Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Institutions and firm formation: An empirical analysis of Portuguese municipalities

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Economics from the NOVA – School of Business and Economics

Schistosoma mansoni: immunodepression of hepatic schistosome granuloma formation in mice infected by Trypanosoma cruzi

Infection by Trypanosoma cruzi in mice depresses hepatic granuloma formation around Schistosoma mansoni eggs. This immunodepressive effect occurred in mice with Chagas' disease at the acute and/or chronic phases, granulomas being signijicantly smaller than those in Controls. Data suggest that Chagas ' disease depresses the delayed hypersensitivity immune response directly.

The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Dissertation for the Master’s Degree in Structural and Functional Biochemistry

Characterization of sol-gel matrices with entrapped cutinase

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Study of multi-component systems in polybenzimidazole membrane formation and their impact on membrane performance

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Molecular tools to dissect the role of Dmrt2a and Dmrt2b in the left-right axis formation in zebrafish

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

Molecular regulation of secondary gorwth: searching for SHORT-ROOT function in cork formation

The analysis of molecular regulators involved in controlling the maintenance and function of plant meristems has been the subject of many studies. Some master regulators of these processes have been identified in Arabidopsis benefiting from the array of tools available for genetic and molecular analysis in this model plant. However, aspects such as secondary growth that are more extensively observed in woody plants, have been less studied. Secondary growth is responsible for the enlargement of the plant stems and roots and results from the activity of the lateral (secondary) meristems, vascular cambium and cork cambium (phellogen), which produce two important renewable natural resources, wood and cork, respectively.(...)

Paracoccidioidomycosis: no genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis

INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.

Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers

Introduction The aim of this study was to investigate the effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Methods Ten C. albicans isolates recovered from denture wearers were tested using 10% fetal bovine serum with or without 4% R. officinalis essential oil. Results The essential oil from R. officinalis completely inhibited germ tube formation in the investigated C. albicans isolates. Conclusions The results demonstrate that the essential oil of R. officinalis modulates C. albicans pathogenicity through its primary virulence factor (i.e., germ tube formation was suppressed).

Antibacterial, anti-swarming and anti-biofilm formation activities of Chamaemelum nobile against Pseudomonas aeruginosa

AbstractINTRODUCTION:Chamomile ( Chamaemelum nobile ) is widely used throughout the world, and has anti-inflammatory, deodorant, bacteriostatic, antimicrobial, carminative, sedative, antiseptic, anti-catarrhal, and spasmolytic properties. Because of the increasing incidence of drug-resistant bacteria, the development of natural antibacterial sources such as medical herbs for the treatment of infectious diseases is necessary. Extracts from different plant parts such as the leaves, flowers, fruit, and bark of Combretum albiflorum, Laurus nobilis , and Sonchus oleraceus were found to possess anti-quorum sensing (QS) activities. In this study, we evaluated the effect of C. nobile against Pseudomonas aeruginosa biofilm formationMETHODS:The P. aeruginosa samples were isolated from patients with different types of infection, including wound infection, septicemia, and urinary tract infection. The flowers of C. nobile were dried and the extract was removed using a rotary device and then dissolved in dimethyl sulfoxide at pH 7.4. The microdilution method was used to evaluate the minimum inhibitory concentration (MIC) of this extract on P. aeruginosa , and biofilm inhibition was assayed.RESULTS:Eighty percent of the isolated samples (16/20) could form a biofilm, and most of these were isolated from wound infections. The biofilm inhibitory concentration of the C. nobile extract was 6.25-25mg/ml, whereas the MIC was 12.5-50mg/ml.CONCLUSIONS:The anti-QS property of C. nobile may play an important role in its antibacterial activity, thus offering an additional strategy in the fight against bacterial infections. However, molecular investigation is required to explore the exact mechanisms of the antibacterial action and functions of this phytocompound.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.