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

Le hasard m’a permis de faire des belles découvertes. La beauté particulière des câbles électriques et leur potentiel plastiques ont ainsi éclairé mon parcours artistique. J’ai fait le choix du câble comme matière première de mon œuvre depuis 1999. Une technique artisanale celle du Crochet, en lien avec la tapisserie contemporaine met l’accent sur l’idée de la connectivité et le tissage des liens sociaux par un seul fil continu sans coupure. Je cherche une transgression de cette pratique au profit d'une compréhension plus large de ce qu’elle représente. Par la répétition gestuelle qui m’a permis de conduire à une forme de ritualisation de ma technique, je passe par une phase de conscience où la perception devient plus aiguisée. Ma pratique est devenue une forme d’existence, d'amélioration et de soulagement pour arriver au constat d’un effet magique qui délivre le corps par une spiritualité. C’est aussi une forme de connectivité, d’échange avec des plasticiens qui ont agrandi mon univers. Par le travail sériel, monochrome (noir) et une analyse épistémologique, je suggère un mé-tissage de certaines disciplines, une forme d’hybridation entre un univers cosmologique, mathématique et physique. Mon corps devient un fil conducteur dessine dans l’espace externe mon intériorité par des vidéos performatives et expressives. Je fais là l’expérience d'une contiguïté issue en réalité de la continuité du fil. Cet espace interne et fusionnel, où je fais l'expérience de mon corps en relation avec le fil continu, revêt souvent une dimension imaginaire, auquel s'ajoute un espace externe, celui de la représentation et de la mise à distance dans le monde physique de mon œuvre. / Chance has allowed me to make beautiful discoveries. The particular beauty of the electric cables and their plastic potential have thus illuminated my artistic career. I made the choice of cable as raw material of my work since 1999. A traditional technique, that of Crochet, in connection with contemporary tapestry emphasizes the idea of connectivity and the weaving of social links by a single continuous thread, an unbroken thread. But I seek a transgression of this practice in favor of a broader understanding of what it represents. By the gestural repetition that allowed me to lead to a form of ritualization of my technique, I go through a phase of consciousness where the perception becomes more acute. My practice has become a form of existence, improvement, and relief to come to the realization of a magical effect, which delivers the body spiritually. It is also a form of connectivity, of exchange with several artists of which I made the collection carefully and which enlarged my universe, then by the serial work, monochrome (black) and an epistemological analysis, I suggest a " miscegenation" of certain disciplines in my work, a form of hybridization between a cosmological universe, mathematical and physical are interlaced. My body becomes a drawing thread in the external space, my interiority by performative and expressive videos. I am experiencing an adjacency that actually comes from the continuity of the thread. This internal and fusional space, where I experience my body in relation to the continuous thread, often takes on an imaginary dimension, to which is added an external space, that of representation and distancing in the physical world of my work.

Ao acompanhar a trajetória intelectual de Jamil Almansur Haddad desde suas primeiras publicações até sua morte, esta Tese de doutorado traz à luz o percurso de um literato pertencente à segunda geração de imigrantes libaneses e suas relações paradoxais com o meio intelectual brasileiro e, em especial, o paulistano. Seu percurso biográfico ilustra um desdobramento do movimento literário árabe da imigração, que desempenhou papel central no processo de renascimento da literatura árabe no final do século XIX. Imerso no contexto cultural de um país em busca da conformação de sua verdadeira identidade nacional e vivendo na cidade de São Paulo - que em meados do século XX foi um dos maiores polos de imigração do mundo Jamil, através de sua experiência biográfica e de seu caminho literário, revela nuances de um processo intermediário de hibridação cultural. / This thesis follows the intellectual trajectory of Jamil Almansur Haddad from his first publications to his death, bringing to light the trajectory of a poet that belongs to the second generation of Lebanese immigrants and his paradoxical relations with the Brazilian intellectual environment and, in particular, the paulistano one. His biographical journey illustrates an unfolding of the Arab literary movement of immigration, which played a central role in the process of rebirth of Arab literature in the late nineteenth century. Immersed in the cultural context of a country in search of the conformation of its true national identity and living in the city of São Paulo - which in the mid-twentieth century was one of the largest poles of immigration in the world - Jamil, through his biographical experience and his literary path, reveals nuances of an intermediate process of cultural hybridization.

Les travaux de cette thèse visent à apporter des éléments de solutions au problème de la durée de vie des systèmes pile à combustible (FCS – Fuel Cell System) de type à « membrane échangeuse de protons » (PEM – Proton Exchange Membrane) et se décline sur deux champs disciplinaires complémentaires :Une première approche vise à augmenter la durée de vie de celle-ci par la conception et la mise en œuvre d'une architecture de pronostic et de gestion de l'état de santé (PHM – Prognostics & Health Management). Les PEM-FCS, de par leur technologie, sont par essence des systèmes multi-physiques (électriques, fluidiques, électrochimiques, thermiques, mécaniques, etc.) et multi-échelles (de temps et d'espace) dont les comportements sont difficilement appréhendables. La nature non linéaire des phénomènes, le caractère réversible ou non des dégradations, et les interactions entre composants rendent effectivement difficile une étape de modélisation des défaillances. De plus, le manque d'homogénéité (actuel) dans le processus de fabrication rend difficile la caractérisation statistique de leur comportement. Le déploiement d'une solution PHM permettrait en effet d'anticiper et d'éviter les défaillances, d'évaluer l'état de santé, d'estimer le temps de vie résiduel du système, et finalement, d'envisager des actions de maîtrise (contrôle et/ou maintenance) pour assurer la continuité de fonctionnement. Une deuxième approche propose d'avoir recours à une hybridation passive de la PEMFC avec des super-condensateurs (UC – Ultra Capacitor) de façon à faire fonctionner la pile au plus proche de ses conditions opératoires optimales et ainsi, à minimiser l'impact du vieillissement. Les UCs apparaissent comme une source complémentaire à la PEMFC en raison de leur forte densité de puissance, de leur capacité de charge/décharge rapide, de leur réversibilité et de leur grande durée de vie. Si l'on prend l'exemple des véhicules à pile à combustible, l'association entre une PEMFC et des UCs peut être réalisée en utilisant un système hybride de type actif ou passif. Le comportement global du système dépend à la fois du choix de l'architecture et du positionnement de ces éléments en lien avec la charge électrique. Aujourd'hui, les recherches dans ce domaine se focalisent essentiellement sur la gestion d'énergie entre les sources et stockeurs embarqués ; et sur la définition et l'optimisation d'une interface électronique de puissance destinée à conditionner le flux d'énergie entre eux. Cependant, la présence de convertisseurs statiques augmente les sources de défaillances et pannes (défaillance des interrupteurs du convertisseur statique lui-même, impact des oscillations de courant haute fréquence sur le vieillissement de la pile), et augmente également les pertes énergétiques du système complet (même si le rendement du convertisseur statique est élevé, il dégrade néanmoins le bilan global). / This thesis work aims to provide solutions for the limited lifetime of Proton Exchange Membrane Fuel Cell Systems (PEM-FCS) based on two complementary disciplines:A first approach consists in increasing the lifetime of the PEM-FCS by designing and implementing a Prognostics & Health Management (PHM) architecture. The PEM-FCS are essentially multi-physical systems (electrical, fluid, electrochemical, thermal, mechanical, etc.) and multi-scale (time and space), thus its behaviors are hardly understandable. The nonlinear nature of phenomena, the reversibility or not of degradations and the interactions between components makes it quite difficult to have a failure modeling stage. Moreover, the lack of homogeneity (actual) in the manufacturing process makes it difficult for statistical characterization of their behavior. The deployment of a PHM solution would indeed anticipate and avoid failures, assess the state of health, estimate the Remaining Useful Lifetime (RUL) of the system and finally consider control actions (control and/or maintenance) to ensure operation continuity.A second approach proposes to use a passive hybridization of the PEMFC with Ultra Capacitors (UC) to operate the fuel cell closer to its optimum operating conditions and thereby minimize the impact of aging. The UC appear as an additional source to the PEMFC due to their high power density, their capacity to charge/discharge rapidly, their reversibility and their long life. If we take the example of fuel cell hybrid electrical vehicles, the association between a PEMFC and UC can be performed using a hybrid of active or passive type system. The overall behavior of the system depends on both, the choice of the architecture and the positioning of these elements in connection with the electric charge. Today, research in this area focuses mainly on energy management between the sources and embedded storage and the definition and optimization of a power electronic interface designated to adjust the flow of energy between them. However, the presence of power converters increases the source of faults and failures (failure of the switches of the power converter and the impact of high frequency current oscillations on the aging of the PEMFC), and also increases the energy losses of the entire system (even if the performance of the power converter is high, it nevertheless degrades the overall system).

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.

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.