Maspina (mammary serpin) foi identificada em 1994 como uma serpina (serine protease inhibitor) que apresenta atividade de supressão tumoral. Foi classificada como uma serpina devido à homologia na sequência de aminoácidos, porém, maspina não apresenta atividade de inibição de serina proteases. Entre os efeitos biológicos de maspina estão a modulação da adesão, a inibição do crescimento e a invasão tumoral, a inibição da angiogênese, o efeito pró-apoptótico e o controle da resposta ao stress oxidativo, propriedades que contribuem para supressão tumoral. Esta diversidade de funções se reflete nos inúmeros ligantes de maspina e na sua localização subcelular, já que é encontrada na membrana plasmática, no citoplasma, núcleo e mitocôndrias. A localização subcelular de maspina guarda importante relação com sua função, já que foi demonstrado que sua localização nuclear está correlacionada com bom prognóstico em diversos tumores e seu efeito supressor de tumor foi observado somente quando maspina está localizada no núcleo. Entre os ligantes de maspina estão a HDAC1, IRF6, GST, HSP90 e HSP70, β1 integrina, uPAR e colágeno tipo I e III. O mecanismo molecular envolvido na regulação dessas atividades não foi elucidado, e até o momento, somente um gene e uma proteína de maspina foram descritos, desta forma alterações pós-traducionais devem estar envolvidas na regulação dessas atividades. Com objetivo de verificar se há modificações pós-traducionais em maspina, utilizamos células MCF10A, que expressam grande quantidade dessa proteína, e submetemos seu extrato proteico à separação por gel bidimensional seguido de western blot. Identificamos quatro formas de maspina com a mesma massa molecular (42kDa), mas pontos isoelétricos distintos. Três destas formas são sensíveis ao tratamento com fosfatase ácida, o que sugere que estas sejam fosforiladas. Utilizamos ainda peroxidovanadato de sódio, um potente inibidor de tirosina fosfatase para investigar o papel da fosforilação de maspina em resíduos de tirosina. Através de western blot e imunofluorescência, observamos que o tratamento das células com o inibidor resultou no aumento dos níveis celulares de maspina assim como no seu acúmulo no citoplasma. Deste modo, concluímos que existem três diferentes fosfoformas de maspina em células MCF10A e ainda a inibição de tirosinas fosfatases aumentam os níveis de maspina e resultam no acúmulo da proteína no citoplasma. Esses resultados sugerem que a fosforilação pode estar envolvida na localização subcelular de maspina e na regulação dos seus níveis proteicos na célula. / Maspin (mammary serpin) was identified in 1994 as a serpin (serine protease inhibitor) which presents tumor suppressor activity. It was classified as a serpin due to its homology in amino acids sequence; however, maspin doesn\'t exhibit serine protease inhibition activity. Among maspin biological effects are modulation of cell adhesion, inhibition of tumor growth, invasion and angiogenesis, a pro-apoptotic effect and control of oxidative stress response, properties which contribute to tumor suppression. This functional diversity reflects maspin numerous ligands and its subcellular localization, since it is found on the plasma membrane, in the cytoplasm, nucleus and in mitochondria. Maspin subcellular localization is closely related to its function, as its nuclear localization correlates with good prognostic in several tumors and maspin tumor suppressor activity is only observed when it is located in the nucleus. Among maspin ligands are histone H1 deacetylase, IRF6, GST, HSP90 e HSP70, β1 integrin, uPAR and type I and III collagen. The molecular mechanisms involved in the regulation of maspin biological activities are poorly understood. So far, only one gene and one protein have been assigned to maspin, so posttranslational modification should be involved. In order to verify posttranslational modification in maspin, we utilized MCF10A cells, which express great amount of this protein, and we submitted its proteic extract to 2D-SDS-PAGE followed by western blot. We identified four maspin forms with the same molecular mass (42kDa), but different isoelectric point. Three of these forms are sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated maspin forms. We also utilized sodium peroxovanadate, a potent tyrosine phosphatase inhibitor to investigate the role of maspin tyrosine phosphorylation. Through western blot and immunofluorescence analyses, we observed that cell treatment resulted in increase in maspin cellular levels as well as its cytoplasmic accumulation. Thus, we concluded that there are three diferente maspin phosphoforms in MCF10A cells and yet tyrosine phosphatase inhibition increases maspin levels and results in accumulation of the protein in the cytoplasm. These data suggest that phosphorylation may be involved in maspin subcellular localization and regulation of its protein levels in the cell.

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder that affects 1:12000 newborns. It is characterized by mental retardation, delayed major motor and cognitive milestones, seizures, absence of speech and excessive laughter. The majority of AS cases arise from deletions or mutations of UBE3A gene located on the chromosome 15q11-13. UBE3A codes for E3-ubiquitin ligase that target specific proteins for degradation. To date, a wide variety of Ube3a substrates has been identified. The accumulation of Ube3a-dependent proteins and their effect on the multitude of signal transduction pathways are` considered the main cause of the AS pathology. While the majority of research has been directed towards target identifications, the overall role of Ube3a in activity-dependent synaptic plasticity has been greatly overlooked. The present work is designed to fill some of these knowledge gaps. Chapter 2 is focused on the activity-dependent aspect of Ube3a expression following neuronal stimulation in vivo and in vitro. We examined total Ube3a expression followed by KCl depolarization in neuronal primary culture. By utilizing a subcellular fractionation technique, we were able to determine which cellular pools are responsive to the depolarization. Next, a fear conditioning paradigm (FC) was used to activate neurons in the paternal Ube3a-YFP reporter mouse brain. This mouse model allowed us to resolve spatial and temporal alterations of the maternal and the paternal Ube3a in hippocampus and cortex followed by FC. In accordance to KCl depolarization results, we observed alterations in Ube3a protein but at later time points. Furthermore, we investigated if the absence of activity-dependent Ube3a changes has any effect on learning and memory kinase activation. We utilized KCl and FC to determine synaptic activity-induced ERK 1/2 phosphorylation in acute hippocampal slices and in CA1 area of hippocampus of wild type (Ube3a m+/p+) and Ube3a deficient mice (Ube3a m-/p+). We demonstrated that Ube3a loss leads to impaired activity-dependent ERK 1/2 phosphorylation. It has been established that Ube3a m-/p+ mice have a profound deficit in LTP, implying the importance of this ligase in excitatory synaptic transmission. The abnormal LTP could be partially explained by an aberrant CaMKII function, decreased activity-dependent ERK 1/2 phosphorylation and reduced phosphatase activity. These proteins have also been implicated in another form of synaptic plasticity such as long-term depression (LTD). Chapter 3, we investigated the contribution of Ube3a to NMDAR - dependent and - independent LTD. Our data showed that Ube3a m-/p+ P21-30 animals exhibit the impairments in both forms of LTD. Next, we focused on elucidating molecular mechanism underlying the reduced mGluR1/5-LTD. We discovered that mGluR1/5 kinase activation such as ERK, mTOR and p38 is not affected by Ube3a loss. In concordance with previous work, we detected increased Arc expression together with abnormal AMPAR distribution in the Ube3a m-/p+ hippocampus. Surprisingly, the mGluR1/5 induced GluR2 trafficking was normal. Our findings infer that elevated Arc levels together with the increased internalization of AMPAR may result in compromised basal state of the synapses leading to a more depression-like state in Ube3a m-/p+ mice. Evidence points that loss of Ube3a produces alterations in a variety of activity-dependent signal transduction cascades that may ultimately result in impaired synaptic plasticity and cognition. Similar to AS, abnormal molecular and behavioral phenotypes have already been observed in other mouse models of human mental retardation such as Fragile X Mental Retardation Syndrome (FXS). Chapter 4 is set to explore if any correlation can be found in between these neurodevelopmental disorders. Analysis of crude synaptoneurosomes of adult Fmr1 KO mice revealed a significant reduction in Ube3a protein. Additionally, a blunted translation of Ube3a in response to mGluR1/5 stimulation was observed. However, we didn't find any evidence of direct interaction between Ube3a mRNA and Fragile X Mental Retardation Protein (FMRP). To examine if some of the pathology seen in Fmr1 KO mice is due to Ube3a downregulation, we performed a rescue experiment by increasing overall levels of Ube3a in hippocampus of FRMP deficient mice. An exhaustive battery of behavioral testing indicated that alterations of Ube3a expression impacted only associative fear conditioning. In summary, the present work has attempted to answer some of the fundamental questions about Ube3a and its role in synaptic plasticity. We have demonstrated that Ube3a expression is modulated by synaptic activation and its activity-dependent alterations are essential for normal brain functioning. Additionally, our data suggest that Ube3a is not only significant for the synaptic excitation but also crucial for the synaptic depression. Finally, our findings indicate that the alteration of Ube3a expression may contribute to the cognitive phenotypes in other neurodevelopmental disorders such as FXS suggesting an advantage of exploring Ube3a function outside the AS research.

Prostate cancer is one of the leading causes of death among males in the United States. In this study, we hypothesized that an activated PKC-iota-dependent anti-apoptotic pathway, drives the cell cycle proliferation and survival of prostate cancer cells. We investigated the role of atypical PKC-iota (PKC-ι) in androgen- independent prostate DU-145 carcinoma, androgen-dependent prostate LNCaP carcinoma compared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent activating kinase (CAK/Cdk7) in androgen-dependent, RWPE-1 and LNCaP cells but not in androgen-independent DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2. In addition, PKC-ι siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis. In DU-145 cells, PKC-ι is anti-apoptotic and still required for cell survival. Treatment with PKC-ι siRNA blocked an increase in cell number, and inhibited G1/S transition. In addition to cell cycle arrest, both RWPE-1 cells and DU-145 cells underwent apoptosis via mitochondria dysfunction and activating apoptosis cascades such as release of cytochrome c, activation of caspase-7, and poly-(ADP-ribose) polymerase (PARP) cleavage. Mechanistic pathways involving aPKCs in the NF-κB survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFα whereas LNCaP cells are sensitive to TNFα treatment and undergo apoptosis. In DU-145 cells, TNFα induced PKC-ι activation of IκB kinase, IKKα/ß, while in RWPE-1 cells, PKC-ζ activates IKKα/ß. Both RWPE-1 and DU-145 show degradation of IκBα allowing NF-κB/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKα/ß was not observed, although there have been reports that LNCaP cells weakly activate IKKα and have NF-κB activation. In vivo kinase assay demonstrates that PKC-ι is the substrate of IKKα/ß. A putative PKC-ι inhibitor (ICA-1) inhibited activation of IKKα/ß in vivo. Hence, PKC-ι is an antiapoptotic protein and this suggests that anti-PKC-ι therapy may be a viable option for prostate carcinoma cells.

The microtubule-associated protein tau is known for its ability to bind to and stabilize microtubules and for its ability to nucleate microtubule assembly. In neurodegenerative tauopathies such as Alzheimer's disease, tau becomes hyperphosphorylated and loses the capacity for microtubule binding, possibly contributing to microtubule destabilization and axonal degeneration. However, evidence now indicates that soluble forms of hyperphosphorylated tau might have a toxic gain of function linked to abnormal signal transduction and cell cycle events in normally post-mitotic neurons. In support of this hypothesis, tau has been found to associate with numerous signaling proteins such as tyrosine kinases, adaptor proteins, and scaffold proteins. During early brain development, fetal tau is also more phosphorylated than tau in the adult brain and weakly binds microtubules, suggesting tau has functions in addition to microtubule stabilization. The aim of this dissertation research is to investigate the possible role of tau in neuronal signaling, using tau-expressing and tau-depleted cell lines. Here, we provide evidence that during growth factor stimulation of neuronal cells, tau functions in advance of the neurite elongation stage. Tau is required for neurite initiation in a manner that does not require its microtubule binding function, and in addition, tau potentiates AP-1 transcription factor activation in response to nerve growth factor (NGF). The effect of tau on AP-1 activation is mediated through the enhanced activation of extracellular signal-regulated kinase (ERK), in response to both NGF and epidermal growth factor (EGF). We show that phosphorylation of tau at Thr231 also occurs in response to NGF and is required for tau to impact on ERK signaling, whereas the ability of tau to bind to microtubules is not required. Together, these findings indicate a new functional role for tau in neuronal signal transduction and have implications for tau function during early brain development and in neurodegenerative disease.

The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts various neuromodulatory functions in mammalian brain. Enhancement of synaptic activity, mediation of chronic inflammatory and neuropathic pain, and neuroprotection in cerebral ischemia reperfusion injury constitute some of the exemplary functions of PACAP. However, it remains unclear whether PACAP signaling can directly influence the function of critical voltage-gated ion channels, which could profoundly alter the excitability of neurons. Voltage-gated K+ (Kv) channels are critical regulators of neuronal excitability. The major Kv channel in the dendrites of mammalian neurons, Kv4.2, contributes most of the fast-activating and rapidly-inactivating K+ currents (IA), and is a key regulator of dendritic excitability, as well as modulation of synaptic inputs. In addition, the major somatic Kv channel Kv2.1 that contributes the bulk of slow-activating and non-inactivating K+ currents (IK), acts as an integrator of neuronal inputs and limits high frequency firing in neurons. As such, it provides homeostatic control of excitability under hyperexcitable and ischemic conditions. Both these Kv channels are known to undergo extensive post-translational modifications mainly by phosphorylation that alters their localization and biophysical properties. PACAP can activate its specific receptor PAC1 that could result in downstream activation of various kinases including protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2). Therefore, I hypothesize that PACAP activation of PAC1 receptor can cause phosphorylation-dependent modulation of somatodendritic Kv4.2 and Kv2.1 channels, resulting in altered neuronal excitability. First, I identified the various PAC1 receptor isoforms expressed in rat and mouse brain and elucidated that their activation by PACAP caused downstream PKA- and PKC-dependent signaling pathways, ultimately converging on ERK1/2 activation. Further, PACAP caused reduction in IA that was mediated by phosphorylation-dependent internalization of the channel protein from the plasma membrane. These effects were mediated by direct phosphorylation of the channel by ERK1/2 at the cytoplasmic C-terminus of the channel. Although PACAP did not significantly alter the voltage-dependence of Kv4.2 channel activation/inactivation, I observed distinct ERK1/2- and PKA-dependent changes in the extent and kinetics of channel inactivation. Next, I observed that PACAP induced dephosphorylation of the Kv2.1 channel in CHN that was mediated by protein phosphatase 2A (PP2A), and was dependent on PKC activation but was independent of the effects of PACAP on Kv4.2 currents. Rapid but reversible dephosphorylation of Kv2.1 was also observed following induction of ischemia in neurons by oxygen-glucose deprivation (OGD). PACAP prolonged the dephosphorylation of Kv2.1 following in vitro ischemia-reperfusion and also reduced neuronal death. My results therefore suggest a novel PACAP/PAC1-PKC-PP2A-Kv2.1 signaling axis that provides neuroprotection during ischemia reperfusion injury. In summary, my results suggest that PACAP can induce direct phosphorylation-dependent modulation of the Kv4.2 and Kv2.1 channel localization and function in mammalian brain neurons. The effect of PACAP on these two critical somatodendritic ion channels occurs via distinct signaling - convergent PKA-PKC-ERK-mediated phosphorylation of Kv4.2 channel, and PKC-PP2A-mediated dephosphorylation of the Kv2.1 channel. Such distinct modulations of these ion channels are presumably responsible for the multifarious roles of PACAP in the central nervous system.

Les mitochondries sont des organelles qui jouent un rôle clé dans le métabolisme cellulaire en centralisant la production d'ATP à partir de nombreux substrats via la phosphorylation oxydative (OXPHOS). Les réactions enzymatiques impliquées dans ce processus régulent la prolifération, la différenciation, l'activation et l'auto renouvellement cellulaire. Le but de mon travail a été d'identifier le rôle de l'OXPHOS dans l'hématopoïèse et les mécanismes d'adaptation métabolique des cellules sanguines de la moelle, des lymphocytes B et des thymocytes à la dysfonction mitochondriale. L'atout majeur de cette étude est la génération de deux modèles murins déficients pour les protéines mitochondriales AIF ou NDUFS4 dans le système hématopoïétique. Nous avons observé que l'absence de ces protéines entraine des dysfonctions de l'OXPHOS sévère (AIF KO) ou modérée (NDUFS4 KO), entrainant des anomalies dans le développement hématopoïétique. Dans les deux modèles, en réponse au stress métabolique induit par la dysfonction de l'OXPHOS, les cellules de moelle activent la glycolyse anaérobie et la biogenèse mitochondriale tandis que les thymocytes favorisent l'assimilation et la dégradation des acides gras. Cette étude multiparamétrique, incluant des approches in vivo, ex vivo et in vitro, souligne l'importance de l'OXPHOS et du métabolisme mitochondrial dans le développement hématopoïétique. / By integrating different biochemical pathways and generating energy in form of ATP, through the electron transfer associated to oxidative phosphorylation (OXPHOS), mitochondria play a key role in cellular metabolism. In the hematopoietic cells, the mitochondrial metabolism appears implicated in proliferation, differentiation, activation and self-renewal regulation. In this context, the aim of my PhD work was to unravel the response of bone marrow (BM) cells, B-cells and thymocytes to OXPHOS dysfunction. To do that, we have developed two original hematopoietic cell-specific murine models deficient in the mitochondrial proteins AIF or NDUFS4. Severe (AIF KO) or moderate (NDUFS4 KO) OXPHOS dysfunction leads to pleiotropic consequences on hematopoietic development, including pancytopenia, BM aplasia, alterations in the development of the B-cell and erythroid lineages and T-cell developmental blockade at the immature stage. Strikingly, in response to OXPHOS dysfunction, BM cells stimulate anaerobic glycolysis and mitochondrial biogenesis, whereas thymocytes favor the assimilation and degradation of fatty acids. Overall my work, which included in vivo, ex vivo and in vitro approaches, underlines the relevance of OXPHOS and mitochondrial metabolism in the development of the hematopoietic cells.

Ce travail concerne la fonctionnalisation des esters méthyliques d'acides gras (EMAG) dans le but de valoriser les ressources halieutiques marocaines, en particulier les huiles de poisson dont ils sont les principaux constituants. Pour cela, des réactions d'hydroboration, d'hydrosilylation, d'hydrogermylation et de phosphorylation ont été réalisées dans les premier, deuxième et troisième chapitres et ont permis d'accéder à une grande variété de composés présentant des motifs EMAG diversement substitués. Dans les deux derniers chapitres, nous avons développé des applications de ces dérivés. Ainsi, des polyesters et des copolymères renfermant un élément du groupe 14 et un motif de type EMAG ont été préparés et caractérisés. Parallèlement, la décomposition thermique en solution d'aminogermylènes nous a permis d'accéder à des nanoparticules de germanium.

Le but de ce travail était de développer des sondes oligonucléotidiques liées à des marqueurs fluorescents émettant des signaux modifiés lors de l'hybridation avec des séquences complémentaires. Des conjugués ODNs-cyanines originaux ont été développés. L'influence de différents paramètres sur le signal fluorescent émis par la sonde ont été étudiés : position de liaisons des marqueurs à l'ODN (5'- ou internucléotidique), structure des cyanines, stéréochimie. Un nouveau réactif de phosphorylation des ODNs a été mis au point et utilisé pour la synthèse des conjugués marqués en position 5'.<br> D'autres conjugués comportant à leur extrémité 5' deux marqueurs identiques possédant des propriétés intercalantes : thiazole orange, pyrène ou pérylène ont été obtenus. Les interactions entre les deux marqueurs varient lors de l?hybridation de la sonde avec la séquence complémentaire, induisant une modification du signal fluorescent dont la nature et l'intensité dépendent du marqueur considéré