Orientadores: José Andrés Yunes, Ana Carolina de Mattos Zeri / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T13:46:58Z (GMT). No. of bitstreams: 1 Canevarolo_RafaelRenatino_M.pdf: 3376497 bytes, checksum: f3e8edf217e0d57089ffcffbe4a8097a (MD5) Previous issue date: 2012 / Resumo: O uso intensivo e combinado de diferentes quimioterápicos tem permitido a cura de 70-80% das leucemias linfóides agudas (LLA) da infância, sendo que a recaída da doença decorre em grande parte da resistência intrínseca das células leucêmicas à quimioterapia. Alguns dos quimioterápicos utilizados na LLA são inibidores metabólicos, como o metotrexato (MTX), antagonista do ácido fólico, que impede a divisão celular ao inibir a síntese de nucleotídeos. Em uma abordagem metabolômica, foi investigada a associação entre linhagens leucêmicas resistentes ou sensíveis ao MTX e metabólitos biondicadores de cada um destes fenótipos. Seis linhagens celulares B-derivadas e oito T-derivadas foram classificadas como sendo resistentes ou sensíveis ao MTX pelo método do 3-(4,5-dimetiltiazol-2il)-2,5-difenil brometo de tetrazolina (MTT) após 48h de co-cultura com diferentes concentrações da droga. Cinco linhagens foram classificadas como resistentes e nove como sensíveis ao MTX. Após 24h de cultura na presença ou ausência de MTX (ambos em triplicata), os metabólitos intracelulares das linhagens foram acessados por ressonância magnética nuclear (RMN) numa abordagem metabolômica. Ao total, oitenta e quatro metabólitos foram quantificados, dos quais 72 foram também identificados. A análise de componentes principais (PCA) não conseguiu segregar as amostras de acordo com sua resistência, ao passo que a análise discriminante por mínimos quadrados parciais (PLS-DA) foi efetiva nesta separação. Os metabólitos mais relevantes para a construção dos modelos de classificação quanto à resistência ao MTX, tanto para amostras tratadas quanto controles foram: ATP, dimetilglicina, fosfocolina e sarcosina (associados à resistência); carnitina, CB-09 (composto não identificado), colato, fumarato, glicocolato, lactato, malato e succinato (associados à sensibilidade ao MTX). A capacidade de classificar corretamente as amostras em sensíveis ou resistentes foi obtida com a construção de curvas da característica operativa do receptor (ROC) para os metabólitos individualmente. Os metabólitos com desempenho bom ou excelente na análise ROC (AUC>0,8) foram selecionados para comporem "testes diagnósticos" de classificação de amostras. De todas as combinações possíveis dentre os metabólitos selecionados, o teste que considerou a combinação de carnitina, sarcosina e succinato em amostras não tratadas com MTX apresentou sensibilidade de 100% (identificou todas as 15 amostras resistentes) e especificidade de 92,3% ao classificar corretamente 24 de 26 amostras sensíveis. O melhor teste diagnóstico para amostras tratadas com MTX considerou as concentrações de CB-MTX, glicocolato, sarcosina e succinato; apresentou sensibilidade de 100% (identificou as 15 amostras resistentes) e especificidade de 85,2%, equivocando-se na classificação de 4 dentre 27 amostras sensíveis. As concentrações metabólicas diferenciais apontaram para uma superativação dos metabolismos energético e de lipídeos em linhagens sensíveis ao MTX, ao passo que linhagens resistentes teriam superativado o metabolismo da glicina. As análises metabolômicas e de integração bioquímica dos metabólitos revelaram interações gênicas, enzimáticas e metabólicas que podem estar alteradas em linhagens sensíveis ou resistentes ao MTX, bem como permitiram a especulação sobre possíveis alvos moleculares que poderiam tornar sensíveis células resistentes ao quimioterápico / Abstract: The intensive use of different and combined chemotherapics has allowed curing 70-80% of pediatric acute lymphoblastic leukemia (ALL), and the relapse of the disease stems largely from the intrinsic resistance of leukemic cells to chemotherapy. Some of the chemotherapics used in ALL are metabolic inhibitors such as methotrexate (MTX), a folic acid antagonist, which prevents cell division by inhibiting the synthesis of nucleotides. The association between leukemic strains resistant or sensitive to MTX and the metabolites associated with each of these phenotypes were investigated. Six B- and eight T-derived cell lines were classified as resistant or sensitive to MTX by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, after 48h in co-culture with different concentrations of the drug. Five lineages were classified as resistant, and nine as sensitive to MTX. After 24 hours of culture in the presence or absence of MTX (both in triplicates), the intracellular metabolites of the lineages were assessed by nuclear magnetic resonance (NMR), in a metabolomic approach. In total, 84 metabolites were quantified, 72 of which were also identified. The principal component analysis (PCA) did not segregate the samples according to their resistance, whereas the supervised partial least square discriminate analysis (PLS-DA) was effective in this separation. ATP, dimethylglycine, sarcosine and phosphocholine were associated with MTX resistance in both models constructed for treated and untreated samples, whereas carnitine, CB-MTX (unidentified compound), cholate, fumarate, glycocholate, lactate, malate and succinate were associated with sensitivity to MTX. The ability to correctly classify the samples into sensitive or resistant groups was checked with the construction of the receiver operating characteristic (ROC) curves for metabolites individually. Metabolites with good or excellent performance in ROC analysis (AUC> 0.8) were selected to compose "diagnostic tests" for classifying samples. Of all the possible combinations among the selected metabolites, the test composed by the comination of carnitine, sarcosine and succinate in untreated samples exhibited sensitivity of 100% (identified all 15 resistant samples) and specificity of 92.3% in classifying correctly 24 of 26 sensitive samples. The best diagnostic test for samples treated with MTX took into consideration concentrations of CB-MTX, glycocholate, sarcosina, succinato. It had a sensitivity of 100% (identified 15 resistant samples) and specificity of 85.2%, classifying incorrectly 4 out of 27 sensitive samples. Differential metabolic concentrations pointed to an over activation of energy and lipids metabolism in MTX-sensitive strains, whereas resistant strains seemed to have overactive the glycine metabolism. Metabolomic and biochemical integration analysis revealed genetic, enzymatic and metabolic interactions that might be altered in strains sensitive or resistant to MTX, as well as allowed speculations about possible molecular targets on which intervention could make resistant cells susceptible to chemotherapy / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Les maladies infectieuses constituent un sérieux problème de santé publique aussi bien dans les pays en développement où elles sont la principale cause de taux de mortalité élevés, que dans les pays industrialisés où les résistances aux antibiotiques existants se développent de façon alarmante. Cette situation engendre un besoin sans cesse croissant de trouver de nouveaux composés antimicrobiens et/ou inhibiteurs de mécanismes de résistances aux antibiotiques. Les plantes médicinales, notamment celles utilisées de façon traditionnelle dans les pays en développement, constituent une source potentielle de ce type de composés. C’est dans ce cadre que l’espèce Cordia gilletii De Wild (Boraginaceae), une plante dont les écorces de racines et les feuilles sont traditionnellement utilisées en République Démocratique du Congo pour combattre les maladies infectieuses, a été étudiée sur le plan tant de ses activités biologiques que de sa composition chimique. Les extraits obtenus à partir des écorces de racines de cette plante ont montré d’intéressantes activités biologiques, (i) un effet antimicrobien direct (bactéricide pour les bactéries gram positif et bactériostatique pour les gram négatif) et indirect (augmentation ou restauration de l’activité des antibiotiques vis-à-vis des souches résistantes); (ii) un effet inhibiteur sur deux des gènes impliqués dans le quorum sensing de Pseudomonas aeruginosa, lasB and rhlA; (iii) un effet antiplasmodial sur une souche chloroquino-sensible de Plasmodium falciparum; (iv) un effet antioxydant mis en évidence par réaction avec le radical libre DPPH. Pour les extraits de feuilles, seule l’activité antiplasmodiale a été observée. <p>Les extraits d’écorces de racines, doués d’activité antimicrobienne directe (extrait méthanolique) et indirecte (extrait n-hexanique) ont été soumis à une série de fractionnements dans le but d’isoler et d’identifier les composés actifs. Pour suivre l’activité lors des fractionnements, le milieu de culture utilisé pour la détection des composés actifs sur une plaque chromatographique (CCM-bioautographie) a été optimisé. Le composé férulaldéhyde, isolé de l’extrait méthanolique, a montré des propriétés antimicrobiennes, antioxydantes et antiplasmodiales. De l’extrait n-hexanique ont été isolés deux composés, l’un actif, le lupéol et l’autre inactif, la friedéline. Le lupéol a montré un effet antimicrobien indirect en réduisant la CMI de certains antibiotiques vis-à-vis d’une souche de MRSA. Ces trois composés, s’ils ont déjà été identifiés dans d’autres plantes, sont décrits pour la première fois dans l’espèce Cordia gilletii ;et ce travail constitue le premier rapport de l’effet antimicrobien indirect du lupéol. <p>Dans le but de s’assurer de l’innocuité des extraits de C. gilletii, une recherche d’alcaloïdes pyrrolizidiniques (APs) a été réalisée par GC-MS. Ces alcaloïdes présentent en effet un réel danger pour la santé humaine et la famille des Boraginaceae, à laquelle appartient l’espèce C. gilletii, est connue comme une des principales sources de ces composés. Aucun AP n’a pu être mis en évidence dans les extraits d’écorces de racines et de feuilles de cette plante jusqu’à une limite de détection de 2 ppm, suggérant ainsi une absence de risque toxicologique en relation avec ces alcaloïdes. Ces résultats rassurants restent à confirmer sur d'autres échantillons obtenus dans des lieux de récolte différents.<p>Le présent travail montre que C. gilletii peut agir contre les microorganismes pathogènes par :(i) son action antimicrobienne directe (due entre autre au férulaldéhyde); (ii) son effet antimicrobien indirect (dû au lupéol), effet permettant d’augmenter ou de restaurer l’activité des antibiotiques vis-à-vis des souches résistantes ;et (iii) son effet inhibiteur de l’expression des gènes du quorum sensing, effet permettant d’atténuer la virulence d’agents infectieux. Ces actions peuvent permettent de faire face aux infections dues notamment à des microorganismes résistants.<p><p><p><p>Infectious diseases remain a serious public health problem both in developing countries, where they are the main cause of the high mortality rates recorded, and in industrialized countries where there is an alarming incidence of antibiotic resistance. There is thus an increasing need for new compounds that can act by a direct antimicrobial effect or by an indirect effect, inhibiting resistance mechanisms of microorganisms. Medicinal plants, particularly those traditionally used against infectious diseases in developing countries, are a probable source for these types of compounds. In this context, Cordia gilletii De Wild (Boraginaceae), a medicinal plant from which root barks and leaves are traditionally used against infectious diseases in Democratic Republic of Congo, was investigated for biological activities and phytochemical composition. Root bark extracts showed interesting biological activities: (i) antimicrobial properties, acting directly (bactericid and bacteriostatic effects against gram positive and gram negative bacteria, respectively) or indirectly (enhancement or restoration of antibiotic activity on resistant strains); (ii) inhibitory effect on the expression of two Pseudomonas aeruginosa QS genes, lasB and rhlA; (iii) antiplasmodial effect against a chloroquine sensitive strain of Plasmodium falciparum; (iv) antioxidant effect determined by the free radical DPPH quenching. Leaves extracts showed only antiplasmodial activity. <p>Root barks extracts with the highest direct (methanol extract) and indirect (n-hexane extract) antimicrobial properties were fractionated to isolate and to identify the active compounds. To bio-guide the fractionation, the culture medium for the detection of active compounds on chromatographic plates (TLC-bioautography) was optimized. The compound ferulaldehyde, isolated from the methanol extract, showed antimicrobial, antioxidant and antiplasmodial properties. From the n-hexane extract two compounds were isolated, lupeol and friedelin. Lupeol showed indirect antimicrobial effect by decreasing the MIC of some antibiotics against MRSA; whereas friedelin was inactive. Although these three compounds have already been described in other plant species, this is the first report of their occurence in Cordia gilletii; the indirect antimicrobial effect of lupeol is described for the first time in this work. <p>As it belongs to the family of Boraginaceae, a family well known as one of the most important sources of pyrrolizidine alkaloids (PAs), Cordia gilletii is susceptible to contain these toxic compounds that were consequently researched. A GC-MS analysis did not reveal the presence of PAs (detection limit, 2 ppm) in root barks and leaves extracts of C. gilletii, suggesting a lack of PA-related toxicity of this plant. This reassuring finding needs to be confirmed with samples harvested at different locations.<p>This work reveals that C. gilletii may act against pathogenic microorganisms by: (i) a direct antimicrobial effect (partly due to férulaldéhyde); (ii) the enhancement or restoration of antibiotic activity against resistant strains (effect of lupeol); and (iii) an inhibitory effect on the expression of quorum sensing regulator genes, decreasing the virulence of microorganisms. These actions could help to fight infections caused by resistant strains. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le programme d’élimination du paludisme de l’OMS est menacé par l’émergence etla propagation potentielle de parasites de l’espèce Plasmodium falciparum résistants à l’artémisinine. Récemment il a été montré que (a) des SNPs dans une région du chromosome 13 subissaient une forte sélection positive récente au Cambodge,(b) plusieurs sous-populations de parasites de P. falciparum résistants et sensibles à l’artémisinine étaient présentes au Cambodge, (c) des mutations dans le domaine Kelch du gène k13 sont des déterminants majeurs de la résistance à l’artémisinine dans la population parasitaire cambodgien et (d) des parasites de sous-populations du nord du Cambodge près de la Thaïlande et du Laos sont résistants à la méfloquine et portent l’allèle R539T du gène de k13.Il est donc nécessaire d’identifier la base génétique de la résistance dans le but de surveiller et de contrôler la transmission de parasites résistants au reste du monde, pour comprendre le métabolisme des parasites et pour le développement de nouveaux médicaments. Ce travail a porté sur la caractérisation de la structure de la population de P. falciparum au Cambodge et la description des propriétés métaboliques des sous-populations présentes ainsi que des flux de gènes entre ces sous-populations. Le but est d’identifier les bases génétiques associées à la transmission et l’acquisition de résistance à l’artémisinine dans le pays.La première approche par code-barre a été développée pour identifier des sous-populations à l’aide d’un petit nombre de loci. Une approche moléculaire de PCR-LDR-FMA multiplexée et basée sur la technologie LUMINEX a été mise au point pour identifier les SNP dans 537 échantillons de sang (2010 - 2011) provenant de 16centres de santé au Cambodge. La présence de sous-populations le long des frontières du pays a été établie grâce à l’analyse de 282 échantillons. Les flux de gènes ont été décrits à partir des 11 loci du code-barre. Le code-barre permet d’identifier les sous-populations de parasites associées à la résistance à l’artémisinine et à la méfloquine qui ont émergé récemment.La seconde approche de caractérisation de la structure de la population de P.falciparum au Cambodge a été définie sur la base de l’analyse de 167 génomes de parasites (données NGS de 2008 à 2011) provenant de quatre localités au Cambodge et récupérés à partir de la base de données ENA. Huit sous-populations de parasites ont pu être décrites à partir d’un jeu de 21257 SNPs caractérisés dans cette étude. La présence de sous-populations mixtes de parasite apparait comme un risque majeur pour la transmission de la résistance à l’artémisinine. L’analyse fonctionnelle montre qu’il existe un fond génétique commun aux isolats dans les populations résistantes et a confirmé l’importance de la voie PI3K dans l’acquisition de la résistance en aidant le parasite à rester sous forme de stade anneau.Nos résultats remettent en question l’origine et la persistance des sous-populations de P. falciparum au Cambodge, fournissent des preuves de flux génétique entre les sous-populations et décrivent un modèle d’acquisition de résistance à l’artémisinine.Le processus d’identification des SNPs fiables a été ensuite appliqué au génome de Babesia microti. Ce parasite est responsable de la babésiose humain (un syndrome de type malaria) et est endémique dans le nord-est des Etats-Unis. L’objectif était de valider la position taxonomique de B. microti en tant que groupe externe aux piroplasmes et d’améliorer l’annotation fonctionnelle du génome en incluant la variabilité génétique, l’expression des gènes et la capacité antigénique des protéines. Nous avons ainsi identifié de nouvelles protéines impliquées dans les interactions hôte-parasite. / The undergoing WHO Malaria elimination program is threatened by the emergenceand potential spread of the Plasmodium falciparum artemisinin resistant parasite.Recent reports have shown (a) SNPs in region of chromosome 13 to be understrong recent positive selection in Cambodia, (b) presence of P. falciparum parasiteresistant and sensitive subpopulations in Cambodia, (c) the evidence that mutationsin the Kelch propeller domain of the k13 gene are major determinants ofartemisinin resistance in Cambodian parasite population and (d) parasite subpopulations in Northern Cambodia near Thailand and Laos with mefloquine drugresistance and carrying R539T allele of the k13 gene.Identifying the genetic basis of resistance is important to monitor and control thetransmission of resistant parasites and to understand parasite metabolism for the development of new drugs. This thesis focuses on analysis of P. falciparum population structure in Cambodia and description of metabolic properties of these subpopulations and gene flow among them. This could help in identifying the genetic evidence associated to transmission and acquisition of artemisinin resistance over the country.First, a barcode approach was used to identify parasite subpopulations using smallnumber of loci. A mid-throughput PCR-LDR-FMA approach based on LUMINEXtechnology was used to screen for SNPs in 537 blood samples (2010 - 2011) from 16health centres in Cambodia. Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Gene flow was described based on the gradient of alleles at the 11 loci in the barcode. The barcode successfully identifies recently emerging parasite subpopulations associated to artemisinin and mefloquine resistance.In the second approach, the parasite population structure was defined based on167 parasite NGS genomes (2008 - 2011) originating from four locations in Cambodia,recovered from the ENA database. Based on calling of 21257 SNPs, eight parasite subpopulations were described. Presence of admixture parasite subpopulation couldbe supporting artemisinin resistance transmission. Functional analysis based on significant genes validated similar background for resistant isolates and revealed PI3K pathway in resistant populations supporting acquisition of resistance by assisting the parasite in ring stage form.Our findings question the origin and the persistence of the P. falciparum subpopulations in Cambodia, provide evidence of gene flow among subpopulations anddescribe a model of artemisinin resistance acquisition.The variant calling approach was also implemented on the Babesia microti genome.This is a malaria like syndrome, and is endemic in the North-Eastern USA. Theobjective was to validate the taxonomic position of B. microti as out-group amongpiroplasmida and improve the functional genome annotation based on genetic variation, gene expression and protein antigenicity. We identified new proteins involved in parasite host interactions.

Although A. fumigatus strains are generally susceptible to azoles, recently, acquired resistance to a number of antifungal compounds has been reported, especially to triazoles possibly due to widespread clinical use of triazoles or through exposure to azole fungicides in the environment. The significant clinical problem of azole resistance has led to study the antifungal resistance mechanisms for developing effective therapeutic strategies. Of 230 clinical A. fumigatus isolates submitted during 2008 and 2009 to the Mycology Reference Centre Manchester, UK (MRCM), 64 (28%) were azole resistant and 14% and 20% of patients had resistant isolates, respectively. Among the resistant isolates, 62 of 64 (97%) were itraconazole resistant, 2 of 64 (3%) were only voriconazole resistant and 78% were multi-azole resistant. The gene encoding 14-α sterol demethylase (cyp51A) was analyzed in 63 itraconazole resistant (ITR-R) and 16 ITR-susceptible clinical and environmental isolates of A. fumigatus respectively. Amino acid substitutions in the cyp51A, the commonest known mechanism of azole resistance in A. fumigatus, were found in some ITR-R isolates. Fifteen different amino acid substitutions were found in the cyp51A three of which, A284T, M220R and M220W, have not been previously reported. In addition, several mutations were found in the cyp51A gene in one of the A. fumigatus environmental isolates. Importantly, a remarkably increased frequency of azole-resistant isolates without cyp51A mutations was observed in 43% of isolates and 54% of patients. Other mechanisms of resistance must be responsible for resistance. In order to assess the contribution of transporters and other genes to resistance, particular resistant isolates that did not carry a cyp51A mutation were studied. The relative expression of three novel transporter genes; ABC11, MFS56 and M85 as well as cyp51A, cyp51B, AfuMDR1, AfuMDR2 AfuMDR3, AfuMDR4 and atrF were assessed using real-time RT-PCR in both azole susceptible and resistant isolates, without cyp51A mutations. Interestingly, deletion of ABC11, MFS56 and M85 from a wild-type strain increased A. fumigatus susceptibility to azoles and these genes showed changes in expression levels in many ITR-R isolates. Most ITR-R isolates without cyp51A mutations showed either constitutive high-level expression of the three novel genes or induction of expression upon exposure to itraconazole. One isolate highly over-expressed cyp51B, a novel finding. Our results are most consistent with over-expression of one or more of these genes in ITR-R A. fumigatus without cyp51A mutations being at least partially responsible for ITR resistance. Multiple concurrent possible resistance mechanisms were found in some isolates. My work probably explains the mechanism(s) of resistance in A. fumigatus isolates with cyp51A mutations. Other ITR resistance mechanisms are also possible. To determine taxonomic relationships among A. fumigatus clinical and environmental isolates, the sequences of the ITS, β-tubulin, actin and calmodulin gene of 23 clinical and 16 environmental isolates were analyzed phylogenetically. Actin and calmodulin sequences proved to be good for species differentiation of A. fumigatus while both ITS, β-tubulin regions did not, in this dataset. Many cryptic species of A. fumigates (complex) were found. All environmental A. fumigates complex isolates were ITR susceptible and no cross resistance was found.

Mycoplasma (M.) bovis est une bactérie pathogène des bovins, à l'origine de signes cliniques divers, comme des mammites, des arthrites, des otites et des bronchopneumonies, ces dernières étant majoritaires en France. Les mycoplasmoses à M. bovis ont un fort coût économique et leur contrôle impose une importante mobilisation sanitaire et un recours très fréquent à l'antibiothérapie. Peu de données étaient disponibles jusque récemment concernant le typage moléculaire et l'antibiosensibilité des souches françaises de M. bovis. Deux études antérieures à ce travail et réalisées au sein de l'UMR « Mycoplasmoses des ruminants » ont montré que les isolats cliniques de M. bovis collectés en France après 2000 appartiennent à un sous-type moléculaire majoritaire (ST2), très homogène et sont par ailleurs multirésistants à la plupart des familles antibiotiques à l'exception des fluoroquinolones. Ces résultats suggèrent la diffusion sur le territoire national d'un clone unique multirésistant. Le premier objectif de cette étude était de déterminer les mécanismes à la base de la perte de sensibilité aux antibiotiques des isolats français. Dans un deuxième temps, les liens entre les différents sous-types moléculaires, les profils d'antibiosensibilité, les maladies associées et le polymorphisme des gènes cibles des antibiotiques ont été investigués. Cette approche a été déployée pour trois familles d'antibiotiques utilisées en pratique vétérinaire: les macrolides, les tétracyclines et également les fluoroquinolones, quoique récemment classées comme molécules critiques. De façon générale, les mutations identifiées dans les cibles des antibiotiques expliquent à elles seules les phénotypes de résistance observés. Des mutations dans les ARNs ribosomaux, cibles des macrolides et des tétracyclines, ont été observées sur des isolats cliniques dès 1978 et sont devenues systématiques sur tous les isolats collectés après 2000 et appartenant au sous-type ST2 majoritaire. En ce qui concerne les fluoroquinolones, la faible augmentation des CMI (concentrations minimales inhibitrices) mesurée chez la plupart des isolats cliniques récents n'a pas été associée à des mutations des QRDR (« Quinolones Resistance-Determining Regions »). Par contre, des altérations cumulées de façon séquentielle dans ces QRDR, associées à une hausse des CMI, ont été mises en évidence lors d'expériences de sélection in vitro et majoritairement pour des souches appartenant à un sous-type récent minoritaire, ST3, apparemment plus variable et plus apte à fixer les mutations. En 2013, le premier isolat clinique présentant une CMI augmentée aux fluoroquinolones a été isolé: il appartient à ce sous-type ST3. L'ensemble des résultats obtenus montrent que les différents sous-types de M. bovis n'évoluent pas de la même façon vers la résistance. Ce constat ajouté à celui de la multirésistance des isolats récents (ST2 ou ST3) met en exergue l'intérêt de la surveillance (sous-typage et antibiosensibilité) et le suivi de l'évolution des isolats de M. bovis circulant en France. Ce suivi permettrait notamment d'anticiper une éventuelle émergence de la résistance aux fluoroquinolones / Mycoplasma (M.) bovis is a bacterial pathogen for cattle, responsible for various clinical signs, like mastitis, arthritis, otitis and respiratory diseases, the latter being the main syndrome present in France. Mycoplasmoses have a great economic impact and their control entails drastic sanitary measures and a frequent use of antibiotherapy. Few data was available until recently on the molecular subtyping and the antimicrobial susceptibility of the French strains of M. bovis. Two previous studies done in the UMR « Mycoplasmoses des ruminants » proved that clinical isolates collected in France after the year 2000 belonged to one major subtype (ST2), which is very homogeneous, and that they were multiresistant to the main antimicrobial families except fluoroquinolones. These results suggested the diffusion of one unique multiresistant clone on the national territory. The first aim of the present study was to decipher the molecular mechanisms underlying the loss of susceptibility to antimicrobials of the French strains. Secondly the links between the molecular subtypes, the antibiotics susceptibility profiles, the clinical origins and the polymorphisms of the target genes were assessed. This approach was used for 3 antimicrobial families currently used in veterinary medicine: macrolides, tetracyclines and fluoroquinolones, although recently classified as critical. Actually, the point mutations observed in the target genes of the antimicrobials accounted for the observed resistance phenotypes. Some mutations in the ribosomal RNAs, targets of the macrolides and the tetracyclines, were observed in clinical isolates as soon as 1978 and they were generalized in all isolates collected after 2000 and belonging to the major subtype ST2. Concerning the fluoroquinolones, the slight increase in MIC (Minimum Inhibitory Concentration) observed in most of the recent isolates was not associated with mutations in the QRDR (Quinolone Resistance-Determining Regions). However alterations that were associated with increased MICs were highlighted and proved to be sequentially cumulated during experiments of in vitro selection under antimicrobials pressure. This was mainly true for strains belonging to a recent and uncommon subtype, ST3, which is apparently more variable and more able to fix the mutations. In 2013 the first clinical strain showing an increased MIC to fluoroquinolones was isolated and proved to belong to ST3. The whole results of this study showed that the different subtypes did not evolve with the same speed towards resistance. This fact, associated with the multiresistant phenotype of the recent isolates (ST2 or ST3), highlights the urge to monitor (subtyping and antimicrobial susceptibility profiles) and to follow-up the evolution of the isolates of M. bovis circulating in France in order to anticipate a potential emergence of the resistance to fluoroquinolones

Medicinal plants have been used as traditional medicine in the treatment of numerous human diseases for thousands of years in many parts of the world. In the developing world, especially in rural areas, herbal remedies continue to be a primary source of medicine. Scientifically, medicinal plants have proven to be an abundant source of biologically active compounds, many of which have already been formulated into useful therapeutic substances or have provided a basis for the development of new lead molecules for pharmaceuticals. Antibiotic resistance, undesireable side effects and expences associated with the use of combination therapy in the treatment of Helicobacter pylori infections have generated a considerable interest in the study of medicinal plants as potential sources of new drugs against this organism. The high complexicity of bioactive compounds accumulated in plants coupled with their broad antimicrobial activity may make it difficult for pathogenic organisms, including H. pylori to acquire resistance during treatment. This study therefore evaluates the antimicrobial potential of selected South African medicinal plants employed in the treatment of H. pylori-related infections, and the subsequent isolation of the plant active principles. An ethnobotanical survey of plants used in the treatment of H. pylori-related infections was conducted in the study area. Crude extracts of Combretum molle, Sclerocarya birrea, Garcinia kola, Alepidea amatymbica and 2 Strychnos species were screened against 30 clinical strains of H. pylori and 2 standard control strains (NCTC 11638 and ATCC 43526). In the preliminary stages of this study, ethyl acetate, acetone, ethanol, methanol and water extracts of the plants were tested against H. pylori by agar well diffusion and micro broth dilution methods. The plant crude extracts that exhibited anti-H. pylori activity with a iv percentage susceptibility of 50 percent and above were considered for the rate of kill assays and the most active crude extracts selected for bio-assay guided isolation of the active ingredient. Preliminary fractionation of the crude extract was achieved by thin layer chromatography (TLC) using different solvent combinations; hexane/diethylether (HDE), ethyl acetate/methanol/water (EMW) and chloroform/ethyl acetate/formic acid (CEF) in order to determine the most suitable combination for column chromatography (CC) and subsequent testing by indirect bioautography. The extract was then fractionated in a silica gel column using previously determined solvent combinations as eluent. Active fractions obtained from column chromatography separations were further fractionated and the compounds identified by gas chromatography/mass spectrometry (GC/MS) analysis. All the plants exhibited antimicrobial activity against H. pylori with zone of inhibition diameters ranging from 0 - 38 mm and minimum inhibitory concentration (MIC) values ranging from 0.06 - 5.0 mg/mL. The most active plant extracts were the acetone extract of C. molle with a percentage susceptibility of 87.1 percent, acetone and aqueous extracts of S. birrea (71 percent each) and the ethanolic extracts of G. kola (53.3 percent). Except for the aqueous extract, these extracts also exhibited a strong bactericidal activity against H. pylori at different concentrations. TLC analysis revealed the presence of 9 components in the acetone extract of S. birrea with the EMW solvent system as opposed to 5 and 8 with HDE and CEF respectively. Bioassay-guided isolation led to the identification of 52 compounds from the acetone extract of S. birrea with n-octacosane being the most abundant (41.68 percent). This was followed by pyrrolidine (38.91 percent), terpinen-4-ol (38.3 percent), n-eicosane (24.98 percent), cyclopentane (16.76 percent), n-triacontane (16.28 percent), aromadendrene (13.63 percent) and α-gujunene (8.77 percent). Terpinen-4-ol and pyrrolidine demonstrated strong antimicrobial activity against H. pylori at all concentrations tested. These results may serve as preliminary scientific validation of the ethnomedicinal uses of the above mentioned plants in the treatment of H. pylori-related infections in South Africa. Terpinen-4-ol and pyrrolidine could be considered for further evaluation as therapeutic or prophylactic agents in the treatment of H. pylori-related infections. However, further investigations would be necessary to determine their toxicological properties, in-vivo potencies and mechanism of action against H.pylori

Garcinia kola is one of the plants used in folklore remedies for the treatment of microbial infections. Bacterial resistance to commonly used antibiotics has necessitated the search for newer and alternative compounds for the treatment of drug resistant microbial infections. This study focuses on the bioactivity of G. kola seeds on Streptococcus pyogenes (ATCC 49399), Staphylococcus aureus (NCTC 6571), Plesiomonas Shigelloides (ATCC 51903) and Salmonella typhimurium (ATCC 13311), organisms which can cause illnesses from mild to severe with potentially fatal outcomes. The crude ethyl acetate, ethanol, methanol, acetone and aqueous extracts were screened by agar-well diffusion method and the activities of the extract were further determined by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. The inhibition zones ranged from 0 - 24 mm, while MIC and MBC of the extract ranged between 0.04 - 1.25 mg/mL and 0.081 - 2.5 mg/mL respectively. Chloroform/ Ethyl Acetate/ Formic acid (CEF) solvent system separated more active compounds followed by Ethyl Acetate/ Methanol/ Water (EMW) and Benzene/ Ethanol/ Ammonium Hydroxide (BEA). The extracts were fractionated by Thin Layer Chromatography (TLC). Bioautography was used to assess the activity of the possible classes of compounds present in the more active extracts. Column chromatography was used to purify the active compounds from the mixture while Gas Chromatography-Mass Spectrometry (GC-MS) was used to identify the phyto components of the fractions. The MIC of the fractions ranged between 0.0006 - 2.5 mg/mL. CEF 3 (F3), CEF 11 (F11) and CEF 12 (F12) revealed the presence of high levels fatty acids Linoleic acid, 1, 2-Benzenedicarboxylic acid and 2, 3-Dihydro-3, 5-dihydroxy-6-methyl, respectively. The results obtained from this study justify the use of this plant in traditional medicine and provide leads which could be further exploited for the development of new and potent antimicrobials.

No description available.

Fluctuations, or "noise", can play a key role in determining the behaviour of living systems. The molecular-level fluctuations that occur in genetic networks are of particular importance. Here, noisy gene expression can result in genetically identical cells displaying significant variation in phenotype, even in identical environments. This variation can act as a basis for natural selection and provide a fitness benefit to cell populations under stress. This thesis focuses on the development of new conceptual knowledge about how gene expression noise and gene network topology influence drug resistance, as well as new simulation techniques to better understand cell population dynamics. Network topology may at first seem disconnected from expression noise, but genes in a network regulate each other through their expression products. The topology of a genetic network can thus amplify or attenuate noisy inputs from the environment and influence the expression characteristics of genes serving as outputs to the network. The main body of the thesis consists of five chapters: 1. A published review article on the physical basis of cellular individuality. 2. A published article presenting a novel method for simulating the dynamics of cell populations. 3. A chapter on modeling and simulating replicative aging and competition using an object-oriented framework. 4. A published research article establishing that noise in gene expression can facilitate adaptation and drug resistance independent of mutation. 5. An article submitted for publication demonstrating that gene network topology can affect the development of drug resistance. These chapters are preceded by a comprehensive introduction that covers essential concepts and theories relevant to the work presented.

Orientador: Katia Borgia Barbosa Pagnano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:42:43Z (GMT). No. of bitstreams: 1 Ribeiro_BeatrizFelicio_M.pdf: 2588401 bytes, checksum: 356f862f97d6467c69db0ac9b13f28d5 (MD5) Previous issue date: 2015 / Resumo: A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa crônica caracterizada pela presença do cromossomo Filadélfia (Ph) e produção da fusão proteica BCR-ABL que possui atividade tirosina quinase. O tratamento da LMC é realizado com inibidores de tirosina quinase (TKIs) e apesar das altas taxas de respostas obtidas com imatinibe, alguns pacientes são resistentes ou intolerantes ao tratamento, sendo necessário a troca para um TKI de segunda geração (nilotinibe ou dasatinibe). O monitoramento das respostas obtidas ao longo do tratamento permite identificar os pacientes que estão em um "estado seguro" (resposta ótima) e os que podem falhar ao tratamento. Os mecanismos de resistência ao tratamento são multifatoriais e a identificação desses mecanismos pode contribuir para o desenvolvimento de novas estratégias para o tratamento dos casos resistentes. Em um trabalho anterior do nosso grupo foram identificados diversos genes e RNAs longos não codificantes diferencialmente expressos em pacientes responsivos e não responsivos ao dasatinibe, entre eles os genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2. O objetivo desse trabalho foi avaliar a resposta molecular em pacientes com LMC em uso de inibidores de tirosina quinase de segunda geração (dasatinibe ou nilotinibe) após falha ou intolerância a um ou dois TKIs e avaliar a expressão dos genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 em pacientes responsivos e resistentes ao dasatinibe. A metodologia utilizada para avaliação dos níveis de trancritos BCR-ABL e da expressão gênica foi o PCR quantitativo em tempo real. A pesquisa de mutações no domínio quinase do BCR-ABL foi realizada nos casos resistentes, através da técnica de sequenciamento direto. Setenta e um pacientes tratados com dasatinibe ou nilotinibe após falha ou intolerância ao imatinibe foram avaliados quanto à resposta molecular. Sessenta e sete porcento, 43% e 33% dos pacientes em fase crônica (FC), acelerada (FA) e crise blástica (CB) obtiveram resposta molecular maior (RMM), respectivamente, ao longo do tratamento. Os pacientes com respostas moleculares precoces (transcritos BCR-ABL <10% aos 3 meses e <1% aos 6 meses) apresentaram maiores SLP e SLE do que os casos que não alcançaram esses níveis de transcritos BCR-ABL aos 3 e/ou aos 6 meses. A avaliação molecular aos 3 meses e aos 6 meses permitiu a melhor identificação dos pacientes com pior prognóstico. Foram também avaliados 25 pacientes tratados com dasatinibe/nilotinibe após falha ou intolerância a dois TKIs. RMM foi obtida em somente 24% dos casos (em FC, um paciente em FA). As taxas de SG e SLP em 5 anos para pacientes em FC foram de 94% e 94%, respectivamente. Poucos pacientes alcançam respostas e essas respostas não são duráveis. Embora seja uma opção para pacientes não elegíveis ao TMO, é necessário o desenvolvimento de um tratamento mais eficaz para esses pacientes. Para avaliação da expressão dos genes ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 foram utilizadas amostras de 9 pacientes ao diagnóstico (sem tratamento prévio), 39 pacientes tratados com dasatinibe (25 responsivos com RCC e 14 resistentes) e 13 doadores saudáves. Não houve diferença de expressão do gene ncSLC44A2 entre os grupos avaliados. Os genes ALOX15B e ncMYLIP estavam hipoexpressos em pacientes com LMC ao diagnóstico em relação aos pacientes com LMC tratados com dasatinibe e ao grupo controle. O gene ncFOXO3A apresentou expressão diminuída em pacientes com LMC ao diagnóstico em relação aos pacientes tratados com dasatinibe. O genes ABCF2 e PAWR estavam hipoexpressos em pacientes ao diagnóstico e nos pacientes tratados com dasatinibe em relação ao grupo controle. Além disso, o gene PAWR estava pouco expresso em pacientes resistentes ao dasatinibe em relação aos pacientes responsivos. Portanto, os genes ABCF2, ALOX15B, PAWR, ncMYLIP e ncFOXO3A foram encontrados com expressão alterada nos grupos estudados e podem estar associados a mecanismos importantes relacionados ao desenvolvimento e resistência da LMC, o que deve ser elucidado em estudos prospectivos / Abstract: Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm characterized by the presence of Philadelphia chromosome (Ph) and production of BCR-ABL fusion protein that has tyrosine kinase activity. Currently, the treatment of CML is accomplished with tyrosine kinase inhibitors (TKIs). Despite the high rates of responses obtained with imatinib, some patients are resistant or intolerant to treatment and need to switch to second generation TKIs (dasatinib or nilotinib). Monitoring responses during treatment allows the identification of patients who are in a "safe haven" (optimal response) and patients who may fail to treatment. Mechanisms of resistance to treatment are multifactorial and identification of these mechanisms may contribute to the development of new strategies for treatment of resistant cases. In a previous report of our group were identified several genes and long non-coding RNAs differentially expressed in CML patients responders and non-responders to dasatinib, including ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP and ncSLC44A2 genes. The aim of this study was to evaluate molecular responses in CML patients treated with second generation TKIs (dasatinib or nilotinib) after failure or intolerance to one or two TKIs and to evaluate the expression of ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP and ncSLC44A2 genes in patients responsive and resistant to dasatinib. The methodology used to evaluate BCR-ABL transcript leves and the gene expression was quantitative real time PCR. BCR-ABL kinase domain mutations analysis was performed in resistant cases by direct sequencing. Seventy-one patients treated with dasatinib/nilotinib after failure or intolerance with imatinib were evaluated according to molecular responses. Sixty-seven percent, 43% and 37% of chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML patients achieved major molecular response (MMR), respectively, during treatment. Patients with early molecular responses (BCR-ABL1<10% at 3 months and <1% at 6 months) had superior PFS and EFS than patients that not achieved these landmarks. The evaluation at 3 and 6 months allows better identication of patients with worse prognosis. We analyzed molecular responses in 25 patients treated with dasatinib/nilotinib after failure or intolerance with two TKIs. MMR was achieved in 24% of cases (all in CP, one in AP). Five-year OS and PFS rates for CP-CML patients were 94% and 94%, respectively. Few patients achieved responses and these responses are not durable. Although, dasatinib/nilotinib had been options for patients not elegible for bone morrow transplantation, its necessary the development of a treatment more effective to these patients. To evaluate the expression of ABCF2, ALOX15B, PAWR, ncFOXO3A, ncMYLIP e ncSLC44A2 genes was used samples of 9 patients newly diagnosed (without treatment), 39 patients treated with dasatinib (25 responsives with CCyR and 14 resistant) and 13 healthy donors. There¿s no difference of ncSLC44A2 gene expression between the groups analized. ALOX15B and ncMYLIP genes were down-regulated in CML patients newly diagnosed in comparison with CML patients treated with dasatinib and control group. ncFOXO3A gene presented decreased expression in CML patients at diagnosis in comparison with patients treated with dasatinib. ABCF2 and PAWR genes were down-regulated in CMl patients newly diagnosed and in CML patients treated with dasatinib in comparison with control group. Moreover, PAWR gene was down-regulated in CML patient¿s resistants to dasatinib in comparison with responsives. ABCF2, ALOX15B, PAWR, ncMYLIP and ncFOXO3A were found with altered expression between the groups evaluated and may be associated with mechanisms related to the development and resistance of CML, which should be elucidated in prospective studies / Mestrado / Clinica Medica / Mestra em Ciências