O genoma humano é composto por diversos tipos de variações estruturais, como por exemplo, as variações no número de cópias (CNVs), que, mesmo sendo muito pequenas, podem gerar diversas alterações clínicas específicas, como as malformações congênitas e o atraso do desenvolvimento neuropsicomotor (MC/ADNPM). Para a detecção destas alterações existem diferentes técnicas citogenômicas dentre elas a FISH (Fluorescent in situ Hibridization) e a MLPA (Multiplex Ligation-dependent Probe Amplification), que investigam um número limitado de regiões do genoma, como as regiões envolvidas nas síndromes de microdeleções/microduplicações mais comuns e as regiões subteloméricas. Outros métodos como a cariotipagem clássica e o array genômico possibilitam uma análise completa do DNA em uma única reação, aumentando a taxa de detecção de desequilíbrios complexos. Alcançar um diagnóstico inequívoco é fundamental para entender a natureza da doença, fornecendo respostas sobre o prognóstico, sobre os riscos de recorrência e direcionando o paciente à terapia específica, o que pode minimizar o custo financeiro dessas doenças e até mesmo possibilitar a inclusão desses indivíduos na sociedade. O projeto teve como objetivo comparar a capacidade diagnóstica destas tecnologias (FISH, MLPA e array) para a elucidação etiológica de pacientes sindrômicos encaminhados para a unidade de genética. A casuística deste trabalho foi composta pela análise dos resultados das técnicas de FISH e/ou MLPA e array, utilizadas no diagnóstico de 78 pacientes com MC/ADNPM. Na técnica de FISH, empregada na análise genômica de 22 pacientes, foram utilizadas sondas locus específicas para as regiões das principais síndromes de microdeleção/microduplicação e para as regiões subteloméricas de cromossomos específicos. Por meio desta metodologia, foram identificados ~18,2% dos pacientes com diferentes alterações. Já a técnica de MLPA, utilizada no diagnóstico dos 78 pacientes, por meio dos kits para as principais síndromes de microdeleção/microduplicação e para as regiões subteloméricas, detectou ~34,6% de pacientes com diversas alterações. A técnica de array, realizada em todos os pacientes utilizando diferentes plataformas (Agilent, Affymetrix ou Illumina) apresentou uma taxa de ~42,3% de detecção de pacientes com pelo menos uma alteração patogênica e ~38,5% de pacientes com alterações benignas ou de significado clínico incerto. Ao avaliar as três técnicas concomitantemente foi verificada uma taxa de ~93,6% de concordância, apesar dos resultados não serem iguais em todos os casos e da técnica de MLPA não detectar ~66,2% das alterações em relação ao array. Os resultados obtidos corroboraram com dados da literatura, mas no geral a taxa de detecção foi superior às taxas descritas, o em que em parte pode ser devido ao critério de seleção dos pacientes, sugerindo fortemente que a hipótese clínica adequada é crucial para o sucesso da detecção de alteração. Embora o array seja a ferramenta mais eficiente para o diagnóstico de pacientes com malformações, seu uso como primeiro teste diagnóstico nem sempre é o mais apropriado devido ao seu custo elevado ou sua limitação em detectar inversões e translocações balanceadas. Portanto todas as técnicas estudadas têm suas vantagens e desvantagens, e poderão ser aplicadas em conjunto para que o diagnóstico molecular seja concluído. Dessa forma, são necessárias uma interação clínico-laboratorial e uma equipe técnica multiprofissional especializada para o direcionamento do diagnóstico molecular mais eficaz em relação ao custo-benefício / The human genome is composed of several types of structural variations, such as copy number variation (CNVs) which, although very small, can generate several specific clinical abnormalities, such as congenital malformations and developmental delay (CM/DD). To detect these changes there are different cytogenomics techniques, among them, FISH (Fluorescent in situ Hybridization) and MLPA (Multiplex Ligation-dependent Probe Amplification) that can investigate a limited number of genomic regions for example the most common microdeletion/microduplications syndromes and subtelomeric regions. Other methods such as classical karyotyping and array provide a complete DNA analysis in a single reaction, increasing the detection rate of complex imbalances. Acquire an unequivocal diagnosis is critical to understand the nature of the disease, providing answers about the prognosis, risks of recurrence and directing the patients to specific therapy, which can minimize the cost of these diseases and even allow the inclusion of these individuals in society. The objective of this project was to compare the diagnostic ability of these technologies (FISH, MLPA and array) for the etiologic diagnosis of syndromic patients referred to the clinical unit of genetics. The casuistry was composed by the results of analysis of 78 patients with CM/DD using FISH and/or MLPA and array. The FISH technique was utilized in genomic analysis for 22 patients and locus specific probes were used for regions of the microdeletion/microduplication syndromes and the subtelomeric regions of specific chromosomes. By this methodology ~18.2% of the patients were identified with different genomic changes. The MLPA technique was used in the diagnosis of 78 patients, with microdeletion/microduplication syndrome and subtelomeric regions, and detected ~34.6% of patients with several changes. The array technique was performed in all patients using different platforms (Agilent, Illumina or Affymetrix) and shows a rate of ~42.3% of detection at least one pathogenic change and ~38.5% of patients with benign or uncertain clinical significance changes. In assessment of the three techniques concomitantly was observed a rate of ~93.6% of concordance, although the results are not the same in all cases and the MLPA technique to detect ~ 66.2% of the changes in relation to the array. The results obtained corroborated with literature data, but the overall detection rate was higher than the rates described in the literature, due in part to the criteria selection of patients. Our results strongly suggesting that appropriate clinical hypothesis is crucial for successful change detection. Although the array is the most efficient tool for the diagnosis of patients with abnormalities, using this test as a first diagnostic approach is not always the most suitable tool because of the high cost or the limitation to detect inversions and balanced translocations. Therefore, all techniques studied have their advantages and disadvantages, and could be applied together for the completed molecular diagnosis. Thus, a clinical laboratory interaction and multidisciplinary skilled technicians is required for targeting the most effective molecular diagnosis in relation to cost-benefit

O genoma humano é composto por diversos tipos de variações estruturais, como por exemplo, as variações no número de cópias (CNVs), que, mesmo sendo muito pequenas, podem gerar diversas alterações clínicas específicas, como as malformações congênitas e o atraso do desenvolvimento neuropsicomotor (MC/ADNPM). Para a detecção destas alterações existem diferentes técnicas citogenômicas dentre elas a FISH (Fluorescent in situ Hibridization) e a MLPA (Multiplex Ligation-dependent Probe Amplification), que investigam um número limitado de regiões do genoma, como as regiões envolvidas nas síndromes de microdeleções/microduplicações mais comuns e as regiões subteloméricas. Outros métodos como a cariotipagem clássica e o array genômico possibilitam uma análise completa do DNA em uma única reação, aumentando a taxa de detecção de desequilíbrios complexos. Alcançar um diagnóstico inequívoco é fundamental para entender a natureza da doença, fornecendo respostas sobre o prognóstico, sobre os riscos de recorrência e direcionando o paciente à terapia específica, o que pode minimizar o custo financeiro dessas doenças e até mesmo possibilitar a inclusão desses indivíduos na sociedade. O projeto teve como objetivo comparar a capacidade diagnóstica destas tecnologias (FISH, MLPA e array) para a elucidação etiológica de pacientes sindrômicos encaminhados para a unidade de genética. A casuística deste trabalho foi composta pela análise dos resultados das técnicas de FISH e/ou MLPA e array, utilizadas no diagnóstico de 78 pacientes com MC/ADNPM. Na técnica de FISH, empregada na análise genômica de 22 pacientes, foram utilizadas sondas locus específicas para as regiões das principais síndromes de microdeleção/microduplicação e para as regiões subteloméricas de cromossomos específicos. Por meio desta metodologia, foram identificados ~18,2% dos pacientes com diferentes alterações. Já a técnica de MLPA, utilizada no diagnóstico dos 78 pacientes, por meio dos kits para as principais síndromes de microdeleção/microduplicação e para as regiões subteloméricas, detectou ~34,6% de pacientes com diversas alterações. A técnica de array, realizada em todos os pacientes utilizando diferentes plataformas (Agilent, Affymetrix ou Illumina) apresentou uma taxa de ~42,3% de detecção de pacientes com pelo menos uma alteração patogênica e ~38,5% de pacientes com alterações benignas ou de significado clínico incerto. Ao avaliar as três técnicas concomitantemente foi verificada uma taxa de ~93,6% de concordância, apesar dos resultados não serem iguais em todos os casos e da técnica de MLPA não detectar ~66,2% das alterações em relação ao array. Os resultados obtidos corroboraram com dados da literatura, mas no geral a taxa de detecção foi superior às taxas descritas, o em que em parte pode ser devido ao critério de seleção dos pacientes, sugerindo fortemente que a hipótese clínica adequada é crucial para o sucesso da detecção de alteração. Embora o array seja a ferramenta mais eficiente para o diagnóstico de pacientes com malformações, seu uso como primeiro teste diagnóstico nem sempre é o mais apropriado devido ao seu custo elevado ou sua limitação em detectar inversões e translocações balanceadas. Portanto todas as técnicas estudadas têm suas vantagens e desvantagens, e poderão ser aplicadas em conjunto para que o diagnóstico molecular seja concluído. Dessa forma, são necessárias uma interação clínico-laboratorial e uma equipe técnica multiprofissional especializada para o direcionamento do diagnóstico molecular mais eficaz em relação ao custo-benefício / The human genome is composed of several types of structural variations, such as copy number variation (CNVs) which, although very small, can generate several specific clinical abnormalities, such as congenital malformations and developmental delay (CM/DD). To detect these changes there are different cytogenomics techniques, among them, FISH (Fluorescent in situ Hybridization) and MLPA (Multiplex Ligation-dependent Probe Amplification) that can investigate a limited number of genomic regions for example the most common microdeletion/microduplications syndromes and subtelomeric regions. Other methods such as classical karyotyping and array provide a complete DNA analysis in a single reaction, increasing the detection rate of complex imbalances. Acquire an unequivocal diagnosis is critical to understand the nature of the disease, providing answers about the prognosis, risks of recurrence and directing the patients to specific therapy, which can minimize the cost of these diseases and even allow the inclusion of these individuals in society. The objective of this project was to compare the diagnostic ability of these technologies (FISH, MLPA and array) for the etiologic diagnosis of syndromic patients referred to the clinical unit of genetics. The casuistry was composed by the results of analysis of 78 patients with CM/DD using FISH and/or MLPA and array. The FISH technique was utilized in genomic analysis for 22 patients and locus specific probes were used for regions of the microdeletion/microduplication syndromes and the subtelomeric regions of specific chromosomes. By this methodology ~18.2% of the patients were identified with different genomic changes. The MLPA technique was used in the diagnosis of 78 patients, with microdeletion/microduplication syndrome and subtelomeric regions, and detected ~34.6% of patients with several changes. The array technique was performed in all patients using different platforms (Agilent, Illumina or Affymetrix) and shows a rate of ~42.3% of detection at least one pathogenic change and ~38.5% of patients with benign or uncertain clinical significance changes. In assessment of the three techniques concomitantly was observed a rate of ~93.6% of concordance, although the results are not the same in all cases and the MLPA technique to detect ~ 66.2% of the changes in relation to the array. The results obtained corroborated with literature data, but the overall detection rate was higher than the rates described in the literature, due in part to the criteria selection of patients. Our results strongly suggesting that appropriate clinical hypothesis is crucial for successful change detection. Although the array is the most efficient tool for the diagnosis of patients with abnormalities, using this test as a first diagnostic approach is not always the most suitable tool because of the high cost or the limitation to detect inversions and balanced translocations. Therefore, all techniques studied have their advantages and disadvantages, and could be applied together for the completed molecular diagnosis. Thus, a clinical laboratory interaction and multidisciplinary skilled technicians is required for targeting the most effective molecular diagnosis in relation to cost-benefit

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

Chez les végétaux supérieurs, lembryogenèse est une phase clé du développement au cours de laquelle lembryon établit les principales structures de la future plante. La compréhension des processus moléculaires et physiologiques menant à la formation de la graine est donc dun intérêt agronomique majeur. Chez Phaseolus la caractérisation moléculaire de lembryogenèse permet de mieux comprendre les mécanismes du développement embryonnaire et de son dysfonctionnement observé chez les hybrides interspécifiques. Cette thèse sinscrit dans ce cadre et vise à identifier et caractériser des gènes clés impliqués dans le développement de l'embryon chez Phaseolus. Des hybridations interspécifiques ont été réalisées entre lespèce P.vulgaris L. (cultivar NI637) utilisée comme parent mâle et lespèce P. coccineus L. (cultivar NI16) utilisée comme parent femelle. Des analyses ont aussi été effectuées sur un mutant obtenu par mutagenèse chimique à l'EMS (Ethyl Méthyl Sulfonate) de graines de la variété BAT93 de P.vulgaris. Une étude histologique comparative a permis de suivre la dynamique de lembryogenèse du haricot commun à partir dembryons prélevés 3 à 12 jours après la pollinisation et provenant de plantes normales et déficients dans la production de graines. Les embryons de P. vulgaris se développent plus rapidement par rapport à ceux issus du mutant EMS. Ces derniers présentent des anomalies au niveau de lembryon et du suspenseur. La caractérisation fonctionnelle de deux gènes candidats MIPS (myo-inositol phosphate synthase) et Sus (sucrose synthase) a été réalisée par RT-PCR quantitative et hybridation in situ suite à une étude spatio-temporelle dexpression de ces deux gènes candidats au cours de développement embryonnaire chez Phaseolus. Lanalyse du profil dexpression de ces deux gènes montre quils sont exprimés différemment au niveau des tissus de lembryon et du suspenseur. Lanalyse in silico nous a permis de sélectionner 22 gènes candidats dont nous avons vérifié l'expression au cours de développement de la graine chez Phaseolus. Des variations au niveau de la méthylation de lADN ont été déterminées chez les hybrides interspécifiques comparativement à leurs parents. La technique de lHSS a permis disoler des fragments dADNs complémentaires différemment exprimés au cours de développement de la graine chez Phaseolus. Lanalyse des séquences de ces ADNs complémentaires montre quils codent pour plusieurs protéines intervenant dans le développement cellulaire et embryonnaire, en particulier le "storage protein activator" (SPA), le "pentatricopeptide repeat-containing protein" (PPR) et lacetyl-CoA carboxylase (ACCase). La caractérisation de ces différents gènes exprimés au cours du développement de la graine, fournit de nouveaux outils susceptibles de mettre en évidence des mécanismes de dysfonctionnement embryonnaire chez le genre Phaseolus.

Aquesta tesi tracta sobre l'efecte de la contaminació per cru de petroli sobre l'ecosistema costaner mediterrani i sobre el paper fonamental dels sediments marins en la regulació i el manteniment dels processos biogeoquímics. L'estudi presta especial atenció a les comunitats bacterianes reductores de sulfat i la seva implicació en la degradació de contaminants orgànics. La diversitat, abundància i fisiologia dels bacteris reductors de sulfat que habiten el sediment arenós del nord de Mallorca (Illes Balears), van ser analitzades mitjançant un enfocament polifàsic, basat en la combinació d'experiments in situ i in vitro, biologia molecular clàssica i d’última generació, cultius i determinació d'activitats metabòliques. Els resultats obtinguts durant aquesta tesi demostren que el sediment mediterrani alberga una microbiota autòctona que podria prosperar després d'un vessament de cru de petroli i el paper de la qual podria ser crucial per a la transformació i l'eliminació de compostos orgànics xenobiòtics en aquest ambient. / Esta tesis trata sobre el efecto de la contaminación por crudo de petróleo en el ecosistema costero mediterráneo y sobre el papel fundamental de los sedimentos marinos en la regulación y el mantenimiento de los procesos biogeoquímicos. El estudio presta especial atención a las comunidades bacterianas reductoras de sulfato y a su implicación en la degradación de contaminantes orgánicos. La diversidad, abundancia y fisiología de las bacterias reductoras de sulfato que habitan el sedimento arenoso del norte de Mallorca (Islas Baleares), fueron analizadas mediante un enfoque polifásico, basado en la combinación de experimentos in situ e in vitro, biología molecular clásica y de última generación, cultivos y determinación de actividades metabólicas. Los resultados obtenidos durante esta tesis demuestran que el sedimento mediterráneo alberga una microbiota autóctona que podría prosperar después de un derrame de crudo de petróleo y cuyo papel podría ser crucial para la transformación y la eliminación de compuestos orgánicos xenobióticos en este ambiente. / This thesis discusses the fate and behave of crude oil contamination in the Mediterranean coastal ecosystem, and the essential role of the marine sediments in the regulation and maintenance of biogeochemical processes. The study pays particular attention to the role of sulphate reducing bacterial communities in the degradation of organic matter and pollutants entering the Mediterranean environment. A polyphasic approach based in the combination of in situ and in vitro experiments, next generation and classical molecular biology, cultivation, and the determination of metabolic activities, provided first insights into the diversity, abundance and physiology of sulphate reducing bacteria inhabiting the undisturbed sandy sediment at the north of Mallorca (Balearic Islands). The results obtained during the thesis demonstrate that the undisturbed Mediterranean sediment harbours an autochthonous microbiota that could prosper after a crude oil spill and which role might be crucial for the transformation and removal of hazardous organic compounds in this environment.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

No description available.

No description available.

Le benzo-a-pyrène (BaP) est un cancérogène reconnu pour l'homme, contaminant présent dans notre environnement. Il cause des dommages à l'ADN que nous avons mesurés dans les lymphocytes exposés à de faibles concentrations de BaP, provenant de 20 jeunes volontaires non fumeurs et en santé. Suite à l’exposition, la fréquence des micronoyaux (MN) augmente significativement et décrit une courbe dose-réponse non linéaire, suggérant le déclenchement du processus de détoxification et la réparation de l’ADN. Des différences entre les individus et entre les sexes sont présentes dans la réponse génotoxique produite par le BaP. Le test des aberrations chromosomiques montre que le pourcentage de chromosomes cassés augmente significativement dans les cellules exposées au BaP. Combinés avec l'augmentation de la fréquence des MN, nos résultats confirment l'effet clastogène du BaP déjà rapporté dans la littérature. L’hybridation in situ en fluorescence (FISH) des MN avec une sonde pancentromérique est aussi utilisée pour établir leur mécanisme de formation. La FISH révèle que la majorité des MN formés après une exposition au BaP contient un centromère et plus, ce qui est significativement différent de la condition non exposée. Plus précisément, dans nos conditions expérimentales, les MN induits par le BaP contiennent surtout trois centromères et plus, indiquant également la présence d'un effet aneugène. L'effet clastogène du BaP est relié à son rôle d'initiateur dans la cancérogenèse, alors que l'effet aneugène le relierait à l'étape de progression. Ces résultats sont importants puisque l'exposition aux composés de la classe du BaP est de longue durée (cigarette, air pollué). / Benzo-a-pyrene (BaP) is a known human carcinogen, contaminating all spheres of our environment. In human cells, BaP can induce various genotoxic effects on DNA, such as micronuclei (MNs) and chromosomal aberrations (CAs). MNs and CAs are measured in human lymphocytes in vitro exposed to low BaP concentrations, taken from 20 young healthy non-smoking subjects. Following BaP exposure, MN frequency increases significantly and shows a non-linear dose-response curve, suggesting the induction of detoxification process and/or DNA repair. Also, interindividual and sex differences in BaP-induced genotoxic damages are present. CA test shows that chromosome breaks increase significantly in cells exposed to BaP even at low concentrations. Combined to the observed MN frequency increase, our results confirm the clastogenic properties of BaP, as already reported in literature. In addition, fluorescence in situ hybridization (FISH) on MN using a pancentromeric probe is done to assess MN content. FISH reveals that most BaP-induced MNs contain centromeres, and specifically three or more centromeres. This difference is significant when compared to the unexposed condition, and suggest presence of an aneugenic effect. Clastogenic effect of BaP is associated with initiation step of carcinogenesis, while the aneugenic effect would link it with cancer progression. These results could be particularly important because exposure to BaP and other member of its chemical class usually last for decades (smoking, air pollution, etc.), and need to be confirm in future studies.

Les noyaux supraoptiques (NSO) et paraventriculaires (NPV) de l’hypothalamus montrent un phénomène réversible de plasticité structurale neurono-gliale dans diverses conditions physiologiques telles que la parturition, l’allaitement ou lors d’une surcharge en sel. En effet, les feuillets astrocytaires qui enveloppent normalement les somas et dendrites des neurones à ocytocine (OT) ou à vasopressine (AVP) se rétractent alors, autour des neurones à OT, laissant place à la formation de nouvelles synapses, surtout GABAergiques. Nous avons émis l’hypothèse voulant que ces mouvements cellulaires soient régulés par des molécules connues pour leurs rôles dans l’adhérence et la motilité cellulaires, notamment les récepteurs Eph et les éphrines (Efn). Nous avons étudié le rôle de l’un de ces récepteurs, EphA4, un récepteur à tyrosine kinase reconnaissant l’ensemble des Efn, A ou B, puis tenté d’identifier les Efn partenaires dans le NSO, à la suite d’une surcharge en sel. Pour démontrer la présence d’EphA4 dans le NSO et déterminer l’effet d’une surcharge en sel sur son expression et sa localisation, nous avons utilisé l’hybridation in situ et l’immunohistochimie en microscopie électronique, sur des coupes de cerveaux de souris ou rats traités ou non à l’eau salée pendant 1-7 j, avec des ribosondes ou des anticorps spécifiques pour EphA4. Ces travaux ont démontré une augmentation de l’expression d’EphA4 dans le NSO, notamment dans des dendrites, après le régime salé. La distribution de cette expression correspondait à celle des neurones OT et était absente de la glia limitans. Nous avons ensuite déterminé l’effet d’une absence d’EphA4 sur les mouvements astrocytaires et la synaptogènese autour des dendrites à OT et AVP, en utilisant des souris EphA4 knockouts et des souris de type sauvage des mêmes portées. Nous avons ainsi mesuré la couverture astrocytaire des dendrites OT ou AVP, identifiées par immunocytochimie anti-OT ou anti-AVP, en microscopie électronique. Ces mesures ont confirmé la rétraction des feuillets astrocytaires et la synaptogenèse autour des dendrites OT, mais pas autour des dendrites AVP, chez les souris de type sauvage, et démontré que la rétraction des feuillets astrocytaires et la synaptogenèse sur les dendrites OT ne se produisait pas chez les souris knockouts soumises à la surcharge en sel. L’ensemble de ces résultats démontre un rôle d’EphA4 dans cette plasticité structurale neurono-gliale. Afin d’identifier l’Efn partenaire d’EphA4 dans cette fonction, nous avons utilisé l’hybridation in situ et l’immunohistochimie pour les EfnB3 et -A3. L’hybridation in situ n’a pas démontré d’expression de l’EfnB3 dans le NSO, tandis que les résultats pour l’EfnA3 restent à quantifier. Cependant, l’immunohistochimie anti-EfnA3 montre un marquage d’astrocytes dans le NSO et la glia limitans, marquage qui semble augmenter après surcharge en sel, mais il reste à démontrer que l’anticorps anti-EfnA3 est bien spécifique et à quantifier les éventuels changements sur un plus grand nombre d’animaux. L’ensemble de ces observations démontre un rôle du récepteur EphA4 dans les mécanismes à la base des changements structuraux neurono-gliaux du NSO et pointe vers l’EfnA3 comme partenaire d’EphA4 dans ce modèle. / The supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus display reversible neurono-glial structural plasticity in various physiological conditions, such as parturition, lactation, or following salt loading. In such conditions, astrocytic leaflets that normally envelop the somas and dendrites of ocytocin (OT) or arginin-vasopressin (AVP) neurons retract from OT processes where they are replaced by new synapses, mainly GABAergic. Our hypothesis proposes that these cellular movements are regulated by molecules known for their roles in cell adhesion and motility, notably Eph receptors and ephrins (Efn). We have examined the role of one of these receptors, EphA4, a tyrosine-kinase receptor recognizing all ephrins, A or B, and then tried to identify the Efn interacting with EphA4 in these functions, following salt-loading. To demonstrate the presence of EphA4 in the SON and determine the effect of salt loading on its expression, we used in situ hybridization and immunohistochemistry in light and electron microscopy, on brain sections from rats or mice treated with salted water during 1-7 d, using riboprobes and antibodies specific for EphA4. These experiments demonstrated that EphA4 is expressed in the SON, with a distribution of its mRNA similar to that of OT neurons, and that it was absent from the glia limitans. Its expression increased following salt loading, particularly in dendrites. We then tested the effect of an absence of EphA4 on astrocytic process retraction and on synaptogenesis, using EphA4 kockout mice and wild-type littermates. We measured the ratio of astrocytic contact, and counted the number of synapses on the circumference of OT and AVP dendrites, identified in electron microscopy by immunocytochemistry, after 7 d of salt loading. The results confirmed the retraction of astrocytic processes from OT dendrites in wild-type animals after salt loading, and no change around AVP dendrites. However, there was no retraction from OT dendrites in EphA4 knockout mice, following salt loading. Altogether, these results constitute strong evidence for a role of EphA4 in the astrocyte leaflet retraction and accompanying synaptogenesis, specifically around OT dendrites. In order to identify the Efn interacting with EphA4 in this function, we used in situ hybridization and immunohistochemistry for EfnB3 and –A3. The in situ hybridization did not show the presence of EfnB3 in the SON, while the results for EfnA3 are currently being quantified. Nevertheless, anti-EfnA3 immunohistochemistry showed labelling in astrocytes and in the glia limitans of the SON, a labelling that seemed to increase following salt loading, although the specificity of the anti-EfnA3 antibody remains to be demonstrated on EfnA3 knockout mice, and its expression requires to be measured on a larger number of mice. The latter observations indicate EfnA3 as the potential partner (receptor/ligand) for EphA4 in the neurono-glial structural plasticity occurring in the SON following salt loading.