<p>B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.</p><p>The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.</p><p>Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs. </p><p>In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.</p><p>Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis. </p>

Marine microbes are an essential component of global biogeochemical cycles. In oligotrophic marine surface waters, the phytoplankton, phototrophic, single-celled (on occasion, colonial) organisms, is often dominated by the picoplankton (cells <2 micrometers in size), which constitute the base of the marine food chain. The picophytoplankton is composed of three main groups of organisms: two genera of cyanobacteria, Prochlorococcus and Synechococcus, and a third group, the picoeukaryotes. Even though numerically less abundant than cyanobacteria, picoeukaryotes can contribute significantly to biomass and primary production in this size fraction. Furthermore, picoeukaryotes are a diverse group but this diversity is still underexplored and their ecological roles and physiology is poorly understood. Here uncultured protists are investigated using 18S rRNA gene clone libraries, phylogenetic analyses, specific fluorescence in situ hybridization (FISH) probes and other methods in tropical and subtropical waters. Gene sequences comprising a unique eukaryotic lineage, biliphytes, were identified in most samples, whether from high (30 degrees Celsius) or low (5 degrees Celsius) temperature waters. Sequences within this uncultured group have previously been retrieved from mid and high latitudes. Phycobilin-like fluorescence associated with biliphyte-specific FISH probed cells indicated they may be photosynthetic. Furthermore, the data indicated biliphytes are nanoplanktonic in size, averaging between 3.0 and 4.1 micrometers. Using the 18S rRNA gene, sequences belonging to a broadly distributed but uncultivated pico-prymnesiophytes were retrieved. We investigated the ecological importance of these natural pico-prymnesiophyte populations and field experiments showed that they could grow rapidly and contributed measurably to primary production. They also appear to form a large portion of global picophytoplankton biomass, with differing contributions in five biogeographical provinces, from tropical to high latitudes. Finally, the physiology of the picoeukaryote Micromonas was studied under a shift from medium to high light and UV radiation. Results showed that the growth of these photosynthetic cells was synchronized with the light: dark period. Forward angle side scatter and red autofluorescence from chlorophyll increased throughout the light period and decreased during the dark period. This is consistent with cell division occurring at the beginning of the dark period. Additionally, genes proposed to have roles in photoprotection were up-regulated under high light and UV, but not in controls.

B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (VH) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load. The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the VH gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed VH genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated VH genes, thus questioning the cell of origin in HCL. Biased usage of particular VH genes was detected in both HCL (VH3-30) and MCL (VH3-21 and VH4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, VH3-21+ MCLs showed a highly restricted Vλ3-19 gene use and they also had a superior outcome compared to other MCLs. Rearrangement analysis of 67 VH3-21+ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the Vλ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in VH3-21+ CLLs. In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse. Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Les sarcomes sont des tumeurs malignes des tissus conjonctifs, représentant moins de 1%des tumeurs malignes de l’adulte, mais près de 8% de l’ensemble des cancers pédiatriques. Enraison de leur rareté, de leur grande variété histologique et de leur potentiel évolutifhétérogène, les sarcomes sont des pathologies difficiles à traiter, tant sur le plan diagnostique,pronostique que thérapeutique. Ces dernières années, l’avènement de techniques d’analyse pangénomiques par biologie moléculaire a permis d’améliorer la prise en charge clinique des sarcomes, mais les microARN sont des biomarqueurs émergents encore peu utilisés. au cours de c e travail de thèse, nous avons cjhoisi d'étudier la valeur des profils d'expression des micrfoARN dans les rhabdomyosarcomes et les ostéosarcomes. Les données brutes des profils d'expression ont été obtenues à l'aidre d'une technologie à moyen débit basée sur des réactions de PCR quantitative. Nous avons tout d'abord développé une méthodologie d'ananlyse permettant d'obtenir des données d'expression précises, reproductibles et à forte valeur ajoutée, à partir de matériel biologique hétérogène.. Dans un second temps, nous avons montré que les profils d'expression de microARN permettent d'améliorer la prise en charge clinique des deuc types de sarcomes étudiés : il est possible d'affiner la classification nosologique des rhabdomyosarcomes, et de prédire la réponse des ostéosarcomes à la chimiothérapie néo-adjuvante. La recherche de nouvelles applications cliniques liées aux profils d'expression des micorARN doit donc être poursuivie, et peut désormais l'être grâce à l'outil robuste que nous avons développé au cours de cette thèse. / Sarcomas are malignant soft tissue tumors, accounting for 1% of adult tumors and 8% of all pediatric malignancies. Sarcomas are rare, and display a variety of histological subtypes and clinical characteristics. Therefore, everyday management is difficult in terms of diagnosis,prognosis and treatment. Recently, the development of pangenomic molecular techniquesimproved the clinical management of sarcomas, but the use of microRNAs as biomarkers is still being investigated.In the present work, we studied the value of microRNA expression profiles inrhabdomyosarcomas and osteosarcomas. Raw data of expression profiles were obtained using amedium throughput technology based on quantitative PCR. We first developed an analysismethodology to gain accurate, reproducible and relevant expression data, starting fromheterogeneous samples. Furthermore, we showed that microRNA expression profiles canimprove the clinical management of both sarcoma entities: they are helpful to upgrade the fine nosological classification of rhabdomyosarcomas, and they are able to predict the response of osteosarcomas to neoadjuvant chemotherapy. Searching for new clinical applications tomicroRNA expression profiles must be pursued.

Loose smut of barley, caused by fungal pathogen Ustilago nuda is one of the major concerns throughout the globe for barley producers. The infection takes place without exhibiting any obvious symptoms and an infected seed lot can only be identified at the heading stage when the fungal teliospores emerge at the place of crop. The percentage losses on yield are directly proportional to the occurrence of infection. Currently available detection methods include seed health testing protocols which are time-consuming and cumbersome. With the globalization of the international market and increased crop demand, development of rapid disease screening methodologies has become an essential focus in the field of plant pathology. The present study sought to develop a rapid probe-based detection method for screening of U. nuda with real-time qPCR. Two U. nuda specific primer pairs were compared using standard PCR alongside optimization of real-time qPCR assay. The advantage of high fidelity DNA polymerase for amplification of U. nuda genomic DNA was recorded. U. nuda genomic DNA was amplified and cloned into a vector which was further used for generation of a quantification curve with a specific probe. The qPCR assay developed in this study was successful in the detection of as little as 43 copies of U. nuda genomic DNA. With studies involving larger sample size and field samples, this assay can be improved for enhanced sensitivity and specificity which can help in monitoring infection from DNA extractions of barley seeds and further improving the current microscopic detection methods.

No description available.

Day care centers and schools are important environments for children, but few epidemiological studies exist from these environments. Mould, dampness, fungal DNA and allergens levels in these environments and respiratory health effects in school children were investigated in this thesis. In the day care centers studies, Allergen Avoidance Day care Centers (AADCs) and Ordinary Day care Centers were included. One third of the Swedish day care centers had a history of dampness or mould growth. Total fungal DNA levels were positively associated with risk construction buildings, reported dampness/moulds, rotating heat exchangers, linoleum floors and allergens (cat, dog, horse allergen) levels. The two school studies included secondary schools in Johor Bahru, Malaysia and elementary schools from five European countries (Italy, Denmark, Sweden, Norway, and France) (HESE-study). In Malaysia, 13 % of the pupils reported doctor-diagnosed asthma but only 4 % had asthma medication. The prevalence of wheeze in the last 12 months was 10 % in Malaysia and 13 % in the HESE-study. Cough and rhinitis were common among children in the HESE-study. There were associations between fungal DNA and reported dampness or mould growth. Fungal DNA levels and viable mould (VM) concentration in the classrooms were associated with respiratory symptoms (wheeze, rhinitis, cough, daytime breathlessness) in school children. In the HESE-study, associations were found between total fungal DNA, Aspergillus/Penicillium DNA and respiratory symptoms among children. Moreover, Aspergillus versicolor DNA and Streptomyces DNA were associated with respiratory symptoms in Malaysia and the HESE-study, as well as reduced lung function [forced vitality capacity (FVC) and forced expiratory volume in 1 second (FEV1)] among children in the HESE-study. In conclusion, fungal DNA and pet allergens were common in day care centers and schools and respiratory symptoms in school children were common. The associations between VM concentration and fungal DNA levels in the schools and respiratory health effects in school children indicated a need for improvement of these environments. Moreover, risk constructions should be avoided and buildings should be maintained to avoid dampness and microbial growth. Health relevance of microbial exposure and biodiversity needs to be further studied using molecular methods.

Les champignons mycorhizien à arbuscules (CMA) sont des organismes pouvant établir des symbioses avec 80% des plantes terrestres. Les avantages d'une telle symbiose sont de plus en plus caractérisés et exploités en agriculture. Par contre, jusqu'à maintenant, il n'existe aucun outil permettant à la fois l'identification et la quantification de ces champignons dans le sol de façon fiable et rapide. Un tel outil permettrait, entre autres, de mieux comprendre les dynamiques des populations des endomycorhizes dans le sol. Pour les producteurs d'inoculum mycorhiziens, cela permettrait également d'établir un suivi de leurs produits en champs et d'avoir un contrôle de qualité de plus sur leurs inoculants. C'est ce que nous avons tenté de développer au sein du laboratoire du Dr. Hijri. Depuis environ une trentaine d'années, des outils d'identification et/ou de quantification ont été développés en utilisant les profiles d'acides gras, les isozymes, les anticorps et finalement l'ADN nucléaire. À ce jour, ces méthodes d’identification et de quantification sont soit coûteuses, soit imprécises. Qui plus est, aucune méthode ne permet à la fois la quantification et l’identification de souches particulières de CMA. L’ADN mitochondrial ne présente pas le même polymorphisme de séquence que celui qui rend l’ADN nucléaire impropre à la quantification. C'est pourquoi nous avons analysé les séquences d’ADN mitochondrial et sélectionné les régions caractéristiques de deux espèces de champignons mycorhiziens arbusculaires (CMA). C’est à partir de ces régions que nous avons développé des marqueurs moléculaires sous forme de sondes et d’amorces TaqMan permettant de quantifier le nombre de mitochondries de chacune de ces espèces dans un échantillon d’ADN. Nous avons ensuite tenté de déterminer une unité de quantification des CMA, soit un nombre de mitochondries par spore. C’est alors que nous avons réalisé que la méthode de préparation des échantillons de spores ainsi que la méthode d’extraction d’ADN avaient des effets significatifs sur l’unité de quantification de base. Nous avons donc optimisé ces protocoles, avant d’en e tester l’application sur des échantillons de sol et de racines ayant été inoculés avec chacune des deux espèces cibles. À ce stade, cet outil est toujours semi-quantificatif, mais il permet 9 l’identification précise de deux espèces de CMA compétentes dans des milieux saturés en phosphore inorganique. Ces résultats , en plus d’être prometteurs, ont permis d’augmenter les connaissances méthodologiques reliées à la quantification des CMA dans le sol, et suggèrent qu’à cause de leurs morphologies différentes, l’élaboration d’un protocole de quantification standardisé pour toutes les espèces de CMA demeure un objectif complexe, qui demande de nouvelles études in vivo. / Arbuscular Mycorrhizal Fungi (AMF) are able to form symbiosis with approximately 80% of plant species, including most important corps. This symbiosis is known as Arbuscular Mycorrhiza which has been largely used in agriculture to promote plant growth by enhancing minerals uptake and protecting plants against biotic and abiotic stresses. Despite the ecological and economical importance of this symbiosis, specific markers for spore quantification of commercial AMF strains by quantitative real-time PCR (qPCR) are extremely limited and none has been rigorously validated for quality control of manufactured products such as biofertilizers. Most of the existing AMF markers developed either for AMF identification or for AMF quantification purposes are based on either morphological characters, on Fatty Acids Methyl Esters (FAME) profiles, on specific isozymes or antibodies, or on nuclear DNA. It has been shown that mitochondrial (mt) genomes are conserved among AMF species. This allowed us to use mt genomes in order to develop efficient isolate-specfic markers. Using the alignments of 11 complete AMF mt genomes, a qPCR assay using a hydrolysis probes designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of the model R. irregularis isolate DAOM197198 and another probe was designed in nad1-cox2 intergene specific to Glomus cerebriforme. The specificity tests performed, using standard PCR and qPCR, clearly showed that the primers specifically amplified the isolate DAOM-197198 or Glomus cerebriforme DAOM220722. Quantification assays were successfully undertaken on unknown samples in liquid suspensions, commercial solid formulations and soil samples to show the accuracy and reproducibility of the assays. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate Glomus cerebriforme DAOM220722. The latter could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.