Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire, responsable d'une infection cosmopolite très répandue, la toxoplasmose. Les différents schémas de traitement de la toxoplasmose reposent sur l'association de la pyriméthamine et d'un sulfamide qui agissent en synergie pour bloquer la voie de synthèse des folates. Cependant des échecs thérapeutiques ont été rapportés dans la littérature, et trois souches naturellement résistantes à la sulfadiazine, TgA 103001 (Type I), TgH 32006 (Type II) et TgH 32045 (Type II variant), ont été décrites. Notre travail a porté sur l'étude des mécanismes de résistance à la sulfadiazine chez T. gondii. Nous nous sommes intéressés dans un premier temps à l'implication des gènes cibles, dhps et dhfr, ainsi que les ABC transporteurs TgABC.B1, TgABC.B2, TgABC.C1 et TgABC.C2 dans la résistance à la sulfadiazine chez T. gondii. Cependant aucun polymorphisme ni surexpression de ces gènes n'a pu être relié aux mécanismes de résistance. Nous avons ensuite comparé les protéomes des souches naturellement résistantes aux protéomes des souches sensibles RH (Type I) et ME-49 (Type II) par DIGE. Parmi les 31 protéines différentiellement exprimées entre souches sensibles et résistantes, quatre protéines, ROP2, MIC2, ENO2 et IMC1, nous ont semblé intéressantes. Afin de s'affranchir des variations liées aux différences de fond génétique des souches, les souches sensibles RH et ME-49 ont été rendues résistantes in vitro à la sulfadiazine par pression médicamenteuse croissante, résistance validée in vitro grâce à la mise au point d'un nouveau test de chimiosensibilité. Nous avons ensuite, par 2-DE, comparé les protéomes des souches de type II sensible (ME-49), et résistantes (ME-49-RSDZ et TgH 32006) sans identifier le ou les candidat(s) impliqué(s) dans la résistance médicamenteuse. Cependant, l'analyse des souches ME-49 sensible et ME-49-RSDZ résistante, par microarrays, nous a permis d'identifier une cible appartenant à la voie de synthèse des folates : la folylpolyglutamate synthase. / Toxoplasma gondii is an obligate intracellular protozoan parasite responsible of a widespread infection, toxoplasmosis. Treatment options for toxoplasmosis are generally limited to combinations of sulfonamide and pyrimethamine which have a synergistic action on T. gondii folate synthesis by inhibiting two major enzymes: dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). However treatment failures have been reported, and three naturally sulfadiazine resistant strains, TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant), have been described. In this work, we studied resistance mechanisms to sulfadiazine on T. gondii. We are interested, in a first time, on the involvement of target genes, dhps and dhfr, and ABC transporters, TgABC.B1, TgABC.B2, TgABC.C1 and TgABC.C2, in the sulfadiazine resistance on T. gondii. However, neither polymorphisms nor overexpression of these genes has been linked to resistance mechanisms. Then, we compared proteomes of naturally resistant strains to sensitive strains RH (Type I) and ME-49 (Type II) by DIGE. Among the 31 proteins differentially expressed between sensitive and resistant strains, four proteins, ROP2, MIC2, ENO2 and IMC1, seemed to be interesting. In order to avoid variations due to differences from genetic background, sensitive strains RH and ME-49 have been made resistant in vitro by gradual increase in sulfadiazine concentration. This resistance was checked in vitro by the development of a new chemosensitivity assay. We compared then, by 2-DE, proteomes of the type II strains, sensitive (ME-49) and resistant (ME-49-RSDZ and TgH 32006), without identifying candidates implicated in sulfadiazine resistance mechanisms. However, analysis of the sensitive strain ME-49 and the resistant strain ME-49-RSDZ, by microarrays, allowed us to identify a candidate belonging to folate synthesis pathways: folylpolyglutamate synthase.

by Alexander Kreymerman. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web. / Sulindac is a known NSAID that has also been shown to have anti-cancer activity that is not related to its ability to inhibit COX 1 and 2. During the past 15 years there have been a large number of studies attempting to elucidate its mechanism of action. Our laboratory has shown that sulindac can both protect normal cells and enhance the killing of cancer cells under oxidative stress from H2O2 and TBHP. However, except for mitochondrial dysfunction and ROS production, the mechanism by which sulindac sensitized the cancer cells to oxidative stress remains unknown. Results of this research project suggest that the effect of sulindac and oxidative stress not only involves mitochondrial ROS production, but also aspects of the preconditioning response. In normal cells this leads to survival by a preconditioning pathway, likely involving PKCε. . However, cancer cells react by initiating a pathway leading to apoptosis involving PKCδ.

Ensaios clínicos indicam haver diferenças regionais na eficácia do tratamento da doença de Chagas com (BZ) e Nifurtimox (NFX). Transportadores ABC desempenham um papel importante na resistência a drogas. Evidências indicam que o transportador TcABCG1 é superexpresso em cepas resistentes a BZ. O objetivo geral foi obter evidências para o envolvimento de TcABCG1 no fenótipo de resistência a BZ. Determinamos a classificação de seis cepas em suscetíveis e resistentes a BZ, e o efeito de três inibidores de transportadores ABC de eucariotos. Transfectamos em CL Brener os genes TcABCG1 de cepas resistentes. Nas culturas transfectadas com os genes de Silvio e YuYu verificamos aumento da resistência a BZ e a NFX, e aumento da abundância relativa de transcritos de TcABCG1. Genes ABC ortólogos aos de T. cruzi foram identificados em tripanossomas africanos e Leishmania spp. Dados referentes à análise filogenética e classificação dos transportadores ABC de T. cruzi nas oito subfamílias das proteínas ABC de eucariotos são apresentados. / Clinical trials indicate regional differences in the efficacy of Chagas disease treatment with (BZ) and nifurtimox (NFX). ABC transporters play an important role in drug resistance. Evidences indicate that TcABCG1 is overexpressed in BZ-resistant strains. The overall aim was to obtain evidence for the involvement of TcABCG1 in the BZ resistance phenotype. We determined the classification of six BZ susceptible and resistant strains and the effect of three eukaryotic ABC transporters inhibitors. We transfected TcABCG1 genes from resistant strains in CL Brener. In cultures transfected with Silvio and YuYu genes we observed an increased resistance to BZ and NFX, and increased relative abundance for TcABCG1 transcripts. ABC orthologue genes to those of T. cruzi were identified in African trypanosomes and Leishmania spp. Data relating to classification and phylogenetic analysis of T. cruzi ABC transporters in eight eukaryotic ABC proteins subfamilies are presented.

A malária é responsável por cerca de 207 milhões de casos e 627 mil óbitos em todo o mundo. No Brasil, em 2013, foram registrados mais de 167 mil casos. Um dos grandes desafios para o controle da doença é o desenvolvimento de resistência aos antimaláricos. Dentre as cinco espécies que podem causar malária em seres humanos, Plasmodium falciparum é a que apresenta maior habilidade de desenvolver resistência a quase todas as classes de medicamentos utilizados no tratamento. Estudos com marcadores moleculares têm associado mutações em diversos genes à resistência aos antimaláricos. A mutação K76T no gene pfcrt é relacionada à resistência à cloroquina. Mutações em pfmdr1 e aumento de seu número de cópias foram associados à resistência a diversos antimaláricos, como quinino, mefloquina e derivados de artemisinina. A resistência aos antifolatos é associada a mutações nos genes pfdhfr e pfdhps. Mutações nos genes pfATPase6 e pfAP2-u têm sido relacionadas à diminuição de sensibilidade à artemisinina. O monitoramento dessas mutações e suas associações às respostas in vivo e in vitro aos antimaláricos pode contribuir para a escolha de terapêutica para o controle da doença. O objetivo deste estudo foi analisar o perfil genético relacionado à resistência em P. falciparum ao longo de 27 anos. Foram analisadas amostras de P. falciparum coletadas no período entre 1984 e 2011, de pacientes atendidos em serviços de saúde nos estados do Pará e São Paulo, com infecções provenientes principalmente de países da América do Sul e África. A análise do gene pfcrt mostrou frequência de 100% da mutação K76T nas amostras de países da América do Sul ao longo das décadas analisadas. Este resultado é concordante com o fenótipo de resistência à cloroquina observado em 100% das amostras testadas in vitro para sensibilidade a este antimalárico. Amostras de países africanos apresentaram o genótipo selvagem em infecções únicas ou mistas. Com relação ao gene pfmdr1, a análise do códon 86 mostrou surgimento do mutante 86Y na última década em amostras da América do Sul e ocorrência de alelos selvagens e mutantes em amostras da África. Na análise do códon 1246 foi observada predominância do mutante 1246Y nas amostras sul-americanas e do selvagem D1246 nas africanas. Com relação ao gene pfdhfr, as amostras brasileiras apresentaram frequência de 100% dos mutantes 51I e 108N. O mutante 59R não foi observado no período 1980-1990, mas estava presente em 22,6% das amostras de 2000-2010. A análise do gene pfdhps das amostras brasileiras mostrou frequência de 100% do mutante 437G em todas as décadas e diminuição da frequência de 540E no período 2000-2010 em relação aos anteriores. O sequenciamento do gene pfATPase6 revelou a ocorrência de mutações que já haviam sido relatadas anteriormente, porém seu papel na resistência aos derivados de artemisinina ainda não foi elucidado. Na análise do gene pfAP2-? foram observadas duas novas mutações. Não foram observadas associações entre as mutações estudadas e a resposta in vivo e in vitro à mefloquina, quinino e derivados de artemisinina. Este estudo permitiu a avaliação do perfil genético de P. falciparum, com análise de marcadores moleculares relacionados à resistência a diversos antimaláricos, em amostras coletadas ao longo de 27 anos em que o parasito foi submetido à pressão de diferentes esquemas terapêuticos adotados no Brasil / Malaria is responsible for 207 million cases and 627 thousand deaths worldwide. In Brazil, in 2013, more than 167 thousand cases were reported. The major challenge for malaria control is the emergence and spread of resistance to antimalarials. Among the five species able to cause malaria in humans, Plasmodium falciparum presents ability to develop resistance to almost all classes of drugs for malaria treatment. Studies with molecular markers have associated mutations in several genes to antimalarial resistance. The mutation K76T in pfcrt is related to chloroquine resistance. Polymorphisms in pfmdr1 and increased copy number were associated to several antimalarials resistance, such as quinine, mefloquine and artemisinin derivatives. Resistance to antifolates is associated to mutations in pfdhfr and pfdhps genes. Mutations in pfATPase6 and pfAP2-u were linked to decreased sensibility to artemisinins. The monitoring of these mutations and their association with in vivo and in vitro responses may contribute to the choice of therapeutics for malaria control. The aim of the present study was to analyze the genetic profile related to resistance in P. falciparum over twenty-seven years. Samples collected from 1984 to 2011 from patients enrolled at health facilities in Para and Sao Paulo States were assessed. The origin of infections was mainly from South America and Africa countries. Pfcrt analysis showed that all samples from South America countries harbored the K76T mutation over the four decades, in agreement with the resistant phenotypic response of samples tested in vitro for chloroquine. African samples presented the wild type in mixed or single infections. Regarding to the pfmdr1 gene, the emergence of the mutant 86Y was observed in the last decade in South American samples and occurrence of both, mutant and wild type, in African ones. The analysis of 1246 showed only the mutant 1246Y in South American samples and the wild type D1246 in samples from Africa. Regarding to pfdhfr gene, the frequency of mutants 51I and 108N was 100% in Brazilian samples. The mutant 59R was not observed in the period 1980-1990 and it was present in 22.6% in samples from 2000-2010. The mutant 437G of pfdhps gene was observed in 100% of Brazilian samples in all decades and the frequency of mutant 540E decreased in the period 2000-2010 when compared to 1980-1990. The sequence analysis of pfATPase6 gene showed mutations previous described, but their role in artemisinin resistance is not well defined. Two novel mutations were observed in pfAP2-? gene. No association between the polymorphisms studied and in vivo or in vitro responses to mefloquine, quinine and artemisinin derivatives was observed. This study enabled the knowledge of P. falciparum genetic profile regarding to mutations related to resistance in samples collected over twenty-seven years, when the parasite was exposed to selective pressure of several therapeutic schemes adopted in Brazil

O fenômeno de amplificação gênica é um mecanismo de auto-preservação celular observado com freqüência no protozoário parasita Leishmania quando submetido a estímulos negativos como, por exemplo, a presença de drogas. A região H do genoma de Leishmania (Leishmania) major é um dos loci mais estudados que leva à amplificação em resposta a drogas não relacionadas, como a terbinafina. Foram isoladas linhagens de L. (L.) major e Leishmania (Viannia) braziliensis resistentes à terbinafina. Análises por corridas curtas de eletroforese em campo pulsado (PFGE) e Southern blotting revelaram que a resistência observada nestas linhagens não foi gerada pela amplificação do locus H. Sendo assim, a resistência ao inibidor da esqualeno epoxidase está sendo gerada por um outro mecanismo uma vez que outros loci podem estar envolvidos na resistência à terbinafina e através da análise diferencial do perfil de proteínas, os mutantes resistentes apresentaram diferenças na expressão de proteínas. O alvo inicial para a elucidação da resistência à terbinafina nas linhagens selecionadas foi a via de biossíntese de ergosterol. Foram escolhidos os genes da 3-cetoacil-CoA tiolase (ERG10), esqualeno sintase (ERG9 ou SQS1), esqualeno epoxidase (ERG1), oxidoesqualeno-lanosterol ciclase (ERG7) e lanosterol 14-demetilase (ERG11), de L. major e L. braziliensis, além do gene YIP1 de L. braziliensis. Para isso foram sintetizados oligonucleotídeos a partir das seqüências geradas pelo projeto genoma destas espécies depositadas em bancos de dados. Os genes ERG10, SQS1, ERG1, ERG7 e ERG11 de L. major e de L. braziliensis além do YIP1 de L. braziliensis amplificados por PCR foram clonados no vetor pGEM-T Easy, que possibilitou a construção de reagentes para ruptura de todos genes pela inserção da marca HYG e, com exceção do gene ERG7 de L. braziliensis, os genes foram subclonados no vetor pXG1. Fragmentos de restrição dos genes clonados no vetor pGEM-T Easy foram utilizados para analisar o nível de seus transcritos por northern blot. Também verificamos através de corridas curtas de PFGE e análises de Southern, que as linhagens em estudo não apresentam amplificação dos loci em estudo. A participação de genes da via de biossíntese de ergosterol de L. major e L. braziliensis e do gene YIP1 de L. braziliensis na resistência ou susceptibilidade à terbinafina e/ou anfotericina B foi verificada através de experimentos funcionais. Com esse objetivo, os genes YIP1, ERG10 e ERG1 de L. braziliensis foram transfectados na linhagem LB2904 de L. braziliensis, e os genes ERG10, SQS1, ERG1, ERG7 e ERG11 e a ruptura do gene ERG1 pelo cassete HYG de L. major foram transfectados na linhagem LT252 de L. major. Foram realizados experimentos para analisar a susceptibilidade à anfotericina B associada ou não à terbinafina, das linhagens selvagens de L. major e L. braziliensis, e a partir destes experimentos iniciais, foram selecionadas linhagens destas duas espécies resistentes à anfotericina B. Para analisar a possível função regulatória dos elementos RIME 5/2/6 de L. braziliensis, foi feita a amplificação por PCR da repetição invertida LbRIME 5/2/6b que permitiu a clonagem deste fragmento no vetor pGEM-T Easy e a sua ruptura pela marca SAT. A clonagem no vetor pGEM possibilitou a análise da interação de proteínas nucleares à repetição LbRIME 5/2/6b através do gel shift. / Gene amplification is a common phenomenon observed in Leishmania cell lines subjected to drug pressure. The H locus of Leishmania (Leishmania) major is normally found amplified in cell lines selected in unrelated drugs, as terbinafine. We selected cell lines of L. (L.) major and Leishmania (Viannia) braziliensis resistant to terbinafine. Short-run Pulsed Field Gel Electrophoresis (PFGE) and Southern blotting analysis showed that this resistance was not generated by H locus amplification. Resistance to squalene epoxidase inhibiter is being generated by other mechanism once others loci can be involved in the terbinafine resistance and through protein partner differential analysis, mutants resistant showed differences in protein expression. The initial target to terbinafine resistance elucidation in the cell lines selected was ergosterol biosynthesis pathway. We choose genes: 3-ketoacyl-CoA thiolase (ERG10), squalene synthase (ERG9 or SQS1), squalene epoxidase (ERG1), oxidosqualene-lanosterol cyclase (ERG7), and lanosterol 14-demethylase (ERG11), of L. major and L. braziliensis, besides YIP1 gene of L. braziliensis. For this, primers were synthesized using the sequences generated by genome project of these species inserted in gene bank. The genes ERG10, SQS1, ERG1, ERG7 and ERG11 of L. major and of L. braziliensis besides YIP1 of L. braziliensis were amplified by PCR and cloned into pGEM-T Easy vector that enabled all genes disruption by insertion of HYG cassette and, with exception of the ERG7 gene of L. braziliensis, genes were subcloned into shuttle-vector pXG1. Restrictions fragments of these genes were used in Northern analysis to verify the transcripts level. We verified through short-run PFGE and Southern analysis that the resistant cell lines do not show amplification of studied loci. The participation of ergosterol biosynthesis pathway genes of L. major and L. braziliensis and YIP1 gene of L. braziliensis in the resistance to terbinafine was verified in functional experiments. With this objective, the genes YIP1, ERG10 and ERG1 of L. braziliensis were transfected into LB2904 cell line of L. braziliensis, and the genes ERG10, SQS1, ERG1, ERG7 and ERG11 and the ERG1 gene disruption by HYG mark of L. major were transfected into LT252 cell line of L. major. Experiments that analyze the susceptibility to amphotericin B associated or not to terbinafine were performed using the wild type cell lines of L. major and L. braziliensis, and with this initials experiments, we selected cell lines of these two species resistant to amphotericin B. In order to analyze the possible regulatory function of the RIME 5/2/6 elements of L. braziliensis, the repeat LbRIME 5/2/6b was amplified by PCR and cloned into pGEM-T Easy vector and with this clone, the element was disrupted with a SAT cassette. The cloning into vector pGEM enabled to analyze the interaction of the nuclear protein with the repeat LbRIME 5/2/6b through gel shift assay.

A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported

Acinetobacter baumannii é um importante agente de infecções relacionadas à assistência à saúde, principalmente nas unidades de terapia intensiva no Brasil, sua resistência a antimicrobianos vem aumentando nas últimas décadas e as opções para o tratamento são restritas. Em 2011, no HC-FMUSP, ocorreu um surto de infecção por A. baumannii resistente a todos os antibióticos e desde então isolados resistentes a todas as classes de antibióticos vêm sendo identificados no hospital. O estudo investigou o efeito de combinações antimicrobianas contra 20 isolados clínicos de A. baumannii, sendo sete isolados resistentes (2011 a 2012) e treze sensíveis a colistina (2002 a 2004), obtidas do banco de cepas do LIM-54 com diferentes mecanismos de resistência. Foram realizados, concentração inibitória mínima dos antibióticos, avaliação da clonalidade por Pulsedfield gel electrophoresis, detecção de mecanismos de resistência por reação de amplificação em cadeia da polimerase, análise de proteínas da membrana externa e baseado na clonalidade e o sequenciamento total do genoma de quinze isolados. Sinergismo foi investigado usando os métodos de checkerboard e time-kill. Para monitorização da expressão do sistema regulatório PmrCAB e dos genes responsáveis pela biossíntese do lipopolissacarídeo, foi realizada reação em cadeia da polimerase quantitativa. Todos os isolados foram resistentes ao meropenem e a rifampicina. OXA- 23 e OXA-143 foram as carbapenemases mais frequentes. Quatro isolados mostraram perda de uma proteína de membrana externa denominada OMP 43kDa. Os isolados sensíveis a colistina pertenciam a diferentes clones e Multilocus Sequence Types, também apresentaram o maior efeito sinérgico com fosfomicina-amicacina. A resistência a colistina foi associada com a superexpressão do gene pmrA. Seis isolados resistentes a colistina, pertenciam ao Complexo Clonal 113 e o maior efeito sinérgico foi observado com combinações de colistina-rifampicina seguido de colistina-vancomicina. Foram encontrados diferentes genes de virulência envolvidos com formação de biofilme, aderência, produção de enzimas e captação de ferro / Acinetobacter baumannii is an important agent of infections related to health care, especially in intensive care units in Brazil, its antimicrobial resistance has increased in recent decades and the options for treatment are restricted. In 2011, in the HC-FMUSP, there was an outbreak of A. baumannii infection resistant to all antibiotics and since then isolates resistant to all classes of antibiotics have been identified in the hospital. The study investigated the effect of antimicrobial combinations against 20 clinical isolates of A. baumannii, seven isolates colistin resistant (2011-2012) and thirteen colistin sensitive (2002-2004) from LIM-54 strains bench with different resistance mechanisms. Minimum inhibitory concentration of antibiotics, clonality evaluation by pulsed-field gel electrophoresis and detection of resistance mechanisms by polymerase chain reaction, outer membrane protein analysis and based on clonality, the whole genome sequencing of fifteen isolates were performed. Synergism was determined using checkerboard and time-kill methods. For expression monitoring of the regulatory system PmrCAB and the genes responsible for the biosynthesis of lipopolysaccharide, it was performed quantitative polymerase chain reaction. All isolates were resistant to meropenem and rifampicin. OXA-23 and OXA-143 were the most frequent carbapenems. Four isolates showed loss of one outer membrane protein called OMP 43kDa. Colistin susceptible isolates belonged to different clones and Multilocus Sequence Types; it also showed the greatest synergistic effect with fosfomycin-amikacin. The colistin resistance was involved in overexpression of the pmrA gene. Six colistin-resistant isolates belonged to Clonal Complex 113 and higher synergistic effect were observed with colistin-rifampicin followed by colistin-vancomycin combinations for these isolates. We found different virulence genes involved in biofilm formation, adhesion, enzyme production and iron uptake

Li Ka Wai Kay. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT (ENGLISH/CHINESE) --- p.ii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF APPENDICES --- p.xiii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Gastric carcinomas --- p.1 / Chapter 1.1a --- Epidemiology --- p.1 / Chapter 1.1b --- Classification --- p.2 / Chapter 1.1c --- TNM staging --- p.3 / Chapter 1.1d --- Prognosis --- p.4 / Chapter 1.1e --- Etiology --- p.6 / Chapter 1.1f --- Genetic alteration in gastric cancer --- p.10 / Chapter 1.2 --- Treatment --- p.16 / Chapter 1.2a --- "Surgery, chemotherapy, and others" --- p.16 / Chapter 1.2b --- Response rate of treatments in previous studies --- p.18 / Chapter 1.2c --- Chemotherapeutic Drugs --- p.21 / Chapter 1.2c (1) --- 5-fluorouracil (5-FU) --- p.22 / Chapter 1.2c (2) --- cis-diamminedichloroplatinum (Cisplatin) --- p.23 / Chapter 1.2c (3) --- Doxorubicin (Adriamycin) --- p.23 / Chapter 1.2c (4) --- Daunorubicin --- p.25 / Chapter 1.2c (5) --- Epirubicin --- p.25 / Chapter 1.2d --- Toxicity of chemotherapeutic drugs --- p.26 / Chapter 1.2d (1) --- Side effects of 5-FU --- p.26 / Chapter 1.2d (2) --- "Side effects of anthracyc lines (adriamycin, daunobicin, epuirbicin)" --- p.27 / Chapter 1.2d (3) --- Side effects of cisplatin --- p.28 / Chapter 1.3 --- Mechanisms of drug resistance --- p.28 / Chapter 1.3a --- Drug resistance --- p.28 / Chapter 1.3b --- P-glycoprotein (MDR1 gene) --- p.29 / Chapter 1.3c --- p53 tumor suppressor gene --- p.35 / Chapter 1.4 --- Chemosensitivity testing --- p.40 / Chapter 1.4a --- Original of chemosensitivity testing --- p.40 / Chapter 1.4b --- Non-clonogentic assay --- p.40 / Chapter 1.4c --- Clonogenic assay --- p.42 / Chapter 2 --- AIM OF MY STUDY --- p.44 / Chapter 3 --- MATERIALS AND METHODS --- p.45 / Chapter 3.1 --- Patients --- p.45 / Chapter 3.2 --- Tumor collection and handling procedure --- p.46 / Chapter 3.2a --- Large tumor tissue from gastrectomy --- p.46 / Chapter 3.2b --- Pseudo-biopsies --- p.47 / Chapter 3.3 --- Chemosensitivity testing --- p.48 / Chapter 3.3a --- Cell Plating --- p.48 / Chapter 3.3b --- Drug testing --- p.49 / Chapter 3.4 --- Chemosensitivity analysis --- p.50 / Chapter 3.5 --- Conformational sensitive gel electrophoresis analysis (CSGE) and single strand conformational polymorphism (SSCP) --- p.51 / Chapter 3.5a --- Preparation of genomic DNA --- p.51 / Chapter 3.5b --- PCR condition for CSGE analysis --- p.51 / Chapter 3.5c --- Scanning PCR products by CSGE --- p.52 / Chapter 3.5d --- PCR condition for SSCP analysis --- p.53 / Chapter 3.5e --- Scanning PCR products by SSCP --- p.53 / Chapter 3.6 --- Reverse transcription-polymerase chain reaction (RT-PCR) for multi-drug drug resistance (MDR1) gene --- p.54 / Chapter 3.6a --- Isolation of RNA --- p.54 / Chapter 3.6b --- cDNA synthesis --- p.55 / Chapter 3.6c --- PCR primers --- p.55 / Chapter 3.6d --- Optimalization of PCR condition for MDR1 gene expression --- p.56 / Chapter 3.6e --- PCR of β2-m gene --- p.57 / Chapter 3.6f --- PCR of MDR1 gene and analysis of its expression --- p.57 / Chapter 3.7 --- Immunohistochemistry --- p.58 / Chapter 3.7a --- Immunostaining by DO-7 --- p.58 / Chapter 3.7b --- lmmunohistochemistochemical analysis of p53 protein expression --- p.59 / Chapter 3.8 --- Statistics --- p.59 / Chapter 4. --- RESULTS --- p.60 / Chapter 4.1 --- Chemosensitivity testing --- p.60 / Chapter 4.1a --- Tests completed --- p.60 / Chapter 4.1b --- Number of cases tested for each drug --- p.60 / Chapter 4.1c --- 〇D reading of the background samples --- p.60 / Chapter 4.1d --- Dose-dependent response curve --- p.61 / Chapter 4.1e --- Unique IC50 for each tumor in each drug test --- p.61 / Chapter 4.1f --- Wide distribution of ic50 for anti-tumor drugs --- p.61 / Chapter 4.1g --- Chemosensitivity and tumor histologic type --- p.63 / Chapter 4.1h --- Correlation of ic50 with tumor stage --- p.63 / Chapter 4.2 --- Immunohistochemical staining of p53 protein (DO-7) --- p.64 / Chapter 4.2a --- p53 protein accumulation in samples --- p.64 / Chapter 4.2b --- Correlation of p53 IHC expression and chemosensitivity --- p.64 / Chapter 4.3 --- SSCP and CSGE --- p.65 / Chapter 4.3a --- Detection of abnormal band movement --- p.65 / Chapter 4.3b --- Correlation of p53 mutations with chemosensitivity --- p.66 / Chapter 4.3c --- Concordance between IHC and SSCP/CSGE --- p.66 / Chapter 4.4 --- MDR1 gene expression --- p.67 / Chapter 4.4a --- MDR1 gene expression in normal and tumors --- p.67 / Chapter 4.4b --- Correlation of MDR1 expression and chemosensitivity --- p.68 / Chapter 4.5 --- Pseudobiopsies --- p.68 / Chapter 5 --- DISCUSSION --- p.70 / Chapter 5.1 --- p53 analysis of the tumors --- p.70 / Chapter 5.1a --- Immunohistochemistry versus mutational analysis --- p.70 / Chapter 5.1b --- Methods of mutational analysis --- p.73 / Chapter 5.1c --- Comparing IHC results with previous findings --- p.77 / Chapter 5.1d --- Comparing SSCP/ CSGE results with previous findings --- p.78 / Chapter 5.1e --- Correlation of IHC and SSCP/CSGE results --- p.81 / Chapter 5.2 --- MDR1 expression --- p.85 / Chapter 5.2a --- Methods for detecting MDR1 expression --- p.85 / Chapter 5.2b --- Comparing MDR1 expression results with published data --- p.88 / Chapter 5.2c --- Correlation between chemosensitivity and MDR1 gene expression --- p.92 / Chapter 5.3 --- Chemosensitivity testing --- p.94 / Chapter 5.3a --- Chemosensitivity testing method --- p.94 / Chapter 5.3b --- The chemosensitivity results --- p.102 / Chapter 5.3c --- Chemosensitivity and MDR1 expression --- p.108 / Chapter 5.3d --- Chemosensitivity and p53 immunohistochemical expression… --- p.110 / Chapter 5.3e --- Chemosensitivity and p53 mutations --- p.112 / Chapter 5.3f --- Limitation of this study --- p.115 / Chapter 5.3g --- Pseudobiopsies and large tumor samples --- p.118 / Chapter 6. --- conclusions --- p.121 / figures / appendices / references

O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

O objetivo do presente estudo clínico foi compreender o efeito do uso prévio de agentes antimicrobianos na diversidade e a estrutura do microbioma procariótico de saliva, biofilme supragengival e canal radicular de dentes com infecção endodôntica aguda. Realizaram-se coletas microbiológicas de saliva (S), biofilme supragengival (BS) e canal radicular (CR) de pacientes que não utilizaram antibióticos (G1: n=6) e de pacientes que utilizaram antibióticos (G2: n=6). Para a caracterização das comunidades de procariotos por meio de sequenciamento de alto rendimento, foram produzidos pools de seis amostras para cada um dos sítios. A região hipervariável V3-V4 do gene 16S rRNA foi amplificada e a plataforma Illumina MiSeq foi empregada para análise das sequências geradas. Foram determinadas a presença e abundância relativa das unidades taxonômicas operacionais (OTUs) em cada amostra. Procedeu-se a análise de alfa-diversidade para cada amostra, considerando-se as métricas de Simpson, dominância, estimativa de riqueza de Chao-I e o Índice de Shannon. Os valores obtidos foram comparados por meio de testes estatísticos. Para a análise dos índices de beta-diversidade, empregou-se o método de agrupamento UPGMA, com jackknifing e método de comparação UniFrac com peso. A representação gráfica tridimensional da beta-diversidade foi realizada por meio de análise de coordenadas principais. A técnica de PCR gene específico foi empregada para determinar a presença de genes relacionados à resistência bacteriana para agentes beta-lactâmicos (blaTEM, blaZ, ampC, mecA), macrolídeos (ermB, ermC, mefA), tetraciclinas (tetM, tetQ, tetW), lincosamidas (lnuB, lsaB) e vancomicina (vanA, vanD, vanE). A similaridade para a presença/ausência de genes de resistência nas amostras de S, BS e CR em G1 e G2 foi determinada por meio de coeficiente de agrupamento, utilizando-se o método UPGMA com distância Euclidiana. Todos os pacientes apresentavam infecção endodôntica aguda, caracterizada pela presença de dor espontânea, necrose pulpar e dor à percussão vertical. Aumento de volume foi observado em 8/12 pacientes. Os pacientes do Grupo 2 utilizaram beta-lactâmicos previamente à consulta (amoxicilina = 5; cefalexina = 1). Há predomínio de integrantes do domínio Bactéria em todas S, BS e CR. Archaea pertencentes ao gênero Methanobrevibacter foram encontradas apenas em amostras de CR, constituindo menos de 1% do total de OTUs (G1-CR = 0,319%; G2-CR = 0,014%). Há predomínio de bactérias dos filos Firmicutes e Bacteroidetes em amostras de S, BS e CR. Há redução intensa no percentual de OTUs pertencentes ao Filo Firmicutes em amostras de saliva, quando antibiótico foi utilizado (G1-S = 75,371; G2-S = 35,242). Comportamento oposto ocorreu neste ecossistema para integrantes do Filo Bacteroidetes (G1-S = 12,657; G2-S = 33,947). Tanto em G1 quanto em G2, bactérias do gênero Streptococcus predominam em amostras de S e BS. Em canais radiculares, maiores percentuais de OTUs foram observados para os gêneros Porphyromonas e Prevotella. As métricas de alfadiversidade indicam que o uso prévio de antimicrobiano parece oportunizar o estabelecimento de espécies antes menos abundantes, especialmente em saliva (dominância: G1-S>G2-S; índice de Shanon: G1-S<G2-S; índice de Simpson: G1- S<G2-S; índice de Chao-1: G1-S<G2-S). A análise de beta-diversidade mostra proximidade entre G1-BS e G2-BS; há distanciamento entre G1-S e G2-S. As amostras G1-CR e G2-CR estão mais distantes de S e BS, sugerindo maior seleção imposta pelo ecossistema do CR aos procariotos. Os genes de resistência mais frequentemente detectados foram tetM, tetQ, tetM, ermB e mefA. Não foram detectados genes vanA, vanD, vanE, blaZ, mecA, lnuB e ermC. O gene ermB foi frequentemente detectado em amostras de S, BS e CR em ambos os grupos. Em pacientes que não utilizaram antibiótico, o gene tetM e o gene tetQ foram detectados simultaneamente em S, BS e CR (tetM = 4/6; tetQ = 3/6). A análise multivariada não demonstrou nível de agrupamento alto entre amostras de um mesmo paciente, de um mesmo ecossistema, ou de um mesmo grupo. A partir dos resultados do presente estudo, observou-se que a utilização de agente antimicrobiano betalactâmico parece alterar parâmetros composicionais de comunidades de procariotos na cavidade bucal. Entretanto, particularidades relativas a cada ecossistema podem modular a extensão deste efeito, parecendo ser mais intensos em amostras de S do que em BS e CR. / The present clinical study aimed to assess the effect of antibiotics over the diversity and structure in prokaryotic communities of saliva, supragengival biofilm and root canal of teeth with acute primary infections. Samples of saliva (S), supragengival biofilm (BS) and root canal of teeth with acute primary infections (CR) were collected from patients, and were grouped according with the previous use of antibiotic (G1 = no antibiotics; G2 = antibiotics). Pooled samples for each community were evaluated. DNA sequencing was performed with MiSeq (Illumina). The V3-V4 hypervariable region from the 16S rRNA gene was amplified. The presence and relative abundance of the operational taxonomic units (OTUs) was determined for each sample. Alpha-diversity analysis was performed with the Simpson’s index, dominance, Chao-1 richness index and Shannon’s index. Statistical analysis was carried out to compare their values for each community.Beta-diversity was computed through UPGMA clustering and jackknifing. The principal coordinate analysis employed weighted UniFrac. Gene-specific PCR was employed to detect resistance genes to lactamics (blaTEM, blaZ, ampC, mecA), macrolides (ermB, ermC, mefA), tetracyclines (tetM, tetQ, tetW), lincosamides (lnuB, lsaB) e vancomycin (vanA, vanD, vanE). The UPGA clustering analysis with Euclidean distance was applied to investigate the existence of similar groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by clustering. All the patients had primary acute endodontic infections, with spontaneous pain, pulp necrosis and tenderness on percussion. Swelling was observed for 8 out of 12 patients. Patients from G2 had lactamics before the urgency appointment (amoxicillin = 5; cephalexin = 1). Bacteria were predominant in S, BS and CR samples. Archaea belonging to the genus Methanobrevibacter were only detected in RC samples and comprised less than 1% of all the OTUs G1-CR = 0.319%; G2-CR = 0.014%). The great majority of the detected OTUs in S, BS, and CR belonged to the phyla Firmicutes and Bacteroidetes. Reduction in the percentage of Firmicutes was observed in G2-S (G1-S = 75.371%; G2-S = 35.242%). A distinct behavior was demonstrated by the Bacteroidetes (G1- S = 12.657%; G2-S = 33.947%). Components belonging to the genus Streptococcus were predominant in S and BS, for both G1 and G2. CR harbored high percentage of species belonging to the genus Porphyromonas and Prevotella. The alpha-diversity indexes demonstrated that the G2-S had an increase in low abundant species (dominance: G1- S>G2-S; Shanon index: G1-S<G2-S; Simpson index: G1-S<G2-S; Chao-1 index: G1-S<G2- S). Beta-diversity showed that G1-BS and G2-BS had close spatial distribution. G1-CR and G2-CR were more distant from S and BS samples. The RC may promote a most intense species selection, due to environmental conditions. The most frequently detected genes associated with resistance to antibiotics were tetM, tetQ, tetM, ermB, and mefA. The genes vanA, vanD, vanE, blaZ, mecA, lnuB, and ermC were not detected in the samples. The gene ermB was frequently detected in S, BS and CR samples. In patients from G1, tetM and tetQ were detected simultaneously in all the three environments (tetM = 4/6; tetQ = 3/6). There was no clustering behavior for samples belonging to different environments in the same patients and between the same environment samples from different groups. An overall analysis from the data allows for suggesting that the use of lactamic agents may alter compositional parameters from prokaryotic communities in the oral cavity to different extents. Specific environmental characteristics from each site may modulate the effect that seemed to be more intense for S than for BS and CR.