http://hdl.handle.net/10362/2064 ITQB 2013-05-23T15:51:19Z http://hdl.handle.net/10362/8594 Title: Quantum dots: synthesis, functionalization and bioconjugation for biological applications Authors: Miguel, Ana Sofia Abstract: The main goal of this Doctoral work was to develop a fluorescent biomarker to identify antigenic proteins associated with specific parasites. Although it is known that fluorescent techniques making use of standard organic dyes are widely used for this purpose, the development of a new method was proposed using nanotechnology; in particular the use of nano optical reporters also called quantum dots (QDs). In order to achieve this goal, the research work was divided into four main tasks: (1) synthesis and characterization of CdSe/ZnS core-shell QDs; (2) design of dihydrolipoic acid (DHLA) ligands appended to oligo and poly (ethylene) glycols (PEG) with different functional groups to generate biocompatible QDs; (3) bioconjugation of QDs to monoclonal antibodies based on sophisticated protocols and (4) some chemical and biological applications of the synthesized non-conjugated and conjugated nanoparticles.(...) Description: Dissertation presented to obtain the Ph.D degree in Engineering Sciences and Technology 2012-09-01T00:00:00Z http://hdl.handle.net/10362/8593 Title: Epidemiological studies of Streptococcus pneumoniae carriage in the post-vaccination era among two risk groups: children and the elderly Authors: Nunes, Sónia Abstract: Streptococcus pneumoniae is a global cause of disease including pneumonia, otitis media, conjunctivitis, sepsis, and bacterial meningitis. These infections are not essential to the transmission or long-term survival of the bacterium; indeed, S. pneumoniae depends on asymptomatic colonization of the human nasopharynx for its dissemination to additional hosts. Considering this, colonization studies are a good way to monitor changes in the pneumococcal epidemiology that may result from the use of antibiotics and vaccines. The molecular characterization of pneumococci is crucial to assess these changes which highlight the need for the development and validation of easier and faster methods of molecular typing. Since 1996 our group has been monitoring the pneumococcal population colonizing children attending day care centers. However, for several years these studies have been confined to the Lisbon area. In this PhD we have addressed this situation by including other regions of Portugal in our study. In addition, we have started to study pneumococcal colonization in the elderly, the other age group where the incidence of pneumococcal infections is high. This thesis summarizes five studies conducted during this PhD. The first four studies were focused on the pneumococcal epidemiology among the two age groups where the rates of pneumococcal disease are highest: children up to six years old and adults older than 60 years. The fifth and last study describes the evaluation and validation of a new genotyping strategy for pneumococci.(...) Description: Dissertation presented to obtain the Ph.D. degree in Biology/ Molecular Biology 2012-11-01T00:00:00Z http://hdl.handle.net/10362/8592 Title: Insights into cell wall synthesis and cell division in Staphylococcus aureus Authors: Jorge, Ana Maria Abstract: Staphylococcus aureus is a gram-positive bacterial pathogen that besides persistently colonizing healthy individuals, is responsible for a large number of hospital-associated bacterial infections. The extraordinary capacity of S. aureus to acquire resistance to antibiotics led to the emergence of highly resistant strains, mainly methicillin-resistant S. aureus (MRSA) strains, that are a major cause of soft skin and tissue infections and bacteremia. In one third of European countries, including Portugal, more than 25% of S. aureus infections are caused by MRSA strains. The capacity of MRSA strains to resist β-lactam antibiotics (such as penicillin) is mainly due to the acquisition of an extra-species penicillin-binding protein (PBP), PBP2A. PBPs are bacterial enzymes involved in the synthesis of the cell wall polymer peptidoglycan. Besides PBP2A, which is present only in MRSA strains, S. aureus has 4 native PBPs (PBP1-4), which catalyze the polymerization (transglycosylation) and the cross-linking (transpeptidation) of glycan chains, forming a strong yet flexible structure that protects the cell from the high internal osmotic pressure. Peptidoglycan is unique to the bacterial kingdom and its biosynthesis is the target of a vast number of clinically important antibiotics such as β-lactams and glycopeptides. β-lactam antibiotics target the transpeptidase domain of PBPs, halting peptidoglycan synthesis and eventually leading to cell lysis. However, in MRSA strains the existence of PBP2A, which has a low affinity for β-lactams, enables cell wall synthesis to continue even in the presence of these antibiotics. Under these conditions, the transpeptidase domain of PBP2A functionally cooperates with the transglycosylase domain of the unique bifunctional PBP, PBP2, to ensure continued cell wall synthesis and cell survival.(...) Description: Dissertation presented to obtain the Ph.D degree in Biology 2012-04-01T00:00:00Z http://hdl.handle.net/10362/8591 Title: Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase Authors: Gonçalves, Susana Abstract: Enzyme evolution is often constrained by aspects of catalysis. Mechanistically diverse enzymes evolved from a common ancestor still preserve those structural signatures essential to the core chemistry retained by all members of the superfamily. Indeed, these shared features allow superfamilies to be accurately classified, while derived features allow nested families and subfamilies to be identified in a hierarchical fashion. Accurate classification has helped elucidate mechanisms promoting functional diversification, for example catalytic promiscuity, and protein engineering by rational design. Nowadays, a holistic view of enzymes` regulatory mechanisms and catalytic proficiency is provided by the identification of conserved features of molecular architecture in combination with aspects of reaction dynamics. My work focused on the structural elucidation and analysis of three enzymes: a glycosyltransferase; a phosphatase and an oxidorreductase. “Snapshots” along the reaction coordinate of each enzyme were obtained by combining X-ray diffraction with “cryo-trapping” ligand-binding methods. These were used to characterize the molecular mechanisms involved in substrate recognition and binding. They were also used to distinguish between models proposed for the catalytic mechanisms of each enzyme, and provide insights into enzyme dynamics essential for catalysis and the stereo and regio-selective strategies at work.(...) Description: Dissertation presented to obtain the Ph.D degree in Biochemistry 2012-05-01T00:00:00Z