Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B. crocidurae. Both diseases differ in their manifestations, duration times and dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known. Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species. The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B. duttonii, B. recurrentis and B. hermsii were compared and their biophysical properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

Negli ultimi anni sono state pubblicate diverse normative, tra le quali ritroviamo la Decisione 2015/495 che ha emesso una lista di contaminanti con problematiche emergenti. Tra questi vi è il Triallate, erbicida pre-emergenza sul quale è stato basato questo progetto. In questo elaborato sono stati messi a confronto i risultati di uno studio triennale, il cui obiettivo era quello di monitorare le concentrazioni nelle acque di falda di due metaboliti del Triallate (DIPA e TCPSA), con i dati emersi dai modelli previsionali (FOCUS-Pearl) e verificare se tali dati erano concordi o se i valori della modellistica fornivano una tutela largamente protettiva nei confronti dell’ambiente, in sede di registrazione. / Recently, several regulations have been published, among which we find Decision 2015/495 which contains a list of contaminants with emerging problems. Among these we find Triallate, an herbicide on which this project was based. In this paper, the results of a three-year study on two metabolites of Triallate (DIPA and TCPSA) were compared with the data emerging from models (FOCUS-Pearl ). At the end we check if these data were in agreement or if the values ​​of the modeling provided a largely protective protection towards the environment.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.

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