Enhanced Reductive Bioremediation (ERB) is a sustainable remediation technology for the in situ treatment of chlorinated solvent contamination in aquifers. However, monitoring efforts employed to measure performance metrics rely on inferences of the subsurface environment from water samples collected at monitoring wells, ignoring the microbial activity that occurs at the granular level of aquifer sediment. This study compared potential bioavailable organic carbon (PBOC) and microbial diversity of two ERB sites. A two-sample t-test and a one-way ANOVA test with Tukey's HSD were performed to show differences between ERB and non-ERB samples and their degree of variability at selected geospatial locations downgradient of ERB treatment. Non-parametric multidimensional scaling (MDS) with similarity analysis was performed along with other data visualization plots to show microbial diversity. At Tinker AFB, results from the t-test showed that the PBOC concentrations from the ERB samples were statistically significantly greater than the samples without treatment (95% confidence; p-value = 0.018). For Dover AFB, results from the ANOVA with Tukey's HSD showed that there is a significant difference between the sample (DV3) collected in the ERB treatment zone to all other samples upgradient and downgradient of the ERB treatment. MDS and similarity analysis performed on relative abundance results from the Illumina MiSeq Sequencing of 16S rRNA genes showed large similarities among the samples within each site and the only observed differences occurred when comparing any sample to DV3, nearest to treatment. / Master of Science

"This We Have Now" is a five-movement work inspired by various spiritual texts, some liturgical, while others more secular in approach. Each one is connected by several motives that are eventually reunited within the final movement. The title comes from a poem under the same name by Rumi (1207-1273), which serves as the crux for the underlying, grander arch of this spiritually inspired work. This synthesis of sacred and secular, traditional with contemporary, is a personal expression of my own beliefs and a musical representation of the interconnecting belief systems throughout the world. / text

In the past few years, interferon-induced transmembrane (IFITM) proteins have been identified as important antiviral factors. The current understanding of IFITMs suggests that they localise within distinct cellular compartments from where they exert their broad antiviral role. For example, IFITM1 localises to the plasma membrane and restricts viruses that do not require endocytosis to infect host cells. In contrast, IFITM2 and IFITM3 are found in the early and late endosomes, respectively, and are potent inhibitors of viruses that depend on endosomal pathways for infection. I begin this dissertation by providing some background on the biology and function of IFITM proteins, including details of in vitro assays that have helped elucidate IFITMs role as antiviral factors. I also describe some early candidate-gene association studies that have attempted to correlate genetic variation within these genes with variation in viral restriction. I also describe how genetic association studies have been used more broadly to understand the biology underlying both infectious and non-communicable diseases. Evidence from in vitro, and in vivo work has demonstrated the IFITMs role as potent antiviral factors, however, no genome-wide association study has reported any significant associations to genetic variant in or around these genes. In Chapter 2, I explore reasons why this may be the case and calculate the coverage of IFITM genes by commercially available genotyping arrays. I show that IFITM2 and IFITM3 are amongst the 7% of all protein coding genes with less than 25% common variant (minor allele frequency > 5%) coverage across all arrays. Poor coverage of genetic variation is therefore one explanation for the lack of IFITM associations in GWAS. The lack of coverage in the genotyping arrays led me to explore other tools to capture variation in the IFITM region. I employ a targeted sequencing method using two different sequencing technologies: short-read sequencing (Illumina MiSeq) and single molecule, real-time sequencing (PacBio RS). Conventional pulldown protocols for targeted sequencing have not been designed for single molecule, real-time sequencing at the time, thus in Chapter 3, I provide some details of the optimisation work required to adapt the targeted method for PacBio sequencing. I then assess the performance of the method for both Illumina and PacBio sequencing. In Chapter 4, I apply the targeted sequencing method described in Chapter 3 to test genetic variants in and around IFITM1, IFITM2 and IFITM3 for association with rapid disease progression in HIV. I also explore the contribution of rare genetic variants (MAF < 1%) to this phenotype by testing for a differential enrichment between cases and controls across each of the three genes. Studies in vitro have also reported that IFITM proteins are potent restrictors of dengue virus infection. In Chapter 5, I use genotype data across a cohort of 2,008 Vietnamese children diagnosed with dengue haemorrhagic fever (DHF) and 2,018 cord blood controls to test if common variants are associated with the disease.

No description available.

L’anthropisation est la principale source de perturbations dans les grottes, et dans la grotte de Lascaux cela a entrainé la prolifération de microorganismes et des altérations de paroi menaçant sa conservation. L’objectif de cette thèse était de mieux comprendre l’écologie des microorganismes colonisant la grotte de Lascaux, en identifiant sa communauté microbienne à différentes échelles spatio-temporelles, de caractériser les facteurs qui structurent cette communauté et d’en étudier la dynamique fonctionnelle en utilisant le séquençage à haut débit d’acides nucléiques, une nouvelle approche à Lascaux. Une comparaison à l’échelle régionale de différentes grottes de Dordogne, plus ou moins anthropisées a été réalisée, puis à une échelle locale avec l’étude de salles de Lascaux, le Passage pour évaluer le rôle des substrats minéraux, et l’Abside qui présente deux types d’altérations (taches noires et zones sombres). Nos résultats montrent que les grottes anthropisées (dont Lascaux) ont des communautés microbiennes particulières. Le substrat minéral structure davantage la communauté du Passage que la présence de taches. Dans l’Abside, bien que les zones sombres soient visuellement différentes des taches noires, les communautés microbiennes présentent des similarités fortes, et notamment le rôle des interactions entre les collemboles, les champignons noirs et des bactéries. Enfin, les profils métatranscriptomiques diffèrent en fonction des salles et de la présence de taches. Ce projet a permis de caractériser l’écologie de la communauté microbienne de Lascaux et permet de mieux comprendre le fonctionnement microbien de la grotte / Anthropisation is the main source of disturbance in the caves, and in the cave of Lascaux it has led to the proliferation of microorganisms and alterations of the wall threatening its conservation.The objective of this thesis was to better understand the ecology of microorganisms colonizing the cave of Lascaux, by identifying its microbial community at different spatio-temporal scales, to characterize the factors that structure this community and to study its functional dynamics in using high throughput sequencing of nucleic acids, a new approach to Lascaux.A regional comparison of different Dordogne caves, more or less anthropised was carried out, then at a local scale with the study of Lascaux rooms, the Passage to evaluate the role of mineral substrates, and the Apse which presents two types of alterations (black spots and dark areas).Our results show that anthropogenic caves (including Lascaux) have particular microbial communities. The mineral substrate structures the Passage community more than the presence of spots. In the Apse, although dark areas are visually different from black spots, microbial communities show strong similarities, including the role of interactions between collembolans, black fungi, and bacteria. Finally, the metatranscriptomic profiles differ according to the rooms and the presence of spots.This project has made it possible to characterize the ecology of the Lascaux microbial community and to better understand the microbial functioning of the cave

O 4-Nonilfenol (4-NF) é o principal produto formado a partir da degradação do Nonilfenol etoxilado, surfactante não iônico amplamente utilizado em formulações de uso doméstico e industrial. Objetivou-se neste estudo desenvolver método de quantificação de 4-NF em HPLC; avaliar a remoção de 4-NF em reatores em batelada com co-substratos (etanol, metanol e fumarato) e avaliar a remoção e degradação de 4-NF em Reator Anaeróbio de Leito Fluidificado (RALF) em escala aumentada (20L), bem como caracterizar a comunidade microbiana estabelecida no material suporte por meio das técnicas de PCR/DGGE do gene RNAr 16S e sequenciamento massivo via plataforma Illumina-Miseq&#174;. O RALF foi preenchido com areia como material suporte, operado com Tempo de Detenção Hidráulico (TDH) de 18,2±1,1 horas e alimentado com meio de cultura acrescido de 4-NF PESTANAL&#174; (Sigma-Aldrich&#174;). Análises de monitoramento da concentração de 4-NF e matéria orgânica, bem como, dos parâmetros físico-químicos foram realizadas para avaliar a estabilidade do reator quanto a remoção e degradação do composto de interesse. A operação do reator foi dividida em distintas etapas, contando com inoculação do RALF em circuito fechado, adaptação ao meio de cultura e subsequentes fases com adição de 4-NF. Para reatores em batelada a adição de 4-NF (de 288,97±96,49 a 469,98±182,42 &#181;g L-1) favoreceu a produção média de metano acumulada (de 2.292,3 para 2.744,7 mol, respectivamente) para todos os co-susbtratos testados, todavia, retardou o tempo médio de início da produção (de 15,9 h para 107,9 h), bem como reduziu a velocidade de produção (de 24,4 para 10,9 &#181;mol d-1). Para ensaio com adição de 4-NF e Fumarato foram verificados os maiores valores de produção acumulada de metano (3.163,68±169,17 &#181;mol) e de remoção de DQO (75,52±0,34% para DQO inicial de 1.242,00±27,48 mg L-1), em relação aos demais ensaios com adição de 4-NF. Para a remoção de 4-NF em reatores em batelada os valores não diferiram significativamente. Para o RALF, foram verificadas eficiências médias de remoção de DQO de 90,34±6,1% (Fase I), 94,0±1,2% (Fase II), 97,0±1,3% (Fase III) e 95±1,5% (Fase IV) e 4-NF de 73,2±11,1% (Fase II), 67,3±7,3% (Fase III) e 77,88±8,9% (Fase IV). As diferentes concentrações de 4-NF aplicadas ao RALF não afetaram a eficiência de remoção de DQO e promoveram a seleção dos microrganismos que compuseram a biomassa do leito. Os gêneros mais abundantes identificados no reator sem adição de 4-NF foram Prolixibacter, Geothrix, Klebsiella, Lactobacillus e Geobacter. Os gêneros com maior abundância relativa identificados após adição de 4-NF foram os seguintes: Geothrix, Holophaga, Elusimicrobium, Paludibacter, Lactobacillus, Aeromonas, Pelobacter, Aquaspirillum, Pseudomonas, Delftia, Acinetobacter, Arcobacter, Ignavibacterium, Treponema, Lysinibacillus e Enterococcus. / 4-Nonylphenol (4-NP) is the main product formed in the Nonylphenol ethoxylate degradation, nonionic surfactant used in formulations of domestic and industrial use. The objective of this study was to develop a method to determine 4-NP in HPLC; to evaluate the 4-NP removal in batch reactors with co-substrates (ethanol, methanol and fumarate) and removal and degradation of 4-NP in anaerobic Fluidized Bed Reactor (AFBR) on an enlarged scale (20L), as well as characterize the microbial community established in the support material by PCR / DGGE techniques of the 16S RNAr gene and massive sequencing by the Illumina-Miseq&#174; platform. The AFBR was filled with sand as carrier material, operated with Hydraulic Retention Time (HRT) of 18.2±1.1 hours and fed with synthetic sewage plus 4-NP PESTANAL&#174; (Sigma-Aldrich&#174;). Monitoring of the 4-NP concentration and organic matter, as well as physical-chemical parameters were performed to evaluate the stability of the reactor for the removal and degradation of the compound of interest. Reactor operation was divided into different stages, with inoculation of the RALF in closed circuit, adaptation to the culture medium and subsequent phases with 4-NF addition. The addition of 4-NP (from 288.97±96.49 to 469.98±182.42 &#181;g L-1) in batch reactors favored the average accumulated methane production (from 2,292.3 to 2,744.7 &#181;mol, respectively) for all tested co-susbtrates, however, delayed the mean time to start production (from 15.9 h to 107.9 h), as well as reduced production rate (from 24.4 to 10.9 &#181;mol d-1). The highest accumulated values of methane production (3,163.68 ± 169.17 &#181;mol) and COD removal (75.52±0.34% for the initial COD of 1,242±27.48 mg L-1) were verified for the addition of 4-NP and Fumarate, compared to the other tests with addition of 4-NP. For the 4-NP removal in batch reactors the values did not differ significantly. Mean values of COD removal for the AFBR were 90.34±6.1% (Phase I), 94.0±1.2% (Phase II), 94.0±1.2% (Phase III) and 97.0±1.3% (Phase IV) and 4-NF of 73.2±11.1% (Phase II), 67.3±7.3% (Phase III) and 77.88±8.9% (Phase IV). Different concentrations of 4-NP applied to the AFBR did not affect the COD removal efficiency and promoted the selection of the microorganisms that composed the bed biomass. The most abundant genera identified in the reactor without addition of 4-NP were Prolixibacter, Geothrix, Klebsiella, Lactobacillus and Geobacter. The genotypes with the highest relative abundance identified after addition of 4-NP were as follows: Geothrix, Holophaga, Elusimicrobium, Paludibacter, Lactobacillus, Aeromonas, Pelobacter, Aquaspirillum, Pseudomonas, Delftia, Acinetobacter, Arcobacter, Ignavibacterium, Treponema, Lysinibacillus and Enterococcus.

Objetivou-se com este trabalho avaliar o potencial agrícola do lodo de esgoto produzido no estado de São Paulo, bem como, verificar a possibilidade de interação entre a composição química e a abundância relativa de bactérias no lodo. Foram realizadas coletas de amostra de lodo de esgoto em 19 estações de tratamento de esgoto, em três épocas distintas. Nas amostras provenientes das três épocas foram determinados as concentrações dos 16 hidrocarbonetos policíclicos aromáticos (HPAs) listados como prioritários no monitoramento ambiental pela USEPA (acenafteno, acenaftileno, antraceno, benzo(a)antraceno, benzo(a)pireno, benzo(b)fluoranteno, benzo(ghi)perileno, benzo(k)fluoranteno, criseno, dibenzo(a,h)antraceno, fenantreno, fluoranteno, fluoreno, indeno(1,2,3-cd)pireno, naftaleno e pireno). Nas amostras da segunda época de coleta, além da presença de HPAs, determinou-se as concentrações de poluentes orgânicos emergentes (hormônios, produtos farmacêuticos e produtos de uso industrial), realizou-se a caracterização completa segundo a Resolução CONAMA 375/2006 (umidade, pH, N-Kjeldahl e inorgânico, carbono orgânico, cálcio, potássio, fósforo, magnésio, enxofre, boro, cobre, ferro, níquel, manganês, molibdênio, selênio, zinco, alumínio, arsênio, bário, cádmio, cromo, chumbo, mercúrio e sódio) e a caracterização da comunidade bacteriana através de metodologia independente de cultivo (sequenciamento illumina). Os macronutrientes em maiores concentrações no lodo de esgoto são: N > Ca > S > P > Mg > K. Os elementos inorgânicos Ni e Zn apresentaram concentração superior à máxima permitida para utilização agrícola pela resolução Conama 375/2006 em 1 e 3 amostras, respectivamente. A substância inorgânica que mais limita o enquadramento do lodo de esgoto como adubo orgânico (Instrução Normativa 27/2006) é o Hg. Os compostos benzilparabeno, bisfenol AF (BPAF), ácido perfluorooctanoico (PFOA) e tetrabromobisfenol A (TBBPA) não foram detectados. Por outro lado, cimetidina, metilparabeno, bisfenol A (BPA) e triclocarban foram detectados nas 19 amostras avaliadas. O composto presente em maior concentração é o triclocarban. As concentrações de hidrocarbonetos policíclicos aromáticos são baixas, de acordo com a norma Europeia. Os filos Proteobacteria e Bacteroidetes estão presentes em maior abundância relativa. Existe uma comunidade bacteriana núcleo nas estações de tratamento de esgoto do estado de São Paulo, composta por 81 gêneros, presentes nas 19 ETEs avaliadas, dos quais, os que estão em maior abundância relativa são Treponema, Clostridium, Propionibacterium, Syntrophus e Desulfobulbus. A elevação do pH a valores próximos de 12 reduz a diversidade microbiana. Considerando a abundância relativa e a composição química do lodo de esgoto, as estações podem ser agrupadas em três grupos distintos, sendo que um deles é influenciado principalmente pelos teores de Ca, Zn e Cu, o outro pelos teores de Fe e S e o terceiro grupo que foi influenciado pelos demais fatores avaliados. / The aim of this work was to evaluate the agricultural potential of sewage sludge produced in the São Paulo state - Brazil, as well as to verify the possibility of interaction between the chemical composition and sewage sludge bacterial abundance. Samples were collected from 19 wastewater treatment plants in three different periods. On the samples from the three times were determined the presence and concentrations of 16 polycyclic aromatic hydrocarbons (HPAs) that are listed as priorities in environmental monitoring by the USEPA (acenaphthene, acenaphthylene, anthracene, benzo (a) anthracene, benzo (a) pyrene, benzo (b) fluoranthene, benzo (ghi) perylene, benzo (k) fluoranthene, chrysene, dibenz (a, h) anthracene, phenanthrene, fluoranthene, fluorene, indeno (1,2,3-cd) pyrene, naphthalene and pyrene). On the samples of the second collect time, besides HPAs, were determined the concentrations of emerging organic pollutants (hormones, pharmaceuticals and industrial products). It was performed the complete characterization according to CONAMA 375/2006 (moisture, pH, Kjeldahl and inorganic Nitrogen, organic carbon, calcium, potassium, phosphorus, magnesium, sulfur, boron, copper, iron, nickel, manganese, molybdenum, selenium, zinc, aluminum, arsenic, barium, cadmium, chromium, lead, mercury and sodium); and characterization of bacterial communities through cultivation-independent methods (Illumina sequencing). Macronutrients in higher concentrations in sewage sludge are: N > Ca > S > P > Mg > K. The inorganic elements Ni and Zn showed up in higher concentration than the maximum allowable for agricultural use by CONAMA Resolution 375/2006, at 1 and 3 samples, respectively. The inorganic element that most limits sewage sludge usage as organic fertilizer (MAPA, 2006) is the Hg. The compounds benzylparaben, bisphenol AF (BPAF), perfluorooctanoic acid (PFOA) and tetrabromobisphenol A (TBBPA) were not detected. On the other hand, methylparaben, cimetidine, bisphenol A (BPA) and triclocarban were detected in all 19 samples. The compound present in highest concentration is triclocarban. The concentrations of polycyclic aromatic hydrocarbons are low, according to the European standard. Proteobacteria and Bacteroidetes phyla are present in greatest relative abundance. There is a bacterial core in the sewage sludge treatment plants of the São Paulo State, comprising 81 genera present in all WWTPs evaluated. Those who are at a higher relative abundance are Treponema, Clostridium, Propionibacterium, Syntrophus and Desulfobulbus. The elevation of pH to values close to 12 reduces the microbial diversity. Considering the relative abundance and chemical composition of sewage sludge, the stations can be grouped into three distinct groups, one of which is influenced mainly by Ca, Zn and Cu, the other by Fe and S and the third group that was influenced by the others evaluated factors.

Objetivou-se com este trabalho avaliar o potencial agrícola do lodo de esgoto produzido no estado de São Paulo, bem como, verificar a possibilidade de interação entre a composição química e a abundância relativa de bactérias no lodo. Foram realizadas coletas de amostra de lodo de esgoto em 19 estações de tratamento de esgoto, em três épocas distintas. Nas amostras provenientes das três épocas foram determinados as concentrações dos 16 hidrocarbonetos policíclicos aromáticos (HPAs) listados como prioritários no monitoramento ambiental pela USEPA (acenafteno, acenaftileno, antraceno, benzo(a)antraceno, benzo(a)pireno, benzo(b)fluoranteno, benzo(ghi)perileno, benzo(k)fluoranteno, criseno, dibenzo(a,h)antraceno, fenantreno, fluoranteno, fluoreno, indeno(1,2,3-cd)pireno, naftaleno e pireno). Nas amostras da segunda época de coleta, além da presença de HPAs, determinou-se as concentrações de poluentes orgânicos emergentes (hormônios, produtos farmacêuticos e produtos de uso industrial), realizou-se a caracterização completa segundo a Resolução CONAMA 375/2006 (umidade, pH, N-Kjeldahl e inorgânico, carbono orgânico, cálcio, potássio, fósforo, magnésio, enxofre, boro, cobre, ferro, níquel, manganês, molibdênio, selênio, zinco, alumínio, arsênio, bário, cádmio, cromo, chumbo, mercúrio e sódio) e a caracterização da comunidade bacteriana através de metodologia independente de cultivo (sequenciamento illumina). Os macronutrientes em maiores concentrações no lodo de esgoto são: N > Ca > S > P > Mg > K. Os elementos inorgânicos Ni e Zn apresentaram concentração superior à máxima permitida para utilização agrícola pela resolução Conama 375/2006 em 1 e 3 amostras, respectivamente. A substância inorgânica que mais limita o enquadramento do lodo de esgoto como adubo orgânico (Instrução Normativa 27/2006) é o Hg. Os compostos benzilparabeno, bisfenol AF (BPAF), ácido perfluorooctanoico (PFOA) e tetrabromobisfenol A (TBBPA) não foram detectados. Por outro lado, cimetidina, metilparabeno, bisfenol A (BPA) e triclocarban foram detectados nas 19 amostras avaliadas. O composto presente em maior concentração é o triclocarban. As concentrações de hidrocarbonetos policíclicos aromáticos são baixas, de acordo com a norma Europeia. Os filos Proteobacteria e Bacteroidetes estão presentes em maior abundância relativa. Existe uma comunidade bacteriana núcleo nas estações de tratamento de esgoto do estado de São Paulo, composta por 81 gêneros, presentes nas 19 ETEs avaliadas, dos quais, os que estão em maior abundância relativa são Treponema, Clostridium, Propionibacterium, Syntrophus e Desulfobulbus. A elevação do pH a valores próximos de 12 reduz a diversidade microbiana. Considerando a abundância relativa e a composição química do lodo de esgoto, as estações podem ser agrupadas em três grupos distintos, sendo que um deles é influenciado principalmente pelos teores de Ca, Zn e Cu, o outro pelos teores de Fe e S e o terceiro grupo que foi influenciado pelos demais fatores avaliados. / The aim of this work was to evaluate the agricultural potential of sewage sludge produced in the São Paulo state - Brazil, as well as to verify the possibility of interaction between the chemical composition and sewage sludge bacterial abundance. Samples were collected from 19 wastewater treatment plants in three different periods. On the samples from the three times were determined the presence and concentrations of 16 polycyclic aromatic hydrocarbons (HPAs) that are listed as priorities in environmental monitoring by the USEPA (acenaphthene, acenaphthylene, anthracene, benzo (a) anthracene, benzo (a) pyrene, benzo (b) fluoranthene, benzo (ghi) perylene, benzo (k) fluoranthene, chrysene, dibenz (a, h) anthracene, phenanthrene, fluoranthene, fluorene, indeno (1,2,3-cd) pyrene, naphthalene and pyrene). On the samples of the second collect time, besides HPAs, were determined the concentrations of emerging organic pollutants (hormones, pharmaceuticals and industrial products). It was performed the complete characterization according to CONAMA 375/2006 (moisture, pH, Kjeldahl and inorganic Nitrogen, organic carbon, calcium, potassium, phosphorus, magnesium, sulfur, boron, copper, iron, nickel, manganese, molybdenum, selenium, zinc, aluminum, arsenic, barium, cadmium, chromium, lead, mercury and sodium); and characterization of bacterial communities through cultivation-independent methods (Illumina sequencing). Macronutrients in higher concentrations in sewage sludge are: N > Ca > S > P > Mg > K. The inorganic elements Ni and Zn showed up in higher concentration than the maximum allowable for agricultural use by CONAMA Resolution 375/2006, at 1 and 3 samples, respectively. The inorganic element that most limits sewage sludge usage as organic fertilizer (MAPA, 2006) is the Hg. The compounds benzylparaben, bisphenol AF (BPAF), perfluorooctanoic acid (PFOA) and tetrabromobisphenol A (TBBPA) were not detected. On the other hand, methylparaben, cimetidine, bisphenol A (BPA) and triclocarban were detected in all 19 samples. The compound present in highest concentration is triclocarban. The concentrations of polycyclic aromatic hydrocarbons are low, according to the European standard. Proteobacteria and Bacteroidetes phyla are present in greatest relative abundance. There is a bacterial core in the sewage sludge treatment plants of the São Paulo State, comprising 81 genera present in all WWTPs evaluated. Those who are at a higher relative abundance are Treponema, Clostridium, Propionibacterium, Syntrophus and Desulfobulbus. The elevation of pH to values close to 12 reduces the microbial diversity. Considering the relative abundance and chemical composition of sewage sludge, the stations can be grouped into three distinct groups, one of which is influenced mainly by Ca, Zn and Cu, the other by Fe and S and the third group that was influenced by the others evaluated factors.

Les aptamères sont des oligomères d'ADN ou d'ARN simple brin qui, en se repliant sous forme de structures tridimensionnelles peuvent avoir des interactions fortes et spécifiques envers un certain nombre de cibles. L'objectif de cette thèse a été de compléter les études existantes sur l'utilisation de l'électrophorèse capillaire (CE) et les aptamères afin de mettre au point une méthode de sélection d'aptamères par CE couplée à la fluorescence induite par laser et le séquençage haut-débit Illumina. Dans un premier temps, nous avons mis au point une méthode de détection et de séparation par électrophorèse capillaire couplée à la double détection UV-LEDIF d'une banque d'ADN en interaction avec une cible : la thrombine. C'est un modèle déjà étudié pour lequel deux aptamères ont fait l'objet de publications. Nous avons utilisé l'aptamère T29 dans le cadre de notre étude car c'est celui qui présente la meilleure affinité. L'électrophorèse capillaire est un puissant outil analytique qui facilite l'efficacité de sélection des aptamères et précise la détermination des paramètres d'interactions. Nous avons ainsi pu déterminer la constante d'affinité KD par CE-UV-LEDIF sur le modèle de base : la thrombine. Par ailleurs, nous montrons également comment l'utilisation du tampon Tris peut dégrader un ADN simple brin en électrophorèse capillaire et nous proposons comme alternative l'utilisation d'un tampon sodium phosphate dibasique qui évite ce phénomène de dégradation. Enfin, nous expliquons la difficulté d'amplification par qPCR et PCR d'un aptamère comme le T29 ayant une structure en G-quadruplex. Nous avons montré que le séquençage haut-débit Illumina nous a permis de trouver une corrélation entre le nombre de molécules séquencées et le nombre de séquences obtenues. L'analyse des séquences obtenues montre une quantité importante (20%) de séquences de T29 qui ne correspondent pas à la séquence de cet aptamère. Cela prouve que les étapes de PCR et de séquençage haut débit pour la détection de G-quadruplex peuvent induire un biais dans l'identification de ces molécules. / Aptamers are oligomers of small single-stranded DNA or RNA which can have strong and specific interactions with some targets when they fold into three-dimensional structures. The objective of this thesis was to complete existing studies on the use of capillary electrophoresis in order to develop a method for the selection of aptamers by CE coupled to laser induced fluorescence and Illumina high-throughput sequencing. In a first step, we developed a method of detection and separation by capillary electrophoresis coupled with the double detection UV-LEDIF of a DNA library interacting with a target: thrombin. It is a model already studied and for which two aptamers have been published. We used aptamer T29 as part of our study because it has the best affinity. Capillary Electrophoresis is a powerful analytical tool that facilitates the selection efficiency of aptamers and specifies the determination of the interaction parameters. We thus were able to determine the affinity constant KD by CE-UV-LEDIF on the basic model: thrombin. Moreover, we also show how the use of Tris buffer can degrade single-stranded DNA during capillary electrophoresis and we propose as an alternative the use of a dibasic sodium phosphate buffer which avoids the phenomenon of degradation. Finally, we explain the difficulty of amplification by qPCR and PCR of an aptamer such as T29 with a G-quadruplex structure. We showed that the Illumina high-throughput sequencing allowed us to find a correlation between the number of sequenced molecules and the number of sequences obtained. Analysis of the sequences obtained shows a significant amount (20%) of T29 sequences which do not correspond to the sequence of this aptamer. This shows that the PCR and high-throughput sequencing steps for the detection of G-quadruplex can induce bias in the identification of these molecules.

Acute myeloid leukaemia (“AML”) is a clonal disease characterised by increased, uncontrolled abnormal white blood cells and the accumulation of leukaemia immature cells in the bone marrow and bloodstream. Chromosomal rearrangements have been detected in almost half of AML cases. It has been proven that the chromosomal rearrangements constitute a marker for the diagnosis and prognosis of AML and have therapeutic consequences. The discovery of these rearrangements has led to a new World Health Organization (“WHO”) classification system. However, small regions of cryptic chromosomal rearrangements have been identified among these cases. Such cryptic rearrangements can be explained by the identification of small regions which cannot be found by conventional chromosome banding techniques. Moreover, approximately 50% of AML cases have been found with normal karyotypes. The improvement of cytogenetic techniques, including fluorescence in situ hybridization (“FISH”) and single nucleotide polymorphism (“SNP”) platforms, have allowed the detection of small rearranged regions (such as copy number changes) both in normal and abnormal karyotype AML. This study identifies: (i) cryptic chromosomal translocations in leukaemia cells of AML patients; (ii) DNA copy number changes in patients with known chromosomal translocations; and (iii) the proliferating state of leukemic cells harbouring chromosomal abnormalities within a series of patients. In the initial study, the FISH technique was performed on 7 AML patient samples to validate a novel three colour probe for the detection of t(7; 12). The results demonstrated that the new three-colour FISH approach used in this study has enabled the detection of a cryptic t(7;12) translocation as part of a complex rearrangement in one patient previously been described as having t(7;16) and ETV6-HLXB9 fusion transcript at the molecular level. To date there are only two cases of a cryptic t(7; 12) translocation reported in the literature. Additionally, the new three-colour FISH approach also enabled identification of t(7; 12) in a new seven year-old AML patient (the first case of childhood leukaemia with an onset after infancy to be found positive for t(7; 12)). In the second study the FISH technique was used to validate three colour probe sets for the detection of 7(q22-q31) and 7(q22-q36.1) regions on several myeloid cell lines. The results indicate that the probes found chromosome 7 rearrangements in myeloid cell lines with complex rearrangements. The three colour probe sets enabled detection of a new rearrangement in the k562 cell line, described as a duplication of 7q36 region, followed by intrachromosomal insertion of long arm material into the short arm of chromosome 7. The intrachromosomal insertion identified in k562 cell line is an uncommon form of chromosomal rearrangement in myeloid leukaemia which has not been previously reported. In the third study, the Illumina BeadArray approach was used to assess copy number alterations (“CNAs”) and copy number loss of hertrozygosity (“CN-LOH”) regions in 22 AML patients samples with inv(16)(p13;q22) and t(8;21)(q22;q22) rearrangements. In order to distinguish between true CNAs and false-positive findings as well as to verify whether CNAs are present in the same clone harbouring inv (16), FISH was used on fixed chromosome and cell suspensions from the same patients. The results showed a low number of copy number losses and copy number gains in 17 (77.27%) out of 22 cases, with an average of 1.86 CNAs per case as well as copy neutral-LOH with an average of 6.7% per patient. Furthermore, interphase FISH was carried out on four cases showing a 7q36.1 deletion, 4q35.1 deletion, 16.13.11 deletion and 8q24.21-q24.3 gain identified by array. The FISH results confirmed CNAs in most cases while CNA was not confirmed in one patient. Moreover, the FISH data analysis showed that the CNAs were found in both cells without inv (16) and cells harbouring the inv (16) rearrangement. In the final study, indirect immunofluorescence (IF) was used to determine the ki67 staining patterns in 8 stimulated and unstimulated peripheral blood samples and k562 cell lines. The results showed a high percentage of ki67 positive staining in the stimulated samples in comparison with unstimulated samples, which showed a low percentage of ki67 positive staining. In addition, a high percentage of proliferating cells were detected in the k562 cell line. ImmunoFISH was performed on five different patient samples and leukaemia cell lines using specific probes in the regions of interest to detect the chromosomal abnormalities and using the ki67 antibody to assess the proliferation state of the cells. The results showed that the proliferation state of the cells carrying chromosomal abnormalities in two patients was higher than the proliferation state of the cells carrying abnormalities in three patients; in other words, most of the cells carrying abnormalities were proliferating in two cases and non-proliferating in three cases.