L’avortement spontané (AS) désigne la perte du produit de conception avant sa viabilité, c'est-à-dire avant la 22e semaine d’aménorrhée, ou un poids fœtal inférieur à 500 g. La cause génétique est à l’origine de plus des deux tiers des AS, les aneuploïdies autosomiques, représentant à elles seules jusqu’à 70% des pertes fœtales du 1er trimestre. Le caryotype présente une très bonne sensibilité en ce qui concerne le dépistage des trisomies autosomiques (13, 18 et 21) et des aneuploïdies affectant les chromosomes sexuels, mais il montre d’importantes limites, d’une part en raison des échecs de culture cellulaire et d’autre part en raison de l’existence de remaniements non détectables au caryotype standard. Actuellement plusieurs techniques moléculaires de dépistage rapides des aneuploïdies liées aux échecs de grossesses ont été vérifiées : 1°) la fluorescence in situ par hybridation (FISH) 2°) l’amplification multiplex de sondes nucléiques dépendant des ligatures (MLPA). Ces deux méthodes présentent l’avantage d’être réalisables, sans culture préalable, sur noyaux en interphase ou sur ADN extrait et de permettre la détection d’anomalies cryptiques. Notre étude repose sur l’étude cytogénétique des produits d’AS pour mettre en évidence les anomalies chromosomiques les plus fréquentes à l’origine de ces pertes fœtales et d’en mieux appréhender les mécanismes de survenue. Elle a été réalisée sur 220 patientes âgées de 19 à 45 ans, et était fondée sur l’analyse directe par FISH sur noyaux interphasiques (AneuVysionTM) de prélèvements de villosités choriales et sur l’analyse de l’ADN extrait de tissus fœtaux par MLPA afin de révéler d’éventuelles aneuploïdies et micro-remaniements. L’âge gestationnel au moment des prélèvements était compris entre 7 et 38 semaines d’aménorrhée. Sur un total de 151 échantillons analysés par AneuVysionTM, 10 anomalies chromosomiques ont été observées: 3 trisomies 21, 1 trisomie 18, 1 trisomie 13, 1 mosaïque 46,XX/47,XX+21, 3 triploïdies et 1 monosomie X (Turner). Par ailleurs, sur les 69 autres échantillons analysés par MLPA, 6 étaient ininterprétables. Les anomalies trouvées par cette technique étaient: 2 monosomies X. Pour les échantillons restants, la MLPA a été négative. Nous avons en parallèle réalisé une étude rétrospective fondée sur l’analyse comparative d’un échantillon recruté à Sidi Bel Abbès, de femmes ayant subi un AS et admises à la maternité del’hôpital Hassani Abdelkader de Sidi Bel Abbès et d’un échantillon recruté à Clermont-Ferrand de femmes ayant subi un AS et pour lesquelles un prélèvement pour établir le caryotype du produit de fausse-couche avait été adressé dans le service de cytogénétique du CHU Estaing de Clermont-Ferrand. Cette étude a couvert une période de six années, allant de janvier 2005 à décembre 2010. Les techniques de FISH et de MLPA représentent des outils simples, rapides et sensibles pour la détection des remaniements chromosomiques. Elles représentent une alternative très intéressante à la culture cellulaire, et permettent le diagnostic de désordres génomiques indécelables par les techniques conventionnelles. / Spontaneous abortion (SA) is the loss of the product of fertilization before its viability, that is, before22 weeks of gestation or fetal weight less than 500 g. Genetic causes account for more than two thirds of SA, autosomal aneuploidies alone accounting for up to 70% fetal loss. Chromosomal cytogenetic techniques show significant limitations on the one hand because of the failures of cell culture, and secondly because of the existence of undetectable alterations to the standard karyotype. It was therefore planned to use molecular techniques :- Fluorescent in situ hybridization (FISH)- Multiplex ligation-dependent probe amplification (MLPA). Both techniques have the advantage of being achievable without prior culture of cores interphase or DNA extracted and to enable detection of cryptic abnormalities. The project is based on cytogenetic study of AS products to highlight the most frequent chromosomal abnormalities causing fetal losses, and to better understand their occurrence. Our study was performed on 220 patients from 19 to 45 years, and was based on the direct analysis by FISH on interphase nuclei (AneuVysionTM) of chorionic villus sampling and analysis of DNA extracted fetal tissue by MLPA to reveal any aneuploidy and rearrangements. The gestational age of the samples ranged from the 7th to the 38th week of gestation. In a total of 151 samples analyzed by AneuVysionTM, 10 chromosomal abnormalities were observed: three trisomies 21, one trisomy 18, one trisomy 13, one mosaic 46,XX/47,XX+21, 3 triploidies and one monosomy X (Turner). In addition, among the other 69 samples analyzed by MLPA, 6 were uninterpretable. The abnormalities found by this technique were 2 monosomies X. For the remaining samples, the MLPA was negative. We conducted a retrospective parallel study based on the analysis of a sample recruited in Sidi Bel Abbes, women who have had an AS and were admitted to the maternity hospital Abdelkader Hassani, Sidi Bel Abbes ; and a sample recruited in Clermont-Ferrand : women who underwent AS for which a levy to establish the karyotype product miscarriage had been addressed in the Department of Cytogenetics of CHU Estaing, Clermont-Ferrand. This study covered a period of six years, from January 2005 to December 2010. The techniques of FISH and MLPA are simple, rapid and sensitive tools for the detection of chromosomal rearrangements. They represent a very interesting alternative to cell culture and allow diagnosis for genomic disorders undetectable by conventional techniques.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-03-22T15:32:25Z No. of bitstreams: 2 Dissertação - Flávio Adalberto Gomes - 2018.pdf: 15036236 bytes, checksum: 6f6f7663415c300c3d9a27bd77d12300 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-23T14:55:24Z (GMT) No. of bitstreams: 2 Dissertação - Flávio Adalberto Gomes - 2018.pdf: 15036236 bytes, checksum: 6f6f7663415c300c3d9a27bd77d12300 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-23T14:55:24Z (GMT). No. of bitstreams: 2 Dissertação - Flávio Adalberto Gomes - 2018.pdf: 15036236 bytes, checksum: 6f6f7663415c300c3d9a27bd77d12300 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-21 / This work presents methodology for construction and optimization of mathematical structures that represent experimental data. A hybrid optimization process between an heuristic method and a direct search deterministic method is used. Input and output values of real systems are used and some mathematical expressions are formulated to be used as test and validation. The results are compared with traditional methods, analyzing the oscillations at the edges. The proposed method presents a feasible solution for experimental data analysis and extrapolation, with reduced mathematical expressions. / Este trabalho apresenta metodologia para construção e otimização de estruturas matemáticas que representem dados experimentais. É utilizado processo de otimização híbrido entre método heurístico e método determinístico de busca direta. São utilizados valores de entrada e saída de sistemas reais e são formuladas algumas expressões matemáticas para serem utilizadas como teste e validação. Os resultados são comparados com métodos tradicionais, analisando as oscilações nas bordas. O método proposto apresenta solução viável para análise de dados experimentais e extrapolação, com expressões matemáticas reduzidas.

Passiflora (Passifloraceae) é um grande gênero de espécies vegetais encontradas, principalmente, na flora tropical. Algumas passifloras são cultivadas como plantas ornamentais, frutíferas ou exploradas pelas suas propriedades medicinais. A principal espécie comercial brasileira, o maracujá-azedo (Passiflora edulis f. flavicarpa, 2n = 18), ocupa 95% da área plantada. Os frutos são consumidos in natura ou processados pela indústria de suco. Estudos genéticos e cromossômicos têm sido gerados para esta espécie. Entretanto, devido ao pequeno tamanho e a similaridade morfológica dos seus cromossomos, as medições do cariótipo convencional do maracujá-azedo têm levado a resultados inconsistentes, sendo necessário o desenvolvimento de marcadores cromossomos-específicos. Estes marcadores são produzidos a partir da identificação de sequências de cópia-única em clones de bibliotecas de BACs (Bacterial Artificial Chromosome), que são utilizadas como sondas em ensaios de FISH (Hibridização in situ Fluorescente). Neste trabalho, foi construída uma biblioteca genômica de maracujá-azedo em BACs contendo 82.944 clones, com tamanho médio dos insertos de 108 kb, e provendo uma cobertura de seis vezes o genoma. A biblioteca apresentou baixa contaminação com cpDNA e mtDNA (~0,04% e 0%, respectivamente), e foi possível o isolamento de oito clones contendo genes putativos de P. edulis f. flavicarpa. Estes clones foram marcados e utilizados como sondas em ensaios de FISH. Destas sondas, quatro apresentaram sinal único de hibridização, e foram mapeadas nos cromossomos 1 (gene ERS), 3 (gene ACCO) e 4 (genes G3PD e CYCD1). As demais sondas (genes LOX, NDID e MIPS) apresentaram sinais de hibridização subteloméricos ou pericentroméricos, indicando a presença de DNA repetitivo nos clones; a sonda contendo o gene EMB não revelou sinal fluorescente. Com base em análises de FISH, definiu-se, no presente trabalho, um novo cariótipo para o maracujá-azedo, com marcadores específicos para os cromossomos 1, 3 e 4, localizando, também, sítios de DNAr 45S nos cromossomos 7 e 8, e um sítio de DNAr 5S no cromossomo 5. A exploração da biblioteca de BAC, bem como o mapa físico aqui estabelecido, representa importantes avanços para guiar pesquisas futuras sobre o gênero Passiflora. / Passiflora (Passifloraceae) is a large genus of plant species essentially found in the tropical flora. Some passiflora are grown as ornamentals, cultivated for their edible fruits, or exploited due to their medicinal properties. The main Brazilian commercial species, the yellow passion fruit (Passiflora edulis f. flavicarpa, 2n = 18) occupies 95% of all planted orchards. The fruits are eaten fresh or used for industrial juice production. Genetic and chromosomal studies have been carried out on the species. However, due to the small size and morphological similarity of their chromosomes, the conventional measures of the karyotype have produced some inconsistent results, being imperative the development of chromosome-specific markers. These markers are produced by identifying BAC (Bacterial Artificial Chromosome) library clones that harbor single copy sequences, which are used as probes in FISH (Fluorescent in situ Hybridization) assays. In the present work, a yellow passion fruit genomic BAC library of 82.944 clones was constructed, with average insert sizes of 108 kb, and covering six times the genome equivalent. The library has shown a low level of cpDNA and mtDNA contamination (~0.04% and 0%, respectively), and it was possible the isolation of eight clones harboring putative genes of P. edulis f. flavicarpa. These clones were labeled and used as probes in FISH assays. Of these probes, four have shown single hybridization signals, and they were mapped on chromosome 1 (ERS gene), 3 (ACCO gene), and 4 (G3PD and CYCD1 genes). The other probes (LOX, NDID and MIPS gene) revealed subtelomeric or pericentromeric signals, suggesting the presence of repetitive DNA sequences in the clones; the probe harboring the EMB gene did not reveal any hybridization signal. Based on FISH analyses, a new karyotype for the passion fruit was established in the present work, with specific markers in chromosomes 1, 3 and 4; we also mapped 45S rDNA sites in chromosomes 7 and 8, and one 5S rDNA site in chromosome 5. The exploitation of the BAC library, as well as the physical map here established, represents novel and essential advances to guide future researches on the Passilfora genus.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-10-10T21:54:56Z No. of bitstreams: 2 Tese - Saulo José Linhares de Siqueira - 2012.pdf: 3918109 bytes, checksum: cfb7931590a50ed74838d6c8cefab1ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-13T20:44:05Z (GMT) No. of bitstreams: 2 Tese - Saulo José Linhares de Siqueira - 2012.pdf: 3918109 bytes, checksum: cfb7931590a50ed74838d6c8cefab1ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-13T20:44:05Z (GMT). No. of bitstreams: 2 Tese - Saulo José Linhares de Siqueira - 2012.pdf: 3918109 bytes, checksum: cfb7931590a50ed74838d6c8cefab1ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-30 / Species of Trichoderma are commercially applied as biological control agents and are antagonists of important plant pathogenic fungi (as Rhizoctonia, Sclerotinia and Fusarium species) due to its mycoparasitic characteristics. Research has been performed to have a better comprehension of molecular aspects of the biocontrol mechanisms performed by Trichoderma and to find isolates with high antagonistic potential against plant pathogens. In the present study the expression of mycoparasitism-related genes was performed T. asperellum T00 and T. harzianum ALL42 (Enzimologia group ICB/UFG fungal collection) that have great potential as biocontrol agent. Each chapter of this work refers to one of the species studied. In Chapter 1 T. asperellum isolate T00, known to produce high levels of cell wall degrading enzymes, has its β-1,3-glucanases enzymes and genes (tag83 and tag27) studied. The gene tag27 was cloned and characterized and codes to an 27kDa endo-β-1,3glucanase with and 285 aminoacids and 96% similar to a glucanases from T.atroviride. The enzyme was detected when T. asperellum was grown in Rhizoctonia solani or Sclerotinia sclerotiorum cell wall-containing media but not in Fusarium oxysporum cell wall-containing media. The tag83 and tag27 genes was repressed in media containing glucose as carbon source and upregulated in cell wall containing media and during plate confrontation tests with pathogenic fungi. Chapter 2 shows T. harzianum isolate ALL42 genes involved in mycoparasitism against R. solani or S. sclerotiorum detected using subtractive hybridization approach. T. harzianum was grown with R. solani or S. sclerotiorum cell wall as carbon source and the RNA used both as tester and driver in each of two subtractive library constructed. Sequencing analysis resulted in 47 genes related with growth in R. solani cell wall media and 114 genes related with growth in S. sclerotiorum cell wall media. To confirm the obtained data, 18 genes were tested by quantitative real time RT-PCR and 9 were differentially expressed in the same condition of the library they were detected. Five of these genes were also differentially expressed during plate confrontation assay with the respective pathogen, two of them expressed during contact with R. solani (cutinase and alginate lyase) and 3 during contact with S. sclerotiorum (hsp98, serin endopeptidase and a hypotetic gene). The results presented in this study provides additional information about the role of 1,3glucanase genes in mycoparasitism and of other genes related to antagonism against specific pathogens, providing helpful insights in the mechanism of biocontrol performed by Trichoderma. / Espécies do gênero Trichoderma são eficientes antagonistas de fungos fitopatogênicos, como as espécies Rhizoctonia, Sclerotinia e Fusarium, e são comercializados como agentes de controle biológico principalmente por sua característica de micoparasita. Muitos estudos têm sido feitos para compreender as bases moleculares dos mecanismos de biocontrole de Trichoderma e também para encontrar espécies com alto potencial de antagonismo contra fitopatógenos. O objetivo deste trabalho foi analisar a expressão de genes relacionados ao micoparasitismo de T. asperellum T00 e T. harzianum ALL42 (Enzimologia ICB/UFG) que possuem potencial para uso como agente de biocontrole. Este trabalho foi dividido em dois capítulos. O Capítulo 1 se refere ao fungo T. asperellum T00 e suas β-1,3-glicanases (TAG83 e TAG27) que degradam componentes da parede celular de fungos fitopatógenos. O gene tag27 codifica para uma endo-β-1,3glicanase de 27kDa que possui 285 resíduos de aminoácidos e apresentou 96% de similaridade com uma enzima de T. atroviride. A enzima foi secretada em meio de cultura contendo parede celular de Rhizoctonia solani e Sclerotinia sclerotiorum, mas não em meio com parede de Fusarium oxysporum. A expressão dos genes tag83 e tag27 foi reprimida na presença de glicose e ativada tanto na presença de parede celular dos fitopatógenos quanto durante o contato entre os fungos em placa. O Capítulo 2 trata do fungo T. harzianum isolado ALL42 e de genes identificados pela técnica de hibridização subtrativa relacionados especificamente ao biocontrole contra R. solani e S. sclerotiorum. Foram construídas duas bibliotecas subtrativas sendo que os RNAs utilizados como condição teste e controle da subtração foram obtidos de T. harzianum crescido em meio contendo parede celular de R. solani ou S. sclerotiorum. As bibliotecas foram sequenciadas resultando em 47 genes relacionados ao crescimento em meio com R. solani e 114 genes relacionados ao crescimento em meio com S. sclerotiorum. Dos 18 genes escolhidos para validação da biblioteca por RT-PCR em tempo real, 9 se mostraram diferencialmente expressos na condição correspondente à biblioteca em que foram identificados. Dentre estes, 5 genes se mostraram também diferencialmente expressos em experimento de confronto em placa, 2 deles mais expressos contra R. solani (cutinase e alginato liase) e 3 contra S. sclerotiorum (hsp98, serina endopeptidase e um de função hipotética). Os resultados obtidos com as duas linhagens fornecem informações adicionais sobre genes de β-1,3-glicanases, conhecidamente envolvidos no processo de micoparasitismo, e sobre genes relacionados ao antagonismo de patógenos específicos, além de contribuir para o conhecimento relacionado ao biocontrole realizado por fungos do gênero Trichoderma.

São freqüentes alterações em vias de sinalização de genes de desenvolvimento, no que diz respeito ao carcinoma epidermóide de boca (CEB). Vinte e nove casos de CEB e tecido não tumoral adjacente à neoplasia foram investigados através da técnica de hibridização “in situ”. Os transcritos dos genes WNT5A e HOXB5 foram observados em todos os casos de tumor. A hibridização “in situ” revelou que o WNT5A estava mais expresso em tumores bem diferenciados. Adicionalmente, observou-se transcritos do WNT5A em glândulas salivares menores, estroma glandular, estroma tumoral e alguns vasos sanguíneos Entretanto, com respeito ao HOXB5 não foi possível estabelecer mudança do padrão do transcrito nos diferentes graus histológicos nas amostras de CEB. O HOXB5 também pôde ser identificado em alguns fragmentos de glândulas salivares menores, tecido muscular e em endotélio. Os resultados do presente estudo sugerem que a expressão dos genes WNT5A e HOXB5 podem estar relacionadas com a diferenciação e progressão do câncer de boca. / Disruption in developmental genes pathway are common in oral squamous cell carcinoma (OSCC). This study investigated the pattern of expression of two developmental genes, WNT5A and HOXB5, in 29 cases of OSCC and adjacent non tumoural tissue using in situ hybridization technique. Transcripts for WNT5A and HOXB5 were detected in all tumoral samples. In situ hybridization technique demonstrated that WNT5A transcripts were mainly detected in well differentiated tumors when compared with moderately and undifferentiated OSCC. WNT5A transcripts were also observed in accessory salivary glands, glandular stroma, and vessels. Therefore, for the HOXB5 transcript it was not possible to stability a relationship with the tumoral histological grade. The expression of HOXB5 transcripts in non tumoral samples was detected in salivary glands, glandular stroma, endothelium, and muscle. Results suggest that WNT5A and HOXB5 genes play a possible role in tumor differentiation and cancer progression.

Neste trabalho investigou-se o desenvolvimento de biofilmes anaeróbios em um sistema de laboratório chamado de \"Modified Robbins Device\" (MRD). O objetivo específico foi o de comparar a organização das células anaeróbias, particularmente daquelas que são comuns em lodos de esgoto, sobre superfícies hidrofílicas (vidro) e hidrofóbicas (polipropileno). A hibridação in situ com sondas fluorescentes complementares ao RNAr 16S específicas para domínio e grupos e a microscopia confocal de varredura a laser foram utilizadas para verificar a composição microbiana dos biofilmes, bem como do inóculo. Foram realizados dois tipos de experimentos, um com culturas puras de metanogênicas e outro com células oriundas de lodo granulado anaeróbio. As culturas puras de metanogênicas, Methanobacterium formicicum (DSM 1535), Methanosaeta concilii (DSM 3671) e Methanosarcina barkeri (DSM 800) foram usadas como inóculo para a formação dos biofilmes no interior do MRD durante 9 dias. Os resultados mostraram que as três espécies colonizaram ambas as superfícies após o segundo e sétimo dia de ensaio. No segundo experimento, o MRD foi inoculado com um consórcio microbiano anaeróbio e a formação do biofilme foi estudada durante 22 dias. As amostras dos biofilmes bem como aquelas retiradas do frasco-reservatório de células apresentaram composição microbiana semelhante, ambas foram dominadas por Archaeae metanogênicas hidrogenotróficas relacionadas com membros da família Methanobacteriaceae, já que foram detectadas com a sonda MB1174. Este grupo contribuiu com cerca de 44 a 90% do total de células coradas com DAPI e foi morfologicamente semelhante à Methanobacterium e Methanobrevibacter. As células detectadas com a sonda específica para membros da ordem Methanomicrobiales (MG1200) representaram cerca de 2 a 18,0% do total de células coradas com DAPI no frasco-reservatório e de 0,1 a 2,0% nas amostras dos biofilmes. Estas células foram ) morfologicamente semelhantes à Methanospirillum, também uma metanogênica hidrogenotrófica. Não foram detectadas células pertencentes à família Methanosarcinaceae, pois a hibridação com a sonda MSMX860 foi negativa. Células que hibridaram com a sonda específica para o Domínio Bacteria (EUB338) representaram cerca de 2 a 18% do total de células coradas com DAPI. Os resultados mostraram que as Archaeae metanogênicas hidrogenotróficas que foram predominantes no inóculo também dominaram os biofilmes que se desenvolveram em ambas as superfícies, vidro e polipropileno. Os dados desse trabalho sugerem que a hidrofobicidade do material suporte não influenciou o desenvolvimento e a composição microbiana dos biofilmes anaeróbios, considerando as condições específicas dos ensaios realizados. / In this study the development of anaerobic biofilms using a laboratory system called modified robbins device (MRO) were investigated. We were especially interested in comparing the organization of anaerobic cells, particularly those that are very common in domestic sewage sludge, in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescence in situ hybridization (FISH) with domain and group speci fie probes that target intracell ular 16S rRNA and confocal laser scanning microscopy (CLSM) were used to investigate the microbial composition of both the inoculum and anaerobic biofilms. Two sets of experiments were carried, one with pure methanogenic organisms and the other with cells from a mesophilic anaerobic granular sludge. The pure methanogenic cultures, Methanobacterium formicicum (OSM 1535); Methanosaeta conci/ii (OSM 3671) and Methanosarcina barkeri (OSM 800) were used to seed the MRD to allow the development of biofilms over 9 days. The results showed that ali the three species were colonizing both surfaces after 2 and 7 days of experimental period. In the second experiment, the biofilm reactor was seeded with a microbial anaerobic consortium and biofilm forrnation was studied during 22 days. Biofilm and culture vessel samples showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MBI174). This group accounted for 44 to 90% of the OAPI-stained cells and morphologically resembled Methanobacterium and Methanobrevibacter. Cells detected with the Methanomicrobiales specific probe (MG 1200) accounted for 2 to 18.0% of the OAPI-stained cells in the culture vessel and 0.1 to 2.0% in the biofilm samples. These cells were morphologically similar to Methanospiriltum, also a hydrogenotrophic methanogen. No cells were detected by the Methanosarcinaceae specific probe (MSMX860). Cells which hybridized to the Bacteria specific probe (EUB338) accounted for the remaining 3 to 18% of the DAPI-stained cells. The results showed that the hydrogenotrophic methanogenic Archaea cells predominated in the inoculum and the biofilms that developed on both surfaces, glass and polypropylene. Our data suggest that the hydrophobicity of the support material did not influence the development and the microbial composition of anaerobic biofilms, considering specific conditions of the experiments.

Made available in DSpace on 2017-07-10T19:25:13Z (GMT). No. of bitstreams: 1 isamara.pdf: 1125655 bytes, checksum: 8f7b2f7606310b4ddb2713e9e4043ec0 (MD5) Previous issue date: 2012-02-13 / Chemical contamination removal in soil and water depends on microbiological community that is able to degrade these compounds. There is a great evolutionary interest on studying microorganisms that metabolize the xenobiotic ones, since they have relatively been seen as new in the last five decades. Little is known about structure variation of microbiological community of soil due do the absence and presence of s-triazine herbicides.Unlike crop dependent methods that require time to detect bacteria, molecular techniques have been developed to identify individual species in mixed populations under natural enviromments. Fluorescence in situ Hibiridization (FISH) technique overcomes some difficulties that are found out in other molecular techniques, as it does not need DNA isolation and amplification steps and allows the identification of specific genes in intact cells. Thus, this study aimed at comparing the absence/presence of atrazine effect on bacteriological community structure in soil according to the phylogenetic aspect. Target probes were used on subdivisions of alpha, beta and gamma Proteobacteria, gram-positive bacteria with high G+C content, ammonia oxidizing bacteria, nitrite oxidizing bacteria and Planctomycetes. It was also used an AtzB1 specific probe to check the atzB gene presence, which makes part of s-triazine degradation. Bacteriological amount was determined by direct counting on epifluorescence microscopy, while the corresponding values to each probe were expressed in percentages of the total count with DAPI for each sample. According to this study, positive cells were found out for all probes used in both soils, but the abundance of all groups was lower in soil contaminated with atrazine herbicide, thereby demonstrating its negative influence. Planctomycetes was the most affected group with 57% lower abundance in contaminated soil. The nitrite oxidizing bacteria was the second most affected group followed by β-Proteobacteria. It was also detected the gene atzB presence, so, it can be inferred that there are potentially degrading s-triazine bacteria in both soils. / A remoção da contaminação química no solo e água é dependente principalmente da presença de uma comunidade microbiana capaz de degradar tais compostos. A existência de microorganismos capazes de metabolizar xenobióticos é de um considerável interesse evolucionário, uma vez que estes compostos são relativamente novos no planeta nas últimas cinco décadas. Pouco se sabe sobre a variação da estrutura da comunidade microbiana do solo em função da ausência e presença dos herbicidas s-triazínicos. Diferentemente dos métodos dependentes de cultivo, que requerem tempo para a detecção de bactérias, técnicas moleculares vem sendo desenvolvidas para o reconhecimento de espécies individuais em populações mistas em ambientes naturais. A técnica de Hibridização Fluorescente in situ (FISH) supera algumas dificuldades encontradas com outras técnicas moleculares, pois dispensa as etapas de isolamento e amplificação de DNA e permite a identificação de genes específicos em células intactas. Em virtude disso, o presente trabalho teve como objetivo comparar o efeito da ausência/presença de atrazina na estrutura da comunidade bacteriana do solo no aspecto filogenético. Foram utilizadas sondas alvo para as subdivisões de Proteobactéria alfa, beta e gama, bactérias Gram-positivas com alto teor de G + C e Betaproteobactérias oxidantes de amônia, Bactérias oxidantes de Nitrito e Planctomicetos. Também foi utilizada uma sonda específica AtzB1 para verificar a presença do gene atzB que está envolvido na degradação das s-triazínas. A abundância bacteriana foi determinada através de contagem direta em microscopia de epifluorescência, e os valores correspondentes a cada sonda foram expressos em porcentagem da contagem total com DAPI para cada amostra. No presente estudo células positivas para todas as sondas utilizadas foram encontradas em ambos os solos, porém a abundância de todos os grupos foi menor no solo contaminado com o herbicida atrazina, demonstrando dessa forma a influência negativa do mesmo, sendo o grupo mais afetado o dos Planctomicetos com uma abundância 57% menor em solo contaminado. O segundo grupo mais afetado foi o das bactérias oxidantes de nitrito seguido pelo grupo das β-Proteobactérias. Foi também detectado no presente estudo a presença do gene atzB demonstrando que em ambos os solos existem bactérias potencialmente degradadoras de s-triazinas.

Made available in DSpace on 2017-05-12T14:48:37Z (GMT). No. of bitstreams: 1 isamara.pdf: 1125655 bytes, checksum: 8f7b2f7606310b4ddb2713e9e4043ec0 (MD5) Previous issue date: 2012-02-13 / Chemical contamination removal in soil and water depends on microbiological community that is able to degrade these compounds. There is a great evolutionary interest on studying microorganisms that metabolize the xenobiotic ones, since they have relatively been seen as new in the last five decades. Little is known about structure variation of microbiological community of soil due do the absence and presence of s-triazine herbicides.Unlike crop dependent methods that require time to detect bacteria, molecular techniques have been developed to identify individual species in mixed populations under natural enviromments. Fluorescence in situ Hibiridization (FISH) technique overcomes some difficulties that are found out in other molecular techniques, as it does not need DNA isolation and amplification steps and allows the identification of specific genes in intact cells. Thus, this study aimed at comparing the absence/presence of atrazine effect on bacteriological community structure in soil according to the phylogenetic aspect. Target probes were used on subdivisions of alpha, beta and gamma Proteobacteria, gram-positive bacteria with high G+C content, ammonia oxidizing bacteria, nitrite oxidizing bacteria and Planctomycetes. It was also used an AtzB1 specific probe to check the atzB gene presence, which makes part of s-triazine degradation. Bacteriological amount was determined by direct counting on epifluorescence microscopy, while the corresponding values to each probe were expressed in percentages of the total count with DAPI for each sample. According to this study, positive cells were found out for all probes used in both soils, but the abundance of all groups was lower in soil contaminated with atrazine herbicide, thereby demonstrating its negative influence. Planctomycetes was the most affected group with 57% lower abundance in contaminated soil. The nitrite oxidizing bacteria was the second most affected group followed by β-Proteobacteria. It was also detected the gene atzB presence, so, it can be inferred that there are potentially degrading s-triazine bacteria in both soils. / A remoção da contaminação química no solo e água é dependente principalmente da presença de uma comunidade microbiana capaz de degradar tais compostos. A existência de microorganismos capazes de metabolizar xenobióticos é de um considerável interesse evolucionário, uma vez que estes compostos são relativamente novos no planeta nas últimas cinco décadas. Pouco se sabe sobre a variação da estrutura da comunidade microbiana do solo em função da ausência e presença dos herbicidas s-triazínicos. Diferentemente dos métodos dependentes de cultivo, que requerem tempo para a detecção de bactérias, técnicas moleculares vem sendo desenvolvidas para o reconhecimento de espécies individuais em populações mistas em ambientes naturais. A técnica de Hibridização Fluorescente in situ (FISH) supera algumas dificuldades encontradas com outras técnicas moleculares, pois dispensa as etapas de isolamento e amplificação de DNA e permite a identificação de genes específicos em células intactas. Em virtude disso, o presente trabalho teve como objetivo comparar o efeito da ausência/presença de atrazina na estrutura da comunidade bacteriana do solo no aspecto filogenético. Foram utilizadas sondas alvo para as subdivisões de Proteobactéria alfa, beta e gama, bactérias Gram-positivas com alto teor de G + C e Betaproteobactérias oxidantes de amônia, Bactérias oxidantes de Nitrito e Planctomicetos. Também foi utilizada uma sonda específica AtzB1 para verificar a presença do gene atzB que está envolvido na degradação das s-triazínas. A abundância bacteriana foi determinada através de contagem direta em microscopia de epifluorescência, e os valores correspondentes a cada sonda foram expressos em porcentagem da contagem total com DAPI para cada amostra. No presente estudo células positivas para todas as sondas utilizadas foram encontradas em ambos os solos, porém a abundância de todos os grupos foi menor no solo contaminado com o herbicida atrazina, demonstrando dessa forma a influência negativa do mesmo, sendo o grupo mais afetado o dos Planctomicetos com uma abundância 57% menor em solo contaminado. O segundo grupo mais afetado foi o das bactérias oxidantes de nitrito seguido pelo grupo das β-Proteobactérias. Foi também detectado no presente estudo a presença do gene atzB demonstrando que em ambos os solos existem bactérias potencialmente degradadoras de s-triazinas.

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La mobilité, en particulier la mobilité urbaine est aujourd’hui un thème d’une grande actualité politique et scientifique, qui soulève des questions et engage des démarches qui vont bien au-delà des problématiques habituelles des transports (Bonnet, Desjeux, 2000, p.201). Au centre de la vie quotidienne, économique et/ou sociale des acteurs locaux, la mobilité est un problème aux multiples enjeux : impact sur le réchauffement climatique, droit à la mobilité, économie, urbanisme et cadre de vie, équilibre entre ville et campagne, sécurité routière et santé publique. Le besoin de mobilité peut être traité, voire satisfait, de plusieurs façons, soit en apportant une réponse au besoin de déplacement soit en apportant une réponse au mode de mobilité. La question de la mobilité est un des enjeux majeurs pour l'accès aux emplois comme aux services dans les territoires et lorsque celle-ci se heurte au manque de transports collectifs, publics et privés, ce manque entraîne l'isolement voire l’exclusion, surtout pour les populations les plus fragiles avec pour corollaire la saturation des infrastructures routières par le recours à l’utilisation intensive des véhicules individuels dans les zones péri-urbaines. Ainsi, le secteur des transports a besoin d’utiliser les technologies de l’information. Ces technologies de l’information (TI) sont au centre de nombreuses réflexions sur la mobilité et la non mobilité, en particulier dans le cadre de la réduction des déplacements physiques rendus nécessaire par la maitrise des émissions de gaz à effet de serre. Les technologies de l’information en hybridant les territoires, peuvent être des outils d’une gestion globale et durable des déplacements territoriaux. Dans cette recherche, nous avons mis l’accent sur le croisement de l’« Intelligence Territoriale et mobilité durable » avec une orientation sur le déplacement partagé, biens et personnes, facteur de communication sociale, et de développement d’équilibre territorial en menant en parallèle une double étude entre l’Afrique de l’Est (la CAE) et l’Euro Méditerranée (PACA et Corse), pour en tirer des enseignements. Bertacchini, Girardot, et Grammacia (2006), présentent l’intelligence territoriale (IT) comme étant une théorie, posture, et démarche ascendante d’intelligence collective fondée sur une approche citoyenne de la valorisation territoriale. Nous avons insisté sur la nécessité de fonder l'action sur une analyse fine des besoins des habitants en matière de déplacement et comment inventer de nouvelles modalités d'organisation des services par le développement des stratégies de communications qui s’inspirent de l’intelligence territoriale (IT), la cohésion sociale, la convivialité, l’équité, les hypothèses de l’IT et la capacité de la communication à promouvoir la médiation territoriale / We actually do make, in our PhD research work, a tough choice on studying mobility at the crossroads of Territorial Intelligence Process and Sustainable Mobility through Communication Science sensitive approach with the help of IT (Information Technologies). Because mobility, or absence of, can lead to isolation (may be seclusion) or even exclusion, especially for the most vulnerable people, is a subject of high political and scientific relevance, raising questions and involving processes far beyond the usual and specific questions of transportation (Bonnet, Desjeux, 2000, p. 201). We stress the point about the link between « Territorial Intelligence and Sustainable Mobility » with a focus on shared displacement, goods and people, which means, and can be seen as, a social communication factor and development of territorial equilibrium; We conducted a simultaneous study both in East Africa and Euro Mediterranean space (PACA and Corsica), to recap learned lessons. Bertacchini, Girardot, and Grammacia (2006), shown Territorial Intelligence (IT) as a theory, posture, and bottom-up approach of collective intelligence based on citizen's approach of territorial development. And for that purpose, we underlined the need of action based on analysis of travel needs of residents and how to create new ways of structuring mobility offer through the development of communications suggested by Territorial Intelligence (IT), social cohesion, conviviality, equity, assumptions of IT and with the ability of communication to promote territorial mediation. For local stakeholders mobility is a matter of life and at the crossroads of their daily economic and social life, mobility is an issue with multiple challenges: impact on global warming, rights to mobility, economy development, jobs accessibility, town planning and environment, road safety and public health. The need for mobility can be addressed or even satisfied by several ways, either by responding to the need for mobility, or by providing a response to the mode of mobility. Thus, the transportation sector needs using information technology. These information technologies are studied through numerous ways on mobility and non-mobility, particularly focusing on how reducing physical displacements made necessary and call for the control of greenhouse gas emissions. Information technologies by hybridizing territories, as described within Territorial Intelligence assumptions, can be tools for a comprehensive and sustainable management of territorial displacements.