Bacterial resistance to antibiotics is an emerging epidemic throughout the world and there is a desperate need for new antibiotics and new strategies to maintain the effectiveness of current agents. ??-Lactams, such as the penicillins and cephalosporins, have been the most important class of antibiotic for several decades and represent half of the global antibacterial market, but the continued use of ??-lactams is threatened by ??-lactamases, enzymes that efficiently inactivate ??-lactams through hydrolysis. Class A, C, and D ??-lactamases use an active-site serine residue for hydrolysis and achieve turnover through an acylenzyme intermediate while the class B metallo-??-lactamases (MBLs) use a zinc-bound hydroxide as the active-site nucleophile. Two successful approaches to combat ??-lactamase-mediated resistance have involved the development of ??-lactam antibiotics which bind poorly to ??-lactamases and the combination of ??-lactams with ??-lactamase inhibitors. These strategies have been effective for overcoming resistance due to class A ??-lactamases, but the ever-increasing prevalence of extended-spectrum ??-lactamases (ESBLs), metallo-??-lactamases, and carbapenemases compromises the effectiveness of current penicillins, cephalosporins, carbapenems, and mechanism-based ??-lactamase inhibitors. Cyclobutanone analogues of ??-lactam antibiotics were explored in the early 1980s as potential inhibitors of ??-lactamases and D-Ala-D-Ala transpeptidases, but simple analogues showed only weak inhibitory activity and this approach was subsequently abandoned. The increasing threat of multidrug-resistant ??-lactamase-producing organisms in recent years, however, has inspired a re-evaluation of these inhibitors since cyclobutanones have the potential to exhibit broad-spectrum inhibition of both serine- and metallo-??-lactamases through the formation of enzyme-bound hemiketals or hydrates. 7,7-Dichloro-2-thia-bicyclo[3.2.0]heptan-6-one-4-carboxylic acid (65), a dichlorocyclobutanone that had shown modest inhibition of the class B and D ??-lactamases IMP-1 and OXA-10 in earlier work in this laboratory, was prepared in an efficient seven-step sequence from triethyl phosphonoacetate (103) with an overall yield of 28%. Initial efforts to improve upon the potency of the cyclobutanones involved functionalization at C3 and a highly stereoselective chlorination with sulfuryl chloride provided the 3??-chloro derivative 117?? in nearly quantitative yield. Elimination of HCl from 117?? was achieved under a variety of conditions and 3-alkoxy derivatives were prepared from 117?? through diastereoselective substitution reactions with alcohols. Cyclobutanones with 3??-OR substituents were found to favour an endo envelope conformation while the 3??-OR derivatives adopt the exo envelope conformation. Evidence from X-ray crystal structures and ab initio molecular orbital calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favours the 3-alkoxy substituent in an axial orientation. In addition, the conformation of the bicyclic system was found to have a dramatic effect on the tendency of the cyclobutanone to undergo hemiketal formation. Cyclobutanone analogues of penicillins, including 3-alkoxy derivatives, and cyclobutanone analogues of penems were evaluated against class A, B, C, and D ??-lactamases and found to be moderate inhibitors of KPC-2, IMP-1, GC1, and OXA-10. The cyclobutanones found to be most potent were those which are hydrated to a larger extent in aqueous solution. Dichlorocyclobutanones were found to be better inhibitors than dechlorinated cyclobutanones and a 3??-methoxy derivative 152??, which favours the exo envelope conformation in which the C4 carboxylate is equatorial, was found to be a better inhibitor than cyclobutanones that favour the endo envelope conformation. A 3,4-unsaturated penem analogue, 153, showed comparable potency to that of 152?? and molecular models of enzyme-inhibitor complexes indicate that an equatorial carboxylate is required for binding to ??-lactamases. An X-ray crystal structure of 152?? bound to the class D ??-lactamase OXA-10 confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. The biochemical data described above demonstrate that cyclobutanones can indeed act as inhibitors of serine- and metallo-??-lactamases and these cyclobutanones represent the first class of reversible inhibitors to show moderate inhibition of all four classes of ??-lactamase. Although the inhibitory potency of these compounds is modest (low micromolar IC50 values), penem analogue 153 was able to enhance the potency of meropenem against carbapenem-resistant MBL-producing clinical isolates of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia and the synergy demonstrated in these antimicrobial assays is encouraging. Synthetic studies toward other C3-alkyl and C3-thioalkyl-substituted inhibitors are described and the design and synthesis of C7-monochloro- and 7??-hydroxymethyl-7??-chloro cyclobutanone derivatives is presented.

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia. Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function. In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 152 pages. Includes Vita. Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-01T18:23:08Z No. of bitstreams: 1 Milena Soares dos Santos Análise da composição clonal dos Streptococcus pneumoniae não susceptíveis a penicilina....pdf: 2536157 bytes, checksum: ee1fe922b8d1366faa0d4a8bea4d3b9f (MD5) / Made available in DSpace on 2012-08-01T18:23:08Z (GMT). No. of bitstreams: 1 Milena Soares dos Santos Análise da composição clonal dos Streptococcus pneumoniae não susceptíveis a penicilina....pdf: 2536157 bytes, checksum: ee1fe922b8d1366faa0d4a8bea4d3b9f (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O Streptococcus pneumoniae permanece como principal causa de doenças infecciosas que conduzem a elevada morbi-mortalidade em todas as faixas etárias, principalmente nas crianças. Desde a década de 1990, a resistência antimicrobiana deste microrganismo tem aumentado mundialmente, representando mais de 30% de resistência à penicilina em todos os isolados de doença pneumocócica invasiva em algumas áreas geográficas. Neste estudo, relatamos os casos identificados através de uma vigilância ativa para a meningite bacteriana em Salvador, Bahia, no período de janeiro de 1996 a dezembro de 2007. Para avaliar o perfil de susceptibilidade aos antimicrobianos dos isolados dos pacientes com meningite por S.pneumoniae, utilizamos a microdiluição em caldo e isolados com CIM de penicilina ≥ 0.125μg/mL foram considerados como não-susceptíveis à penicilina (PNSP). A diversidade clonal foi estudada por Box-A PCR, PFGE e MLST e a distribuição de pili foi investigada em 133 amostras, selecionadas de forma aleatória em cada grupo clonal, através das técnicas de PCR e seqüenciamento do gene rlrA. Um total de 748 pacientes com meningite por S.pneumoniae foi identificado durante os 12 anos de vigilância. Foram encontrados 135 (19%) isolados de S. pneumoniae não-susceptíveis à penicilina sendo que destes, 85 (63%) eram crianças <5 anos de idade e 28 (20,7%) dos pacientes apresentavam alguma doença precedendo a meningite. A incidência média anual de PNSP foi estimada em 2,47 por 100.000 habitantes para todas as faixas etárias, 1,69 casos/100.000 habitantes para crianças menores de 5 anos e 1,37 casos/100.000 habitantes para crianças menores de 1 ano. A taxa de letalidade obtida para todas as idades foi de 39,2%. Os sorotipos mais prevalentes entre os isolados de PNSP foram: 14 (46,7%; 63/135) 23F (17,8%; 24/135), 6B (14,8%; 20/135), 19F (8,1%; 11/135) e 19A (4,4%; 6 / 135). Os isolados do sorotipo 14 foram identificados como o grupo clonal predominante 32,6% (44/135) e foram caracterizados como ST66 e como ST156 com alta resistência à ceftriaxona. Os outros sorotipos apresentaram maior diversidade clonal e novos STs foram encontrados, entre outros sorogrupos. Foram detectados 22% (29/133) de isolados portadores de pili, independente de composição clonal ou perfil de resistência. Baseada na vacina pneumocócica conjugada 10-valente, prevista para ser implementada no Brasil este ano, esperamos 89% de proteção contra PNSP em crianças menores de 5 anos de idade. As alterações previstas na população pneumocócica, ao longo dos anos após a implementação desta vacina, ressaltam a importância do monitoramento através de vigilância ativa. / Streptococcus pneumoniae remains an important cause of infectious diseases leading to high morbidity and mortality in all age groups, especially in children. Since the 1990s, resistance of this organism to penicillin has emerged worldwide accounting for >30% of all invasive pneumococcal isolates in some geographic areas. In this study, active surveillance for bacterial meningitis was performed from January 1996 to December 2007. Antimicrobial susceptibility testing used broth microdilution and isolates with penicillin MIC ≥ 0.125μg/mL were considered penicillin non-susceptible. Clonal diversity was studied by Box-A PCR, PFGE and MLST and pili detection was determinated by PCR and sequencing of rlrA gene. A total of 748 patients with pneumococcal meningitis were identified during 12 years of surveillance. We found 135 (19%) S. pneumoniae isolates to be penicillin-nonsusceptible, which 85 (63%) were children < 5 years age and 28 (20.7%) patients had previous acute illness. The annual incidence of PNSP was estimated to 2.47 per 100,000 for all age groups, 1.69 cases/100.000 population for children younger than 5 years, and 1.37 cases/100.000 inhabitants for children under 1 year. The case-fatality rate obtained for all ages was 39.2%. The most prevalent serotypes PNSP isolates were among the 14 (46.7%; 63/135), 23F (17.8%; 24/135), 6B (14.8%; 20/135), 19F (8.1%; 11/135) and 19A (4.4%; 6/135). Serotype 14 isolates were identified as the predominant clonal group [32.6% (44/135)] and were characterized as ST66 and as ST156 with high resistance to ceftriaxone. The other serotypes were more diverse and new ST´s were found among others serogroups. We detected 22% (29/133) of pili positive among the isolates, independently of clonal patterns or susceptibility profile. Based on 10-valent pneumococcal vaccine, which will be implemented in Brazil in 2010, we expect 89% protection against PNSP in children < 5 years of age. Changes in the pneumococcal population over the coming years following implementation of this vaccine should be monitoring throughout active surveillance.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-04-16T19:40:46Z No. of bitstreams: 1 raquel_simbalista_de queiroz. Aspectos da admissao. 2010.pdf: 39174617 bytes, checksum: e35efd5722282aa850fa314a2345a654 (MD5) / Made available in DSpace on 2013-04-16T19:40:46Z (GMT). No. of bitstreams: 1 raquel_simbalista_de queiroz. Aspectos da admissao. 2010.pdf: 39174617 bytes, checksum: e35efd5722282aa850fa314a2345a654 (MD5) Previous issue date: 2010 / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, Bahia, Brasil / Fundação OSwaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A pneumonia na infância permanece um assunto relevante, tendo em vista a sua elevada taxa de mortalidade mundial, principalmente nos países em desenvolvimento. Objetivo: Descrever o resultado da hospitalização de crianças internadas com suspeita diagnostica de pneumonia. Desenho do estudo: Coorte retrospectiva. Material e métodos: Foi realizado acompanhamento retrospectivo de pacientes internados com suspeita de pneumonia em um centro pediátrico, de outubro de 2002 a outubro de 2005. A partir dos prontuários médicos, dados demográficos, de história clínica, do exame físico, do tratamento, da evolução e do desfecho foram coletados e registrados em formulário específico para o estudo. Todos os casos incluídos tiveram as radiografias de tórax avaliadas por radiologista cego às informações clínicas, com o objetivo de definir a presença ou não de infiltrado pulmonar e avaliar a presença de alterações radiológicas outras. A população do estudo foi alocada -“m quatro grupos diferente? para que pudessem ter suas variáveis comparadas entre pacientes com características semelhantes. Resultados: No grupo das crianças > 2 meses de idade, internadas com diagnóstico clínico-radiológico de pneumonia e tratadas com penicilina cristalina, as freqüências de febre (46,4% vs. 26,3%, /’=0,002), taquipnéia (73,6% vs. 59,4%, ^=0,003), tiragem subcostal (29,4% vs. 12,7%, /’<0,001) e aleteo nasal (10,2% vs. 1,6%, 7^=0,001) diminuíram de forma significante entre a admissão e o primeiro dia de tratamento. A penicilina foi substituída após 48 horas por outros antibióticos em 28 (18,2%) dos pacientes, nos quais houve redução significante da taquipnéia entre o primeiro e o segundo dia de tratamento (86,4% ví. 50,0%, /*=0,008). Nas crianças com idade < 2 meses, internadas com diagnóstico clínico ou clínico-radiológico de pneumonia e tratadas com antibióticos diversos, o esquema antibiótico mais utilizado foi a monoterapia com penicilina cristalina e derivados ou associação com cefalosporinas (68,9%). A antibioticoterapia inicial foi modificada em 8,9% dos casos, tendo 62,5% e 58,5% recebido alta após cura ou melhora, respectivamente. Entre aqueles que não modificaram a terapêutica inicial, 58,5% foram classificados como cura e 41,5% como melhora. Nas crianças com idade > 2 meses, com diagnóstico clínico ou clínico-radiológico de pneumonia, tratadas com antibióticos diversos, excluindo-se aquelas pertencentes aos outros grupos, a escolha inicial por penicilina cristalina foi mais frequente. Com relação ao desfecho, 191 (64,1%) dos pacientes receberam alta após cura e 107 (35,2%) após melhora clínica. 13 Finalmente, entre as crianças com idade > 2 meses internadas com diagnóstico clínico ou clínico-radiológico de pneumonia tratadas com penicilina cristalina, o que incluiu aquelas do primeiro grupo, todas foram consideradas curadas (72,6%) ou com melhora clínica (27,4%) no momento da alta hospitalar. A freqüência de cura foi maior entre os pacientes que não modificaram a antibioticoterapia inicial (p<0,001). Conclusões principais: Pôde-se observar que em todos os grupos, indistintamente, a antibioticoterapia concordante permaneceu como uma conduta fundamental para o tratamento da pneumonia. Além disso, a aderência à antibioticoterapia empírica conforme as diretrizes foi considerável, principalmente quando o diagnóstico clínico foi confirmado pelo radiológico, sendo essa conduta uma das chaves para o tratamento eficaz da pneumonia na faixa etária pediátrica. / Childhood pneumonia remains a relevant issue, due to its mortality rates worldwide, especially in developing countries. Objective: The present study has the main purpose to describe the outcome among children hospitalized with suspected pneumonia. Design: Retrospective cohort. Methods: This is a retrospective review of suspected cases of pneumonia in children hospitalized in a pediatric center of Salvador, North-east Brazil, from October/2002 to October/2005. A standardized form containing data on demographics and clinical history, physical examination, treatment, evolution and outcome during the first five or seven days of hospitalization was filled in for each patient, according to medical charts. The radiological reading was performed by a pediatric radiologist blind to clinical information, to define the presence of pulmonary infiltrate or other findings. The study population was allocated in four distinct groups, to compare each group with homogeneous aspects. Results: Among the group of children aged ¿ 2 months hospitalized wi" radiographically diagnosed pneumonia, treated with intravenous aqueous penicillin G, fever (46.4% vs. 26.3%, P=0.002), tachypnea (73.6% v^. 59.4%, P=0.003), chest indrawing (29.4% V5. 12.7%, /'<0.001) and nasal flaring (10.2% vs. 1.6%, F-O.OOl) frequencies significantly decreased from admission to first day of treatment. Penicillin was substituted by other antibiotics in 28 (18.2%) patients in whom the sole significant decrease was on tachypnea frequency from first to second day of treatment (86.4% vs. 50.0%, P=0.008). Among children aged < 2 months hospitalized with suspected pneumonia, treated with several antibiotic schemes, the most frequent first antibiotic scheme were penicillin and derivates alone or associated with cephalosporines (68.9%). The antibiotic had been changed after 48 hours in 8.9% of cases during hospitalization, and 62.5% were discharged after cure, against 37.5% improvements. From those who did not switched therapy, 58.5% were cured and 41.5% improved. In the group of children aged > 2 months old hospitalized with suspected pneumonia, excluding those of the other three groups, the choice for penicillin G had higher frequencies than the other schemes registered, among all age groups. According to outcome, 191 (64.1%) those children > 2 months old hospitalized with suspected pneumonia, treated with intravenous aqueous penicillin G, what includes children from the first group, all were considered improved (27.4%) or cured (72.6%) at the time of hospital discharge, and there was significantly more cure among those who 15 did not switched therapy (p<0.001). Main conclusions: From the aforementioned data, it is possible to observe that guideline-concordant empiric antibiotic remains an important procedure to an effective treatment of pneumonia among children of all age groups. Besides, the adherence of empiric antibiotic therapy was good among physicians in our study, especially when there was concordance between the clinical and radiological diagnosis; and this approach is one of the keys to an effective treatment of childhood pneumonia.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Alternanthera brasiliana (L.) Kuntze (Amaranthaceae) is a species known as penicilina, terramicina, doril or carrapichinho. Studies have proved that the extract of the leaves has analgesic, anti-inflammatory and healing. Based on the medicinal use of this species work aimed to propagate vegetatively in vitro and ex vitro A. brasiliana, and to evaluate the genotoxic, anti-proliferative and anti-mutagenic aqueous extract of the leaves of plants from four cropping systems through the Allium cepa test as well as the quantification of the content of polyphenols, flavonoids and chlorogenic acid. Propagation by cuttings of new shoots ex vitro were used, apical, medium and basal cuttings treated medium MS (0 and 20%) with addition of AIB to the liquid medium (0.0 and 2.5 mg L-1). For micropropagation were used apical and nodal segments were inoculated on MS medium with combinations of NAA (0.0 and 0.05 mg L-1) and BAP (0.0 and 0.5 mg L-1). Evaluations of potential genotoxic, anti-proliferative and anti-mutagenic, roots of Allium cepa bulbs were immersed for 24 hours in aqueous extracts by infusion of the dried leaves of A. brasiliana four sources: Campus UFSM; micropropagation, cuttings and greenhouse, all with concentrations of 5 and 20 g L-1. The same extracts were used for the quantification of secondary metabolites, flavonoids and polyphenols, through spectrophotometer and chlorogenic acid by high performance liquid chromatography. Apical cuttings, median and basal can be used for propagation presenting rooting and survival above 62%. In general, plants from middle and basal cuttings show better results than the apical, suggesting that these would be the most suitable for the production of seedlings. In vitro micropropagation apices showed significantly higher results for nodal segments and the use of growth regulators naphthaleneacetic acid (NAA) (0.05 mg L-1) and benzylaminopurine (BAP) (0.5 mg L-1) was not significant. Plants regenerated in vitro acclimatized showed no significant difference between treatments, with the highest survival percentage (72.57%) occurred in plants from the apical medium supplemented with 0.05 mg L-1 NAA and 0, 5 mg L-1 BAP. In Allium cepa was observed that the mitotic index is superior in cell subjected to leaf extracts from the field (5 g L-1) and leaf extract of cuttings (20 g L-1) did not differ from the negative control in distilled water. The other treatments showed potential anti-proliferative with reduced mitotic index did not differ from the positive control in glyphosate 5%. Only the extract of the leaves of the greenhouse (20 g L-1) cells to genotoxic effects of A. cepa, where the number of cells with chromosomal abnormalities did not differ from the positive control. Occurred anti-mutagenic activity of the leaf extract of Campus UFSM (20 g L-1), on the cells of A. cepa, undergoing changes in glyphosate 5%, recovering from the mutagenic effect. Regarding the quantification of metabolites produced higher concentration of polyphenols and flavonoids chlorogenic acid in the samples collected from the field. / Alternanthera brasiliana (L.) Kuntze (Amaranthaceae) é um espécie conhecida como penicilina, terramicina, doril ou carrapichinho. Trabalhos comprovam que o extrato das folhas apresenta atividade analgésica, anti-inflamatória e cicatrizante. Com base no uso medicinal desta espécie o trabalho visou propagar vegetativamente in vitro e ex vitro A. brasiliana, e avaliar o potencial genotóxico, anti-proliferativo e anti-mutagênico do extrato aquoso das folhas de plantas provenientes de quatro sistemas de cultivo, através do Teste Allium cepa, bem como a quantificação do conteúdo de polifenóis, flavonoides e ácido clorogênico. Na propagação por estaquia de ramos aéreos ex vitro foram utilizadas estacas apicais, medianas e basais tratadas em meio nutritivo MS (0 e 20%) com acréscimo de AIB ao meio liquido (0,0 e 2,5 mg L-1). Para a micropropagação foram utilizados segmentos apicais e nodais inoculados em meio MS com as combinações de ANA (0,0 e 0,05 mg L-1) e BAP (0,0 e 0,5 mg L-1). Nas avaliações do potencial genotóxico, anti-proliferativo e anti-mutagênico, raízes de bulbos de Allium cepa foram imersas por 24 horas em extratos aquosos por infusão das folhas secas de A. brasiliana de quatro proveniências: Campus da UFSM; micropropagação; estaquia e casa de vegetação, todos com concentrações de 5 g L-1 e 20 g L-1. Estes mesmos extratos foram utilizados para a quantificação dos metabólitos secundários, polifenóis e flavonoides, através de espectrofotômetro e ácido clorogênico através de cromatografia líquida de alta eficiência. Estacas apicais, medianas e basais podem ser utilizadas na propagação, apresentando porcentagem de enraizamento e sobrevivência acima de 62%. Em geral, plantas provenientes de estacas medianas e basais apresentam melhores resultados que as apicais, sugerindo que estas seriam as mais adequadas na produção de mudas. Na micropropagação in vitro os segmentos apicais apresentaram resultados significativamente superiores aos segmentos nodais e o uso de fitorreguladores Ácido naftalenoacético (ANA) (0,05 mg L-1) e Benzilaminopurina (BAP) (0,5 mg L-1) não foi significativo. As plantas regeneradas in vitro aclimatizadas não apresentaram diferença significativa entre os tratamentos, sendo que a porcentagem de sobrevivência máxima (72,57%) ocorreu em plantas provenientes de segmentos apicais com meio acrescido de 0,05 mg L-1 de ANA e 0,5 mg L-1 de BAP. No teste Allium cepa foi observado que o índice mitótico é superior em células de A. cepa submetidas aos extratos de folhas do campo (5 g L-1) e extrato de folhas da estaquia (20 g L-1), não diferindo do controle negativo em água destilada. Os demais tratamentos apresentaram potencial anti-proliferativo com a redução do índice mitótico, não diferindo do controle positivo em glifosato 5%. Apenas o extrato das folhas da casa de vegetação (20 g L-1) apresentou efeito genotóxico às células de A. cepa, onde o número de células com alterações cromossômicas não diferiu do controle positivo. Ocorreu atividade anti-mutagênica do extrato de folhas do Campus da UFSM (20 g L-1), sobre as células de A. cepa, submetidas à alteração em glifosato 5%, recuperando-se do efeito mutagênico. Quanto à quantificação dos metabólitos ocorreu maior concentração de polifenóis flavonoides e ácido clorogênico nas amostras coletadas do campo.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / IntroduÃÃo. A leptospirose à uma doenÃa endÃmica no Nordeste, sendo caracterizada por complicaÃÃes potencialmente fatais como a lesÃo renal aguda (LRA). O objetivo deste estudo foi avaliar as manifestaÃÃes clÃnicas, a classificaÃÃo da LRA e os fatores de risco para Ãbito em pacientes com a forma grave de leptospirose. MÃtodos. Foi realizado estudo retrospectivo em pacientes com a forma grave de leptospirose internados em hospitais terciÃrios na cidade de Fortaleza, nordeste do Brasil. Foram avaliadas as manifestaÃÃes clÃnicas, os exames laboratoriais na admissÃo e durante a internaÃÃo e o tratamento instituÃdo. LRA foi definida de acordo com as classificaÃÃes RIFLE e AKIN, sendo comparados os pacientes nas diferentes classes. Foram comparados os pacientes que usaram com aqueles que nÃo usaram penicilina, assim como os pacientes que sobreviveram com os que foram a Ãbito. AnÃlises univariada e multivariada foram usadas para a investigaÃÃo dos fatores de risco para Ãbito. A anÃlise estatÃstica foi feita pelo programa SPSS versÃo 10.0. Resultados. Foram incluÃdos 287 pacientes, com mÃdia de idade de 36,8Â15,6 anos, sendo 80,8% do sexo masculino. Os principais sinais e sintomas apresentados foram febre (96,2%), mialgia (90,6%), icterÃcia (85,7%), cefaleia (74,2%), vÃmitos (70,7%), desidrataÃÃo (54%) e calafrios (53,7%). LRA foi observada em 237 pacientes (82%) pelo critÃrio RIFLE e 242 (84%) pelo AKIN. A mortalidade geral foi de 13%. A mortalidade foi semelhante nos pacientes que usaram e que nÃo usaram penicilina (11,6% vs. 13,7%, p=0,60). Aumento da mortalidade foi observado de acordo com as piores classificaÃÃes: RIFLE-R (2%), RIFLE-I (8%) e RIFLE-F (23%), assim como AKIN 1 (2%), AKIN 2 (8%) e AKIN 3 (23%), p < 0,0001. Os pacientes com oligÃria tiveram maior mortalidade (20%), em comparaÃÃo com os pacientes sem oligÃria (5%), p=0,02. Os fatores de risco independentes para Ãbito foram: RIFLE-F (OR=10,5, IC 95%=1,3-80,8, p<0,001), AKIN 3 (OR=7,5, IC 95%=2,2-25,2 p<0,001) e necessidade de diÃlise (OR=3,5, IC 95%=1,1-11,01, p=0,01). ConclusÃes. A LRA à uma complicaÃÃo frequente na leptospirose, com mortalidade significativa. Houve associaÃÃo entre as classificaÃÃes RIFLE e AKIN com a mortalidade na leptospirose. Os fatores de risco independentes para Ãbito sÃo classificaÃÃo RIFLE-F, AKIN 3 e necessidade de diÃlise. / Introduction. Leptospirosis is en endemic disease in Northeast of Brazil, which is characterized by potential fatal complications such as acute kidney injury (AKI). The aim of this study was to evaluate the clinical manifestations, the AKI classification and the risk factors for death in patients with the severe form of leptospirosis. Methods. A retrospective study was conducted in patients with severe form of leptospirosis admitted to tertiary hospitals in Fortaleza city, Northeast of Brazil. The clinical manifestations, laboratory tests at admission and during hospital stay, as well as treatment, were evaluated. AKI was defined according to the RIFLE and AKIN classifications, and the patients in each category were compared. Patients who used and who did not use penicillin, as well as survivors and non-survivors, were compared. Univariate and multivariate analysis were performed to investigate the risk factors for death. Statistical analysis was done with SPSS program version 10.0. Results. A total of 287 patients were included, with a mean age of 36.8Â15.6 years, and 80.8% were male. The main signs and symptoms at admission were fever (96.2%), myalgia (90.6%), jaundice (85.7%), headache (74.2%), vomiting (70.7%), dehydration (54%) and chills (53.7%). AKI was observed in 237 patients (82%) according to the RIFLE criteria and 242 (84%) according to AKIN. General mortality was 13%. Mortality was similar in patients who used and who did not use penicillin (11.6% vs. 13.7%, p=0.60). An increase in mortality was observed according to the worst classifications of RIFLE and AKIN: RIFLE-R (2%), RIFLE-I (8%) e RIFLE-F (23%), AKIN 1 (2%), AKIN 2 (8%) e AKIN 3 (23%), p<0.0001. Patients with oliguria had a higher mortality (20%), when compared to those without oliguria (5%), p=0.02. Independent risk factors for death were: RIFLE-F (OR=10.5, 95% CI=1.3-80.8, p<0.001), AKIN 3 (OR=7.5, 95% CI=2.2-25.2 p<0.001) and need of dialysis (OR=3.5, 95% CI=1.1-11.01, p=0.01). Conclusions. AKI is a frequent complication in leptospirosis, with significant mortality. There was association between RIFLE and AKIN classifications with mortality. Independent risk factors for death were RIFLE-F, AKIN 3 and need of dialysis.

Orientadores: Telma Teixeira Franco, Adrianus Johannes Straanhof, Lucas Antonius Maria van der Wielen / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-05T06:02:03Z (GMT). No. of bitstreams: 1 Ferreira_JulianadeSouza_D.pdf: 5552134 bytes, checksum: f2234304e4f228a88bb1b83073350bfa (MD5) Previous issue date: 2004 / Resumo: Este trabalho estuda a hidrólise enzimática da penicilina G (PenG) em ácido 6-aminopenicilânico (6-APA) e ácido fenil acético (PAA). Em um reator contra-corrente multi-estágio e em baixos valores de pH, a reação enzimática ocorre na fase aquosa e os produtos são separados entre a fase aquosa e a fase orgânica (acetato de butila). Além disso, em pH baixo, a cristalização do 6-APA ocorre quando concentrações de PenG são altas. Ambos fenômenos deslocam o equilíbrio no sentido de conversão do substrato, promovendo alta produtividade. A primeira etapa deste trabalho refere-se à avaliação da atividade e estabilidade da penicilina amidase a baixo pH e na presença de acetato de butila (BuAc). A enzima apresentou máxima atividade na faixa de pH 8,0 - 9,0 e permaneceu estável mesmo em pHs baixos (3,0 - 6,0) num período de incubação de até 32 dias. Embora a atividade enzimática sofra um decréscimo de aproximadamente 80%, isto não representa empecilho para sua utilização no emprego da hidrólise de PenG em processo contínuo e bifásico (água e BuAc) em pH baixo. O efeito de PenG, PAA e BuAc na cristalização do 6-APA e os parâmetros cinéticos de cristalização também foram avaliados. Os resultados mostraram que as impurezas não exerceram efeito sobre a cristalização de 6-APA, na faixa de pH entre 4 e 5 e nas concentrações de impurezas de 0,55 mM - 3,0 mM. A avaliação da cinética de cristalização possibilitou o uso de um modelo que pode predizer as taxas de cristalização de 6-APA. Um modelo quantitativo foi desenvolvido para o cálculo do pH e das concentrações do substrato e dos produtos nos estágios do reator contra-corrente. Os dados fornecidos pelo modelo podem ser utilizados para otimizar as condições de operação como: estágio de alimentação, vazão volumétrica das fases e concentração inicial do substrato. Na última etapa deste trabalho foi feita uma revisão bibliográfica sobre biorreatores extrativos em que são apresentadas as vantagens de cada configuração e as restrições dos processos biocatalíticos. Através desta revisão, foi verificado que o uso de um sistema, formado por agitadores acoplados a hidrocic1ones em série, pode representar uma opção adequada de reatores multi-estágio contra-corrente para a hidrólise de PenG em escala de laboratório / Abstract: This work studies the enzymatic hydrolysis of penicillin G (PenG) into 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). In a multi-stage countercurrent reactor and at low pH, the enzymatic reaction takes place in the aqueous phase and the products are separated between the aqueous phase and organic phase (butyl acetate - BuAc). Furthermore, 6-APA crystallization occurs at low pH when PenG concentrations are high. Both phenomena shift the equilibrium towards conversion of substrate, favoring high productivity. The first step of this work, concerns the evaluation of activity and stability of penicillin amidase at low pH and in the presence of butyl acetate (BuAc). The enzyme presented the maximum activity in the pH 8.0 - 9.0 and remained stable at low pHs (3.06.0) during at least 32 days. Although the enzyme activity decreased by 80%, this does not represent a drawback in the application of a biphasic (water and BuAc) and continuous PenG hydrolysis at low pH. The effect of PenG, PAA and BuAc in APA crystallization and the kinetic parameters were also analyzed. The results showed that impurities have no effect on APA crystallization, in the pH range 4 - 5 and in the impurity concentrations of 0.55 mM - 3.0 mM. The evaluation of crystallization kinetics allowed the use of a mo del that predicts the APA crystallization rates. A quantitative model was developed in order to calculate the pH and substrates and products concentrations in the countercurrent reactor. The data provided by the model can be used to optimize the operation conditions: stage of feed, flow rate of phases and initial substrate concentration. In the last step of this work, a literature review concerning extractive bioreactor was made. This review presents the advantages of each configuration and the restrictions of the biocatalytic processes. Through this review, a integrated system of mixers and hydrocydone was suggested as an appropriate option of multi-stage countercurrent reactor for PenG hydrolysis in laboratory scale / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.