Nanopores have been shown to be a useful analytical tool for single molecule detection. They have been used to study the composition of DNA and other molecules of interest. These pores are usually α-hemolysin which is a toxin from Staphylococcus aureus or more recently nanoscale synthetic solid state pores. Now we are beginning to look at other molecules or proteins by sending them into the nanopores and measuring a characteristic partial current blockade. In this thesis we look at polyethylene glycol (PEG) as it enters and blocks current through a single alpha hemolysin pore. We report the effects of ionic strength, PEG size, and applied voltage on the depth and duration of the current blockades. We also apply autocorrelation analysis on the arrival times of PEG molecules to the pore see if we can identify if the PEG is translocating through the pore or escaping from the same side it enters. This suggests a new approach to current blockade analysis.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-06-28T17:52:25Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Melo-MCA_dissertacao.pdf: 6271738 bytes, checksum: ce55245b114aac78286d16fa3cabf5d4 (MD5) / Made available in DSpace on 2016-06-28T17:52:25Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Melo-MCA_dissertacao.pdf: 6271738 bytes, checksum: ce55245b114aac78286d16fa3cabf5d4 (MD5) Previous issue date: 2015-07-12 / CAPES / O Staphylococcus (S.) aureus é responsável por inúmeros casos de infecções em animais e seres humanos. Essa bactéria secreta uma grande variedade de exoproteínas, incluindo quatro hemolisinas: alfa, beta, gama e delta. A atividade hemolítica da alfa-toxina (α-HL) se deve a formação de poros em membranas celulares. A α-HL é um monômero solúvel em água, que se torna mais estável ao se oligomerizar e formar poros heptaméricos transmembranares. Devido sua importância na patogênese bacteriana, essa proteína tem sido alvo de estudos no desenvolvimento de agentes terapêuticos. Sabe-se que os compostos de base de Schiff têm apresentado significativos resultados em testes de atividade biológica, incluindo antibacteriana. Neste contexto, avaliou-se a atividade de complexos de cobre-base de Schiff no bloqueio de poros da α-HL de S. aureus. Simultaneamente, foram realizados estudos conformacionais da δ-toxina em solução aquosa. Experimentos com eritrócitos de coelho expostos a α-HL foram utilizados na seleção dos compostos. Membranas lipídicas planas artificiais com canais individuais de α-HL, em condições de fixação de voltagem, foram utilizados para entender o mecanismo de ação dos compostos. Finalmente, avaliou-se através de docking molecular a interação entre os compostos e o canal de α-HL, visando corroborar o mecanismo de bloqueio. No estudo da δ-toxina, dinâmica molecular foi usada para determinar sua conformação em solução aquosa. Observou-se que os complexos de cobre inibiram parcialmente a atividade hemolítica da α-HL, devido ao bloqueio parcial dos canais desta toxina. O mecanismo molecular desse bloqueio ocorre pela interação dos compostos com a região de constrição do poro. Observou-se ainda que a δ-toxina em solução aquosa apresenta de 5 a 14 resíduos enovelados. Sugere-se que os complexos de cobre são potenciais candidatos a futuros agentes coadjuvantes no tratamento de infecções por S. aureus. / Staphylococcus (S.) aureus is responsible for a great number of infecctions in animals and humans. This bacteria produces several exoproteins and virulence factors, including four hemolysins: alfa, beta, gama and delta. The alfa-toxin (α-HL) hemolytic activity occurs due to the formation of pores in cellular membranes. α-HL is a soluble monomer in water, but becomes more stable when oligomerize and form transmembranar heptameric pores. Considering its importance for bacterial patogenesis, this protein has been target of studies on new antimicrobial agents. For example, base Schiff copper compounds have been observed to show significative results in biological activity assays, including antibacterial. In this context, the present work intended to evaluate the activity of a base Schiff-copper compounds to block the S. aureus α-HL ion channel. The most effective compounds were selected based on the exposition of rabbit erythrocytes to α-HL, and tested against control conditions. The mechanism of action of these compounds were evaluated through single channel experiments in planar lipid bilayrs, through eletrophisiological techniques. Finally, molecular docking studies were employed to confirm the blocking mechanism of such compounds. Moreover, molecular dynamics simulations were used to determine δ-toxin conformation in aqueous solutions. The obtained results indicated that the copper(II) compounds were capable of partially blocking the α-HL hemolytic activity, due to partial blockade of the ionic channels. The molecular mechanis of pore blockade was identified to mainly occur due to the interaction of the compounds to the pore constriction. Additionally, it was observed that δ-toxin in aqueous solution presents from 5 to 14 amino acids showing α-helical content. Based on these results, we suggest that such compound may be further evaluated as a coadjuvante agent for the treatment of staphylococcal infections

Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.