Les lignines sont des hétéropolymères phénoliques de la paroi cellulaire, principalement à base de p-coumaryl, coniférylique et sinapylique alcools. Ces monolignols sont synthétisées dans le cytoplasme de la voie des phénylpropanoïdes, peut ensuite transportées vers les parois des cellules où ils sont polymérisés par oxydation en p-hydroxyphényl (H), guaiacyle (G) et syringyle (S) des unités de lignine. Cette étape de polymérisation par oxydation est conduite par les peroxydases dépendantes H₂O₂ et / ou laccases dépendantes O₂. Dans cette étude, nous avons signalé pour la première fois que les laccases sont également impliqués dans la lignification du les herbes. Un gène de laccase spécifique (BdLAC5) a été identifié parmi les 29 gènes de laccase non redondants dans Brachypodium génome, qui est responsable de la lignification dans les tiges de Brachypodium distachyon, une plante modèle pour les graminées. BdLAC5 gène a été retrouvé fortement exprimé dans les organes lignifiées (internodal, nœud et le pédoncule) et mal exprimé dans les organes avec faible niveau de lignine (jeunes feuilles et épillet), ni dans les tissus non-lignifiée (endosperme). Deux autres laccases BdLAC6 et BdLAC8 sont également trouvés coexprimés avec BdLAC5 et curieusement ils appartiennent à la même clade phylogénétique. BdLAC6 et BdLAC8 sont orthologues de Arabidopsis LAC4 et LAC2 respectivement. Dans les expériences d'hybridation in situ ont démontré le signal le plus intense dans les fibres interfasciculaires de l'entre-nœud a été détectée avec des sondes BdLAC5. En outre, des essais ont révélé que les protéines immunomarquages BdLAC5 pourraient être sécrétés dans la matrice de la paroi cellulaire, car nous avons détecté des particules fluorescentes dans ou à proximité de la paroi cellulaire. Le double mutant laccase touchée BdLAC5 et BdLAC8 a clairement montré que la lignification dans les fibres interfasciculaires impliqué différents gènes / protéines que la lignification dans les cellules métaxylème de Brachypodium. Métaxylème cellules ont été que faiblement affectés dans le double mutant lorsque les fibres interfasciculaires montré diminution dramatique de Wiesner coloration. Les différents mécanismes de lignification entre xylème et fibres est discutée. L'interaction physique et la synergie entre réglementation spécifique R2R3-MYB, bHLH et WDR protéines est bien étudiée dans la biosynthèse des flavonoïdes, racine des cheveux, trichomes et le développement en mucilage de graines de différentes espèces de plantes. Dans cette étude, nous avons essayé de comprendre les rôles de MYB-bHLH-WDR pour la régulation de la biosynthèse de la lignine. / Lignins are cell wall phenolic heteropolymers, mainly made from p-coumaryl, coniferyl, and sinapyl alcohols. These monolignols are synthesized in the cytoplasm from the phenylpropanoid pathway, then may transported to the cell walls where they are oxidatively-polymerized into p_hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units. This oxidative polymerization step is driven by H₂O₂-dependent peroxidases and/or O₂-dependent laccases. In this study we reported for the first time that laccases are also involved in lignification in grasses. A specific laccase gene (BdLAC5) was identified among 29 non-redundant laccase genes in Brachypodium genome, which is responsible for the lignification in stems of Brachypodium distachyon, a model plant for grasses. BdLAC5 gene was found highly expressed in lignified organs (internode, node and peduncle) and poorly expressed in organs with low lignin level (young leaf and spikelet) nor in non-lignified tissue (endosperm). Two other laccases BdLAC6 and BdLAC8 are also found coexpressed with BdLAC5 and interestingly enough they belong to the same phylogenetic clade. BdLAC6 and BdLAC8 are close orthologues of Arabidopsis LAC4 and LAC2 respectively. In situ hybridization experiments demonstrated the most intense signal in the interfascicular fibers of the internode was detected with BdLAC5 probes and then for BdLAC8 and BdLAC6 probes. Furthermore, immunolabelling assays revealed that BdLAC5 proteins might be secreted into the cell wall matrix because we detected some fluorescent particles close to or in the cell wall. The double laccase mutant affected in BdLAC5 and BdLAC8 (5ho8ho) clearly showed that the lignification in interfascicular fibers involved different genes/proteins than the lignification in metaxylem cells of Brachypodium. Metaxylem cells were only poorly affected in the double mutant when interfascicular fibers showed dramatic decrease of Wiesner staining. The different mechanisms of lignification between xylem and fibers is discussed. The physical interaction and regulatory synergy between specific R2R3-MYB, bHLH and WD repeat protein is well studied in the biosynthesis of flavonoids, root hair, trichome and seed mucilage development in different plant species. In this study, we were trying to figure out the roles of MYB- bHLH-WDR for the regulation of lignin biosynthesis.

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.

Lymphomas are heterogenous class of diseases characterized by proliferation of a malignant lymphocyte clone. MicroRNA miR-155 was found to be a key molecule in immune response, namely in inflammation and germinal reaction of B cells. On the other hand, miR-155 can drive lymphoproliferation in mouse and its levels were found to be elevated in certain lymphoma types in human. MiR-155 down-regulates expression of its target gene PU.1, a hematopoietic transcription factor important for B cell differentiation. Expression of the gene encoding miR-155, known as MIR155HG, is controled by several transcription factors, among them MYB, a member of an oncogenic E-box protein family. Levels of MYB itself are controled by microRNA miR-150. In this study, we measured levels of miR-155, PU.1, MYB and miR-150 in lymph nodes of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, N=20), diffuse large B-cell lymphoma (DLBCL, N=24), follicular lymphoma (FL, N=29), Hodgkin lymphoma (HL, N=25), marginal zone lymphoma (MZL, N=13), and mantle cell lymphoma (MCL, N=10). We also measured levels of these molecules in lymph nodes with the finding of strong inflammation (N=4). We found that patients of all the diagnoses except of MCL display heterogeneously elevated levels of miR-155 and correspondingly...

abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

High expression of B-Myb (encoded by MYBL2), an oncogenic transcription factor, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adaptor for assembly of both the DREAM and MMB complexes, by a mechanism that requires the S28 phosphorylation site in LIN52. To validate our cellular findings, we determined the effect of B-Myb levels on DREAM target gene expression in HGSOC tissue samples and corresponding patient outcomes. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification and establish the rationale for targeting B-Myb to restore cell cycle control.

Les télomères sont des complexes nucléoprotéiques localisés à l'extrémité des chromosomes des cellules<br />eucaryotes. L'ADN télomérique humain contient entre 500 et 3000 repétitions de la séquence d(T2AG3)n (brin-G) et de son brin complémentaire C3TA2 (brin-C). Le brin G est terminé par une extrémité simple brin contenant de 50 à 200 nucléotides. Les télomères jouent des rôles essentiels au niveau cellulaire en permnettant la protection des chromosomes.<br />Un modèle proposé pour expliquer le rôle protecteur des télomères est le modèle de la boucle-T (télomérique) / boucle-D (déplacement). Dans ce modèle, on observe le repliement de I'ADN télomérique double brin et I'insertion de la partie simple brin dans la partie double brin. La formation de ces boucles fait intervenir, directement ou indirectement, denombreuses protéines comme les protéines TRF1 et TRF2. Néanmoins les mécanismes moléculaires et notamment le rôle de la protéine TRF2 dans la formation de ces boucles restent spéculatifs.<br />Le travail de thèse présenté dans ce manuscrit expose l'étude par Résonance Magnétique Nucléaire du domaine<br />de fixation à I'ADN de la protéine TRF2 (domaine Myb) et de ses interactions avec-l'ADN télomérique ainsi que<br />l'étude de modèles de boucles-D.<br />Après la détermination de la structure tridimensionnelle et l'étude de la dynamique interne du domaine Myb de<br />TRF2, nous avons réalisé l'étude des interactions du domaine Myb avec I'ADN télomérique. Ainsi, deux complexes entre le domaine Myb de TRF2 et I'ADN télomérique ont été étudiés par RMN et ont permis de mieux comprendre le rôle du domaine Myb en montrant sa grande spécificité pour I'ADN télômérique double brin. Ensuite quatre modèles de boucles D différents ont été étudiés par RMN afin d'en déterminer les structures tridimensionnelles.

The relationship between an organism’s genome, developmental stage, and environment is complex. The aim of the research presented herein was to provide experimental evidence to contribute to the annotation of the P. trichocarpa genome and to test two major hypotheses addressing the interaction between drought and time of day in A. thaliana and in two hybrid Populus clones. In order to generate data to address these aims, three separate experiments were undertaken. First, all members of the R2R3-MYB family of transcription factors in the P. trichocarpa genome were characterised by phylogenetic analysis and their transcript accumulation patterns across a range of tissues and organs were assessed using whole genome poplar microarrays. Results of this analysis indicated that expansion and diversification of the R2R3-MYB family may have contributed to phenotypic innovation in the Populus lineage. Second, drought-responsive transcriptome adjustments of two hybrid poplar clones, DN34 (P. deltoides X P. nigra) and NM6 (P. nigra X P. maxiomowiczii) were assessed for time-of-day and genotype dependent patterns. For each genotype, each of four time points was characterised by discrete sets of drought-responsive genes. Furthermore, while a number of genes were identified that were responsive to drought in both genotypes, a much larger number of genotype-dependent, drought-responsive transcriptome changes were detected. Finally, the drought-responsive transcriptome adjustments A. thaliana plants were assessed for time-of-day dependent accumulation patterns. Results of this analysis indicate that time-of-day-dependent differences in the drought response were manifest as changes of different magnitudes for a conserved set of genes across the four time points measured. These results emphasise the complex interplay of a plant’s genome, developmental stage, and environment in shaping the observed transcriptome.

The relationship between an organism’s genome, developmental stage, and environment is complex. The aim of the research presented herein was to provide experimental evidence to contribute to the annotation of the P. trichocarpa genome and to test two major hypotheses addressing the interaction between drought and time of day in A. thaliana and in two hybrid Populus clones. In order to generate data to address these aims, three separate experiments were undertaken. First, all members of the R2R3-MYB family of transcription factors in the P. trichocarpa genome were characterised by phylogenetic analysis and their transcript accumulation patterns across a range of tissues and organs were assessed using whole genome poplar microarrays. Results of this analysis indicated that expansion and diversification of the R2R3-MYB family may have contributed to phenotypic innovation in the Populus lineage. Second, drought-responsive transcriptome adjustments of two hybrid poplar clones, DN34 (P. deltoides X P. nigra) and NM6 (P. nigra X P. maxiomowiczii) were assessed for time-of-day and genotype dependent patterns. For each genotype, each of four time points was characterised by discrete sets of drought-responsive genes. Furthermore, while a number of genes were identified that were responsive to drought in both genotypes, a much larger number of genotype-dependent, drought-responsive transcriptome changes were detected. Finally, the drought-responsive transcriptome adjustments A. thaliana plants were assessed for time-of-day dependent accumulation patterns. Results of this analysis indicate that time-of-day-dependent differences in the drought response were manifest as changes of different magnitudes for a conserved set of genes across the four time points measured. These results emphasise the complex interplay of a plant’s genome, developmental stage, and environment in shaping the observed transcriptome.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 168 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

The aim of this work was to identify potential mRNA targets of PTGS triggered by viroid-derived small RNAs (vsRNAs) in PSTVd-infected tomato plants (S. lycopersicum L.). We selected 47 possible gene targets using data provided by Prof. Dr. Steger (Heinrich Heine Universität, Düsseldorf, Germany) - the list of 1633 possible target mRNAs from tomato based on vsRNA:mRNA duplex prediction. The vsRNA sequences were obtained by Illumina sequencing of small RNA libraries from healthy and PSTVd-infected tomato plants. By qRT-PCR analysis we identified 6 genes with significantly altered levels of mRNA in PSTVd-infected tomato plants: CUL1 (protein ubiquitination), ERF4 (transcription factor of abiotic stress signalling pathway), H/ACA1 (rRNA pseudouridylation), NPH3 (transcription factor of fototropic signalling pathway), Sl-MYB (transcription factor regulating leaf development) and TCP3 (transcription factor regulating leaf development). The binary vector pLV07 with inserted expression cassette containing coding sequence of Sl-MYB was prepared for experiments in ?leaf factory? system in N. benthamiana plants. Expression analyses in ?leaf factory? system after 1,5 DPI using qRT-PCR and RNA blots revealed strong inhibition of expression of Sl-MYB in leaf sectors infiltrated with severe PSTVd AS1 strain, while mild PSTVd QFA strain showed minimal change in expression comparing to control sectors. Moreover, the overexpression of Sl-MYB in leaf sectors resulted in development of necroses after 2,5-3 DPI, in presence of silencing suppressor p19 after 2 DPI. The development of necroses was largely inhibited in PSTVd AS1-infiltrated leaf sectors in comparison with PSTVd QFA- and control-infiltrated sectors.