Homeostatic and circadian contribution to EEG and molecular state variables of sleep regulation.

STUDY OBJECTIVES: Besides their well-established role in circadian rhythms, our findings that the forebrain expression of the clock-genes Per2 and Dbp increases and decreases, respectively, in relation to time spent awake suggest they also play a role in the homeostatic aspect of sleep regulation. Here, we determined whether time of day modulates the effects of elevated sleep pressure on clock-gene expression. Time of day effects were assessed also for recognized electrophysiological (EEG delta power) and molecular (Homer1a) markers of sleep homeostasis. DESIGN: EEG and qPCR data were obtained for baseline and recovery from 6-h sleep deprivation starting at ZT0, -6, -12, or -18. SETTING: Mouse sleep laboratory. PARTICIPANTS: Male mice. INTERVENTIONS: Sleep deprivation. RESULTS: The sleep-deprivation induced changes in Per2 and Dbp expression importantly varied with time of day, such that Per2 could even decrease during sleep deprivations occurring at the decreasing phase in baseline. Dbp showed similar, albeit opposite dynamics. These unexpected results could be reliably predicted assuming that these transcripts behave according to a driven damped harmonic oscillator. As expected, the sleep-wake distribution accounted for a large degree of the changes in EEG delta power and Homer1a. Nevertheless, the sleep deprivation-induced increase in delta power varied also with time of day with higher than expected levels when recovery sleep started at dark onset. CONCLUSIONS: Per2 and delta power are widely used as exclusive state variables of the circadian and homeostatic process, respectively. Our findings demonstrate a considerable cross-talk between these two processes. As Per2 in the brain responds to both sleep loss and time of day, this molecule is well positioned to keep track of and to anticipate homeostatic sleep need. CITATION: Curie T; Mongrain V; Dorsaz S; Mang GM; Emmenegger Y; Franken P. Homeostatic and circadian contribution to EEG and molecular state variables of sleep regulation. SLEEP 2013;36(3):311-323.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS: Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS: 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION: Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Molecular and Functional Characterization of Neuroblastoma-Initiating Cells

Neuroblastoma (NB) is the most common extracranial malignant tumor in young children and arises at any site of the sympathetic nervous system. The disease exhibits a remarkable phenotypic diversity ranging from spontaneous regression to fatal disease. Poor outcome results from a rapidly progressive, metastatic and drug-resistant disease. Recent studies have suggested that solid tumors may arise from a minor population of cancer stem cells (CSCs) with stem cell markers and typical properties such as self-renewal ability, asymmetric division and drug resistance. In this model, CSCs possess the exclusive ability to initiate and maintain the tumor, and to produce distant metastases. Tumor cell subpopulations with stem-like phenotypes have indeed been identified in several cancer including leukemia, breast, brain and colon cancers. CSC hypothesis still needs to be validated in the other cancers including NB.NB originates from neural crest-derived malignant sympatho-adrenal cells. We have identified rare cells that express markers in conformity with neural crest stem cells and their derived lineages within primary NB tissue and cell lines, leading us to postulate the existence of CSCs in NB tumors.In the absence of specific markers to isolate CSCs, we adapted to NB tumor cells the sphere functional assay, based on the ability of stem cells to grow as spheres in non-adherent conditions. By serial passages of spheres from bone marrow NB metastases, a subset of cells was gradually selected and its specific gene expression profile identified by micro-array time-course analysis. The differentially expressed genes in spheres are enriched in genes implicated in development including CD133, ABC-transporters, WNT and NOTCH genes, identified in others solid cancers as CSCs markers, and other new markers, all referred by us as the Neurosphere Expression Profile (NEP). We confirmed the presence of a cell subpopulation expressing a combination of the NEP markers within a few primary NB samples.The tumorigenic potential of NB spheres was assayed by in vivo tumor growth analyses using orthotopic (adrenal glands) implantations of tumor cells into immune-compromised mice. Tumors derived from the sphere cells were significantly more frequent and were detected earlier compared to whole tumor cells. However, NB cells expressing the neurosphere-associated genes and isolated from the bulk tumors did not recapitulate the CSC-like phenotype in the orthotopic model. In addition, the NB sphere cells lost their higher tumorigenic potential when implanted in a subcutaneous heterotopic in vivo model.These results highlighted the complex behavior of CSC functions and led us to consider the stem-like NB cells as a dynamic and heterogeneous cell population influenced by microenvironment signals.Our approach identified for the first time candidate genes that may be associated with NB self-renewal and tumorigenicity and therefore would establish specific functional targets for more effective therapies in aggressive NB.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

Integrative molecular bioinformatics study of human adrenocortical tumors : microRNA, tissue-specific target prediction, and pathway analysis.

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Evolutionary history of the greater white-toothed shrew (Crocidura russula) inferred from analysis of mtDNA, Y, and X chromosome markers.

We investigate the evolutionary history of the greater white-toothed shrew across its distribution in northern Africa and mainland Europe using sex-specific (mtDNA and Y chromosome) and biparental (X chromosome) markers. All three loci confirm a large divergence between eastern (Tunisia and Sardinia) and western (Morocco and mainland Europe) lineages, and application of a molecular clock to mtDNA divergence estimates indicates a more ancient separation (2.25 M yr ago) than described by some previous studies, supporting claims for taxonomic revision. Moroccan ancestry for the mainland European population is inconclusive from phylogenetic trees, but is supported by greater nucleotide diversity and a more ancient population expansion in Morocco than in Europe. Signatures of rapid population expansion in mtDNA, combined with low X and Y chromosome diversity, suggest a single colonization of mainland Europe by a small number of Moroccan shrews >38 K yr ago. This study illustrates that multilocus genetic analyses can facilitate the interpretation of species' evolutionary history but that phylogeographic inference using X and Y chromosomes is restricted by low levels of observed polymorphism.

Characterization of mandarin citrus germplasm from Southern Brazil by morphological and molecular analyses

The objective of this work was to characterize mandarin (Citrus spp.) germplasm from Southern Brazil by morphological and molecular analyses. Thirty seven cultivars from 34 distinct mandarin varieties were evaluated by morphological and agronomic traits of leaves, flowers and fruits, and by microsatellite markers. The morphological and agronomic characteristics suggested that almost all varieties can be produced for commercial use, and some, as the Satsuma variety, are recommended for breeding programs. Pooled DNA samples from 1-5 plants belonging to each cultivar were tested. Eight of the nine primers detected polymorphisms. Specific markers were found for some accessions. The dendrogram constructed with the morphological results divided the 37 cultivars into four groups, while that obtained with the microsatellites clustered 35 of the 37 cultivars into three groups only. Generally, intervarietal differences are not high, and this lack of agreement in the two multifactorial analyses indicates that diverse evolutionary factors are acting at these two levels of investigation.

Genetic characterization of the Moxotó goat breed using RAPD markers

The objective of this study was to verify the genetic diversity between and within seven populations of Moxotó goat (n = 264) from the States of Pernambuco, Paraíba and Rio Grande do Norte, using RAPD (Random Amplified Polymorphic DNA). Moxotó, as well as other naturalized breeds, suffers genetic losses due to the indiscriminate miscegenation with breeds raised in the Northeast Region of Brazil. The genetic characterization of these genetic resources is essential to conservation and breeding programs. DNA was extracted from lymphocytes using a non-organic protocol. The 16 primers used were selected from 120 decamer oligonucleotide primers and generated 56 polymorphic bands. The analysis of molecular variance (AMOVA) showed that the greater part of total genetic variability (71.55%) was due to differences between individuals within populations, while 21.21% was among populations. The analysis of variance among the pairs of populations demonstrated that the populations located in Floresta, PE x Angicos, RN presented a smaller value of intrapopulational differentiation (8.9%), indicating low genetic variability among them. Nei's genetic distances varied between 0.0546 and 0.1868 in the populations. The dendrogram generated showed that the Canindé breed, used as outgroup, clustered with the populations of Moxotó, indicating a possible common origin of the naturalized goat breeds.

Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.

Diversidade genética estimada com marcadores ISSR em populações brasileiras de Zabrotes subfasciatus

O objetivo deste trabalho foi estimar a diversidade genética de populações de Zabrotes subfasciatus, por meio de marcadores moleculares ISSR. Foram avaliadas 12 populações, provenientes de oito estados brasileiros, no total de 269 indivíduos. Cinco iniciadores ISSR permitiram a obtenção do total de 51 fragmentos polimórficos. A percentagem média de locos polimórficos, dentro de cada população, foi de 83,8%. A heterozigosidade corrigida de Nei esperada variou de 0,23 a 0,33, com média de 0,29, e o índice de diversidade genética de Shannon e Weaver variou de 0,29 a 0,48, com média de 0,42. No nível de espécie, estes dois índices apresentaram valores de 0,36 e 0,54, respectivamente. A análise de variância molecular mostrou que 66% da variância molecular total pode ser atribuída a diferenças intrapopulacionais e que os 34% restantes podem ser atribuídos a diferenças interpopulacionais. O teste de Mantel mostrou baixa correlação entre: distância geográfica e diferenciação genética, identidade genética e diferenciação genética e, distância genética de Nei e diferenciação genética. As populações brasileiras de Z. subfasciatus possuem baixa diferenciação genética e fraca estruturação geográfica.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Genetic variability of Brazilian rice landraces determined by SSR markers

The objective of this study was to evaluate the genetic variability of rice (Oryza sativa) landraces collected in Brazilian small farms. Twelve simple sequence repeat (SSR) markers characterized 417 landraces collected in 1986, 1987 and 2003, in the state of Goiás, Brazil. The number of landraces with long and thin grain type increased in the evaluated period, probably due to market demand. Based on the molecular data, the genetic variability increased during this period and, as per to the factorial correspondence analysis, most of the accessions were grouped according to the year of collection. The incorporation of modern rice cultivars in landrace cultivation areas and the selection carried out by small farmers are the most probable factors responsible for increasing landrace genetic variability, during the evaluated period. Genotype exchange between farmers, selection practice and local environmental adaptation are able to generate novel adapted allele combinations, which can be used by breeding programs, to reinitiate the process.

Agronomical and molecular characterization of banana germplasm

The objective of the present work was to characterize banana accessions from the Germplasm Bank at Embrapa Mandioca e Fruticultura Tropical (Brazil), using agronomical, physical and physicochemical characteristics of fruit and simple sequence repeats (SSR) markers. Twenty-six accessions were analyzed, in which high genetic variability was found, especially for the agronomical characters number of fruit and weight of bunch. Accessions with high contents of carotenoids (diploid 'Jaran'), polyphenols (triploid 'Caipira' and tetraploid 'Teparod') and vitamin C (diploid 'Tuugia' and an unknown triploid AAA) in the fruit were identified. Thirteen microsatellite primers revealed an average of 7.23 alleles, which showed high variability. A dendrogram was prepared using the Gower algorithm for the distance matrices obtained from the agronomical, physical and physicolchemical analysis of fruit and SSR markers. Adopting the average genetic divergence as the cut-off point, three clusters were found: G1, formed by the diploids 'Jaran', 028003-01 and M-48; G2, by the diploids 'Malbut' and 'Ido 110'; and G3, by 21 tri-and tetraploid accessions, including one diploid, 'Tuugia'. The triploids with the B genome 'Thap Maeo', 'Walha', 'Pacha Nadan' and 'Champa Madras' were grouped in G2. Results from this work can be used for breeding hybrids with good agronomical traits and fruit quality.

ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers

The objectives of this work were to investigate the genetic variation in 79 soybean (Glycine max) accessions from different regions of the world, to cluster the accessions based on their similarity, and to test the correlation between the two types of markers used. Simple sequence repeat markers present in genomic (SSR) and in expressed regions (EST-SSR) were used. Thirty SSR primer-pairs were selected (20 genomic and 10 EST-SSR) based on their distribution on the 20 genetic linkage groups of soybean, on their trinucleotide repetition unit and on their polymorphism information content. All analyzed loci were polymorphic, and 259 alleles were found. The number of alleles per locus varied from 2-21, with an average of 8.63. The accessions exhibit a significant number of rare alleles, with genotypes 19, 35, 63 and 65 carrying the greater number of exclusive alleles. Accessions 75 and 79 were the most similar and accessions 31 and 35, and 40 and 78, were the most divergent ones. A low correlation between SSR and EST-SSR data was observed, thus genomic and expressed microsatellite markers are required for an appropriate analysis of genetic diversity in soybean. The genetic diversity observed was high and allowed the formation of five groups and several subgroups. A moderate relationship between genetic divergence and geographic origin of accessions was observed.