Salmonella spp. é reconhecida como uma das bactérias que mais causam doenças de origem alimentar no mundo. Dentre as diversas sorovariedades de Salmonella, a Typhimurium é uma das sorovariedades de maior ocorrência no mundo. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas com o intuito de se delinear a epidemiologia e diversidade genotípica de Salmonella Typhimurium. Entretanto, a tipagem fenotípica é muitas vezes limitada por sua baixa capacidade de diferenciação de subtipos pertencentes a uma mesma sorovariedade de Salmonella, um problema minimizado pelos métodos genotípicos. No Brasil, foram realizados poucos estudos que genotiparam linhagens de S. Typhimurium. Os objetivos deste estudo foram caracterizar linhagens de S. Typhimurium isoladas de humanos, alimentos, animais e ambiente do animal no Brasil quanto ao seu potencial patogênico, perfil de resistência a antimicrobianos e diversidade genotípica. Foram estudadas 119 linhagens de S. Typhimurium, isoladas de material clínico de humanos (43), alimentos diversos (49), material clínico de suínos (22) e do ambiente de suínos (5), entre 1983 e 2013, provenientes de várias Estados do Brasil. A presença de 12 genes de virulência foi pesquisada por PCR. O perfil de resistência a 13 antimicrobianos foi realizado pelo método de discodifusão. A tipagem molecular foi realizada por Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variablenumber tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) e sequenciamento do genoma completo para 92 linhagens de S. Typhimurium isoladas de humanos (43) e alimentos (46). As metodologias PFGE, ERIC-PCR e MLVA foram realizadas para 70 linhagens de S. Typhimurium isoladas de humanos (43), animais (22) e ambiente do animal (5). Todas as 119 linhagens apresentaram os genes sipA, flgK, flgL e invA. O gene sipD e o gene sopE2 foram encontrados em 118 (99,2%) linhagens. O gene fljB foi encontrado em 117 (98,3%) linhagens. O gene sopD foi presente em 114 (95,8%) linhagens, o gene sopB em 111 (93,3%) linhagens, o gene ssaR em 102 (85,7%) linhagens, o gene sifA em 86 (72,3%) linhagens e 45 (37,8%) linhagens apresentaram o gene plasmidial spvB. De um total de 119 linhagens, 64 (62,2%) linhagens foram resistentes a pelo menos um dos 13 antimicrobianos testados, sendo que 36 (30,3%) linhagens foram multi-droga resistentes (MDR). Na comparação dos isolados de humanos e alimentos, as linhagens isoladas de humanos antes de meados 1990, ficaram alocadas nos grupos PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 e G1, G2, H para CRISPRMVLST. As linhagens isoladas de humanos após esse período ficaram alocadas nos grupos PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 e G2. As linhagens isoladas de alimentos ficaram alocadas nos grupos PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVAB2, G1 e G2. Por MLST, do total de 92 linhagens isoladas de humanos e alimentos, 77 linhagens foram tipadas como ST19. Pelo sequenciamento do genoma completo, as linhagens isoladas de alimentos e humanos ficaram alocadas no grupos I e J independente das datas de isolamento. Na comparação dos isolados de humanos e animais, as linhagens das duas origens ficaram alocadas nos grupos PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 e MLVA-D. ii Conclui-se que a grande prevalência de genes de virulência nas linhagens de S. Typhimurium estudadas reforça o potencial das mesmas causarem doenças em humanos, bem como, os riscos de sua presença em alimentos, animais para consumo humano e ambiente. A ocorrência de S. Typhimurium multi-droga resistentes isoladas de alimentos diversos e de suínos para consumo é um alerta para o possível risco de humanos ingerirem alimentos contaminados por tais linhagens. Em conjunto os resultados de PFGE, ERIC-PCR, MLVA, CRISPR-MVLST sugerem que as linhagens de S. Typhimurium isoladas de humanos eram geneticamente mais diversificadas antes de meados de 1990, o que pode sugerir a seleção de um subtipo de S. Typhimurium mais adaptado, depois que Salmonella Enteritidis tornou-se a sorovariedade de maior ocorrência no Brasil após esse período. Com relação às linhagens isoladas de alimentos, os resultados de PFGE, ERIC-PCR, MLVA e CRISPR-MVLST sugerem que durante o período estudado houve a circulação de mais de um subtipo no país. Os resultados de MLST sugerem que tais linhagens tenham uma origem filogenética comum. Os resultados do sequenciamento do genoma completo sugerem que houve a circulação de mais de um subtipo de S. Typhimurium no país, com relação às linhagens de humanos e alimentos. Também alerta para o possível risco de linhagens MDR isoladas de alimentos contaminarem humanos e/ou disseminarem genes de resistência a antibióticos para linhagens de origem clínica e não clínica. Na comparação dos isolados de humanos e animais, os resultados de PFGE, ERICPCR e MLVA sugerem que algumas linhagens isoladas de suínos e humanos podem descender de um subtipo comum. Ademais, as linhagens MDR isoladas de suínos e do ambiente de suínos alertam para o possível risco de porcos usados para consumo contaminarem humanos, o ambiente e outros porcos. / Salmonella spp. is recognized as one of the most involved bacteria that cause food-borne diseases in the world. Among the various serovars of Salmonella, Typhimurium is one of the most frequent serovars worldwide. Several phenotypic and genotypic typing methods have been developed in order to delineate the epidemiology and genotypic diversity of Salmonella Typhimurium. However, phenotypic typing is often limited by its low capacity to differentiate subtypes belonging to the same serovar of Salmonella, a problem minimized by genotypic methods. In Brazil, few studies have been conducted that genotyped S. Typhimurium strains. The aims of this study were to characterize S. Typhimurium strains isolated from humans, food, animals and animal\'s environment in Brazil regarding its pathogenic potential, antimicrobial resistance and genotypic diversity. We studied 119 S. Typhimurium strains isolated from human clinical material (43), different foods (49), clinical material from pigs (22) and pigs environment (5), between 1983 and 2013 from various States of Brazil. The presence of 12 virulence genes was investigated by PCR. The resistance profile against 13 antimicrobial was performed by the disk diffusion method. Molecular typing was performed by Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) and whole genome sequencing for 92 S. Typhimurium strains isolated from humans (43) and food (46). PFGE, ERIC-PCR and MLVA methods were performed for 70 S. Typhimurium strains isolated from humans (43), animals (22) and the animal\'s environment (5). All 119 strains showed the sipA, flgK, flgL and invA genes. The sipD and sopE2 genes were found in 118 (99.2%) strains. The fljB gene was found in 117 (98.3%) strains. The sopD gene was present in 114 (95.8%) strains, the gene sopB in 111 (93.3%) strains, the ssaR gene in 102 (85.7%) strains, the gene sifA in 86 (72.3%) strains and 45 (37.8%) strains showed the plasmid gene spvB. From a total of 119 strains, 64 (62.2%) strains were resistant to at least one of the 13 antimicrobials tested, and 36 (30.3%) strains were multi-drug resistant (MDR). In the comparison of isolates from humans and food, the strains isolated from humans before mid-1990s were allocated in PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 and G1, G2, H for CRISPR-MVLST. The strains isolated from humans after this period were allocated in PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 and G2 clusters. The strains isolated from food were allocated in PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2, G1 and G2 clusters. By MLST, of the total of 92 strains isolated from humans and food, 77 strains were typed as ST19. By whole genome sequencing, the strains isolated from food and humans were allocated in I and J clusters independently of its isolation date. In the comparison of isolates from humans and animals, strains of the two origins were allocated in PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 and MLVA-D clusters. In conclusion, the high frequency of virulence genes in the S. Typhimurium strains studied reinforces their potential hazard to cause disease in humans, as well as the risk of its presence in food, animals for human consumption and the environment. The occurrence of S. Typhimurium multi-drug iv resistant isolated from various food and pigs for consumption is an alert of the possible risk for humans to ingest contaminated food with those strains. Together the results of PFGE, ERIC-PCR, MLVA e CRISPR-MVLST suggest that S. Typhimurium strains isolated from humans were genetically more diverse before mid-1990s, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding the strains isolated from food, the results of PFGE, ERIC-PCR, MLVA and CRISPR-MVLST suggest that during the studied period there was circulation of more than one subtype in the country. The MLST results suggest that these strains have a common phylogenetic origin. The results of the whole genome sequencing suggest that there may be more than one subtype circulating in the country, with respect to the strains of human and food origins. Also, alerts for the possible risk of MDR strains isolated from food to contaminate humans and/or disseminate antibiotic resistance genes for strains of clinical and non-clinical origin. In the comparison of isolates from humans and animals, the results of PFGE, ERIC-PCR and MLVA suggest that some strains isolated from pigs and humans may descend from a common subtype. In addition, the MDR strains isolated from pigs and pig environment warn for the possible risk of pigs used for human consumption to contaminate humans, the environment and other pigs.

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido / Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène important, entrainant des pertes économiques de 130 millions de dollars annuellement au Canada. La surveillance est effectuée par séquençage Sanger du gène ORF5 mais nous croyons que le séquençage du génome entier (SGE) du VSRRP permettrait une meilleure surveillance épidémiologique comparé au séquençage du gène ORF5. Pour développer une méthode efficace de SGE du VSRRP, 149 échantillons (sérums, poumons, tissus, autres) d’animaux malades ou récoltés pour fin de surveillance ont été analysés. L'ARN viral a été concentré par enrichissement d'ARN à queue poly (A) et le séquençage effectué sur une plateforme Illumina. Le SGE a été efficace dans 67,11% des échantillons, réussissant dans certains échantillons de poumons et de sérums possédant une valeur de quantification (Cq) du virus par RTqPCR jusqu’à 26,50 et 34.13, respectivement. La méthodologie développée de SGE du VSRRP a été 4650 fois plus sensible que les méthodes décrites précédemment. Pour quantifier l’impact du SGE, 88 échantillons (dont le SGE a réussi) ont été utilisés pour comparer le SGE au séquençageORF5. Deux génomes de VSRRP différents ont été trouvés dans quatre échantillons différents (taux de coinfection de 4,55%). Six génomes de VSRRP (6,52% des souches) ont été classés différemment par rapport à la classification ORF5. Ainsi, le SGE du VSRRP a permis une meilleure caractérisation de 9,10% des échantillons VSRRP positifs comparé au séquençage ORF5. Donc, le SGE du VSRRP est à la fois sensible et plus précis que la classification par l’ORF5. / Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen, costing over 130 million dollars annually in Canada. Surveillance is done by Sanger sequencing of the ORF5 gene, but we hypothesized that whole genome sequencing (WGS) of PRRSV genome will allow a better epidemiological monitoring of PRRSV compared to ORF5 gene sequencing. To develop an efficient method of PRRSV WGS, 149 PRRSV samples (sera, lungs, pool of tissues and others) collected for surveillance or from sick animals were tested. Viral RNA was concentrated using a poly(A) tailed RNA enrichment method, and sequencing was done on an Illumina platform. WGS was successful in 67.11% of cases. WGS was successful in some tissues and lungs samples with RT-qPCR cycle quantification (Cq) values up to 26.50, and in some sera with Cq value up to 34.13. The developed WGS methodology was 4650 times more sensitive for PRRSV WGS than previously described methods. To quantify the impact of WGS, 88 successful samples for the WGS of PRRSV were used to compare efficiency of WGS and ORF5 sequencing. Two different full-length genomes of PRRSV were found in four of those samples (coinfection rate of 4.55%). Six full-length PRRSV genomes (6.52% of PRRSV strains) were found to cluster differently compared to ORF5 sequencing. WGS of PRRSV also enabled a better classification or characterisation of 9.10% of the PRRSV infected samples compared to ORF5 sequencing. Thus, WGS can be both sensitive and more accurate then ORF5 classification for the characterisation of PRRSV strains.

Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.

Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) places the fourth position in the ranking of most reported Salmonella serovars in Europe. During the last decade, a multi-drug resistant (MDR) S. Infantis population has rapidly increased and widespread in European and non-European broiler production. The study proposed here aimed to i) implement and evaluate the performance of a bioinformatics pipeline named WGSBAC for Salmonella in silico serotyping, and ii) identify the genetic determinants for the increased emergence of broiler-derived S. Infantis observed in Germany. First, we conducted an evaluation of WGSBAC and three bioinformatic tools (SISTR, SeqSero, and SeqSero2) for the characterization and genoserotyping of 43 Salmonella strains of 26 different serovars. Second, we performed sequencing of 30 broiler-derived S. Infantis isolates collected from two distant decades (the 1990s and the 2010s). We applied the WGSBAC pipeline and external bioinformatics software to i) assess and control the quality of the sequenced reads ii) assembly and quality control of assemblies, and iii) annotation, typing by classical MLST, cgMLST, genoserotyping, SNPs-based phylogenetic reconstruction and in silico phenotype prediction including antimicrobial resistance genes (AMR), virulence genes and plasmid replicons detection. To detect possible clonal relatedness with other S. Infantis clones from Europe, we performed a further comparative genome analysis using 17 public genomes of other S. Infantis clones circulating in Europe. WGSBAC was feasible for the serovar prediction of most of the 43 Salmonella strains. The tool SISTR reported the highest correlation (79.1%) followed by SeqSero2 (72.1%) and SeqSero (60.5%). The study of the S. Infantis strains revealed that in contrast to the isolates from the 1990s, the majority of the strains from the 2010s revealed the presence of a megaplasmid that carried a multidrug-resistant genes (MDR) pattern, a virulence genes pattern, and several fitness-associated determinants. We termed the MDR gene pattern “ESIr” and it coded for at least three antimicrobial families: ant(3”)-Ia (aminoglycosides), sul1 (sulfonamides), and tet(A) (tetracyclines). Besides, we termed the virulence pattern as “ESIv” which includes genes for fimbriae cluster, yersiniabactin siderophore, mercury resistance, and antitoxin/antitoxin systems. Furthermore, the genotyping analysis revealed the presence of a novel sequence type (ST2283) among the majority of the strains from the 2010s and ST32 and ST1032 within the strains from the 1990s. This genetic traits may promote the rapid incidence and dissemination of a novel MDR S. Infantis population. Following a WGS-based approach, this study evidences that MDR S. Infantis ST2283 strains carrying a pESI-like plasmid have emerged during the last decade and are currently circulating in the German poultry production chain. This event results in an urgent public health hazard, thus, control measures and the support of epidemiological studies are needed to prevent the entrance, transmission, and further dissemination of this clonal population in the food chain. / Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) nimmt die vierte Position in der Rangliste der am häufigsten gemeldeten Salmonella-Serovare in Europa ein. Während des letzten Jahrzehnts hat das Auftreten einer multiresistenten Salmonella enterica subspecies enterica serovar Infantis (S. Infantis)-Population rapide zugenommen und ist in der europäischen und außereuropäischen Broilerproduktion weit verbreitet. Die Studie zielte darauf ab, i) eine bioinformatische Pipeline für die in silico-Serotypisierung von Salmonellen zu implementieren und deren Leistungsfähigkeit zu bewerten, und ii) die genetischen Determinanten für das in der deutschen Broilerproduktion während des letzten Jahrzehnts beobachtete vermehrte Auftreten von S. Infantis zu identifizieren. Zunächst wurde eine Bioinformatik-Pipeline (WGSBAC) und drei bioinformatische Tools (SISTR, SeqSero und SeqSero2) zur Genoserotypisierung von 43 Salmonella spp.-Stämmen von 26 verschiedenen Serovaren durchgeführt. Zweitens führten wir die Genomsequenzierung von 30 S. Infantis Broiler-Isolaten durch, die in zwei unterschiedlichen Jahrzehnten (den 1990er und den 2010er Jahren) gesammelt wurden. Wir setzen die WGSBAC-Pipeline und externe Bioinformatik-Software ein, um i) die Qualität der sequenzierten Reads zu bewerten und zu kontrollieren, ii) Assemblierungen und Qualitätskontrollen von und iii) Annotation, Typisierung durch klassischen MLST, cgMLST, Genoserotypisierung, phylogenetische Rekonstruktion mittels Einzelnukleotidänderungen (SNPs) und in-silico-Phänotyp-Vorhersage, einschließlich antimikrobieller Resistenzgene (AMR), Virulenzgene und Plasmid-Replikons zu erkennen. Die Bioinformatik-Pipeline WGSBAC ist geeignet, das antigene Profil der meisten der in der Studie verwendeten Salmonella-Stämme zu bestimmen. Das Tool SISTR zeigte dabei die höchste Übereinstimmung (79,1 %), gefolgt von SeqSero2 (72,1 %) und SeqSero (60,5 %). Die Untersuchung der S. Infantis-Stämme ergab, dass im Gegensatz zu den Isolaten aus den 1990er Jahren, die Mehrheit der Stämme aus den 2010er Jahren das Vorhandensein eines Megaplasmids zeigte, das homolog zu dem pESI-Plasmid aus Israel und anderen pESIähnlichen Plasmiden aus europäischen Isolaten ist. Das deutsche pESI-ähnliche Plasmid kodierte für ein Muster von multiresistenten Genen (MDR), ein Muster von Virulenzgenen und mehrere Fitness-assoziierte Determinanten, welches wir 'ESIr' nannten. Dieses korrelierte mit mindestens drei antimikrobiellen Familien: ant(3')-Ia (Aminoglykoside), sul1 (Sulfonamide) und tet(A) (Tetracycline). Der Genotyp korreliert hier vollständig mit dem antimikrobiellen Phänotyp. Außerdem bezeichneten wir das Virulenzmuster als 'ESIv', welches Gene für Fimbrien-Cluster, Yersiniabactin-Siderophore, Quecksilberresistenz und Antitoxin/Antitoxin- Systeme mit einschließt. Die Genotypisierungsanalyse ergab das Vorhandensein eines neuen Sequenztyps (ST2283) bei der Mehrzahl der Stämme aus den 2010er Jahren und ST32 und ST1032 bei den Stämmen aus den 1990er Jahren. Anhand eines auf Gesamtgenomsequenzierung-basierten Ansatzes zeigte diese Studie, dass MDR S. Infantis ST2283 Stämme, die ein pESI-ähnliches Plasmid tragen, während des letzten Jahrzehnts entstanden sind und derzeit in der deutschen Geflügelproduktionskette zirkulieren. Der Erwerb eines Megaplasmids, das für Resistenzen, Virulenz-assoziierte Determinanten und Fitnessmechanismen kodiert, könnte dieses schnelle und besorgniserregende epidemiologische Ereignis erklären. Dieses Ereignis stellt eine Gefahr für die öffentliche Gesundheit dar. Daher sind Kontrollmaßnahmen und die Unterstützung epidemiologischer Studien erforderlich, um den Eintritt, die Übertragung und die weitere Verbreitung dieser klonalen Population in der Lebensmittelkette zu verhindern.

Ce travail de thèse comporte deux volets ayant pour trait commun l’analyse a posteriori d’une épidémie d’infection invasive à méningocoque (IIM) survenue en Normandie entre 2003 et 2012 et liée à l’expansion d’un clone hypervirulent particulier (B:14:P1.7,16/ST-32 ). Le premier travail (publié dans Virulence : Sevestre et al 2018 ;9 :923-929) s’est focalisé sur les déterminants de la virulence de « B14 » en comparant 6 isolats identifiés les uns d’IIM, les autres de portage pharyngé sain (ces derniers exprimant ou non la capsule). Apparemment identiques selon le typage classique (immunotypage et génotypage par MLST), ces 3 groupes bactériens se sont révélés distincts après analyse génomique et comparaison gène par gène (plus de 600 gènes au profil génétique variable entre les groupes) conduisant à identifier le rôle majeur de l’acquisition du fer dans la virulence et en particulier celui du système HmbR, un récepteur de l’hémoglobine. Dans un modèle murin (souris transgéniques rendues sensibles à l’infection humaine) les 3 groupes de souches sont aussi apparus distincts, avec une hiérarchie des marqueurs d’infectiosité (titres bactériens, taux de cytokines). La restauration du système HmbR (souches de portage capsulées « Off » dérivées en « On ») a restauré le pouvoir invasif in vitro et chez l’animal. Si le fer était déjà connu comme facteur de virulence pour différentes espèces bactériennes, l’originalité ici est d’avoir identifié le rôle de la variation de phase du gène hmbR au sein d’un même clone épidémique, permettant l’adaptation au portage, condition sine qua non de la transmission d’individu en individu. Le second travail (publié dans Vaccine : Sevestre et al 2017 ;35 :4029-4033) s’est intéressé à la durabilité et à l’ampleur de la protection vaccinale du MenBvac®, vaccin à base de vésicules de membranes externes (Outer Membrane Vesicles, OMV) utilisé jadis pour contrôler l’épidémie. Ceci a pu être réalisé grâce à deux cohortes d’enfants vaccinés par un schéma à 4 doses et prélevés pour les uns 1 an après la dernière dose et pour les autres 4 an après. L’immunogénicité (étude de l’activité bactéricide du sérum vis-à-vis du clone ciblé) s’est avérée de durabilité moyenne avec 48% des enfants protégés à 1 an et 31% à 4 ans, un résultat en phase avec les données de la littérature sur les vaccins OMV. Un effet bactéricide fut observé très au-delà de « B14 », du fait d’une immunité croisée aux souches avec une homologie au moins partielle de la porine PorA (principal déterminant antigénique des vaccins OMV) soit pour le MenBvac® 15% des clones virulents B actuellement circulants en France, un résultat davantage original car ayant jusqu’alors que peu investigué. / This thesis work includes two studies which both contribute to analyze a posteriori an outbreak of invasive meningococcal diseases (IMD) that had occurred in Normandy from 2003 to 2012 due to the expansion of a single hypervirulent clone (B:14:P1.7,16/ST-32 ). The first work (published in Virulence: Sevestre et al 2018 ;9 :923-929) focused on the virulence determinants of “B14” by comparing 6 isolates, either from IMD or from asymptomatic carriage (these latter expressing or not the capsule). Apparently identical on the basis of classical typing methods (immunotyping and MLST genotyping), these 3 groups of isolates were markedly different by whole genome analysis and on gene by gene comparison (more than 600 genes presenting a variable genetic profile). This analysis leaded to identify the crucial implication of iron acquisition in virulence and in particular the place of the HmbR system, an hemoglobin receptor. In a murine model (transgenic mice made susceptible to infection), these 3 groups also appeared separated, with a distinct infectivity hierarchy (bacterial counts, levels of cytokines). The restoration of the HmbR system in the capsulated carriage isolates (switch from Off phase to On phase) also restored their invasiveness in vitro and in vivo. Even if iron is already known to be a determining factor in the virulence of many bacterial species, our results clearly indicate the importance of the hmbR phase variation among clonal epidemic isolates, allowing adaptation to carriage, sine qua non condition for people to people transmission. The second work (published in Vaccine: Sevestre et al 2017 ;35 :4029-4033) concerned the durability and the cross-protection of the MenBvac®, an OMV vaccine (Outer Membrane Vesicles), used in the past to control the outbreak. This work has been done thanks to 2 cohorts of children vaccinated with 4 doses and sampled either 1 year or 4 years after the last dose. The efficacy (serum bactericidal activity against the epidemic strain) was short lasting, with 48% of children protected after 1 year and 31% after 4 years, a result in accordance with OMV literature. A bactericidal effect was observed far beyond “B14”, by cross-immunity with strains harboring homologies, even partials of the porin PorA (main antigenic determinant in OMV vaccine), indicating a coverage for 15% of virulent isolates B circulating in France, an original result as until then not so far investigated.

Les dysplasies frontonasales (DFN) sont un groupe de malformations rares de la face résultant d’une anomalie de développement du processus frontonasal, et se manifestant cliniquement par l’association variable d’une fente faciale médiane, d’un hypertélorisme, et d’anomalies nasales. Elles s’intègrent généralement dans un cadre syndromique, la plupart étant peu spécifiques et non caractérisées. Une douzaine d’entités ont été décrites ; les bases moléculaires sont connues pour sept d’entre elles seulement. Pour les autres entités, les hypothèses initiales étaient celles de pathologies autosomiques dominantes liées à des mutations de novo.Ce travail s’est fondé sur une cohorte de 80 patients présentant une DFN, recrutés au niveau national et international, avec l’objectif d’identifier les bases moléculaires de plusieurs entités de DFN. Une caractérisation phénotypique a d’abord été effectuée. Deux stratégies moléculaires ont ensuite été poursuivies en parallèle : une approche « phenotype-first », visant à étudier les patients classés par cohortes homogènes cliniquement, et une approche « genotype-first », visant à réaliser des études moléculaires chez des patients présentant un tableau aspécifique.Lorsque le diagnostic était celui d’un syndrome dont le gène causal était connu, nous avons réalisé un séquençage ciblé dudit gène (EFNB1, ZSWIM6). Nous avons effectué un séquençage haut-débit d’exome (SHD-E) chez 11 patients présentant un syndrome de Pai (5 en trio, 5 en solo, un en profondeur sur tissu atteint en paire), trois patients avec syndrome oculoauriculofrontonasal (en trio), et respectivement un patient avec syndrome oculocérébrocutané et syndrome de Teebi (en trio). Nous avons réalisé un séquençage haut-débit de génome (SHD-G) chez 3 patients avec un syndrome de Pai, ainsi qu’une patiente avec syndrome oculoauriculofrontonasal. Enfin, nous avons séquencé les gènes ALX1, ALX3 et ALX4 chez 13 individus avec DFN aspécifique ; réalisé un SHD-E en profondeur chez une patiente avec DFN et hypomélanose d’Ito, ainsi que trois SHD-E en trio chez des patients avec DFN aspécifique ou non classée.Nous avons réalisé un travail de caractérisation phénotypique des patients de la cohorte, décrivant les diagnostics différentiels principaux et les chevauchements phénotypiques. Nous avons décrit une nouvelle entité de DFN, identifiée chez 4 de nos patients, sans base moléculaire à l’heure actuelle. Nous avons confirmé les diagnostics cliniques lorsque le gène causal était connu, chez 5 patients. Nous avons retrouvé des variants candidats dans deux gènes de la voie du TGFβ chez quatre patients avec syndrome de Pai : un de novo dans le gène TGFBRAP1, deux de novo et un hérité d’un parent asymptomatique dans le gène PCSK7. Nous avons identifié les mutations dans TFE3 comme étant à l’origine d’un syndrome neurocutané de mosaïcisme pigmentaire chez une patiente et 6 patients additionnels. Enfin, nous avons identifié un variant dans le gène POLR2A chez un fœtus avec DFN aspécifique. Au total, 34 individus avec un syndrome connu et 34 avec une DFN aspécifique restent à l’heure actuelle sans piste moléculaire identifiée.Au total, ce travail a permis de démontrer l’intérêt d’une meilleure connaissance clinique de ces syndromes rares, pour le diagnostic et le conseil génétique. Il a permis l’individualisation et la description de deux syndromes pouvant s’accompagner d’une DFN – dont un à l’échelle moléculaire –, et la démonstration du rôle du gène TFE3 dans le développement. Enfin, des hypothèses sont émises concernant les résultats incertains et négatifs : celles d’un oligogénisme, d’une anomalie épigénétique, d’une mutation post-zygotique ou de l’influence de l’environnement. / Frontonasal dysplasias (FND) are a group of rare facial malformations due to an abnormal development of the frontonasal process, clinically resulting in the variable association of median facial cleft, hypertelorism and nasal anomalies. Most of them are syndromic, but non-specific and not characterized. A dozen entities have been described; molecular bases are known for only seven of them. Regarding the other entities, the hypothesis of dominant disorders due to de novo mutations had been raised.This work was based on a cohort of 80 patients presenting with FND, nationally and internationaly recruited with the goal of identifying molecular bases of FND entities. We first phenotypically characterized the individuals. Then two molecular strategies were performed in parallel: a “phenotype-first” approach, aiming to study clinically homogeneous cohorts of patients, and a “genotype-first” approach, aiming to perform molecular studies on patients with an aspecific phenotype.When the causative gene for the disorder was known, we performed targeted sequencing of the gene (EFNB1, ZSWIM6). We performed whole-exome sequencing (WES) in 11 patients presenting with Pai syndrome (5 trio WES, 5 solo WES, one deep-sequencing pair-WES on affected tissue), 3 individuals presenting with oculoauriculofrontonasal syndrome (OAFNS) (trio WES), and respectively one patient with oculocerebrocutaneous syndrome and Teebi syndrome (both trio WES). We performed whole-genome sequencing (WGS) on 3 patients with Pai syndrome and one patient with OAFNS. Finally, we performed ALX1, ALX3 and ALX4 genes sequencing in 13 individuals with aspecific FND; deep-WES in one patient with FND and Ito hypomelanosis; as well as 3 trio WES in individuals with aspecific or non characterized FND.We achieved a phenotypic characterization of the patients from the cohort, describing major differential diagnosis and clinical overlaps. We described a new FND entity, identified in 4 individuals, with no molecular basis so far. We confirmed the clinical diagnosis when the causative gene was known, for 5 patients. We identified candidate variants in two genes from the TGFβ pathway in four patients with Pai syndrome: one de novo variant in the TGFBRAP1 gene, two de novo and one inherited from an asymptomatic parent in the PCSK7 gene. We identified mutations in TFE3 as causative for a neurocutaneous syndrome of pigmentary mosaicism in a female patient and six additional individuals. Finally, we identified a variant in the POLR2A gene in a fetus with aspecific FND. A total of 34 individuals with a known syndrome and 34 with an aspecific FND still have no identified molecular basis so far.In conclusion, this work allowed proving the importance of a better clinical knowledge of these rare disorders, in terms of diagnosis and genetics counseling. It allowed the delineation of two syndromes with FND – one at a molecular level –, and the demonstration of a role for TFE3 in development. Finally, four hypotheses are raised regarding the negative or uncertain results: oligogenism, epigenetic anomaly, post-zygotic mutation or environmental influence.

No description available.

[ES] La biotecnología moderna se basa en la aplicación de una mezcla de herramientas experimentales y computacionales para llevar a cabo de forma dirigida la ingeniería genética. El objetivo es obtener células (re)programadas que implementen nuevas funciones o que sirvan como herramientas para el estudio de sistemas biológicos. En este contexto, el uso de bacterias en biotecnología está muy extendido. Sin embargo, la implementación de circuitos genéticos para el aprovechamiento de estos seres vivos puede verse limitada por procesos biológicos naturales; es decir, los circuitos diseñados (o naturales) pueden verse afectados por el transcurso del tiempo o por cambios en el entorno en el que crecen las bacterias. En esta tesis, nos propusimos seguir un enfoque integrador para estudiar cómo las bacterias responden en el tiempo y el espacio a los cambios genéticos y ambientales, que pueden afectar la funcionalidad de los circuitos de interés biotecnológico. Usamos Escherichia coli como organismo modelo, explotando una variedad de herramientas experimentales para trabajar con él. En primer lugar, estudiamos cómo los cambios ambientales y genéticos afectan la funcionalidad de un circuito genético sintético que implementa un comportamiento lógico sofisticado. Descubrimos que hay amplios rangos de concentración de entrada que el sistema puede procesar correctamente, que el circuito diseñado es bastante sensible a los efectos de la temperatura, que la expresión de pequeños ARN heterólogos es costosa para la célula y que una reorganización genética adecuada del sistema para reducir la cantidad de ADN heterólogo en la célula puede mejorar su estabilidad evolutiva. En segundo lugar, estudiamos el crecimiento bacteriano en entornos en los que existen materiales nanoestructurados. Descubrimos que las poblaciones bacterianas se pueden controlar en gran medida mediante el uso de marcos organometálicos, ya que estos materiales nanoestructurados pueden descomponerse lentamente en medios biológicos liberando agentes antimicrobianos (metales y compuestos orgánicos, incluidos los antibióticos). Analizamos la respuesta bacteriana espaciotemporal siguiendo un enfoque experimental y teórico combinado en un entorno tan complejo y desafiante en medios líquidos y sólidos. Además de las variaciones en el rendimiento debido a cambios ambientales, también se debe considerar que esos circuitos genéticos evolucionarán con el tiempo debido a la acumulación estocástica de mutaciones. Estas mutaciones pueden dar lugar a cambios en la funcionalidad de los circuitos reguladores. Por tanto, en tercer lugar, realizamos un experimento de evolución a largo plazo para estudiar la contribución de un sistema de chaperonas de proteínas en la modulación de la estabilidad evolutiva. En los últimos años, se ha demostrado que los sistemas de chaperonas, como GroES/EL, pueden amortiguar o purgar mutaciones. Realizamos la secuenciación del genoma completo en diferentes líneas con diferentes niveles de expresión de GroEL y también medimos la tasa de crecimiento de las células al principio y al final del experimento evolutivo. Sin embargo, nuestros resultados no fueron concluyentes, por lo que se necesita más investigación para comprender completamente el papel de GroES/EL en la evolución y evaluar su utilidad potencial en biotecnología. En conjunto, esta tesis intenta avanzar en nuestro conocimiento sobre cómo las bacterias, y E. coli en particular, se comportan como se espera cuando el entorno se altera, la fisiología cambia y pasa mucho tiempo, para posibles aplicaciones industriales o (pre)clínicas. / [CA] La biotecnologia moderna es basa en l'aplicació d'una mescla d'eines experimentals i computacionals per a realitzar de forma dirigida l'enginyeria genètica. L'objectiu és obtindre cèl·lules (re)programades que implementen noves funcions o que servisquen com a eines per a l'estudi de sistemes biològics. En aquest context, l'ús de bacteris en biotecnologia està molt estés. No obstant això, la implementació de circuits genètics per a l'aprofitament d'aquests éssers vius pot veure's limitada per processos biològics naturals; és a dir, els circuits dissenyats (o naturals) poden veure's afectats pel transcurs del temps o per canvis en l'entorn en el qual creixen els bacteris. En aquesta tesi, ens vam proposar seguir un enfocament integrador per a estudiar com els bacteris responen en el temps i l'espai als canvis genètics i ambientals, que poden afectar la funcionalitat dels circuits d'interés biotecnològic. Usem Escherichia coli com a organisme model, explotant una varietat d'eines experimentals per a treballar amb ell. En primer lloc, estudiem com els canvis ambientals i genètics afecten la funcionalitat d'un circuit genètic sintètic que implementa un comportament lògic sofisticat. Descobrim que hi ha amplis rangs de concentració d'entrada que el sistema pot processar correctament, que el circuit dissenyat és bastant sensible a l'efecte de la temperatura, que l'expressió de xicotets ARN heteròlegs és costosa per a la cèl·lula i que una reorganització genètica adequada del sistema per a reduir la quantitat d'ADN heteròleg en la cèl·lula pot millorar la seua estabilitat evolutiva. En segon lloc, estudiem el creixement bacterià en entorns en els quals existeixen materials nanoestructurats. Descobrim que les poblacions bacterianes es poden controlar en gran manera mitjançant l'ús de marcs organometàlics, ja que aquests materials nanoestructurats poden descompondre's lentament en medis biològics alliberant agents antimicrobians (metalls i compostos orgànics, inclosos els antibiòtics). Analitzem la resposta bacteriana espai-temporal seguint un enfocament experimental i teòric integrador en un entorn tan complex i desafiador en mitjans líquids i sòlids. A més de les variacions en el rendiment degut a canvis ambientals, també s'ha de considerar que aqueixos circuits genètics evolucionaran amb el temps degut a l'acumulació estocàstica de mutacions. Aquestes mutacions poden donar lloc a canvis en la funcionalitat dels circuits reguladors. Per tant, en tercer lloc, realitzem un experiment d'evolució a llarg termini per a estudiar la contribució d'un sistema de chaperones de proteïnes en la modulació de l'estabilitat evolutiva. En els últims anys, s'ha demostrat que els sistemes de chaperones, com GroES/EL, poden esmorteir o purgar mutacions. Realitzem la seqüenciació del genoma complet en diferents línies amb diferents nivells d'expressió de GroEL i també mesurem la taxa de creixement de les cèl·lules al principi i al final de l'experiment evolutiu. No obstant això, els nostres resultats no van ser concloents, per la qual cosa es necessita més investigació per a comprendre completament el paper de GroES/L en l'evolució i avaluar la seua utilitat potencial en biotecnologia. En conjunt, aquesta tesi intenta avançar en el nostre coneixement sobre com els bacteris, i E. coli en particular, es comporten com s'espera quan l'entorn s'altera, la fisiologia canvia i passa molt temps, per a possibles aplicacions industrials o (pre)clíniques. / [EN] Modern biotechnology is based on applying a mix of experimental and computational tools to perform in a directed way genetic engineering. The aim is to obtain (re)programmed cells that implement new functions or that serve as tools for the study of biological systems. In this context, the use of bacteria in biotechnology is widespread. However, the implementation of genetic circuits for the use of these living beings may be limited due to natural biological processes; that is, the engineered (or natural) circuits may be affected by the course of time or by changes in the environment in which bacteria grow. In this thesis, we proposed to follow an integrative approach to study how bacteria respond in time and space to genetic and environmental changes, which may affect the functionality of the circuits of biotechnological interest. We used Escherichia coli as a model organism, exploiting a variety of experimental tools to work with it. Firstly, we studied how environmental and genetic changes affect the functionality of a synthetic genetic circuit that implements a sophisticated logic behavior. We found that there are wide input concentration ranges that the system can correctly process, that the engineered circuitry is quite sensitive to temperature effects, that the expression of heterologous small RNAs is costly for the cell, and that a proper genetic reorganization of the system to reduce the amount of heterologous DNA in the cell can improve its evolutionary stability. Secondly, we studied of bacterial growth in environments in which there are nanostructured materials. We found that bacterial populations can be greatly controlled through the use of metal-organic frameworks, as these nanostructured materials can slowly decompose in biological media releasing antimicrobials (metals and organic compounds, including antibiotics). We analyzed the spatiotemporal bacterial response following a combined experimental and theoretical approach in a such a complex and challenging environment in both liquid and solid media. In addition to variations in performance due to environmental changes, it must also be considered that those gene circuits will evolve over time due to the stochastic accumulation of mutations. These mutations can lead to changes in the functionality of the regulatory circuits. Then thirdly, we performed an experiment of long-term evolution to study the contribution of a protein chaperone system in modulating evolutionary stability. In recent years, it has been shown that chaperone systems, such as GroES/EL, can buffer or purge mutations. We performed whole-genome sequencing over different lines with varying expression levels of GroEL, and also measured the growth rate of the cells at the beginning and the end of the evolutionary experiment. However, our results were not conclusive, so further research is needed to fully understand the role of GroES/EL in evolution and to assess its potential utility in biotechnology. Taken together, this thesis tries to advance our knowledge on how bacteria, and E. coli in particular, behave as expected when the environment is perturbed, the physiology changes, and long time passes, for potential industrial or (pre)clinical applications. / Montagud Martínez, R. (2023). Study of Spatiotemporal Responses of Bacterial Cells [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/193030

La régulation de l’expression des gènes est fondamentale pour coordonner la synthèse, l’assemblage et la localisation des complexes macromoléculaires dans les cellules. Cette expression est régulée à divers niveaux. Elle commence dans le noyau où les facteurs de transcription se lient à des séquences spécifiques d’ADN et recrutent les ARN polymérases pour la synthèse des ARN. La régulation à ce niveau est dite transcriptionnelle. Les protéines de liaison à l’ARN s’associent avec l’ARN en cours de synthèse et opèrent divers modifications comme l’addition d’une coiffe en 5’, l’épissage, l’édition et la poly-adénylation en 3’. Les transcrits sont alors exportés vers le cytoplasme où ils vont être adressés et stockés dans des régions subcellulaires. Les ARNm s’assemblent avec des facteurs de traduction et les ribosomes pour initier la synthèse protéique de manière contrôlée. Enfin, les ARNm sont dégradés. Les régulations qui touchent chacune de ces étapes sont dites post-transcriptionnelles. Le développement récent d’outils d’analyse à l’échelle génomique ont permis une meilleure compréhension globale des programmes de régulation des gènes au niveau transcriptionnel. Cependant, l’architecture globale des systèmes qui régulent les étapes post-transcriptionnelles d’expression des gènes est encore peu connue. Un tel système de régulation post-transcriptionnelle doit être contrôlé par des centaines de protéines de liaison à l’ARN et de microARN (miARN) encodés dans les génomes eucaryotes. C’est pourquoi il est important de disposer d’outils et de plateformes adaptés à l’étude de cette régulation à l’échelle génomique. Dans cette thèse, nous nous sommes intéressés à deux programmes de la régulation post-transcriptionnelle chez Caenorhabditis elegans : l’épissage alternatif et la régulation par les miARN. Nous avons utilisés des vers rapporteurs de l’épissage alternatif exprimant la double fluorescence GFP et RFP afin d’étudier l’architecture de cette régulation et l’identification ou la validation des facteurs en trans et des éléments en cis par génétique classique en utilisant la mutagenèse aléatoire, l’automatisation du crible grâce au COPAS biosorter et le séquençage des génomes entiers. Nous avons également modifiés en profondeur le module ReFlx du cytomètre en flux adapté aux organismes de grande taille (COPAS Biosorter) afin d’éliminer les problèmes de contamination et diviser par sept le temps nécessaire au traitement dans le but de mener une étude de génétique inverse à haut débit par ARN interférence. Nous avons enfin générer des lignées fluorescentes bi-colores pour étudier la régulation dépendante de la région 3’ UTR grâce aux microARN. / The regulation of gene expression is fundamental to coordinate the synthesis, assembly and localization of macromolecular complexes in cells. This expression is regulated at various levels. It begins in the nucleus where transcription factors bind to specific DNA sequences and recruit RNA polymerases to synthesize RNA. Regulation at this level is called transcriptional. RNA binding proteins associate with RNA during synthesis and operate various modifications such as the addition of a 5' cap, splicing, editing and polyadenylation at the 3'. The transcripts are then exported to the cytoplasm where they will be sent to subcellular regions and stored. mRNA are then associated with translation factors and ribosomes to initiate protein synthesis in a controlled manner. Finally, mRNAs are degraded. Regulations that affect each of these steps are called post-transcriptional regulations. The recent tools developments for genomic scale analysis have allowed a better overall understanding of gene regulation programs at the transcriptional level. However, the overall architecture of systems that regulate post-transcriptional steps of gene expression is still misunderstood. Such a system of post-transcriptional regulation must be controlled by hundreds of RNA binding proteins and microRNA (miRNA) encoded in eukaryotic genomes. This is why it is important to have tools and platforms suited to the study of the post-transcriptional regulation on a genomic scale. During this thesis, we have focused our work on two post-transcriptional regulation programs in Caenorhabditis elegans : alternative splicing and miRNAs regulation. We used GFP and RFP double fluorescent alternative splicing reporter lines to study the architecture of this regulation and to identify trans factors and cis-elements by using forward genetics, random mutagenesis, automated screen through COPAS biosorter and whole genome sequencing. We also extensively modified the ReFlx module of the COPAS to fix carry over problems and divide by seven the time required for processing in order to conduct a High throughput reverse genetic study using RNA interference. We finally generate bi-color fluorescent lines to study 3 'UTR regulation mediated by microRNAs.