As metalo-β-lactamases (MBL) são capazes de hidrolisar os carbapenêmicos, a classe de antimicrobianos com maior potência para o tratamento de infecções graves e de maior uso clinico. Dentre as MBL, o grupo mais recentemente descrito e que apresentou rápida disseminação em todo o mundo é o da New-Delhi-Metalo- β-lactamases (NDM). Nas enterobactérias, os genes que codificam essas enzimas estão mais frequentemente localizados em plasmídeos. O estudo da estabilidade de plasmídeos que albergam o gene blaNDM-1 é importante para entender a predominância de espécies que carregam esses plasmídeos, desvendar mecanismos moleculares envolvidos na sua persistência e para desenvolver novas drogas que possam diminuir a sua persistência. Estudos recentes sobre estabilidade plasmidial evidenciaram que a maprotilina é capaz de induzir perda plasmidial de até 90% em E. coli K12. Neste trabalho, foi estudado o efeito da maprotilina na indução de cura de plasmídeos, que albergam o gene blaNDM-1, em diferentes espécies da família Enterobacteriaceae. Nove isolados pertencentes a diferentes espécies foram incluídas no estudo. Os plasmídeos foram caracterizados quanto ao seu tamanho por eletroforese e por sequenciamento de DNA no sistema Illumina. A persistência plasmidial foi determinada pelo método de contagem em placa em LB ágar com e sem tratamento com maprotilina em concentrações sub-inibitórias (50mg/L). O experimento foi conduzido por 10 dias, representando aproximadamente 100 gerações. Neste estudo evidenciou-se que o grupo das enterobactérias estão envolvidas na disseminação de plasmídeos com blaNDM-1, sendo que plasmídeos do grupo IncF estão mais relacionados a essa dispersão. A maprotilina teve efeito de cura plasmidial em todos os isolados exceto em E. hormaechei \"subsp. oharae\" e C. freundii. O isolado P. rettgeri apresentou maior taxa de perda plasmidial e a análise comparativa da sequência nucleotídica do plasmídeo indicou que a presença da IS5 pode estar relacionada com a diminuição da persistência plasmidial. Diferenças na persistência plasmidial, quando tratados com maprotilina, entre E. hormaechei \"subsp. steigerwaltii\" e E. hormaechei \"subsp. oharae\" sugerem que E. hormaechei \"subsp. oharae\" pode ser um possível disseminador de plasmídeos albergando blaNDM-1, devido a processos de adaptação co-evolutivos. / Metallo- β-lactamases (MBL) are able to hydrolase carbapenems, an antimicrobial class in clinical use with high potency in the treatment of severe infections. The most recently decribed group of MBL is the New-Delhi-Metallo-β-lactamases (NDM). This group is mostly correlated to the spread of resistance mediated by plasmid in Enterobacteriaceae. Understanding the plasmid persistence pattern is important in order to understand the predominance of a given species related to antimicrobial resistance plasmids spread, to unveil molecular mechanisms involved in the increase of plasmid persistence and to develop new drugs which could decrease its persistence. Recent studies have associated maprotiline to a decrease in 90% of plasmid persistence in E. coli K12. In this work, we evaluated the effect of maprotiline in curing plasmids carrying blaNDM-1 in different species of Enterobacteriaceae. Nine isolates belonging to different species were evaluated. Plasmids were characterized by agarose gel electrophoresis and by DNA sequencing with Illumina platform. The plate counting method was used to determine plasmid persistence, with and without sub-inhibitory (50 mg/L) concentration of maprotiline during 10 days, representing approximately 100 generations. We found that Enterobacteriaceae are involved in the spread of NDM-1 plasmid-mediate resistance and the IncF group is the plasmid incompatibility group more frequently involved in this dissemination. Maprotiline showed a plasmid-curing effect in all isolates, except against plasmids of E. hormaechei \"subsp. oharae\" and C. freundii. The P. rettgeri isolate had the highest plasmid-curing rate. Sequencing analysis revealed an IS5 in the plasmid, which could be associated to a decrease in plasmid persistence. The difference between plasmid persistence pattern of plasmids isolated from E. hormaechei \"subsp. steigerwaltii\" and E. hormaechei \"subsp. oharae\", when treated with maprotiline, suggest that E. hormaechei \"subsp. oharae\", could be associated to the spread of plasmids carrying blaNDM-1 due to co-evolution adaptation.

Depression is a physiological disorder that may be treated by increasing the body¡¦s amount of one or a few of the following neurotransmitters: serotonin, dopamine and norepinephrine. Although there are seven distinct classes of antidepressants, selective serotonin reuptake inhibitors (SSRIs) and tetracyclic antidepressants are widely prescribed and generally regarded as the first-line drugs in the treatment of depression. However, many physiological roles of some SSRIs appear to be dissociated with the inhibition of serotonin reuptake. For instance, paroxetine, a member of SSRIs and maprotiline, a member of tetracyclic antidepressant, have been shown to induce apoptosis or to prevent other agents from inducing apoptosis in several cell lines. Thus the effects of these two drugs on the apoptosis are still controversial. The aim of this study is to investigate the molecular mechanisms of paroxetine and maprotiline in induction of cell death in human osteosarcoma and murine neuroblastoma cells. First, WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of paroxetine and maprotiline. Then immunoblotting was used to measure the activity of apoptotic markers caspase-3 and mitogen-activated protein kinases (MAPKs) to survey the apoptotic pathways induced by these two antidepressants. The experimental results may be helpful to understand the pharmacological and toxicological effects of these two antidepressants in cells from important organs. Results showed that paroxetine caused apoptosis via the activation of caspase-3 in cultured human osteosarcoma cells (MG63). Although paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine was also found to induce [Ca2+]i increases but pretreatment with BAPTA/AM, a Ca2+ chelator, prevented paroxetine-induced [Ca2+]i increases, and thus did not protect cells from death. These results suggest that paroxetine caused Ca2+-independent apoptosis via the activation of p38 MAPK-associated caspase-3 in MG63 cells. Maprotiline was also found to induce apoptosis through increased caspase-3 activation in murine neuroblastoma Neuro-2a cells. Induction of JNK phosphorylation contributed to the activation of caspase-3 resulting in maprotiline-induced Neuro-2a cell apoptosis. Thus, it appears that maprotiline induced apoptosis via JNK/caspase-3-dependent signaling pathways. Blockage of activation of ERK was found to increase the activation of caspase-3 leading to an enhancement of maprotiline-induced apoptosis. These data suggest that ERK was a survival signal to oppose maprotiline-caused apoptotic effect in Neuro-2a cells. Thus the activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. [Ca2+]i measurement in the presence of maprotiline showed that the antidepressant induced [Ca2+]i increases. Interestingly, pretreatment with BAPTA/AM could suppress maprotiline-induced ERK phosphorylation, enhance caspase-3 activation and increase maprotiline-induced apoptosis. In conclusion, this study demonstrates that maprotiline induced apoptosis in murine neuroblastoma cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca2+- and ERK-dependent. This thesis contains some published data in the journal of Toxicology and Applied Pharmacology and some data were submitted in the journal of Toxicology Letters.

As metalo-β-lactamases (MBL) são capazes de hidrolisar os carbapenêmicos, a classe de antimicrobianos com maior potência para o tratamento de infecções graves e de maior uso clinico. Dentre as MBL, o grupo mais recentemente descrito e que apresentou rápida disseminação em todo o mundo é o da New-Delhi-Metalo- β-lactamases (NDM). Nas enterobactérias, os genes que codificam essas enzimas estão mais frequentemente localizados em plasmídeos. O estudo da estabilidade de plasmídeos que albergam o gene blaNDM-1 é importante para entender a predominância de espécies que carregam esses plasmídeos, desvendar mecanismos moleculares envolvidos na sua persistência e para desenvolver novas drogas que possam diminuir a sua persistência. Estudos recentes sobre estabilidade plasmidial evidenciaram que a maprotilina é capaz de induzir perda plasmidial de até 90% em E. coli K12. Neste trabalho, foi estudado o efeito da maprotilina na indução de cura de plasmídeos, que albergam o gene blaNDM-1, em diferentes espécies da família Enterobacteriaceae. Nove isolados pertencentes a diferentes espécies foram incluídas no estudo. Os plasmídeos foram caracterizados quanto ao seu tamanho por eletroforese e por sequenciamento de DNA no sistema Illumina. A persistência plasmidial foi determinada pelo método de contagem em placa em LB ágar com e sem tratamento com maprotilina em concentrações sub-inibitórias (50mg/L). O experimento foi conduzido por 10 dias, representando aproximadamente 100 gerações. Neste estudo evidenciou-se que o grupo das enterobactérias estão envolvidas na disseminação de plasmídeos com blaNDM-1, sendo que plasmídeos do grupo IncF estão mais relacionados a essa dispersão. A maprotilina teve efeito de cura plasmidial em todos os isolados exceto em E. hormaechei \"subsp. oharae\" e C. freundii. O isolado P. rettgeri apresentou maior taxa de perda plasmidial e a análise comparativa da sequência nucleotídica do plasmídeo indicou que a presença da IS5 pode estar relacionada com a diminuição da persistência plasmidial. Diferenças na persistência plasmidial, quando tratados com maprotilina, entre E. hormaechei \"subsp. steigerwaltii\" e E. hormaechei \"subsp. oharae\" sugerem que E. hormaechei \"subsp. oharae\" pode ser um possível disseminador de plasmídeos albergando blaNDM-1, devido a processos de adaptação co-evolutivos. / Metallo- β-lactamases (MBL) are able to hydrolase carbapenems, an antimicrobial class in clinical use with high potency in the treatment of severe infections. The most recently decribed group of MBL is the New-Delhi-Metallo-β-lactamases (NDM). This group is mostly correlated to the spread of resistance mediated by plasmid in Enterobacteriaceae. Understanding the plasmid persistence pattern is important in order to understand the predominance of a given species related to antimicrobial resistance plasmids spread, to unveil molecular mechanisms involved in the increase of plasmid persistence and to develop new drugs which could decrease its persistence. Recent studies have associated maprotiline to a decrease in 90% of plasmid persistence in E. coli K12. In this work, we evaluated the effect of maprotiline in curing plasmids carrying blaNDM-1 in different species of Enterobacteriaceae. Nine isolates belonging to different species were evaluated. Plasmids were characterized by agarose gel electrophoresis and by DNA sequencing with Illumina platform. The plate counting method was used to determine plasmid persistence, with and without sub-inhibitory (50 mg/L) concentration of maprotiline during 10 days, representing approximately 100 generations. We found that Enterobacteriaceae are involved in the spread of NDM-1 plasmid-mediate resistance and the IncF group is the plasmid incompatibility group more frequently involved in this dissemination. Maprotiline showed a plasmid-curing effect in all isolates, except against plasmids of E. hormaechei \"subsp. oharae\" and C. freundii. The P. rettgeri isolate had the highest plasmid-curing rate. Sequencing analysis revealed an IS5 in the plasmid, which could be associated to a decrease in plasmid persistence. The difference between plasmid persistence pattern of plasmids isolated from E. hormaechei \"subsp. steigerwaltii\" and E. hormaechei \"subsp. oharae\", when treated with maprotiline, suggest that E. hormaechei \"subsp. oharae\", could be associated to the spread of plasmids carrying blaNDM-1 due to co-evolution adaptation.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / No presente estudo avaliou-se os efeitos dos antidepressivos, amitriptilina (amt), clomipramina (clm) e maprotilina (mpt) em reaÃÃes inflamatÃrias. Ratos machos Wistar, 150-200g, foram divididos em cinco grupos (n=6), em experimentos de edema de pata (EP) e migraÃÃo de neutrÃfilos (MN) em bolsas de ar subcutÃnea. Amt, mpt (v.o.) e clm (i.p),10, 30 e 90mg/kg, foram administradas previamente ao estÃmulo inflamatÃrio (EI), carragenina (Cg-500 Â g/pata), fMLP (10â6M) ou dextran (Dx-300 Âg/pata). O edema foi aferido por hidropletismometria (Ugo Basile 7140), imediatamente antes (âtempo zeroâ) e 1, 2, 3 e 4h apÃs a aplicaÃÃo de Cg ou Dx. O aumento no volume da pata (volume do edema) foi calculado pela diferenÃa do volume apÃs a injeÃÃo do EI. Os dados demonstraram uma inibiÃÃo dose-dependente nos edemas de pata induzidos por Cg e Dx, na presenÃa dos antidepressivos. Amt, na maior dose, inibiu o EP induzido por Cg em 51,34% (p<0,05), e em 49% (p<0,05) o EP induzido por Dx. Clm, na maior e menor doses, reduziu o EP da Cg em 100% (p<0,001) e 42,45% (p<0,05), respectivamente, e em 97,26% (p<0,001) e 36% (p<0,05) o EP do Dx, respectivamente. A mpt inibiu o EP por Cg, em 60,9% (p<0,05) para a maior dose, e em 57,49% (p<0,01) e 34,42% (p<0,05) nas duas maiores doses, em ordem decrescente, no EP por Dx. Adicionalmente, amt e clm inibiram de forma dose-dependente a MN, na sexta hora, apÃs administraÃÃo de Cg. Amt na maior dose inibiu a MN induzida por Cg em 49,19% (p<0,05) e em 96,31% (p<0,001) a MN induzida por fMLP. A mpt (40 mg/kg, i.p.) inibiu a MN por Cg em 49,26% (p<0,001), e em 92,38% (p<0,001) a migraÃÃo induzida por fMLP. Do mesmo modo, a clm nas doses de 90. 30 e 10 mg/kg (i.p.), inibiu a MN por Cg em 76,46% (p<0,001), 64,4% (p<0,01) e 49,13% (p<0,05), respectivamente, e em 100% (p<0,001) a MN induzida por fMLP, na maior dose. Na avaliaÃÃo do efeito dos AD na degranulaÃÃo de mastÃcitos, observamos que a clm (90 mg/kg/i.p.) e a mpt (40 mg/kg/i.p.) reverteram em 100% a degranulaÃÃo induzida pelo composto 48/80. Amt (90 mg/kg/v.o.) tambÃm reverteu significativamente a degranulaÃÃo de mastÃcitos, alcanÃando 90,19% (p<0,001) de inibiÃÃo. Tais dados sugerem que amt, clm e mpt possuem significativa atividade antiinflamatÃria evidenciada por seus efeitos inibitÃrios na MN e edema / In the present study it was evaluated the effects of antidepressants amitriptiline (amt), clormipramine (clm) and maprotiline (mpt) in the inflammatory reaction. Male Wistar rats, 150-200g, were divided into five groups (n=6) in experiments of hind paw edema (HE) and neutrophils migration (NM) in subcutaneous air pouches. Amt, mpt (v.o.) and clm (i,p), 10, 30 and 90 mg/kg, were administrated before the inflammatory stimulus carragenin (Cg-500 Âg/paw), fMLP (10 â6M) or dextran (Dx-300 Âg/paw). Paw edema was measured with a hydroplethysmometer (Hugo Basile 7140 Plethysmometer) immediately before ( time equal zero ) and 1, 2, 3 and 4 h after the Cg or Dx challenges. The increase in paw volume (edema volume) was obtained by subtracting the paw volume measured before stimulus injection. The results demonstrate that the antidepressants induce a dose dependent reduction in the HE induced by Cg and Dx. Amt in largest dose used, inhibited the Cg-induced HE by 51,34% (p< 0,05), and the Dx-induced HE by 49% (p<0,05). Clm, in biggest and smallest dose, inhibited Cg-induced HE by 100% (p<0,001) and 42,45% (p< 0,05), respectively, and by 97,26% (p< 0,001) and 36% (p<0,05) the Dx-induced HE, respectively. The largest dose of mpt inhibited the Cg-induced HE by 60,9% (p<0,05) and at the two greatest dose, in decreasing order, inhibited the Dx-induced HE, by 57,49% (p<0,01) and 34,42% (p<0,05), respectively. Additionally, amt e clm inhibited the MN in a dose-dependently manner, in the sixth hour after Cg administration. Amt in largest dose (90 mg/kg/v.o.) inhibited the Cg-induced NM by 49,19% (p<0,05) and by 96,31% (p<0,001) the fMLP-induced NM. The mpt (40 mg/kg/i.p.) inhibited the Cg-induced NM by 49,26% (p<0,001), and by 92,38% (p<0,001) the fMLP-induced NM. In the same way, clm at the dose 90, 30 e 10 mg/kg (i.p.) inhibited Cg-induced NM by 76,46% (p< 0.001), 64,4% (p<0,01) and 49,13% (p<0,05), respectively, but the fMLP-induced NM was inhibited just by the biggest dose by 100% (p<0,001). We observed that clm (90 mg/kg/i.p.) and the mpt (40 mg/kg/i.p) completely reverted the mast cell degranulation induced by the compound 48/80. Amt (90 mg/kg/v.o.) also significantly reverted the mast cell degranulation by 90,19% (p<0,001). These data suggest that amt, clm e mpt have a significant antiinflammatory activity demonstrated by their inhibitory effects on NM and edema