Les travaux présentés dans ce mémoire ont pour objectif d’approfondir la compréhension de l’altération de la réponse à la stimulation des récepteurs β-adrénergiques dans plusieurs contextes physiopathologiques. La première étude confirme l’existence d’une dysfonction β-adrénergique à l’échelle du cardiomyocyte au cours de la sénescence. Elle met en lumière le rôle de la protéine MRP4 (multidrug resistance associated protein 4) dans cette diminution de réponse inotrope positive à l’isoprotérénol. La deuxième étude évalue la réponse à la stimulation des récepteurs β-adrénergiques dans le syndrome métabolique et montre que la dysfonction est modérée dans ce contexte même en cas de diabète associé à l’obésité. Ces résultats fonctionnels sont expliqués par la diminution d’expression des récepteurs β1- et β2-adrénergiques mais l’absence de surexpression du récepteur β3-adrénergique comme observée dans le diabète de type 1. La troisième étude analyse le rôle de l’atorvastatine sur la réponse β-adrénergique chez les diabétiques et les mécanismes de modulation de cette réponse par une étude du transcriptome cardiaque. Elle montre également que l’inhibition de la production d’oxyde nitrite améliore la réponse β-adrénergique. La quatrième étude a expliqué une part de la dysfonction β-adrénergique chez les diabétiques par la surexpression de MRP4. L’inhibition de MRP4 a permis de restaurer la réponse à l’isoprotérénol au cours de la cardiopathie diabétique. Au total, l’ensemble de nos travaux poursuit la description des mécanismes de la dysfonction β-adrénergique dans la sénescence et le diabète et souligne le rôle de MRP4. / The studies presented in this report looked for a better understanding of the altered response to stimulation of the β-adrenergic receptors in several physiopathological contexts. The first study confirms the alteration of the β-adrenergic response at the cardiomyocyte level in the senescent cardiomyopathy. The role of MRP4 (multidrug resistance associated protein 4) in the reduced inotropic response to isoproterenol is emphasized. The second study evaluates the response to β-adrenoceptors stimulation in the metabolic syndrome and shows mild dysfunction in this context even in obesity associate with diabetes. These functional results are explained by a reduced expression of β1- and β2-adrenergic receptors but no overexpression of β3-adrenoceptor as observed in type 1 diabetes. The third study analyzes the role of atorvastatin on the β-adrenergic response in the diabetic cardiomyopathy and the mechanisms involved by study of the cardiac transcriptome. The inhibition of nitrite oxide production improves the response to β-adrenoceptors stimulation in diabetic heart. The fourth study explained part of the β-adrenergic dysfunction in the diabetic cardiomyopathy by the overexpression of MRP4. The inhibition of this protein restored the response to isoproterenol during diabetic cardiomyopathy. All together the present results carry on with description of the mechanisms involved in the β-adrenergic dysfunction in aging and diabetes and underline the role of MRP4.

La résistance pléiotropique (MDR) est une propriété de certaines cellules relative à la capacité de rejeter ou d’évacuer une très large variété de substances potentiellement toxiques. Les pompes à l’origine de ce rejet sont des protéines membranaires appartenant à la superfamille ABC (ATP-Binding Cassette). Deux membres de cette famille ABC confèrent la propriété de résistance pléiotropique, P-gp (P-glycoprotein) et MRP1 (Multidrug Resistance-associated Protein). Nous avons mené une étude fonctionnelle sur l’activité de ces deux transporteurs dans la muqueuse olfactive à la fois chez le rat et la souris. Nous avons employé le test fluorométrique à la calcéine-AM sur des tranches coronales de la muqueuse olfactive incubées en présence d’inhibiteurs spécifiques des transporteurs de la résistance pléiotropique, vérapamil et cyclosporine A comme inhibiteurs de Pgp ainsi que probénécide et MK571 comme inhibiteurs de MRP1. Chacun de ces quatre inhibiteurs provoque une augmentation significative de l’intensité de la fluorescence.Afin de savoir si les transporteurs de la résistance pléiotropique peuvent être impliqués dans la réponse olfactive nous avons examiné les réponses évoquées par des odorants seuls ou mélangés à l’aide d’enregistrements d’électro-olfactogrammes (EOG). En présence des deux inhibiteurs de MRP1, l’amplitude maximale des EOG est significativement réduite pour chaque stimulus odorant testé, tandis que les inhibiteurs de Pgp n’ont qu’un effet modéré ou nul. L’expression des gènes codant pour Pgp et MRP1 dans l’épithélium olfactif ont ensuite été confirmées par RT-PCR. L’ensemble de ces résultats suggère que les transporteurs MRP1 et Pgp sont présents et fonctionnels dans l’épithélium olfactif principal des rongeurs et sont impliqués dans la réponse aux odorants. Leur fonction précise dans l’olfaction reste à élucider / Multidrug resistance (MDR) is a property of various cells associated with the capacity to reject or efflux a wide range of potentially harmful substances out of the cell. Pumps that effect such efflux are membrane proteins and belong to the ATP- binding cassette (ABC) superfamily. Among the members of the ABC family two are conferring MDR, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP1). In this study we investigated the functional activity of MDR transporters in olfactory mucosa of two species, rat and mouse. We used the fluorometric calcein-AM uptake assay on olfactory mucosal slices incubated with specific inhibitors of the MDR-transporters, verapamil and cyclosporin A as Pgp-inhibitors, and probenecid and MK571 as MRP-inhibitors. All four inhibitors caused significant increases in fluorescence intensities. To test if MDR transporters may be involved in the olfactory response we examined odorant evoked responses to single and mixed odorants by means of electro-olfactograms recordings (EOG). In the presence of the two MRP inhibitors, maximum EOG amplitudes were significantly reduced for all odorants tested, while Pgp inhibitors had only a moderate or no effect. Expression of Pgp and MRP1 encoding genes in the olfactory epithelium was further confirmed by RT-PCR. The results together suggest that MRP and Pgp transporters are present and functional in the main olfactory epithelium of rodents and are implicated in the olfactory response. The precise functional role in olfaction remains to be elucidated.

4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...

Background: Negative outcomes from fetal exposure to maternal dug use include Neonatal Abstinence Syndrome (NAS) and altered development, the unpredictability of which suggests a biological element as yet not accounted for. The manner in which the human placenta protects the fetus from xenobiotics such as drugs of misuse is not completely characterised. However, Adenosine Triphosphate Binding Cassette (ABC) transporters in placentae have demonstrated their ability to efflux xenobiotics away from the fetal vascular compartment leading to lower concentrations than in the maternal compartment and some commonly used drugs have been shown to be substrates for these proteins, e.g. methadone. It is suggested that polymorphisms in the genes that encode these transporter proteins may alter their expression and/or function. Hypothesis- Polymorphisms (SNPs) in the ABC transporters ABCB1, ABCG2, ABCC1 and ABCC2 change protein expression and/or function leading to increased fetal exposure demonstrated by increased signs of NAS and/or altered development. Objectives: To determine if genotype alters protein expression and whether there is a relationship between the level of placental multidrug resistance protein P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP), Multidrug Resistance Associated Proteins (MRP1 and MRP2) expression and neonatal and/or developmental outcomes. Methods: Drug using women were recruited. In the immediate postnatal period placental tissue, cord blood and maternal hair samples were taken. Hair was analysed to determine drug use in the preceding 3 months, immunoblotting determined the level of P-gp, BCRP, MRP1 and MRP2 protein expression. Sequenom MassExtend Array produced genotypes from DNA obtained from cord blood. Infants were assessed for NAS at birth, 3 days and 3 weeks. At 8 months and 1 year development was assessed using the Griffiths Mental Development Scales. Plink was used to determine statistically significant associations between genotype and outcome phenotypes. Results- The level of fetal drug exposure did not predict the need for pharmacological treatment for NAS. 32 polymorphisms with significant associations to outcome measures were identified: 4 SNPs significantly altered protein expression, (3 for P-gp and 1 for MRP1). 41 SNPs were associated with changes across 4 of the 5 GMDS subscales. Discussion: No clear relationship between MDRP protein expression and neonatal outcome was noted. However, fetal genotype did influence the expression of P-gp and MRP1 and genotype across all four proteins was associated with significant changes in the measures of infant development. This was a small study and as such generation of susceptible haplotypes was not possible. However the data generated do support the concept. Further larger and longer term prospective studies, building on the experience reported in this thesis, are necessary to generate more data in order to identify haplotypes leading to increased fetal susceptibility to drug exposure.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

Orientador: Angelo Berchieri Junior / Resumo: Salmonella Heidelberg (SH) é um dos sorovares do gênero Salmonella capaz de infectar tanto seres humanos quanto animais. Surtos infecciosos em seres humanos estão relacionados principalmente à ingestão de produtos de origem avícola e o tratamento é realizado com o uso de antimicrobianos. Entretanto, estas drogas também são utilizadas na produção avícola como melhoradores de desempenho, o que resulta em resistência bacteriana em SH, já relatada em literatura. Com isso, o objetivo deste estudo foi caracterizar 62 isolados de SH quanto ao fenótipo de resistência a 20 antimicrobianos; identificar o genótipo de resistência aos β-lactâmicos por meio da PCR; e avaliar a similaridade dos isolados utilizando as metodologias ERIC-PCR, REP-PCR, BOX-PCR e PFGE. Todas as estirpes de SH foram confirmadas por PCR e submetidas aos testes de sensibilidade a 20 antimicrobianos. A resistência foi observada em 16 dos 20 antimicrobianos utilizados, ressaltando a elevada resistência aos β-lactâmicos (CEF, FOX, AMP, CTX, AMX, AMC e IMP) com a identificação de 41 estirpes multirresistentes. A partir da PCR identificou-se genes codificadores de enzimas ESBL e AmpC, havendo predominância do gene blaCMY-2. O PFGE demonstrou ser a metodologia com a maior diversidade em comparação as demais utilizadas, com a maioria das estirpes sendo agrupadas de acordo com a fonte de isolamento. A partir dos resultados deste estudo é possível observar a diversidade dos isolados de SH no Brasil albergando genes AmpC e a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Salmonella Heidelberg (SH) is one of the serovars of the Salmonella genus capable of infecting both humans and animals. Infectious outbreaks in humans are mainly related to the consumption of products of poultry origin and the treatment is carried out with the use of antimicrobials. However, these drugs are also used in poultry production as performance enhancers, which results in bacterial resistance in SH, already reported in the literature. Thus, the aim of this study was to characterize 62 SH isolates regarding the phenotype of resistance to 20 antimicrobials; identify the genotype of resistance to β-lactams by means of PCR; and to evaluate the similarity of the isolates using the ERIC-PCR, REP-PCR, BOX-PCR and PFGE methodologies. All strains of SH were confirmed by PCR and subjected to sensitivity tests to 20 antimicrobials. Resistance was observed in 16 of the 20 antimicrobials used, highlighting the high resistance to β-lactams (CEF, FOX, AMP, CTX, AMX, AMC and IMP) with the identification of 41 multidrug resistant strains. From the PCR, genes encoding ESBL and AmpC enzymes were identified with a predominance of the blaCMY-2 gene. The PFGE proved to be the methodology with the greatest diversity compared to the others used with the majority of strains being grouped according to the isolation source. From the results of this study, it is possible to observe the diversity of SH isolates in Brazil harboring AmpC genes and presenting different profiles of phenotypic multid... (Complete abstract click electronic access below) / Mestre

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2019 / The emergence of drug resistance to the first line drugs complicates the treatment of tuberculosis (TB), especially in parts of sub-Saharan Africa where accessibility to quality health care is limited. The search for alternative medication has been the centre of research for years due to challenges posed by infectious organisms including drug resistance, lengthy treatment periods and lack of quality health care in developing countries. Stomatostemma monteiroae is used in traditional medicine to treat TB and related symptoms. The aim of this study was to isolate and characterise compounds with antimycobacterial activity from Stomatostemma monteiroae. The plant materials were collected from Ga-Madiga village in Limpopo province of South Africa. Different plant parts namely: leaves, twigs, roots, tuber and tuber-peels were separated, washed, dried and milled to a fine powder. Several solvents (n-hexane, dichloromethane, acetone and methanol) were used to extract the plant material using various extraction methods such as maceration, defatting, and extract enrichment procedure and phytochemical analysis was done using standard chemical tests and thin layer chromatography. The qualitative antioxidant activity was determined by the thin layer chromatography (TLC) based 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity and quantitative antioxidant activity was determined using colorimetric DPPH free radical scavenging and ferric reducing power assay. Antimycobacterial activity of the extracts was assessed using bioautography and micro dilution method tested on Mycobacterium smegmatis (ATCC 1441), Mycobacterium tuberculosis (ATCC 25177) and M. tuberculosis H37Rv (ATCC 27294). The cytotoxic effects of the extracts were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. The compounds with antimycobacterial activity were isolated using bioassay-guided fractionation and purified using preparative thin layer chromatography and thereafter identified using NMR spectroscopy to elucidate the structure. Various phytochemical constituents were detected in different plant parts, with the leaves and twigs possessing more of the phytoconstituents analysed. The TLC profile of S. monteiroae indicated that more compounds are non-polar to intermediate in polarity. The antioxidant activity analysis on TLC plates indicated that all the plant parts have low antioxidant activity, this was also confirmed by xxii quantitative tests. The leaves of S. monteiroae had antimycobacterial activity when analysed using bioautography, while other plant parts had no active bands. The minimum inhibitory concentration values were much higher than the positive control rifampicin and the roots (0.31 mg/mL) followed by the leaves (0.83 mg/mL) had lower inhibitory concentrations when tested against M. smegmatis. The MIC values of extracts against TB causing strains varied greatly, the leaves and the roots had even higher MIC value. Toxicity analysis indicated that all plant parts were non-toxic towards Vero cells (LC50 > 0.02 mg/mL). Bioassay-guided fractionation enabled isolation of one antimycobacterial pure compound from the leaves extracts. The isolated compound was identified using NMR and was found to be a sitosterol derivative 8,9-dehydro-4-methyl-24-vinylobtusifoliol. This compound had a noteworthy activity against M. smegmatis. The present study validates the use of S. monteiroae in the treatment of TB related symptoms traditionally. Further studies are required to analyse the cytotoxic effects of the isolated compound and also testing the antimycobacterial activity of the isolated compound on TB causing pathogens. / National Research Foundation (NRF)

<p>Multidrug resistance is a major issue in treatment and eradication of diseases. There are many mechanisms by which pathogens develop multi drug resistance. Here we focus on the ability of pathogens to evade drug treatment by establishing multi drug resistant reservoirs. In the case of HIV, the virus is able to evade drug treatment and forms both latent and active replicating reservoirs throughout the body. In the case of many bacterial pathogens, multidrug resistance reservoirs are established within mammalian cells, such as macrophages. Many classes of antibiotics are unable to penetrate mammalian cells, making intracellular bacteria difficult to clear</p><p>Previously our research group has developed a Trojan horse strategy to deliver antivirals to HIV cellular reservoirs. Ester based prodrug dimers of abacavir, a reverse transcriptase inhibitor, acted to both inhibit efflux transporters at the BBB and revert to the monomeric therapy in the reducing environments of the cell. Herein we present a new group of sterically hindered carbonate based disulfide linkers that shows improved payload delivery of abacavir and maintain the stability of prodrug molecules towards hydrolysis. We employed these linker molecules to synthesize prodrug dimers of the HIV latency reversal agent prostratin with the hope of targeting latent HIV reservoirs. Payload release studies as well as latency reversal experiments with a latently infected T-helper cell model confirmed that the prostratin carbonate homodimers (<b>ProS₂Me₂</b> and <b>ProS₂Me₄</b>) were able to revert to monomeric prostratin and reverse HIV latency. We next sought to synthesize a prostrain-protease inhibitor heterodimer. While our initial study of a prostratin-lopinavir heterodimer employing this linker strategy (<b>ProLpvS₂Me₂</b>) did not show significant HIV latency reversal activity, we hope to expand our heterodimer studies to achieve dual therapeutic molecules that can both reverse HIV latency and deliver antivirals to HIV reservoirs.</p><p>In order to combat intracellular bacteria our group has focused on development of a novel class of cell penetrating peptides with intrinsic broad spectrum antimicrobial activity that are based on a repeating amino acid triad which forms a cationic amphiphilic polyproline helix (CAPH) scaffold. The first member of this class, <b>P14LRR</b>, exhibited clearance of intracellular bacteria and concentration dependent co-localization within mammalian cells. In efforts to optimize antimicrobial activity we have expanded the CAPHs library by adjusting the chain length between the proline backbone and the guanadinium groups of the cationic amino acids. The first peptide from this expanded library, <b>P14GAP</b> showed much greater cell penetration and antimicrobial activity against a wide range of pathogenic bacteria. However, <b>P14GAP</b> also showed greater toxicity towards mammalian cells, increased hemolysis, and greater membrane binding with mammalian cells as compared to <b>P14LRR</b>. Here we describe the design and synthesis of <b>P14GAP-C1</b>, which contains a methylene between the proline backbone and the guanadinium group. This new analogue decreased the hemolysis activity as compared to <b>P14GAP</b>, although similar membrane binding with mammalian cells was observed. This improvement in hemolysis activity and a slight improvement in cell viability may allow us to use higher concentrations of peptide to treat multidrug resistant bacterial infections.</p><p> </p>

Le phénomène émergent de la tuberculose multirésistante et ultrarésistante est un problème de santé publique à l’échelle mondiale. Dans les pays en développement, ce problème est accru du fait que les laboratoires de diagnostic de la tuberculose manquent d’équipement et d’outils de diagnostics pour identifier ces cas pour prescrire une chimiothérapie adaptée. La première partie de ce travail de doctorat a permis à travers le séquençage du locus pncA, de mettre en évidence que la résistance au Pyrazinamide survient généralement et de manière significative lorsque la souche est multirésistante, c’est-à-dire après l’acquisition de la résistance à la Rifampicine et à l’Isoniazide. Le pourcentage des souches résistantes au PZA est même plus élevé chez les souches MDR résistantes aux FQs. Dans la seconde partie de l’étude, nous proposons une méthode alternative à la culture de bacilles dans un environnement confiné de type P3. À partir d’échantillons cliniques non cultivés (expectoration) et grâce au GeneXpert MTB/RIF, au séquençage de gènes et au spoligotypage, nous avons pu identifier 19 souches multirésistantes, une transmission active de souches sensibles appartenant aux clades LAM10, T1, MANU, H3 et enfin une épidémie sous-jacente de 5 souches Beijing multirésistantes. / The emerging phenomenon of the MDR and XDR-TB is a worldwide public health issue. In developing countries, this problem is amplified due to the fact that TB diagnostic laboratories lack equipment and diagnostic tools to identify these cases and therefore prescribe appropriate chemotherapy. In the first part of this doctoral work, the sequencing of the pncA gene allowed us to show that the resistance to Pyrazinamide occurs significantly when the strain is MDR, corresponding to the acquisition of resistance to Rifampicin and Isoniazid; and that after the acquisition of Fluoroquinolones and to injectable antibiotics of second line (Amykacine, Kanamycine, Capreomycine) resistance by MDR strains, this rate increases even more. In the second part of the study, we propose an alternative method to the culture of bacilli in a BSL3 confined environment. From uncultivated clinical samples (sputum) and through GeneXpert MTB/RIF, sequencing of genes and spoligotyping, we identified 19 MDR strains, active transmission of sensitive strains belonging to clades LAM10, T1, MANU, H3 and finally as well as an underlying epidemic of 5 Beijing MDR strains.In the first study, 272 retrospective samples of Mycobacterium tuberculosis isolates were selected from two large cosmopolitan cities: Northern Paris (Bichat-Claude Bernard Hospital, 101 strains) and Southwest of Shanghai (Songjiang district, 171 Strains). These strains were selected according to their known phenotypic sensitivity to Rifampicin (RIF) and Isoniazid (INH). These phenotypic resistances were confirmed by the HAIN genotype analysis tools MTBDRplus and by the sequencing of the rpoB and katG/inhA genes. To determine the extensively drug resistance strains (XDR), we sequenced the gyrA/gyrB and rrs genes to identify genetic mutations associated with resistance to Fluoroquinolones (FQs) and second-line injectable antibiotics: Amikacin (AMK)-Kanamycin ( KAN)-Capreomycin (CAP), respectively. Finally, we sequenced the pncA gene of all isolates to identify the genetic mutations associated with resistance to Pyrazinamide (PZA). The strains were genotyped by spoligotyping and MIRU-VNTR.In the second study, from October 2014 to February 2015, 159 morning sputum samples with smear-positive smear after Ziehl-Neelsen staining were collected at the three main diagnostic laboratories for tuberculosis in Libreville, Gabon. These clinical samples were transported to the National Laboratory of Public Health in Libreville for analysis with the GeneXpert MTB/RIF automaton to confirm the microscopic diagnosis and to determine the resistance of bacilli to Rifampicin. Of the 159 samples, 29 samples had a sputum volume less than 1 ml, the minimum required according to the manufacturer's recommendations. For the 130 sputum samples analyzed by the GeneXpert automaton, 375 μl of the remaining GeneXpert solution not introduced into the cartridge was introduced into a 50 ml conical tube containing 25 ml of phosphate buffer (autoclaved solution) to neutralize the pH of the GeneXpert solution. The conical tube is centrifuged for 15 minutes at 4,500 rpm, the pellet is taken up in 100 μl of TE and then transferred to a 100 μl microtube which is subsequently heated for 30 minutes at 90°C. After a cycle of freezing (-40 ° C. for 1 h)-defrosting, the microtube is briefly centrifuged and the supernatant is transferred to a new microtube. From this new microtube we amplified by PCR and then sequenced the rpoB, katG/inhA, pncA, gyrA, rrs and rpsL genes to identify mutations associated with resistance to Rifampicin, Isoniazid, Pyrazinamide, Fluoroquinolones, Antibiotics in second lines: Amikacin-Kanamycin-Capreomycin and Streptomycin (SM), respectively. All the samples were genotyped by the multiplexed spoligotyping applied to the Luminex MagPix.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18326号 / 農博第2051号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4833(農学部図書室) / 31184 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM