http://hdl.handle.net/10362/3496 Sun, 26 May 2013 01:59:00 GMT 2013-05-26T01:59:00Z http://hdl.handle.net/10362/8583 Title: The role of Di-iron proteins in pathogen resistance Authors: Baptista, Joana Morais Abstract: Reactive oxygen and nitrogen species (ROS and RNS) are produced by phagocytic cells of the human immune system to attack invading pathogens due to their ability to damage DNA and the metal centres of proteins. In order to survive inside the host, bacteria activate genes that encode detoxifier enzymes, like the Escherichia coli nitric oxide-reductase flavodiiron protein, also known as flavorubredoxin (FlRd), and repairing proteins, such as the E. coli YtfE di-iron protein involved in the recovery of damaged Fe-S centres. Using E. coli and Staphylococcus aureus, the work presented in this thesis aimed at unravelling: i) the role of E. coli FlRd in bacteria exposed to a combination of oxidative and nitrosative stresses, ii) the identification and characterisation of S. aureus YtfE homologue, iii) the study of E. coli YtfE mechanisms that allow the repair of damaged Fe-S clusters, and iv) the identification of proteins that interact with E. coli YtfE. To analyse the role of E. coli FlRd in cells submitted to both hydrogen peroxide and nitric oxide, the transcription and expression of norV was explored by means of β-galactosidase and immunoblotting assays, respectively. Under these conditions, it was observed that the norV transcription and expression were hindered. To identify if the lack of norV expression was related to its regulator, the NorR transcription factor, the gene was cloned and expressed, and the protein was purified and the binding of nitric oxide to NorR in the presence of hydrogen peroxide was studied. EPR experiments revealed that upon incubation of NorR with nitric oxide and hydrogen peroxide the oxidation promoted by H2O2 of the monoiron centre of NorR impairs the ligation of nitric oxide.(...) Description: Dissertation presented to obtain the Ph.D. degree in Biochemistry Sat, 01 Sep 2012 00:00:00 GMT http://hdl.handle.net/10362/8583 2012-09-01T00:00:00Z http://hdl.handle.net/10362/5156 Title: Unravelling novel modes of antimicrobial action Authors: Nobre, Lígia S. Abstract: The work presented in this thesis aimed at unravelling novel modes of antimicrobial action through: i) the study of Staphylococcus aureus defences against nitric oxide, an antimicrobial weapon of the innate immune system, namely by performing the biochemical characterisation of S. aureus NO-detoxifying flavohaemoglobin and analysing the conditions under which the enzyme is operative; ii) the identification of the mechanisms beyond the antibacterial activity of azole antibiotics towards S. aureus that were shown to include the increase of endogenous reactive oxygen species triggered by the binding of imidazoles to flavohaemoglobin; and iii) the discovery of a novel type of bactericides, the carbon monoxide-releasing molecules, and identification of its potential cellular targets via analysis of the transcriptional response of Escherichia coli to CORM-2, a carbon monoxide-releasing molecule known to mimic the physiological function of carbon monoxide.(...) Description: Dissertation presented to obtain the Ph.D. degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Mon, 01 Nov 2010 00:00:00 GMT http://hdl.handle.net/10362/5156 2010-11-01T00:00:00Z http://hdl.handle.net/10362/4082 Title: Anaerobic bacteria: an investigation of metabolic important enzymes: a novel type of oxygen reductase and enzymes of the tetrapyrrole biosynthesis Authors: Lobo, Susana André Abstract: Desulfovibrio desulfuricans was the first species of a sulphatereducing bacterium to be isolated, in 1895. Since that time, many questions were raised in the scientific community regarding the metabolic and ecological aspects of these bacteria. At present, there is still a myriad of open questions remaining to be answered to enlarge our knowledge of the metabolic pathways operative in these bacteria that have implications in the sulfur cycle, in biocorrosion, namely in sewers and in oil and gas systems, and in bioremediation of several toxic metals. The work presented in this dissertation aimed at contributing with new insights of enzymes involved in two different metabolic systems on Desulfovibrio species, namely enzymes that play a role in the response to oxidative stress and that are involved in the haem biosynthetic pathway.(...) Fri, 01 May 2009 00:00:00 GMT http://hdl.handle.net/10362/4082 2009-05-01T00:00:00Z