Synthesis, characterization and applications of new schiff base fluorescent chemosensors for metal and DNA interactions: conventional and "green" approaches

Dissertação apresentada para a obtenção do Grau de Doutor em Química Sustentável, especialidade de Química-Física Inorgânica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

The Role of Actin-Capping Protein and Src signalling in tissue growth and apoptosis during Drosophila wing development

Dissertation presented to obtain the Ph.D degree in Developmental Biology

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry

Synthesis and characterization of sugar containing hydrophilic and hydrophobic polymers with potential application in medicine

Dissertation toobtaina Master of Science degree in Bioorganics

Synthesis and characterization of innovative luminescent glasses for artistic applications

Dissertação para obtenção do Grau de Doutor em Química Sustentável

Synthesis of new enzyme stabilisers inspired by compatible solutes of hyperthermophilic microorganisms

Dissertation presented to obtain the Ph.D degree in Engineering Sciences and Technology.

Evaluation of intrathecal synthesis of specific IgG antibodies against Toxoplasma gondii in the diagnosis assessment of presumptive toxoplasma encephalitis in aids patients

The diagnosis of neurotoxoplasmosis in patients with acquired immunodeficiency syndrome is mainly based on tomographic or magnetic resonance findings and on the response to specific treatment. We studied 55 patients with AIDS and neurotoxoplasmosis according to these diagnostic criteria (group 1), 37 patients with AIDS and neurological involvement of other etiology (group 2), and 16 anti-HIV-negative individuals with neurological manifestations (group 3). Serum and cerebrospinal fluid were examined for the presence of anti-T. gondii IgG, by indirect immunofluorescence. In 72 of them, the total amounts of these antibodies were determined in order to assess local production of anti-T. gondii antibodies in the central nervous system and to correlate their titers with infection activity in patients with AIDS and neurotoxoplasmosis. IgG titers > 1/64 in cerebrospinal fluid reached 100% specificity for the diagnosis of neurotoxoplasmosis in AIDS. Evidence of local synthesis of these antibodies was detected in 42.8% of patients of group 1, in 29.1% of patients of group 2 and in no patient of group 3. The test showed 70.8% specificity and therefore was not useful in our study for the differential diagnosis of neurotoxoplasmosis in patients with AIDS.

Green synthesis of 2-Oxazoline-based polymers with antimicrobial activity using scCO2

Using a green methodology, 17 different poly(2-oxazolines) were synthesized starting from four different oxazoline monomers. The polymerization reactions were conducted in supercritical carbon dioxide under a cationic ring-opening polymerization (CROP) mechanism using boron trifluoride diethyl etherate as the catalyst. The obtained living polymers were then end-capped with different types of amines, in order to confer them antimicrobial activity. For comparison, four polyoxazolines were end-capped with water, and by their hydrolysis the linear poly(ethyleneimine) (LPEI) was also produced. After functionalization the obtained polymers were isolated, purified and characterized by standard techniques (FT-IR, NMR, MALDI-TOF and GPC). The synthesized poly(2-oxazolines) revealed an unusual intrinsic blue photoluminescence. High concentration of carbonyl groups in the polymer backbone is appointed as a key structural factor for the presence of fluorescence and enlarges polyoxazolines’ potential applications. Microbiological assays were also performed in order to evaluate their antimicrobial profile against gram-positive Staphylococcus aureus NCTC8325-4 and gram-negative Escherichia coli AB1157 strains, two well known and difficult to control pathogens. The minimum inhibitory concentrations (MIC)s and killing rates of three synthesized polymers against both strains were determined. The end-capping with N,N-dimethyldodecylamine of living poly(2- methyl-2-oxazoline) and poly(bisoxazoline) led to materials with higher MIC values but fast killing rates (less than 5 minutes to achieve 100% killing for both bacterial species) than LPEI, a polymer which had a lower MIC value, but took a longer time to kill both E.coli and S.aureus cells. LPEI achieved 100% killing after 45 minutes in contact with E. coli and after 4 hours in contact with S.aureus. Such huge differences in the biocidal behavior of the different polymers can possibly underlie different mechanisms of action. In the future, studies to elucidate the obtained data will be performed to better understand the killing mechanisms of the polymers through the use of microbial cell biology techniques.

Avaliação da expressão de interleucina 1 beta (IL-1β) e antagonista do receptor de interleucina 1 (IL-1Ra) em pacientes com hanseníase

A hanseníase é uma doença infectocontagiosa espectral que acompanha-se por uma série de eventos imunológicos desencadeados pela resposta do hospedeiro frente ao agente etiológico, o Mycobacterium leprae. Evidências sugerem que a indução e manutenção da resposta imune/inflamatória na hanseníase estão vinculadas a interações de múltiplas células e fatores solúveis, particularmente através da ação de citocinas. Nesse estudo, foram mensurados níveis de IL-1β e IL-1Ra de 37 casos novos de hanseníase acompanhados ao longo do tratamento e 30 controles sadios pelo teste ELISA. A coleta de sangue periférico foi realizada em quatro tempos para os casos de hanseníase (pré-tratamento com PQT, 2ª dose, 6ª dose e pós-PQT) e em único momento para os controles. Na comparação dos níveis das moléculas de casos no pré-PQT e controles, houve diferença estatisticamente significativa somente para IL-1β. Nossos resultados sugerem a participação dessa citocina no processo imune/inflamatório.

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

Detecção de metalo-beta-lactamase em Pseudomonas aeruginosa isoladas de pacientes hospitalizados em Goiânia, Estado de Goiás

Pseudomonas aeruginosa é uma bactéria frequentemente isolada no ambiente hospitalar. Este estudo teve como objetivo avaliar o perfil de suscetibilidade de Pseudomonas aeruginosa previamente isoladas de pacientes internados em um hospital de Goiânia (Goiás-Brasil); realizar a triagem fenotípica para a produção de metalo-beta-lactamase e detectar os genes das mesmas pela técnica de "Polimerase Chain Reaction". Foram avaliadas 75 Pseudomonas aeruginosa isoladas no período de janeiro de 2005 a janeiro de 2007. A identificação bioquímica foi realizada pelo sistema API 20E® e o antibiograma pelo método de Kirby-Bauer. Entre os 62 isolados que foram resistentes ao imipenem e à ceftazidima, 35 (56,4%) apresentaram produção de metalo-beta-lactamase e em 26 (74,3%) destes, foi detectado o gene blaSPM-1. A frequência de Pseudomonas aeruginosa produtoras de metalo-beta-lactamase sugere um maior controle da disseminação de resistência no ambiente hospitalar.

Synthesis of Polymeric Nanoparticles for biomedical delivery applications

Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behaviors provided by their small size. Such materials could be used in a wide range of applications such as diagnostics and drug delivery. Advantages of PNPs include controlled release, protection of drug molecules and its specific targeting, with concomitant increasing of the therapeutic index. In this work, novel sucrose and cholic acid based PNPs were prepared from different polymers, namely polyethylene glycol (PEG), poly(D,L-lactic-co-glycolic acid) (PLGA) and PLGA-co-PEG copolymer. In these PNP carriers, cholic acid will act as a drug incorporation site and the carbohydrate as targeting moiety. The uptake of nanoparticles into cells usually involves endocytotic processes, which depend primarily on their size and surface characteristics. These properties can be tuned by the nanoparticle preparation method. Therefore, the nanoprecipitation and the emulsion-solvent evaporation method were applied to prepare the PNPs. The influence of various parameters, such as concentration of the starting solution, evaporation method and solvent properties on the nanoparticle size, size distribution and morphology were studied. The PNPs were characterized by using atomic force microscopy (AFM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) to assess their size distribution and morphology. The PNPs obtained by nanoprecipitation ranged in size between 90 nm and 130 nm with a very low polydispersity index (PDI < 0.3). On the other hand, the PNPs produced by the emulsion-solvent evaporation method revealed particle sizes around 300 nm with a high PDI value. More detailed information was found in AFM and SEM images, which demonstrated that all these PNPs were regularly spherical. ζ-potential measurements were satisfactory and evidenced the importance of sucrose moiety on the polymeric system, which was responsible for the obtained negative surface charge, providing colloidal stability. The results of this study show that sucrose and cholic acid based polymeric conjugates can be successfully used to prepare PNPs with tunable physicochemical characteristics. In addition, it provides novel information about the materials used and the methods applied. It is hoped that this work will be useful for the development of novel carbohydrate based nanoparticles for biomedical applications, specifically for targeted drug delivery.

Technical optimization of the synthesis of flavours

The goal of this thesis is the investigation and optimization of the synthesis of potential fragrances. This work is projected as collaboration between the University of Applied Sciences in Merseburg and the company Miltitz Aromatics GmbH in Bitterfeld‐Wolfen (Germany). Flavoured compounds can be synthesized in different ways and by various methods. In this work, methods like the phase transfer catalysis and the Cope‐rearrangement were investigated and applied, for getting a high yield and quantity of the desired substances and without any by‐products or side reactions. This involved the study of syntheses with different process parameters such as temperature, solvent, pressure and reaction time. The main focus was on Cope‐rearrangement, which is a common method in the synthesis of new potential fragrance compounds. The substances synthesized in this work have a hepta‐1,5‐diene‐structure and that is why they can easily undergo this [3,3]‐sigma tropic rearrangement. The lead compound of all research was 2,5‐dimethyl‐2‐vinyl‐4‐hexenenitrile (Neronil). Neronil is synthesized by an alkylation of 2‐methyl‐3‐butenenitrile with prenylchloride under basic conditions in a phase‐transfer system. In this work the yield of isolated Neronil is improved from about 35% to 46% by according to the execution conditions of the reaction. Additionally the amount of side product was decreased. This synthesized hexenenitrile involved not only the aforementioned 1,5‐diene‐structure, but also a cyano group, that makes this structure a suitable base for the synthesis of new potential fragrance compounds. It was observed that Neronil can be transferred into 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid by a hydrolysis under basic conditions. After five hours the acid can be obtained with a yield of 96%. The following esterification is realized with isobutanol to produce 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid isobutyl ester with quantitative conversion. It was observed that the Neronil and the corresponding ester can be converted into the corresponding Cope‐product, with a conversion of 30 % and 80%. Implementing the Cope‐rearrangement, the acid was heated and an unexpected decarboxylated product is formed. To achieve the best verification of reaction development and structure, scrupulous analyses were done using GC‐MS, 1H‐NMR and 13C‐ NMR.

Synthesis of precursors of the rare 3-O-methylmannose polysaccharides present in Nontuberculous Mycobacteria

3-O-methylmannose polysaccharides (MMPs) are cytoplasmic carbohydrates synthesized by mycobacteria, which play important intracellular roles, such as for example in metabolism regulation. An important way to confirm if the inhibition of the synthesis of these polysaccharides will critically affect the survival of mycobacteria is the study of the biosynthetic pathways from these molecules on these microorganisms. The purpose of this work is the efficient synthesis of three saccharides, which are rare cellular precursors from the biosynthesis of the mycobacterial polysaccharides, allowing its study. In order to obtain these molecules, a chemical strategy to connect two precursors was used. This process is called chemical glycosylation and its importance will be highlighted as an important alternative to enzymatic glycosylation. The first objective was the synthesis of the disaccharides Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside and (3-O-Methyl-α-D-mannopyra- nosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose. The mannose precursors were prepared before the glycosylation reaction. The same mannosyl donor was used in the preparation of both molecules and its efficient synthesis was achieved using a 8 step synthetic route from D-mannose. A different mannosyl acceptor was used in the synthesis of each disaccharide and their syntheses were also efficient, the first one a 4 step synthetic route from α-methyl-D-mannose and the second one as an intermediate from the synthesis of the mannosyl donor. The stereoselective preparation of these disaccharides was performed successfully. The second and last objective of the proposed work was the synthesis of the tetrasaccharide methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyra- nosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside. The disaccharide acceptor and donor to be linked through a stereoselective glycosidic reaction had to be first synthesized. Several synthetic strategies were studied. Neither the precursors nor the tetrasaccharide were synthesized, but a final promising synthetic route for its preparation has been proposed.