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Les terres rares (TRs) sont des métaux stratégiques du XXIe siècle dont la demande croissante résulte de leurs propriétés essentielles, notamment dans les domaines des énergies renouvelables, de la médecine et des hautes technologies. Ils sont classés en TR lourdes (HTRs), terres légères légères (LTRs) et non-lanthanides. Leur dissémination dans l'environnement, associée à une faible recyclabilité, fait des TRs des contaminants émergents pour lesquels les études de toxicité sont jusqu'à présent très disparates. Afin d’établir une base générale de la réponse cellulaire et moléculaire à un stress TRs, nous avons premièrement utilisé des stratégies complémentaires et à haut débit pour étudier la réponse, ainsi que l’absorption des TRs chez le modèle eucaryote Saccharomyces cerevisiae. Les deletome, transcriptome, protéome et ionome de la levure ont été analysés et approfondis par des analyses physiologiques ciblées. Bien que des réponses communes aux TRs et à d'autres métaux aient été mises en évidence, les réponses spécifiques aux TRs étaient prédominantes. La composition de la paroi cellulaire, la biosynthèse des sphingolipides, la voie ESCRT et l'endocytose sont des éléments clés de la réponse aux TRs. Deuxièmement, nous avons exploré les effets des TRs sur le transcriptome et le ionome du modèle végétal Arabidopsis thaliana. L'exposition des TRs a négativement impacté l'architecture racinaire, comme l'a révélé la modulation de gènes liés à l'auxine. De plus, le ionome a été modifié et les gènes liés à une carence en Fe largement représentés parmi les gènes les plus différentiellement exprimés. Afin d'identifier de nouvelles plantes modèles accumulant des TRs, des espèces de Phytolacca et de fougères ont été criblées. Malgré un trait d’accumulation des TRs conservé chez quelques genres de fougères et Phytolacca, un enrichissement en HTRs chez Phytolacca et en LTRs chez les fougères a été observé. Cependant, plusieurs espèces de Dryopteris présentent des teneurs contrastées en TRs dans les frondes et représentent de nouveaux modèles pertinents pour décrypter les mécanismes d’accumulation et de tolérance aux TRs. Globalement, des divergences ont été mises en évidence dans la réponse aux différentes TRs, en fonction de leur rayon ionique. La composition de la paroi cellulaire, la détoxification vacuolaire, mais aussi l’accumulation et le fractionnement des TRs ont souligné ces différences. Nous avons confirmé que les LTRs empruntaient les canaux calciques, tandis que de nouvelles preuves ont été données sur le rôle des transporteurs de Fe dans l'accumulation de HTRs. En conclusion, nous apportons ici de nouveaux éléments sur la toxicité et les spécificités des TRs, ainsi que des explications moléculaires pour certains effets déjà connus. Ce travail constitue un premier travail de base complet et multi-approches pour de futures études afin d’approfondir la compréhension de la toxicité des TRs chez les organismes vivants. / Rare earth elements (REEs) are strategic metals whose demand in the 21st century is increasing as a result of their essential properties useful to the fields of renewable energies, medicine, and high-technologies. They are classified as heavy REEs (HREEs), light REEs (LREEs) and non-lanthanides. Their dissemination in the environment, together with poor recyclability, leads REEs to be considered emerging contaminants, for which toxicity studies are currently very fragmented. To build a strong general foundation on the cellular and molecular response to REEs, we first adopted high-throughput and complementary strategies to study the REE stress response and their uptake in the unicellular eukaryotic model Saccharomyces cerevisiae. The deletome, transcriptome, proteome and ionome of yeast were analysed together with in-depth physiological experiments. Although common responses between REEs and other metals were highlighted, REE-specific responses were predominant. Cell wall composition, sphingolipid biosynthesis, the ESCRT pathway and endocytosis were emphasized as key elements in the cellular response to REEs. Second, we explored how REEs affect the transcriptome and ionome of the plant model Arabidopsis thaliana. REE exposure negatively affected the root architecture, as revealed by the modulation of auxin-related genes. REEs impaired the ionome, and Fe deficiency-related genes were largely represented among the most differentially expressed genes in both roots and leaves. Additionally, to identify new REE-accumulating plant models, collections of ferns and Phytolacca species were screened. Despite a conserved REE accumulation trait for Phytolacca and a few fern genera, HREE enrichment was observed in Phytolacca, while LREEs were preferentially transferred into the fronds of all fern species. However, several Dryopteris species harboured contrasting REE contents in the fronds. The latter species will be of great importance in deciphering the mechanisms of REE accumulation and tolerance. Overall, the response towards REEs differed according to their ionic radius. The cell wall composition, vacuolar detoxification, and the accumulation and fractionation of REEs notably accounted for these differences. Our findings support LREE-mediated entry through calcium channels, while new evidence was provided for the role of Fe transporters in the accumulation of HREEs. In conclusion, we have provided new insights into REE toxicity and specificities, together with the molecular elucidation of REE effects that have not previously been mechanistically explained. This work is a first multi-approach comprehensive groundwork that will be used for future studies to deepen the understanding and assessment of REE toxicity in organisms.

Master of Science / Department of Computing and Information Sciences / Doina Caragea / Recent work has shown that bidirectional genes (genes located on opposite strands of DNA, whose transcription start sites are not more than 1000 basepairs apart) are often co-expressed and have similar biological functions. Identification of such genes can be useful in the process of constructing gene regulatory networks. Furthermore, analysis of the intergenic regions corresponding to bidirectional genes can help to identify regulatory elements, such as transcription factor binding sites. Approximately 2500 bidirectional gene pairs have been identified in Arabidopsis thaliana and the corresponding intergenic regions have been shown to be rich in regulatory elements that are essential for the initiation of transcription. Identifying such elements is especially important, as simply searching for known transcription factor binding sites in the promoter of a gene can result in many hits that are not always important for transcription initiation. Encouraged by the findings about the presence of essential regulatory elements in the intergenic regions corresponding to bidirectional genes, in this thesis, we explore a motif-based machine learning approach to identify intergenic regulatory elements. More precisely, we consider the problem of predicting the transcription pattern for pairs of consecutive genes in Arabidopsis thaliana using motifs from AthaMap and PLACE. We use machine learning algorithms to learn models that can predict the direction of transcription for pairs of consecutive genes. To identify the most predictive motifs and, therefore, the most significant regulatory elements, we perform feature selection based on mutual information and feature abstraction based on family or sequence similarity. Preliminary results demonstrate the feasibility of our approach.

Chez les mammifères, l'infection par l'adénovirus conduit à l'activation de la transcription de certains éléments SINE Alu. Il s'agit d'un mécanisme conservé car il est observé avec d'autres familles de virus. Adénovirus code pour une protéine, E1A qui est capable d'interagir avec la protéine Rétinoblastome (RB). Cette interaction provoque l'inactivation de RB ce qui provoque probablement l'activation transcriptionelle des éléments Alu. Ainsi, chez les mammifères, certaines protéines virales agissent négativement sur RB ce qui a pour conséquence de déréguler le cycle cellulaire, la transcription pol III de manière générale et la transcription des éléments SINE en particulier. Chez les plantes, l'activation de la transcription des éléments SINE à la suite d'un stress comme l'infection virale reste à démontrer. Cependant, le virus FBNYV possède au sein de son génome une protéine, CLINK, qui est entre autre constituée d'un domaine de liaison à RB similaire à celui retrouvé chez E1A d'adénovirus. La première étape de ce travail de thèse a porté sur l'analyse de la protéine CLINK et plus particulièrement l'effet de cette protéine sur le cycle cellulaire, sur la transcription pol III en général et sur la transcription des SINE endogènes de plantes. La seconde partie de cette étude porte sur la fonction des éléments SINE de plantes au sein de la cellule. Chez les mammifères, l'élément SINE Alu est capable de jouer un rôle dans la physiologie de la cellule en réponse à certains stress. En effet, la transcription de ces éléments est activée à la suite d'une infection par certaines familles de virus. Les transcrits ainsi produits sont alors capables d'interagir avec la protéine PKR, une kinase d'eIF2α. Ainsi, les éléments SINE sont capables d'intervenir dans des processus clefs de la cellule comme le mécanisme de régulation de la traduction. La seule kinase d'eIF2α identifiée chez les plantes est la protéine GCN2. Ainsi, nous avons choisit de caractériser la fonction de cette protéine chez Arabidopsis. Nous avons déterminé les mécanismes de régulation de la protéine en mettant en évidence certains inducteurs spécifiques aux plantes. Ce travail a permis de montrer l'importance de la protéine pour la plante et de découvrir des fonctions potentielles de la protéine dans des voies de stress typiques des végétaux. Enfin, l'impact des SINE de plantes sur l'activité de GCN2 a été analysé.

Les télomères sont des structures nucléoprotéiques spécialisées dont l'un des rôles est d'empêcher le raccourcissement progressif de l'extrémité des chromosomes suite à la réplication et à l'instabilité génomique due à la recombinaison de l'extrémité de chromosomes. Malgré le rôle des télomères dans la protection de l'extrémité des chromosomes contre les mécanismes de réparation de l'ADN et de recombinaison, de nombreuses protéines de ces voies jouent des rôles essentiels dans l'homéostasie télomérique et la stabilité des chromosomes. Parmi elles, la protéine RAD50 appartenant au complexe MRE11/RAD50/XRS25(NBS1) et l'endonucléase structure spécifique XPF/ERCC1 sont localisées aux télomères ; ces deux complexes connus pour leur rôle dans les voies de réparation de l'ADN ainsi que dans les études sur la recombinaison. Nous avons identifié deux rôles différents pour la protéine RAD50 dans la maintenance télomérique et dans la protection des extrémités des chromosomes, en contexte de présence et absence de la télomérase. L'absence d'AtRAD50 augmente significativement le nombre de fusions chromosomiques impliquant des télomères raccourcis. Nous proposons que ce rôle protecteur des télomères raccourcis de RAD50 est le résultat de sa fonction de contraindre la recombinaison entre chromatides soeurs et ainsi d'éviter les évènements de fusions par les extrémités. Nous avons recherché le ou des mécanismes impliqué(s) dans ces évènements de fusions chromosomiques chez les mutants atrad50 en réalisant des croisements entre les plantes déficientes pour ATRAD50 et des plantes déficientes pour des gènes codant des protéines des voies de réparation par recombinaison non-homologue et homologue. Au contraire de la situation en cellules de mammifères, nous n'avons pas observé d'instabilité chromosomique chez les plantes mutantes correspondantes pour XPF (AtRAD1) or ERCC1 (AtERCC1). Cependant, en absence de la télomérase, la mutation de l'un de ces deux gènes entraîne une augmentation précose et significative de l'instabilité chromosomique sans accélération générale de la perte des répétitions télomériques, mais associée à la présence de fragments ADN extrachromatiques visibles en cytologie. Une analyse intensive par FISH a permis d'identifier ces ADN comme des bras entiers spécifiques de deux chromosomes. Nos données indiquent un rôle protecteur de RAD1/ERCC1 comme l'invasion de l'ADN simple brin télométrique dans des séquences télomériques interstitielles. Le fait que les mutations de rad1 (ou ercc1) augmentent dramatiquement l'instabilité chromosomique des mutants télomérase a des implications très importantes pour les modèles des rôles de la recombinaison aux télomères.

Doctor of Philosophy / Department of Biology / Ruth Welti / The molecular mechanisms by which plants respond to environmental stresses to sustain growth and yield have great importance to agriculture. Lipid metabolites are a major element of plant stress responses. The model plant Arabidopsis thaliana is well-suited to study stress-driven compositional dynamics, metabolism, and functions of lipid metabolites. When Arabidopsis plants were subjected to wounding, infection by Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr), infection by Pseudomonas syringae pv. maculicola (Psm), and low temperature, and 86 oxidized and acylated lipids were analyzed using mass spectrometry, different sets of lipids were found to change in level in response to the various stresses. Analysis of plant species (wheat versus Arabidopsis), ecotypes (Arabidopsis Columbia 0 versus Arabidopsis C24), and stresses (wounding, bacterial infection, and freezing) showed that acylated monogalactosyldiacylglycerol was a major and diverse lipid class that differed in acyl composition among plant species when plants were subjected to different stresses. Mass spectrometry analysis provided evidence that oxophytodienoic acid, an oxidized fatty acid, is significantly more concentrated on the galactosyl ring of monogalactosyldiacylglycerol than on the glycerol backbone. A mass spectrometry method, measuring 272 lipid analytes with high precision in a relatively short time, was developed. Application of the method to plants subjected to wounding and freezing stress in large-scale experiments showed the method produces data suitable for lipid co-occurrence analysis, which identifies groups of lipid analytes produced by identical or inter-twined enzymatic pathways. The mass spectrometry method and lipid co-occurrence analysis were utilized to study the nature of lipid modifications and the roles of lipoxygenases and patatin-like acyl hydrolases in Arabidopsis during cold acclimation, freezing, and thawing.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Lawrence C. Davis / Synthetic dyes are capable of producing the whole color spectrum on account of their structural diversity but this diversity poses challenges in the degradation of dyeing wastes. Laccases and peroxidases from bacterial or fungal sources and parts of plants in the presence of hydrogen peroxide (H₂O₂) plus a mediator have been exploited in the bioremediation of synthetic dyes. However, intact plants have not found much favor despite their phytoremediation potential. The goal of this research was to further clarify ways by which whole plants bring about decolorization of different types of synthetic dyes. Hydroponically cultivated plants from two dicot families namely Arabidopsis thaliana and sunflowers (Helianthus annuus) were exposed to representative dyes from several classes: monoazo (Methyl Red and Methyl Orange), disazo (Trypan Blue, Evans Blue and Chicago Blue 6B), and arylmethane (Brilliant Blue G, Bromocresol Green, Malachite Green and Phenol Red). Tests were done in presence or absence of externally added H₂O₂, with or without a free radical mediator, 1-hydroxybenzotriazole, using UV-Visible spectrophotometry. The initial rate of decolorization and the overall percentage decolorization was calculated for each dye in the different treatments. Decolorization of the dyes from different classes varied between plant species and depending on the treatment. Except for Methyl Red, all dyes required added H₂O₂ as well as mediator to achieve rapid decolorization. Added H₂O₂ was found to be the limiting factor since it was degraded by plants within a few hours. Both species were able to slowly decolorize dyes upon daily addition of fresh dye even in the absence of added H₂O₂ and mediator, provided that nutrients were supplied to the plants with the dye. A. thaliana was found to be more effective in dye decolorization per gram tissue than sunflower when treated under similar conditions. Analysis of the residual dye solution by ESI/MS did not reveal any potential by-products following the decolorization treatment with plants, suggesting that the plant roots might be trapping the by-products of dye decolorization and preventing their release into the solution. All these findings support the potential application of whole plants for larger scale remediation.

Orientador: Fábio Silveira Nogueira / Resumo: O sistema radicular (SR) é importante pela ancoragem e obtenção de água e nutrientes. Em eudicotiledôneas, como Arabidopsis, o crescimento da raiz primária (RP) é afetado por fitormônios, especialmente pelo balanço entre auxina que controla a divisão celular, e citocinina que modula a diferenciação celular; também, os microRNAs (miRNAs), um sub-conjunto de pequenos RNAs que regulam pós-transcricionalmente seus alvos, regulam o crescimento da RP. O microRNA156 (miR156) e seus alvos, membros da família SQUAMOSA Promoter-Binding Protein-Like (SPL), constituem uma via genética que regula vários processos do desenvolvimento, incluindo desenvolvimento da raíz; porém, durante o crescimento da RP, não foi observado o efeito da via miR156/SPL, e da interação com auxina e citocinina; assim, foi avaliada essa interação durante o crescimento da PR regulado pelo tamanho do meristema da raiz (TMR) em Arabidopsis. Usando ferramentas genéticas e moleculares foi analizada a expressão de genes MIR156 e SPLs, o comprimento da RP, o TMR, as taxas de divisão celular, e as respostas de auxina e citocinina durante o crescimento da RP. Os genes MIR156 e SPLs possuem padrões de expressão opostos. Níveis altos do miR156 (nas plântulas p35S :: MIR156A), leva a menor comprimento da RP, TMR reduzido, menores taxas de divisão celular, respostas mais baixas e altas à auxina e citocinina respectivamente; em contraste, níveis severamente reduzidos do miR156 maduro disponível (nas plantas MIM156) conducem a e... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

<p>In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy.</p><p>In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function.</p><p>I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained.</p><p>The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light.</p><p>Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role.</p><p>The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants.</p><p>A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.</p>

Genomic instability underlies diseases of unregulated cell growth that result in cancers and developmental abnormalities in humans. Similar genome destabilizing mechanisms are used to create genetic variety in crops for use in breeding and trait development. Errors that occur during DNA replication may cause mutations if they are not corrected before further cell divisions. DNA mismatch repair (MMR) corrects misinsertions and insertion/deletion DNA loop-outs that arise during DNA replication in plants, animals, prokaryotes, and some archaea, all of which incur mutations at rates 100 to 1,000-fold greater when subjected to inherited or somatic-mismatch repair deficiencies. An understanding of the effects of mismatch repair on somatic and germ-line cells in Arabidopsis thaliana is critical to the development of this plant as a model system for the study of genomic instability. Insertions and deletions of multiples of two base pairs in dinucleotide repeat sequences (microsatellites) occur more frequently in the absence of mismatch repair, and the mismatch-repair status of an individual, tissue, or cell may be inferred on the basis of microsatellite mutation frequency. Single-template PCR analysis measured microsatellite mutation frequencies in leaves and shoot-apical-meristem stem cells, and allowed me to address for the first time an important question: Do plants relax mismatch repair in vegetative tissues relative to meristematic germ-line and floral tissue? Analyses of four microsatellite loci in mismatch repair-deficient and wild type plants surprisingly suggest that there is little difference in mismatch repair activity between leaves and seeds. Mismatch-repair-deficient leaves displayed only two-fold higher microsatellite mutation frequency compared to wild type, and wild-type leaves also displayed a two-fold higher microsatellite mutation frequency compared to shoot-apical- meristems. The high frequency of microsatellite mutation in these wildtype tissues is unexpected, and it suggests that plants relax mismatch repair in differentiated tissues while maintaining genetic fidelity in a small set of stem cells in the shoot apical meristem (SAM). Genome sequencing of msh2⁻/⁻ mutation accumulation A. thaliana lines provides an estimated germ-line mutation rate of 3.9 × 10⁻⁷ in the absence of mismatch repair. Comparison of the rates of base substitution mutation per chromosome in mismatch repair-deficient plants with rates reported for wild-type plants suggests mismatch repair is more efficient on chromosome 5 than on chromosomes 1-4. Bias towards G:C → A:T mutations among transitions is maintained but increased nearly 100-fold in the absence of mismatch repair. / Graduation date: 2012