 6e46b2f1d3699849f939c805ae26d7f9 (MD5) / Made available in DSpace on 2018-05-03T17:40:08Z (GMT). No. of bitstreams: 1 DISS_2015_Anne Francis Agostini Santos.pdf: 973075 bytes, checksum: 6e46b2f1d3699849f939c805ae26d7f9 (MD5) Previous issue date: 2015-04-22 / CAPES / Objetivou-se avaliar o desempenho silvicultural de 18 clones do gênero Eucalyptus em duas condições edafoclimáticas diferenciadas, do estado de Mato Grosso. Os plantios experimentais foram instalados em 2010 no delineamento de blocos ao acaso com quatro repetições. Avaliou-se a altura total, altura dominante, DAP, área transversal média, área basal, e a morfometria de copa dos 18 clones nas duas regiões. Os dados obtidos foram submetidos a análise de variância e, posteriormente, teste de média de Scott-knott a 1% de probabilidade. Comportamento distinto foi encontrado entre os materiais testados nas áreas avaliadas. Os clones que apresentaram características superiores em Sinop foram os S-0102, S-0206 e S-0302em Chapada dos Guimarães foram os S-0410, S-0411, S-0412. O clone S-0402 apresentou características silviculturais superiores nas duas regiões de estudo. / This study aimed to evaluate the silvicultural performance of 18 clones of Eucalyptus in two different soil and wheather conditions, in the state of Mato Grosso.The experimental plantings were installed in 2010, with a randomized block design with four replications. It was evaluated the total height, dominant height, DBH, average cross-sectional area, basal area, and the morphometry canopy of the 18 clones in the two regions. The data obtained were subjected to analysis of variance and, subsequently, to the average test of Scott-Knott 1% probability. Distinct behaviors were found among the materials tested in the evaluated areas. The clones that showed superior characteristics in Sinop were the S-0102, S-0206 and S-0302 and Chapada dos Guimarães were the S-0410, S-0411, S-0412. The S-0402 clone presented sivicultural characteristcs superior in both study areas.

La protéine p53 (« Gardien du génome ») doit être altérée pour que le cancer puisse se développer. Les nombreuses thérapies anti-cancéreuses disponibles sont très efficaces mais la réponse clinique est souvent transitoire et les cancers disséminés rechutent ou progressent du fait de l'évolution de sous-populations cancéreuses résistantes au traitement, impliquant souvent TP53 qui est le gène le plus muté dans les formes agressives de nombreux cancers. Nous l’avons étudié dans la leucémie lymphoïde chronique (LLC) et les syndromes myélodysplasiques avec délétion 5q (SMD del(5q)). Grâce à l’étude rétrospective longitudinale de 40 patients atteints de SMD del(5q), nous avons généré des données de NGS ciblé et montré que le statut mutationnel de TP53 au diagnostic ne permettait pas de prédire la progression tumorale, contrairement à ce qui avait été publié précédemment (Jädersten et al., JCO 2011). Nous avons montré que c’était l’évolution clonale du gène TP53 qui était l’élément clé de la progression des SMD del(5q). Nous avons observé de nombreuses émergences de clones mutés entre le stade diagnostique et un stade ultérieur de la maladie, toujours après initiation du traitement par lénalidomide.Le lénalidomide a été approuvé comme nouveau traitement spécifique et très efficace contre l’anémie liée aux SMD del(5q), permettant à la plupart des patients d’être indépendants des transfusions sanguines. Le lénalidomide permet souvent d’éradiquer le clone tumoral porteur de l’anomalie génétique del(5q) isolée, induisant une rémission clinique. Malheureusement, cette rémission est courte avec une durée médiane de 2 ans, puis, dans environ 1 cas sur 2, survient une transformation en leucémie aiguë secondaire de pronostic péjoratif.Nous avons étudié un possible lien entre le traitement par lénalidomide et l’évolution clonale de TP53 par annotation clinico-bio-thérapeutiques des résultats de séquençage de TP53 chez les 24 patients dont les échantillons séquentiels avaient été analysés. Dans notre étude, les patients avec progression tumorale (dont 10 évolutions clonales de TP53 et 1 évolution clonale de RUNX1) avaient reçu une dose cumulée de lénalidomide supérieure à celle reçue par les patients dont la tumeur était restée stable (p = 0.036). Nous avons observé chez plusieurs patients que l’éradication de la tumeur n’était pas utile à l’amélioration de la qualité de vie des patients. La non-éradication semblait même permettre un maintien de l’équilibre clonal et une compétition entre les différents sous-clones de la tumeur, résistants ou non au lénalidomide.Nous discutons de l’évolution de l'écologie de la tumeur au cours du traitement, i.e., l’évolution de ses interactions avec son micro-environnement qui se modifie après chaque nouvelle dose de traitement. Un modèle évolutif dit théorie de la thérapie adaptative, développée récemment remet en question les protocoles conventionnels de thérapie anti-cancéreuse qui préconisent souvent d'administrer la dose maximale tolérée par le patient (Gatenby, 2009). Elle suggère que la dose minimale efficace présenterait l’avantage de ne pas éradiquer les cellules cancéreuses sensibles au traitement pour qu'elles restent en compétition avec les cellules cancéreuses résistantes et limiter la progression ou la rechute. Nous suggérons de prendre en compte également la diminution des effets indésirables pour le patient, améliorant ainsi sa qualité de vie, et enfin la diminution des dépenses de santé pour la collectivité. A ce jour, peu d’études cliniques évaluent l’intérêt de l’adoption de tels protocoles de thérapie adaptative.Néanmoins, des modèles in vivo (xénogreffes) et in silico (modèles statistiques) ont permis d’analyser la dynamique évolutive des populations tumorales en fonction du traitement reçu. Ces modèles prédisent que la survie de l’hôte peut être maximisée par la mise en place d’une thérapie adaptative. / P53 protein is named «guardian of the genome » because it must be altered to let cancer grow.TP53 is the most mutated gene in agressive cancers.Numerous systemic therapies are successful for treatment of disseminated cancers. However, clinical response is often transient, and cancer undergo relapse or progression due to emergence of resistant populations. These latter often harbour TP53 mutations. We studied TP53 in chronic lymphoid leukemia (CLL) and lower-risk myelodysplastic syndroms with del(5q), MDS del(5q). We conducted a retrospective longitudinal study in 40 patients suffering from MDS del(5q). We obtained targeted NGS data showing that TP53 mutational status at diagnosis could not predict tumor progression, by contrast with previously published data (Jädersten et al., JCO 2011). We show that TP53 clonal evolution is the key feature of tumor progression in MDS del(5q). We observed numerous mutated sub-clones emerging between diagnosis and follow-up. In our study, this emergence always followed onset of lenalidomide treatment. Lenalidomide was recently approved as a new therapy specifically improving anemia in patients with MDS del(5q). It allows most patients to become red-blood-cells-transfusion independent. Lenalidomide often eradicates the major tumor clone harbouring the isolated genetic abnormality deletion (5q) and allows clinical remission. Unfortunately, this remission is short (median, 2 years) and is followed, in 1 case out of 2, by a secondary acute leukemic transformation with a very poor prognosis.We studied the issue of a possible link between lenalidomide therapy and TP53 clonal evolution by annotating TP53 sequencing results with acute biological, clinical and therapeutic features in the 24 patients with sequential samples analyzed. In our study, patients with tumor progression (10 TP53 clonal evolution and 1 RUNX1 clonal evolution) were given a higher cumulative dose of lenalidomide compared to patients with stable disease (p = 0.036). Similarly to « adaptive therapy theory »(Gatenby 2009), we observed that eradication of the tumor wasn’t useful for improvement of quality of life. Absence of eradication might even allow to maintain a clonal equilibrium and a clonal competition between the distinct tumor sub-clones, resistant to lenalidomide or not, and therefore maintain stable disease.This theory of adaptive therapy questions the classical protocols of treatments against cancer, in which the maximal tolerated dose is preferred to the minimal effective dose. The latter might however slow down cancer progression or cancer relapse, with decreased side effects in patients, and decreased health costs.To date, few clinical trials (if any) questions such protocols of adaptive therapy. However, in vivo experiments (xenografts) and in silico statistical models allowed to study evolutionary dynamics of tumor sub-populations with and without therapy.The models predict that host survival can be maximized if “treatment-for-cure strategy” is replaced by “treatment-for-stability.” Specifically, the models predict that an optimal treatment strategy will modulate therapy to maintain a stable population of chemosensitive cells that can, in turn, suppress the growth of resistant populations under normal tumor conditions, Dr Gatenby said.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Several immunological mechanisms are responsible for the control of Toxoplasma gondii infection. The immune response occurs initially because of the participation of innate immune cells, in which macrophages play an important role. The existence of atypical strains of T. gondii with a genetic pattern different from clonal strains lead concern worldwide, principally because these strains cause severe symptoms especially in immunocompromised individuals and congenitally infected newborn. In this sense, we investigated the in vitro polarization of human macrophages to M1 or M2 profile after the infection with RH, ME49 clonal strains and TgChBrUD2 Brazilian strain. THP-1 cells were treated with PMA to induce their differentiation into macrophages. The cells were plated and infected with T. gondii tachyzoites from TgChBrUD2, RH or ME49 strains at a 2:1 (parasites: host cell) ratio or incubated only with medium (control uninfected cells). The supernatant was collected for cytokine analysis and measurement of nitrite. The macrophages were used for quantification of urea, evaluation of parasitism, analysis of morphology and surface markers. Our results showed an up-regulation of MIF during infection by TgChBrUD2, RH and ME49, and low levels of IL-10 compared to control. In addition, TgChBrUD2-infected macrophages show high levels of IL-6 production compared to non-infected macrophages and RH infection. Regarding secretion of nitrite, the infection by TgChBrUD2 exhibited high levels of nitrite compared to control and RH infection. The urea production showed a down-regulation in macrophages infected with TgChBrUD2 and ME49 in comparison to control. The analysis of morphology revealed that TgChBrUD2-infected macrophages induced the cells to change their morphology to spindle shaped. On the other hand, PCR analysis showed an increased amount of T. gondii DNA in RH-infected macrophages compared to infection with TgChBrUD2 and ME49. Thus, our results showed that RH strain produced an insuficient pro-inflamatory response that influenced the proliferation of parasite, in contrast, TgChBrUD2 and ME49 induced characteristic factors of polarization to M1 macrophages, in which the atypical strain showed high production of pro-inflamatory mediators that helped to countain the replication of this strain. / Vários mecanismos imunológicos são responsáveis pelo controle da infecção por Toxoplasma gondii. A resposta imune a este patógeno ocorre inicialmente pela participação de células da imunidade inata, na qual macrófagos têm papel importante. A existência de cepas atípicas de T. gondii com padrão genético diferente das clonais desperta grande preocupação mundial, uma vez que, geralmente, causam sintomas graves particularmente em indivíduos imunocomprometidos e naqueles infectados congenitamente. O objetivo deste estudo foi comparar, in vitro, a polarização de macrófagos humanos derivados da linhagem THP-1 para um perfil M1 ou M2 após infecção com as cepas clonais RH, ME49 e a cepa atípica TgChBrUD2. Células THP-1 foram tratadas com PMA para induzir sua diferenciação em macrófagos funcionais. Após a diferenciação, esses macrófagos foram plaqueados e infectados com as cepas TgChBrUD2, RH ou ME49 de T. gondii em diferentes abordagens experimentais. Como controle foram utilizados macrófagos não infectados. Os sobrenadantes de cultura de macrófagos nas diferentes condições experimentais foram coletados para análise de citocinas pelo teste ELISA e dosagem de nitrito pela reação de Griess. As células foram utilizadas para avaliação da morfologia, quantificação da ureia por espectrofotometria, dos marcadores expressos na superfície celular por citometria de fluxo e do parasitismo por PCR. Nossos resultados mostraram um aumento das citocinas MIF durante infecção pelas cepas TgChBrUD2, RH e ME49, bem como uma diminuição de IL-10 quando comparado aos macrófagos não infectados. Além disso, macrófagos infectados pela cepa atípica apresentaram altos níveis de secreção de IL-6 em relação ao controle. Em relação a produção de nitrito, apenas os macrófagos infectados por TgChBrUD2 apresentaram aumento em relação aos macrófagos controle e aos infectados por RH. A dosagem de ureia revelou uma redução da atividade de arginase na infecção por TgChBrUD2 e ME49 comparado aos macrófagos não infectados. A análise da morfologia celular mostrou que a infecção por TgChBrUD2 induziu os macrófagos a mudarem seu formato de circulares para fusiformes. Por outro lado, a análise de PCR mostrou que a cepa RH proliferou significativamente mais nos macrófagos em comparação com as cepas TgChBrUD2 e ME49. Desse modo, nossos resultados mostraram que a cepa RH induziu resposta pró-inflamatória insuficiente que influenciou na proliferação do parasito, por outro lado, TgChBrUD2 e ME49 induziram fatores característicos da polarização para macrófagos M1, de modo que a cepa atípica mostrou maior liberação de mediadores pró-inflamatórios que auxiliaram a conter a replicação desta cepa. / Mestre em Imunologia e Parasitologia Aplicadas

Cytogenetic analysis of cells of chronic lymphocytic leukemia (CLL) is difficult because of their low proliferative activity. To obtain sufficient number of mitoses for performing chromosomal analysis a suitable stimulation of cell division is needed. Using DSP30/IL2 stimulated cultivation 391 CLL samples were investigated in 5 years' period. The cultivation was showed to have high success rate (96%; 375/391) with also high rate of detection of pathological clones by both karyotype and metaphase FISH analyses (in 84% of samples; 329/391). Almost in half of samples (44%; 171/391) other aberrations than recurrent FISH (i.e. 13q14 deletion, trisomy 12, TP53, ATM genes deletions) were found. Also high frequency of translocations (37%; 144/391), complex karyotypes (28%; 111/391) and clonal evolution, which was detected in one third of all samples (34% of samples with presence of more than two clones; 133/391) and like a new event in disease duration even more frequently (in 39% of samples repeatedly investigated after stimulated cultivation; 21/54), was revealed. The presence of translocations, complex karyotypes and clonal evolution was associated with progressive form of disease (P 0,000003, resp. P 0,0002 and P 0,05/P 0,04). In cases of the recurrent deletions the detailed analysis of metaphase...

Les plantes vivent en association avec une grande diversité de microorganismes qui forment son microbiota. Ce microbiote fournit des fonctions clés qui influencent tous les aspects de la vie d'une plante, de l'établissement à la croissance jusqu'à la production. Cette thèse a pour intention de déterminer les règlent d'assemblage du microbiote et ses conséquences pour le phénotypel l'adaptation et l'évolution des plantes. Pour atteindre cet objectif nous avont utilisé différentes approches expérimentales comprenant des plantes clonales comme organismes modèles ainsi que des mésocosmes prairiaux pour analyses à l'échelle des communautés. Nos résultats ont démontré i) que les Champignons Mycohiziens à Arbuscules induisent d'important es variations phénotypiques pour les traits des plantes clonales impliqués dans l'exploration de l'espace et l'exploitation des ressources. Ces changements dépendent de l'identité des symbiontes et altèrent les capacités des plantes à développer des réponses plastiques à l'hétérogénéité environnementale. ii) Les plantes ont évolué un méchanisme permettant la transmission d'une partie de leur microbiote a leur descendance, assurant la qualité de leur habitat. iii) Le contexte spécifique des communautés de plantes est un facteur majeur structurant l'assemblage du microbiota des plantes à échelle locale. L'abondance de certaines espèces de plante dans le voisinage d'une plante cible augmente ou diminue la diversité de son microbiote, déterminant in fine ses performances. De manière générale, cette thèse démontre l'importance des organismes symbiotiques dans la compréhension de l'adaptation et de l'évolution des plantes. / Plants live in association with a wide diversity of microorganisms forming the microbiota. The plant microbiota provides a variety of key functions that influence many aspects of plant's life comprising establishment, growth and reproduction. The present thesis aims at determining the assembly rules of the plant microbiota and its consequences for plant phenotype, adaptation and evolution. To fulfill this objective, we used different experimental approaches using either clonal plants as model organisms or grassland mesocosms for community-wide analyses. Our results demonstrated i) that Arbuscular Mycorrhizal Fungi induce important phenotypic variations in clonal plants traits involved in space exploration and resources exploitation. These changes depended on the identity of the symbionts and altered the plants ability to produce plastic responses to environmental heterogeneity. ii) Plants have evolved a mechanism allowing the transmission of a part of their microbiota to their progeny, ensuring thus their habitat quality. iii) The plant community context is a major factor structuring local plant microbiota assembly. Particular plant species identity in the neighborhood increase or decrease the microbiota diversity and ultimately determine the focal plant performance. This thesis overall demonstrates the importance of symbiotic microorganisms in the understanding of the plant adaptation and evolution. From the knowledges acquired we developed a novel understanding of symbiotic interactions in clonal plants by extending the holobiont theory to the meta-holobiont theory.

Abstract Cryopreservation enables storage of genetic resources at ultra-low temperatures (<-150°C) while maintaining viability and regeneration capability. The method is especially suitable for long-term preservation of plant materials that cannot be stored as seeds. The genetic resources of potato (Solanum tuberosum L.), one of the most important food crops in the world, are preserved almost entirely by vegetative preservation. Cryopreservation is therefore increasingly applied for securing potato genetic resources in plant genebanks. A major challenge is, however, that recovery percentages can extensively vary among different genotypes. Light spectral quality is among the most important factors affecting plant growth and morphogenesis, but its effect with regard to cryopreservation has not been studied. In the present thesis, I studied the effect of six different light qualities on cryopreservation success of five potato cultivars before and after cryopreservation. I also explored how the different light conditions affect gene transcript abundance of recovering potato shoot tips. The results indicate that light spectral quality significantly affects the cryopreservation success of potato shoot tips in vitro. Prior to cryopreservation, cultivation under blue LEDs resulted in high initial survival, while post-cryopreservation exposure to a combination of red and blue LEDs (90% red, 10% blue) doubled the regeneration percentages. Concurrently, for most cultivars, red LEDs had adverse effects both before and after cryopreservation. The transcriptome analysis of potato shoot tips revealed the complex and extensive effect of cryopreservation on transcript abundance. Moreover, the expression level of stress- and defence-responsive genes was affected by light spectral quality. The positive effect of red-blue LEDs on shoot formation could tentatively be associated with a higher level of morphogenesis-related transcripts and lower level of stress and defence-responsive transcripts. The present thesis reveals that light spectral quality is an additional non-cryogenic factor, which can significantly increase the cryopreservation efficiency of plant germplasm. / Tiivistelmä Syväjäädytys mahdollistaa geenivarojen säilytyksen erittäin alhaisissa lämpötiloissa (<-150°C), siten että niiden elin- ja uusiutumiskyky säilyvät. Menetelmä soveltuu erityisesti sellaisten kasvimateriaalien pitkäaikaissäilytykseen, joita ei voida säilöä siementen avulla. Peruna (Solanum tuberosum L.) on yksi maailman tärkeimmistä ruokakasveista ja sen geenivaroja säilytetään lähes pelkästään vegetatiivisesti. Syväjäädytysmenetelmää käytetäänkin kasvavissa määrin perunan geenivarojen taltioimiseen geenipankeissa. Haasteena on kuitenkin syväjäädytyksen jälkeinen suuri vaihtelu elpymisprosenteissa eri genotyyppien välillä. Valon laatu on yksi tärkeimmistä kasvien kasvuun ja kehitykseen vaikuttavista tekijöistä, mutta sen vaikutusta syväjäädytyksen yhteydessä ei ole tutkittu. Väitöskirjassani tutkin kuuden erilaisen valonlaadun vaikutusta sekä syväjäädytystä edeltävän että sen jälkeisen kasvatuksen aikana viiden perunalajikkeen selviytymiseen. Lisäksi tutkin, miten erilaiset valo-olosuhteet vaikuttivat geenien ilmenemiseen elpyvissä perunan versonkärjissä. Tutkimukseni osoitti, että valon laatu vaikuttaa merkittävästi perunan versonkärkien kykyyn elpyä syväjäädytyksestä in vitro -olosuhteissa. Kun perunan versonkärkiä kasvatettiin sinisten LED-valojen alla ennen syväjäädytystä, niiden elävyysprosentit olivat korkeita, kun taas syväjäädytystä seuraava kasvatus sinipunaisten LED-valojen (90 % punaista, 10 % sinistä) alla kaksinkertaisti uusiutumisprosentit. Samanaikaisesti suurimmalle osalle lajikkeista punaisilla LED-valoilla oli epäsuotuisat vaikutukset selviytymiseen sekä syväjäädytystä edeltävän, että sitä seuraavan kasvatuksen aikana. Perunan kärkisilmujen geeniekspressioanalyysi osoitti, että syväjäädyttäminen aiheuttaa laajoja ja monitahoisia vaikutuksia kasvin geenien toiminnassa. Valon laatu vaikutti erityisesti stressi- ja puolustusgeenien ilmenemiseen. Tulokset viittaavat siihen että sinipunaisten LED-valojen uusiutumista edistävä vaikutus voi liittyä morfogeneesissä toimivien geenituotteiden runsauteen ja toisaalta stressi- ja puolustusgeenituotteiden määrän laskuun. Kaiken kaikkiaan tutkimukseni osoitti, että valon laatu on tärkeä ei-kryogeeninen tekijä, joka voi lisätä kasvien syväjäädytyksen tehokkuutta.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Molekulární biologie, genetika a virologie Bc. Jan Röslein Mutační a substituční tempo u sexuálních a klonáních forem: možný klíč k vysvětlení persistence sexu u modelové skupiny sekavců Mutation AND substitution rates in sexual and asexual forms: a clue to the persistence of sex in a model group of Cobitis? Typ závěrečné práce Diplomová Vedoucí závěrečné práce: Mgr. Karel Janko, Ph.D. Praha, 2015 Velký dík náleží mému školiteli Mgr. Karlu Jankovi, Ph.D. za velmi nápomocné, direktivní vedení práce. Též bych rád poděkoval panu Mgr. Janu Pačesovi, Ph.D. za více než vzdělávací rozměr v oblasti bioinformatické analýzy a Mgr. Ladislavu Pekárikovi, Ph.D., Mgr. Janu Kočímu za pomoc při analýze vybraných kapitol. Také bych rád poděkoval rodině za podporu. Všem participantům na této diplomové práci se hluboce omlouvám za způsobenou psychickou újmu. Prohlášení: Prohlašuji, že jsem závěrečnou práci zpracoval/a samostatně a že jsem uvedl/a všechny použité informační zdroje a literaturu. Tato práce ani její podstatná část nebyla předložena k získání jiného nebo stejného akademického titulu. V Praze dne 12. 8. 2015 Podpis: Abstrakt Klíčová slova: Abstract Key words: Obsah 1...