Orientadores: Lucila Costallat Ricci, Antonio Fernando Pestana de Castro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T19:36:32Z (GMT). No. of bitstreams: 1 Ferraz_MirtisMariaGianciani_M.pdf: 6199459 bytes, checksum: 5178264e436b95d9608ded8b8db43e6f (MD5) Previous issue date: 2003 / Resumo: E. coli é um dos patógenos relacionados a casos de diarréia. Em nosso trabalho analisamos 27 amostras de E. coli isolados em 14 crianças portadoras do HIV, com diarréia persistente. Em 51,8% das fezes foi observada a presença de EAEC, sendo que estas amostras não apresentam nenhum fator de virulência quando analisadas por PCR, e outros testes realizados com exceção de duas amostras que apresentam o gene ast. Observou-se que 51,1% destas amostras apresentam invasão em células HeLa. Entretanto, tais amostras puderam ser caracterizadas como pertencentes ao grupo EIEC, através de provas sorológicas, bioquímicas, biológicas e moleculares. A sorogrupagem não evidenciou a presença de sorogrupos patogênicos entre estas amostras / Abstract: E. coli is pathogen relatd to episodes of diarrhea. In our study, 27 strains from 14 HIV + children with persistent diarrhea were analyzed. 51.8% of the patients showed EAEC. These E. coli did not show any virulent factors in the PCR tests, except two that presented ast gen and was not pathogenic serotype. However, 5.1% had invasion in the HeLa cells can not considered EIEC in serological, biochemistry, biological and molecular tests. The sosotype of these strains did not present pathogen serotype / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Doctora en Ciencias de la Ingeniería, Mención Química / Cada día hay un mayor interés por generar productos con valor agregado a partir de diferentes tipos de residuos. Uno de estos productos es el ácido hialurónico (hyaluronic acid, HA), polímero con variados usos en la medicina y la cosmetología. El HA es sintetizado a partir de una enzima llamada Hialuronan Sintasa o HAS. Esta enzima es una proteína de membrana que sintetiza HA a partir de 2 precursores: Ácido UDP-Glucurónico y UDP-N-Acetilglucosamina, compuestos que se encuentran naturalmente en las bacterias Escherichia coli. El presente trabajo tuvo como objetivo la construcción de una Escherichia coli recombinante capaz de sintetizar HA a partir de xilosa como fuente de carbono alternativa a la glucosa. Este azúcar es uno de los más abundantes en los residuos hemicelulósicos de la industria forestal chilena. Para mejorar el consumo de xilosa por parte de la bacteria nativa E. coli Top10 se usó la técnicade la Evolución Adaptativa de Laboratorio de Palsson (Adaptive Laboratory Evolution, ALE). Este proceso se realizó mediante un cultivo continuo en medio R2 y xilosa como única fuente de carbono. La cepa evolucionada fue transformada con el gen hasA de Streptococcus equisimilis (sehasA) obteniendo la cepa Top10 Evo:pMBAD-sseAB. Para el análisis del cambio generado por la ALE se realizó un Análisis de Balance de Flujos (Flux Balance Analysis, FBA) a partir de un Modelo a Escala Genómica (Genome Scale Model, GSM) del microorganismo. Además, se optimizó el cultivo de la cepa Top10 Evo:pMBAD-sseAB en cuanto a las siguientes condiciones: tiempo de adición de inductor y su concentración concentración, control de pH, temperatura de crecimiento post-inducción y concentración de azúcar suplementada. El ALE realizado tuvo una duración de 48 días. Como resultado se obtuvo que la velocidad especí fica de crecimiento en xilosa de la cepa E. coli Top10 aumentó un 36%: de 0,24 h-1 a 0,33 h-1. Se determinó que un mayor rendimiento de HA se obtiene al hacer 2 adiciones de inductor al cultivo. Anteriormente en la literatura se había observado este fenómeno, pero nunca en una expresión recombinante de proteína asociada a un vector regulado con el promotor PBAD, correspondiente al del presente trabajo. Una temperatura de crecimiento post-inducción de 28,5 °C presentó los mejores rendimientos. Además, se de nió que la adición de xilosa a una concentración de 20 g/L 2 horas después de la inducción, y de 12 g/L a las 24 horas de cultivo (el doble de lo presentado en la literatura) aumenta el rendimiento de HA en cultivos en xilosa. Finalmente, las condiciones de cultivo seleccionadas fueron: inducción con 2 adiciones de 0,05 g/L de L-arabinosa, temperatura de crecimiento post-inducción de 28,5 C y concentración de azúcar suplementada de 20 g/L después de la inducción y 12 de g/L a las 24 horas de cultivo. El rendimiento de HA obtenido en estas condiciones fue de 195,8 mg/g.cel, más del doble que el rendimiento obtenido en condiciones basales, el que fue de 92,6 mg/g.cel. Con el FBA realizado fue posible corroborar que la cepa evolucionada tenía mejoras metabólicas en comparación con la cepa no evolucionada, cambio producido por el ALE. Estas mejoras permitieron que los ujos asociados a las vías de las pentosas fosfato, la glicólisis y el ciclo de ácidos tricarboxílicos fueran mayores en la cepa evolucionada en un 12% y 29% en promedio, durante el consumo de xilosa y manosa, respectivamente. Además, dado que las diferencias observadas entre las cepas evolucionada y no evolucionada se observaron tanto en xilosa como en manosa, y permitieron una mayor producción de HA y de biomasa, se in rió que la ALE impactó alguna enzima de la glicólisis.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Jianfa Bai / Cattle feces are a major source of six Shiga toxin-producing E. coli (STEC) serogroups, O26, O45, O103, O111, O121, and O145, called non-O157 STEC, responsible for >70% of non-O157 STEC-associated human illnesses. Another E. coli serotype, O104:H4, a hybrid pathotype of enteroaggregative and STEC, was responsible for a large outbreak of foodborne illness in Germany. Studies were conducted to develop and validate culture- and PCR-based methods to detect and or quantify six non-O157 E. coli serogroups and E. coli O104 in cattle feces, and genetically assess their virulence potential, based on DNA microarray and whole genome sequencing (WGS). Two multiplex quantitative PCR (mqPCR) assays (assay 1: O26, O103 and O111; assay 2: O45, O121 and O145), targeting serogroup-specific genes, were developed and validated for the detection and quantification of six non-O157 E. coli in cattle feces and was compared to culture-based and end-point PCR methods. The mqPCR assays detected higher proportion of fecal samples as positive for one or more non-O157 E. coli serogroups compared to culture-based and end-point PCR methods. Spiral plating method was validated to quantify six non-O157 E. coli serogroups in cattle feces, and was compared to mqPCR assays. The mqPCR assays quantified higher proportion of fecal samples positive for one or more non-O157 E. coli serogroups compared to spiral plating method, however, unlike mqPCR, spiral plating method quantifies serogroups positive for virulence genes. Quantification by either mqPCR or spiral plating identified a subset of cattle that was shedding non-O157 E. coli at high concentrations (≥ 4 log CFU/g of feces), similar to E. coli O157. Identification of Shiga toxin subtypes associated with non-O157 E. coli serogroups isolated from cattle feces revealed a variety of subtypes, with stx1a and stx2a being the most predominant. Microarray-based analysis of six non-O157 E. coli serogroups isolated from cattle feces revealed the presence of stx, LEE-encoded, and other virulence genes associated with human illnesses. Analysis of WGS of STEC O145 strains isolated from cattle feces, hide and human clinical cases revealed similarity in virulence gene profiles, suggesting the potential of cattle E. coli O145 strains to cause human illnesses. Shiga toxin 1a was the most common stx subtype, followed by stx2a, and stx2c. The strains also carried LEE-encoded, and plasmid-encoded virulence genes. Model adjusted prevalence estimates of E. coli O104 in cattle fecal samples collected from feedlots (n=29) were 0.5% and 25.9% by culture and PCR methods, respectively. Cattle harbor O104 serotypes other than H4, with O104:H7 being the predominant serotype and only a small proportion of them carried stx. DNA microarray and WGS analysis revealed absence of LEE-encoded virulence genes in bovine and human O104 strains. Escherichia coli O104:H7 has the potential to be a diarrheagenic foodborne pathogen in humans, since they possess stx1c and genes that code for enterohemolysin and a variety of adhesins. Data on prevalence, concentration and virulence potential of non-O157 E. coli serogroups, including O104, isolated from cattle feces are essential to design effective intervention strategies to reduce the potential to cause human foodborne illness outbreaks.

Prof. Paul Jagals Dr. Hafsatou Ndama Traoré

>Magister Scientiae - MSc / Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe2O4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally “soft” magnetic materials which can only be used for some applications, while other applications require “hard” magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni2+ or Co2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni2+ or Co2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni2+ or Co2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl2 or CoCl2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni2+ uptake permease the hoxN gene or Co2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni2+ or Co2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and Ni2+ or Co2+ uptake gene for mass production of magnetosome with altered magnetic properties.

No description available.

Legionaminic acid (Leg5,7Ac2) is a nonulosonic acid similar to sialic acid (Neu5Ac), which can be found in the extracellular glycoconjugates of several bacterial pathogens. Due to the similarity in stereochemistry of the two compounds, legionaminic acid has great potential in the production of pharmaceutical drugs. A novel biosynthetic pathway to produce legionaminic acid was created to overcome the limitations of organic synthesis. This is the first study involving the scale-up of legionaminic acid production by high cell density fermentation processes. In this work, fed-batch cultivations of recombinant Escherichia coli BRL04 were carried out in shake flasks and 5-L bioreactors. The final process was optimized by determining the effects of different carbon sources, induction temperatures, pH, dissolved oxygen (DO) content, induction optical density and N-acetylglucosamine (GlcNAc) feed rate on the production of legionaminic acid. Overall, results showed that the titer, yield and productivity for legionaminic acid production achieved relatively high levels, which were 5.53 g/L, 73.29% and 0.092 g/(Lh), respectively. It is hoped that this study accelerates research into the production of legionaminic acid for therapeutic treatments as well as for further study in glycobiology.

Low concentrations of the group IIA metals Mg²⁺, Ca²⁺, and Ba²⁺ stimulated energy-independent transhydrogenase activity. High concentrations of Mg²⁺ inhibited this activity. Transhydrogenase requires Mg²⁺-complexed NADP(H) rather than free NADP(H) as its substrate. High concentrations of Mg2+, however, may change the conformation of the enzyme to inhibit its enzymatic reaction by binding directly to the NADP(H) site. Upon transhydrogenation between NADPH and 3-acetylpyridine dinucleotide, E. coli pyridine nucleotide transhydrogenase can establish a proton gradient across the cell membrane. The primary component of the proton gradient for energization of transhydrogenase was found to be the pH gradient and not the membrane potential. A similar conclusion was drawn for the ATP-driven transhydrogenase reactions. In strains of E. coli that harbored plasmids to give the cells elevated levels of transhydrogenase, it was found that uncouplers stimulated the aerobic-driven transhydrogenase reaction. This is a chemiosmotic anomaly and is in contrast to the non-plasmid containing parent strains where uncouplers inhibited the activity. Further investigation revealed that the plasmid strains contained a much lower NADH oxidase activity than the non-plasmid strains and that neither KCN nor QNO can inhibit the aerobic-dependent activity in both types of strains even though they were effective in blocking the respiratory chain. These effects prompted us to inquire whether the anomaly was due to differences in the respiratory chain, but no differences were found between the NADH dehydrogenase activities, quinone and cytochrome contents of the plasmid and non-plasmid strains. The bacterial cells with amplified transhydrogenases induce extra intracellular tubular membrane structures to accomodate the extra proteins (Clark, D.M., Pyridine Nucleotide Transhydrogenase, PhD thesis, University of British Columbia, 1986). Separation of the E. coli membrane vesicles on a shallow sucrose gradient, however, did not reveal any differences between the vesicles of the plasmid and non-plasmid strains. Therefore, it seems unlikely that the anomaly is due to the plasmid strains performing a unique form of energization on these induced structures. Finally, it was established by SDS-PAGE and Western blot using anti-transhydrogenase antisera that the plasmid strains express a much higher level of transhydrogenase enzymes in their cell membranes than do the non-plasmid strains. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Orientador : Nelson E. Duran Caballero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-07-16T15:17:16Z (GMT). No. of bitstreams: 1 Marcucci_MariaCristina_D.pdf: 11251332 bytes, checksum: df4d73814510400eee18b38cc9d2a93a (MD5) Previous issue date: 1986 / Resumo: O malonaldeído e o ácido indol-3-acético são oxidados aerobicamente pela peroxidase, produzindo oxigênio singlete em ambos os processos, e no último caso também indol-3-aldeído excitado. Estes processos são chamados de biofotoenergizados. Entretanto, existem outras vias de geração de oxigênio singlete como processos fotoquímicos, pelo uso de sensibilizadores. Foi comprovada a similaridade entre os efeitos de oxigênio singlete gerado fotoquimica e enzimaticamenle sobre o ácido ribonucleico de transferência de Escherichia coli. Os métodos de luminescência, dicroísmo circular e absorção no ultra-violeta, foram utilizados neste estudo. Estes efeitos foram comprovados pela proteção por histidina, pelo aumento da fotodegradação na presença de água deuterada, e o uso de supressores específicos. A estrutura do ácido ribunucleico de transferência é protegida destas espécies pelos íons Mg A oxidação de 4-tiouridina nestes processos tanto por auto-sensibiIização ou por transferência de energia de sistemas biofotoenergizados, produz uridina. O indol-3-aldeído triplete transfere sua energia à 4-tiouridina no ácido ribonucleico. A 4-tiouridina excitada sofre dois processos importantes: a) transfere sua energia ao oxigênio, gerando oxigênio singlete, que volta a reagir com a 4-tiouridina formando uridina e b) interage com a citidina na posição 13 do ácido ribonucleico,formando um fotoaduto [8-13]. Estes processos descritos anteriormente no ácido ribonucleico de transferência e 4-tiouridina isolada foram estudados em diferentes cepas de Escherichia coli. As linhagens de Escherichia coli deficientes em 4-tiouridina apresentaram maior sobrevivênica do que o tipo selvagem, que contém a 4-tiouridina, quando submetidas ao tratamento com o indol-3-aldeído biofotoenergizado. Estes resultados sugerem que a energia de ex- citação do indol-3-aldeído triplete pode ser transferida do ácido ribonucleico de transferência ao ácido desoxirribonucleico, originando quebras de fitas simples.A possibilidade de que estados excitados atuem em sistemas celulares indica o avanço das pesquisas nesta área para esclarecimento de processos mutagênicos espontâneos. / Abstract: Both malonaldehyde and indole-3-acetic acid undergo aerobic peroxidase-catalysed oxidation to produce singlet oxygen, while in lhe second reaction, excited indole-3-aldehyde is algo produced. These reactions occur by means of biophotoenergetic processes another means to produce singlet oxygen is by use of sensitzers in a photochemical process. Singlet oxygen produced by both processes (namely the biophotoenergized and photochemical processes) interact similarly on transfer ribonucleic acid from Escherichia coli. In these studies, luminescence, circular dichroism and ultra-violet absorption techniques were used. The singlet oxygen effect on tRNA was shown by way of measurements: a) protection by histidine; b) observed increases in photodegration in the presence of D2O and c) lnfluence of specific scavengers. The tRNA is protected from these species by Mgions. The oxidation of 4-thiouridine, auto-sensitized, photo-sensitized or through energy transfer, produces uridine. Energy transfer from triplet indole-3-aldehyde to 4-thiouridine on tRNA was observed. The excited 4-thiouridine undergoes two important processes: (a) energy transfer to oxygen, generating singlet oxygen which may then reinitiation the 4-thiouridine transformation to urldine. (b) interaction with cytidine on the 13th position of tRNA resulting in [8-13] photoadduct formation. These processes, which occur in tRNA and in isolated 4-thiouridine, were studied in different strains of E. coli mutant. A mutant strain of E.coli difficient in 4-thiouridine exhibited a higher survival factor than the wild type, when treated with biophotoenergized idole-3-aldehyde. These results suggest that the energy from excited triplet indole-3-aldehyde might be transfered to tRNA and then, to DNA, producing single strand breaks, with subsequent mortality. The possibility that these biophotonergyzed processes participate in cellular species indicates an important step describing the processes of spontaneous mutagenesis. / Doutorado

Para se determinar o efeito da adição de baixas concentrações de ácidos na resistência de microrganismos ao calor úmido, cultura de E. coli IZ-923 foi aquecida a 55°C e 60°C em caldo nutriente contendo 0,1 ou 0,2% dos ácidos acético, lático, cítrico e fosfórico por 12min. A intervalos de 4 min amostras foram retiradas, resfriadas, plaqueadas em nutriente ágar (NA) e ágar com bile vermelho violeta (VRBA) e incubadas por 48h a 37°C ± 0,5°C. Com o número de colônias desenvolvidas no nutriente ágar foram calculadas as porcentagens de redução e os valores D55 e D60. Com aquelas desenvolvidas no ágar com bile e vermelho violeta, calculou-se as porcentagens de danificação celular. As conclusões foram: 1. Na maioria das vezes a porcentagem de redução da E. coli IZ-923 aumentou com o aumento da concentração de ácidos e com a temperatura; 2. Durante o aquecimento tanto com a adição de 0,1% como 0,2% de ácido em ambas as temperaturas, pôde-se estabelecer a seguinte ordem de redução: acético > fosfórico > lático > cítrico; 3. As porcentagens da danificação foram semelhantes para todos os ácidos em ambas as concentrações de ácidos e temperaturas empregadas; 4. Os valores D55 e D60 diminuíram com o aumento da temperatura de aquecimento; 5. Nas condições do presente trabalho não foi possível caracterizar uma diferença expressiva entre os ácidos empregados com relação aos seus efeitos na resistência térmica de E. coli IZ-923 / In order to determine the effect of the addition of low concentrations of acids in the microorganism heat resistance, E. coli IZ-923 culture, was heat at 55°C and 60°C in nutrient broth, containing 0.1% and 0.2% acetic, lactic, citric and phosphoric acids for 12 minutes. Every four minutes samples were taken, cooled, plated on nutrient agar and violet-red bile agar and incubation at 37°C ± 0.5°C for 48 hours. The D55 e D60 values and the reduction percentage were calculated from the number of developed colonies on nutrient agar. The cellular injure percentages were calculated from the developed colonies on violet-red bile agar. The conclusions are: 1. The E. coli IZ-923 populations reduction percentage, in the most although variable, raised as the added acid concentration and temperature increased; 2. Either heating temperatures and any of the concentrations showed the following acid reduction power: acetic > phosphoric > lactic > citric; 3. The cellular injure percentages were similar for all acids in both concentrations and temperatures employed; 4. D55 e D60 values decreased as the acid concentration and heating temperature increased; 5. AT working conditions it was not possible to stablish any great difference regarding to the effect of all acids used on the E. coli IZ-923 heat resistance