The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.

Nucleosomes, the primary unit of chromatin structure, are positioned either statistically or specifically. The statistical positioning denotes the arbitrary positioning of nucleosomes on DNA agreeing with the nucleosome’s broad coverage of the genome—however, there is evidence that nucleosomes are also positioned specifically at controlled positions. DNA sequences determine the specific nucleosome positions, and the presence or depletion of nucleosomes affects the availability of the DNA region to other proteins. The DNA sequences of H2A and H2A.Z nucleosomes in Drosophila were analysed in search of nucleosome positioning patterns. Dinucleotide patterns with 10 bp periodicity were identified from the DNA sequences of H2A nucleosomes. Compared with the yeast patterns, the Drosophila patterns had the same periodicity but different dinucleotides near the dyad, which was related to the different H3 structure between them. The nucleosome positioning patterns from the H2A.Z nucleosomes implied the specific histone-DNA interaction as a result of the deviations of the patterns where the different amino acids of H2A and H2A.Z interact with the DNA. The Ly49 gene cluster was selected as a model system to study the interplay between nucleosomes and transcription factors. Ly49 proteins, the surface receptors on NK cells, display variegated expression patterns, and the bidirectional promoter Pro-1 is known as a key determinant of the stochastic expression of each Ly49 gene. The systematic analysis of nucleosome positions based on the genome sequences in the Ly49 gene cluster revealed that the repressing Pro-1 reverse promoters are open, while the activating forward Pro-1 promoters were covered by nucleosomes. Furthermore, specific nucleosome positions covered transcription factor binding sites. The covered factor binding sites were further examined by their periodic appearances on the nucleosome-covered sequences, which revealed the accessibility to the sites. The sequence analysis predicted that the regulation by the transcription factor AML-1 would be sensitive to the nucleosome coverage; the prediction was confirmed by cell line experiments. The 10 bp periodic nucleosome positioning patterns interact with histones specifically. The long nucleosome positioning patterns coexist with the short sequence motifs for transcription factor binding sites adding another layer of the control to the transcriptional regulation.

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las. / Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent.

O presente trabalho tem como objetivo caracterizar três agrupamentos de genes da alfa proteobactéria Caulobacter crescentus envolvidos na resposta a metais e antibióticos. Analisamos o agrupamento composto pelos genes CC1637-CC1640, que contém um sistema BlaR/BlaI de transdução de sinal, e realizamos uma análise comparativa de dois sistemas de efluxo da família RND composto pelos genes CC2720-CC2725 e CC2388-CC2390 que estariam envolvidos na resposta a metais cádmio e zinco. Mutantes simples e duplos com deleção em fase foram obtidos, e o estudo da atividade promotora foi realizado através de ensaio de <font face=\"Symbol\">b-galactosidase utilizando gene repórter lacZ . Ensaios de RT-PCR e atividade <font face=\"Symbol\">b-galactosidase mostraram que o gene CC1638 provavelmente não possui promotor próprio e que os genes CC1637- CC1640 podem consituir um operon. A atividade promotora do gene CC1640 não responde a H2O2, Cd2+ e Zn2+, mas a linhagem <font face=\"Symbol\">DCC1640 apresentou baixa viabilidade na presença de Cd2+. As linhagens <font face=\"Symbol\">DCC1637 e <font face=\"Symbol\">DCC1638 mostraram sensibilidade a t-butil-hidroperóxido. A linhagem <font face=\"Symbol\">DCC1640 mostrou-se sensível aos antibióticos CTX e PPT, e ensaios de <font face=\"Symbol\">b-galactosidase em placa e em meio liquido mostraram indução da expressão pelos antibióticos CTX e CFE. Observamos uma auto-regulação do operon pela proteína codificada pelo gene CC1640 (BlaI), confirmado por ensaios de EMSA. A presença de BlaR inibe a ligação da proteína BlaI ao promotor, sugerindo que ambas BlaI/BlaR regulem em conjunto o promotor do gene CC1640. Análise in silico do consenso TTACGNNCGTAA localizado no promotor de CC1640 identificou esta sequência na região promotora de outros genes. A análise da expressão relativa sugere que os genes CC1568, CC1230 e CC2661 são regulados pela proteína BlaI, sugerindo que BlaI regula a expressão de outros genes possivelmente envolvidos na resposta a antibióticos <font face=\"Symbol\">b-lactâmicos. Ensaios de atividade <font face=\"Symbol\">b-galactosidase da região intergênica CC2720-CC2721 mostraram que esta não possui atividade promotora, e análise por RT-PCR confirmou que os genes CC2720-CC2721 são co-transcritos e que fazem parte do operon CC2720-CC2725. A expressão do operon mostrou indução significativa na presença de Cd2+, moderada indução na presença de Zn2+ e Co2+, e pouca indução na presença de Ni2+. A expressão do operon CC2388-CC2390 é altamente induzida na presença de níquel e cobalto, não é induzida por cádmio e moderadamente induzida por zinco. A linhagem <font face=\"Symbol\">DCC2724 não é sensível a zinco, cobalto ou níquel. A linhagem <font face=\"Symbol\">DCC2390 é sensível a cobalto, pouco sensível a níquel e não sensível a zinco, e ambas as linhagens foram sensíveis a cádmio. A obtenção do duplo mutante, assim como sua complementação, foram realizadas, e os resultados sugerem que se trata de dois sistemas de efluxo com diferentes respostas a metal. / The aim of this work is to characterize three clusters of genes from the alpha proteobacterium Caulobacter crescentus involved in metal and antibiotics response. We analyzed the cluster comprising genes CC1637-CC1640, which contains a BlaR/BlaI signal transduction system, and we performed a comparative analysis with two RND efflux systems consisting on genes CC2720-CC2725 and CC2388-CC2390, which are probably involved in cadmium and zinc response. Mutant strains for one or two of these genes were obtained, and the study of promoter activity was performed by <font face=\"Symbol\">b-galactosidase activity assays using lacZ as reporter gene. RT-PCR and <font face=\"Symbol\">b-galactosidase activity assays revealed that the CC1638 gene probably does not possess an exclusive promoter, and that the genes CC1637-CC1640 may constitute an operon. The promoter of CC1640 does not respond to H2O2, Cd2+ and Zn2+, but the <font face=\"Symbol\">DCC1640 strain presented low viability in the presence of Cd2+. The <font face=\"Symbol\">DCC1637 and <font face=\"Symbol\">DCC1638 strains showed sensitivity to t-butyl-hydroperoxide. The <font face=\"Symbol\">DCC1640 strain showed sensitivity to the antibiotics CTX and PPT, and <font face=\"Symbol\">b-galactosidase activity assays performed both on plates and liquid medium showed induction of the expression by the presence of antibiotics CTX and CFE. We observed auto-regulation of the operon by the protein encoded by the CC1640 gene (BlaI), which was confirmed by EMSA assays. The presence of BlaR inhibits the binding of the BlaI protein to the promoter, suggesting that both BlaI/BlaR regulate the CC1640 gene promoter. In silico analyses for the TTACGNNCGTAA consensus, located on CC1640 promoter, identified this sequence in promoter regions of other genes. Relative expression analyses indicate that the genes CC1568, CC1230 and CC2661 are regulated by the BlaI protein, suggesting that BlaI regulates the expression of other genes, possibly involved in <font face=\"Symbol\">b-lactamic antibiotics response. <font face=\"Symbol\">b-galactosidase activity assays of the intergenic region CC2720-CC2721 showed that it does not possess promoter activity, and a RT-PCR analysis confirmed that the genes CC2720-CC2721 are co-transcribed and belong to the CC2720-CC2725 operon. The expression of the operon showed significant induction in the presence of Cd2+, moderate induction in the presence of Zn2+ and Co2+, and a slight induction in the presence of Ni2+. The expression of the CC2388-CC2390 operon is highly induced in the presence of nickel and cobalt, not induced by cadmium and moderately induced by zinc. The <font face=\"Symbol\">DCC2724 strain is not sensitive to zinc, cobalt or nickel. The <font face=\"Symbol\">DCC2390 strain is sensitive to cobalt, slightly sensitive to nickel and not sensitive to zinc, and both strains are sensitive to cadmium. A double mutant was constructed, as well as a complemented strain, and results suggest that these are two efflux systems with distinct metal responses.

Atualmente, as cepas de S. cerevisiae utilizadas na produção industrial de álcool combustível no Brasil são leveduras diplóides que metabolizam a sacarose através da sua hidrolise extracelular pela invertase periplasmática, seguida pelo transporte para o interior da célula das moléculas de glicose e frutose formadas, e posterior metabolização pela glicólise. Neste trabalho, utilizando técnicas de engenharia genética e posteriores cruzamentos, foram desenvolvidas cepas diplóides de S. cerevisiae capazes de consumir este açúcar por meio da sua captação direta pelo transportador codificado pelo gene AGT1, e hidrólise intracelular pela invertase citoplasmática codificada por uma versão modificada do gene SUC2. Estas modificações permitiram alcançar um rendimento 10% maior na produção de etanol, além de aumentar a velocidade de consumo da sacarose e diminuir a quantidade de açúcares residuais ao final do processo. Estes resultados abrem novos horizontes para tornar a produção de etanol no Brasil mais eficiente. / Currently, the S. cerevisiae strains used in industrial production of fuel alcohol in Brazil are diploid yeasts that of metabolize sucrose through its extracellular hydrolysis by the periplasmic invertase, followed by the transport of the formed glucose and fructose into the cells, and further metabolism through glycolysis. In this study, utilizing genetic engineering techniques and further crosses, diploid strains of S. cerevisiae were developed that are able to consume this sugar through its direct uptake by the transporter encoded by the AGT1 gene, and intracellular hydrolysis by the cytoplasmic invertase encoded by a modified version of the SUC2 gene. These modifications allowed achieving a 10% higher yield in the production of ethanol, besides increasing the sucrose consumption rate and decreasing the amount of residual sugars at the end of the process. These results open new horizons to turn ethanol production in Brazil more efficient.

Saccharomyces cerevisiae é o organismo de escolha para produção de etanol combustível, apresentando-se adaptadas ao ambiente hostil das dornas de fermentação. Adaptações como floculação, formação de espuma e de biofilme são características fenotípicas indesejáveis ao processo brasileiro. Por isso, a pronta identificação destas características faz-se necessária no intuito de minimizar perdas de rendimento e diminuir o custo da produção. Sabe-se que proteínas codificadas pela família de genes FLO estão relacionadas a estes fenótipos, os quais são regulados pela proteína Flo8p, a qual possui papel fundamental na apresentação destas características fenotípicas indesejáveis. Desta forma, o presente trabalho teve como objetivo analisar o fenótipo e o comportamento metabólico, relacionando-os com a regulação via Flo8p. Os resultados obtidos revelam o papel fundamental de Flo8p na regulação positiva destes fenótipos em linhagem isolada do ambiente industrial (FT02). Verificou-se uma forte tendência da participação do gene FLO11 na formação de espuma, via Flo8p. Além disso, através da deleção do FLO8 da linhagem FT02, observou-se a influência positiva de Flo8p na floculação e hidrofobicidade celular, filamentação e poder invasivo. / Saccharomyces cerevisiae is the chosen organism for fuel ethanol production, presenting adapted to the hostile environment of the fermentation vats. Adaptations like flocculation, d foam formation and biofilm are undesirable phenotypes for the Brazilian process. Hence, the early identification of these characteristics is necessary to minimize yield losses and lower the production cost. Proteins codified by FLO gene family are related with these phenotypes, which are positively regulated by Flo8p protein, showing fundamental role in the expression of the undesirable phenotypes. So, the present work aimed to analyze the phenotypes and the metabolic behavior of an industrial strain isolated directly from Brazilian ethanol production process (FT02), relating them with the Flo8p regulation. Our results showed that the Flo8p plays a fundamental role in the regulation of these characteristics in the industrial strain FT02. We also verified a strong tendency of the FLO11 gene participation in the foam formation, by Flo8p. Moreover, by FLO8 deletion in FT02 strain, we could identify the positive influences of the Flo8p in flocculation and cellular hydrophobicity, filamentation and invasive growth.

This thesis consists of two related studies directed at aspects of M. tuberculosis biology. The first focuses on deciphering gene-regulatory circuits from ChIP-seq data, and the second focuses on alternative strategies for combatting drug-resistant infections. The first study describes Binding Resolution Amplifier and Cooperative Interaction Locator (BRACIL), a post-peak-caller computational method that predicts transcription-factor (TF) binding sites with high-resolution as well as cooperative TF interactions derived from ChIP-seq data. BRACIL integrates ChIP-seq coverage with motif discovery from a signal-processing perspective and uses a blind-deconvolution algorithm that predicts binding-site locations and magnitudes. BRACIL also explicitly considers a second-order signal, represented by DNA fragments with two sites bound simultaneously, and uses it to predict cooperative interaction. Cooperative interaction indicates that the binding to a first site influences the probability of binding to a second site. This method estimates the probability of a binding configuration from the ChIP-seq coverage and performs a likelihood ratio test to predict cooperative interaction. As a proof of principle, I validated this method using M. tuberculosis transcription factor DosR. The second study focuses on strategies to fight antibiotic resistance. In particular, recent reports have shown the existence of treatment conditions (called "antiR") that select against drug-resistant strains. I used a mathematical model of infection dynamics and immunity to simulate the growth of resistant and sensitive pathogens under different treatment conditions (no drugs, antibiotic present, and antiR), and could show how a precisely timed combination of treatments can defeat resistant strains. This analysis suggested that a time- scheduled, multi-treatment therapy could lead to complete elimination of both sensitive and resistant strains. Also, my results indicated that the time necessary to turn a resistant infection into a sensitive one ("tclear") depends on the experimentally measurable rates of pathogen division, growth and plasmid loss. Additionally, I estimated tclear for a specific case, using available empirical data, and found that resistance may be lost up to 15 times faster under antiR treatment as compared to a no-treatment regime. Finally, an extension of these findings to population models provides quantitative support for therapeutic plans to clear antibiotic-resistant infections, including novel drug-cycling strategies.

Cannabinoids and cannabinoid receptors have been shown to play important roles in immune regulation particularly as modulators of anti-inflammatory cytokines and antibody production. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB2), which is robustly expressed in B cells. Utilizing a combination of bioinformatics, 5' RACE, real time RT-qPCR, and reporter assays, we showed that human B cells from peripheral blood mononuclear cells (PBMC) expressed one CB2 transcript while mouse B cells from spleen express three CB2 transcripts. Alignment of the sequenced B cell RACE products to either the mouse or human genome, along with the GenBank mRNA sequences, revealed that the transcripts isolated in this study contained previously unidentified transcriptional start sites (TSSs). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB2 exon 1 and 2 transcripts showed a significant increase of promoter activity. Bioinformatics analysis for cis-sequences in the promoter regions identified DNA binding sites for NF-kB, STAT6, and Elk1 transcription factors activated by LPS, IL-4 and anti-CD40. Regarding variations in CB2 transcript expression among the immune cell subtypes, RACE analysis showed that the exon 1b transcript is seen in B cells but not in T cells, dendritic cells or macrophages. Furthermore, RT-qPCR showed variations in transcript expression during B cell development as well as in resting versus LPS or IL-4/anti-CD40 stimulated B cells. The exon 1a transcript was predominant in pre-, immature and resting B cells whereas the exon 1b and 2 transcripts were enhanced in mature and activated B cells. These data showed for the first time that human B cells use one TSS for CB2 expression while mouse B cells use multiple TSSs for the expression of three CB2 transcripts, in which the expression of the individual transcript is related to immune cell type and/or cell activation state. Additionally, this is the first report in mouse B cells defining TSSs that are in genomic areas with promoter activity thus suggesting the location of two promoter regions. Defining the CB2 transcript expression during various stages of B cell activation provide clues to therapeutic methods.

La transition florale résulte d’un jeu complexe d’interactions entre des signaux endogènes et environnementaux. Les feuilles jouent un rôle crucial dans ce processus en percevant les changements associés à la lumière et en produisant les photosynthétats qui participant à la signalisation de la floraison. Toutefois, notre connaissance des changements se produisant dans les feuilles lors de la transition florale reste limitée. Nous avons caractérisé les événements morphologiques, moléculaires et transcriptionnels en relation avec la floraison florale dans les feuilles matures chez Arabidopsis, en exploitant un système de transfert de conditions en jours courts vers des jours longs, transfert qui permet d’induire et synchroniser la floraison. Nous avons identifié la fenêtre temporelle de la transition florale, mesuré la croissance foliaire, et observé un accroissement de la ploïdie au cours du processus. Par une approche de RNA-seq, nous avons étudié la dynamique transcriptionnelle des réseaux de gènes dans la feuille, et comparé avec des données dans la racine et le méristème pour avoir une vue plus intégrée de la floraison dans la plante. De plus, nous avons analysé le mode d’action de LHP1 (LIKE HETEROPROTEIN 1), une sous unité du complexe PRC1, en exploitant des lignées transgéniques avec des modifications conditionnelles du dosage de LHP1 et en analysant les effets sur la chromatine et la transcription des gènes impliqués dans la floraison. Une modulation courte du dosage en LHP1 modifie le dépôt des marques H3K27me3 et H3K4me3, démontrant une interaction fonctionnelle entre LHP1 et le complexe PRC2, et suggérant aussi un nouveau rôle dans la formation de régions chromatiniennes de type bivalent. Enfin, étant donné le rôle clé de l’organisation nucléaire dans la régulation génique, nous avons recherché et identifié des déterminants de l’architecture nucléaire en utilisant de nouveaux outils de statistiques spatiales. / The transition to flowering results from a complex interplay between endogenous and environmental cues. The leaves play a key role in this process, by perceiving the light changes and producing photosynthates, which participate to the floral signalling. However, our knowledge on the changes occurring in leaves during floral transition is still limited. We characterised the morphological, molecular and transcriptional events related to floral transition in mature leaves in Arabidopsis, using a short-day to long-day shift to induce a synchronized flowering. We identified the temporal window of the floral transition, monitored the leaf growth and observed an increase in their ploidy level during the process. By RNA-seq we studied the transcriptional dynamics of the leaf gene network, and compared with events occurring in roots and meristems to get an integrated view of floral transition in the whole plant. Furthermore, we investigated the mode of action of LIKE HETEROPROTEIN 1 (LHP1), a PRC1 subunit, by exploiting transgenic lines with conditional alterations of LHP1 dosage and analysing the effects on chromatin and transcription of flowering genes. A short-term modulation of LHP1 dosage altered the deposition of H3K27me3 and H3K4me3, showing a functional interaction between LHP1 and PRC2, and also suggesting a new role in the formation of bivalent chromatin regions. Finally, since nuclear organisation plays a key role in gene regulation, we searched and identified determinants of the nuclear architecture by using innovative spatial statistical tools.