Esofagopatia chagásica na área endêmica de Bambuí - M.G., Brasil

São estudados clínica e radiologicamente 566 chagásicos crônicos não selecionados, da área endêmica de Bambuí, Minas Gerais. Utilizou-se o método do tempo de trânsito esofágico, em abreugrafias de 70 mm, incidência perfil esquerdo ou oblíqua anterior direita. Verificou-se uma prevalência global de 8,83% de disperistalse esofágica, especialmente a partir da terceira década de vida sem diferença quanto aos sexos. Observou-se 72% de esofagopatias no grau I, 18% no grau II e 10% no grau III. Para 80 indivíduos soro-negativos verificou-se apenas um caso de disperistalse, no grau I. Para 115 pacientes com a fase aguda da tripanossomíase detectada há 27 anos, em média, a prevalência global da esofagopatia foi de 18,3%, sendo crescente a proporção de alterações com o tempo de evolução da doença. Verificou-se diferença significativa entre estes números e a prevalência encontrada nos pacientes sem forma aguda detectada, discutindo-se se a maior proporção de esofagopatias nos indivíduos com fase aguda mais intensa não se deveria à maior desnervação ocorrida nestes casos. Verificou-se a concomitância de cardiopatia crônica chagásica em pelo menos 50% dos casos de disperistalse de esôfago, em acordo com outros Autores. Aparentemente esta associação é mais evidente naqueles casos de esofagopatias de graus mais avançados. Ressalta-se a simplicidade do método empregado e seu baixo custo operacional quando utilizada a abreugrafia postal, adequado portanto à realidade social das áreas endêmicas de doença de Chagas no Brasil.

Desenvolvimento de um ensaio de hibridação múltipla (MHA-MULTIPLE REGION HYBRIDIZATION ASSAY) para identificação presumível de vírus da imunodeficiência humana do tipo 1 (HIV-1) dos subtipos B, G e de formas recombinantes CRF14_BG e CRF02_AG circulantes em Portugal

A maioria dos métodos utilizados na caracterização genética do HIV-1 baseia-se na análise de regiões específicas do genoma viral, fornecendo informação parcial sobre o mesmo e, por consequência, revelando-se inadequados para a identificação de vírus recombinantes. O único método que permite uma caracterização integral do genoma viral passa pela sua sequenciação completa. No entanto, este é um método dispendioso, laborioso e de difícil implementação quando se pretende a análise de elevados números de amostras. Como alternativa a este último, o conjunto de métodos genericamente designados de MHA (Multiple Region Hybridization Assay) baseiam-se na amplificação, por PCR em tempo-real, de várias regiões ao longo do genoma viral e na sua caracterização com sondas específicas (TaqMan). Tendo este modelo por base, o objectivo deste estudo foi o desenvolvimento de um ensaio de hibridação múltipla (MHABG0214) passível de ser aplicado ao estudo de um elevado número de amostras. Este método foi desenvolvido tendo como objectivo a genotipagem as estirpes circulantes dominantes na epidemia Portuguesa, nomeadamente os subtipos B, G e formas genéticas recombinantes CRF02_AG e CRF14_BG. Com base em alinhamentos de sequências de referência de genoma completo, delinearam-se primers universais e subtipo-específicos para a amplificação de diversas regiões codificantes distribuídas ao longo do genoma do HIV-1 (Gag, Protease, Transcriptase Reversa, Integrase, Rev, Gp120 e Gp41). A optimização foi efectuada, inicialmente, para um conjunto de amostras de referência e seguidamente avaliada num conjunto de 50 amostras clínicas. O MHABG0214 foi implementado numa estratégia de PCR em tempo-real, numa detecção dependente de SYBR® Green I para todas as regiões ou, como alternativa, usando sondas TaqMan (Gp41). Apresentamos ainda uma estratégia em que a análise de resultados se baseia, simplesmente, numa abordagem usando PCR/gel de agarose convencional. Estas abordagens constituem ferramentas úteis na identificação das estirpes de HIV-1 em Portugal.

Development of chitosan-based microparticles for pulmonary drug delivery

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

Schottky principal G-bundles over compact Riemann surfaces

Fundação para a Ciência e a Tecnologia (FCT)- PhD grant SFRH/BD/37151/2007; projects PTDC/MAT/099275/2008; PTDC/MAT/119689/2010; PTDC/MAT/120411/2010; PTDC/MAT-GEO/0675/2012

G and P rotavirus genotypes in stool samples from children in Teresina, State of Piauí

A total of 123 stool specimens collected in Teresina, Piauí between 1994 and 1996, from 0 to 2-year-old children with diarrhea, were used for this study. Molecular characterization of the G and P rotavirus genotypes was performed using the reverse transcriptase polymerase chain reaction. The following results were obtained for the P genotypes: P[8] (17. 1%), P[1] (4. 9%), P[4] (3. 3%), P[6, M37] (2. 4%) and mixtures (27. 6%). The P[1]+P[8] mixture was found in 19. 5% of the samples. For the G genotypes, the results were: G1 (25. 2%), G5 (13. 8%), G2 (2. 5%), G4 (2. 5%), G9 (0. 8%) and mixtures (41. 5%). G1+G5 was the mixture most frequently found (12. 1%). Our results showed unusual combinations such as P[1]G5 and P[1]+P[8]G5. The high percentage of mixtures and unusual combinations containing mixtures of human and animal rotavirus genotypes strongly suggests the possibility of gene reassortment and interspecies transmission.

Influence of the molecular weight in mechanical properties and degradation kinetics of chitosan inverted colloidal crystals

Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

Gender influence on treatment of chronic hepatitis C genotype 1

INTRODUCTION: Although various studies have been published regarding the treatment of chronic hepatitis C (CHC) with peginterferon (Peg-IFN) and ribavirin, little is known regarding the real impact of gender on the characteristics that influence the effectiveness and safety of antiviral treatment for CHC patients. The objective of this study was to evaluate the influence of gender on HCV treatment outcomes. METHODS: A retrospective analytical study was conducted among selected carriers of CHC genotype 1, who were treated with Peg-IFN α-2b at a dose of 1.5 μg/kg or Peg-IFN α-2a at a dose of 180 μg/week plus a ribavirin dose of 1,000-1,250 mg/day, according to weight, between 2001 and 2007. RESULTS: Among 181 patients undergoing treatment, the mean age was 46.4 ± 11.0 years and 46% were women. At baseline, 32% of the patients had advanced fibrosis (F3-F4 Scheuer), and 83% of the subjects had viral load > 400,000 IU/ml, without significant difference between the genders (p = 0.428 and p = 0.452, respectively). When compared with men, women had higher incidence of many adverse events such as anemia (p < 0.001) and higher need for dose reduction, for both Peg-IFN (p = 0.004) and ribavirin (p = 0.006). However, the rate of sustained virological response (SVR) did not differ between the genders: 45% (female) vs 41% (male); p=0.464. CONCLUSIONS: This study suggests that women and men react differently to combined therapy, especially in relation to the incidence of adverse events and the need for dose modification. Nevertheless, these differences do not influence the SVR rate.

G-quadruplex ligands for cancer therapy

DNA may fold into a diversity of structures and topologies such as duplexes and triplexes. Some specific guanine-rich DNA sequences may even fold into a higher order structures denominated guanine G-quadruplexes (G4). These G-quadruplex forming sequences have shown biological interest since were found in telomeres and in promoter region of oncogenes. Thus, these G4 forming sequences have been explored as therapeutic targets for cancer therapy, since G4 formation was demonstrated to inhibit RNA-polymerase and telomerase activity. However, the G4 structures are transient and are only formed under specific conditions. Hence the main objective of this work is to develop new G4-specific ligands which may potentially find applications in the therapeutic area. Several potential G4-binding ligands were synthesized and characterized. The synthesis of these compounds consisted on a procedure based on van Leusen chemistry and a cross-coupling reaction through C-H activation, affording phenanthroline compounds (Phen-1, 50%; Phen-2, 20%), phenyl (Iso-1, 61%; Iso-2, 21%; Ter-1, 85%; Ter-2, 35%), and quinolyl (Quin-1, 85%; Quin-2, 45%) compounds. Screening assays for selecting the potential G4 compounds were performed by FRET-melting, G4-FID, CD-melting and DSF. Qualitative biophysical studies were performed by fluorescence and CD spectroscopy. Two high-specific G-quadruplex ligands, Phen-1 and Phen-2, were found to effectively bind telomeric and c-myc G4 structures. Phen-1 was found to stabilize parallel telomeric 22AG and c-myc sequence by 4.1 and 4.3 ˚C, respectively. Phen-2 also displayed high affinity towards 22AG (

Chitosan-based magnetic nanoparticles for osteosarcoma theranostic

Cancer is a well-known disease with a significant impact in society not only due to its incidence, more evident in more developed countries, but also due to the expenses related to medical treat-ments. Cancer research is considered an increasingly logical science with great potential for the development of new treatment options. Advances in nanomedicine have resulted in rapid devel-opment of nanomaterials with considerable potential in cancer diagnostics and treatment. The combination of diagnosis and treatment in a single nano-platform is named theranostic. In this PhD thesis a theranostic system for osteosarcoma was proposed, composed by a magnetic core, a polymeric coating, and a chemotherapeutic drug. The presence of a specific targeting agent, in this case a monoclonal antibody, provides high specificity to the proposed theranostic system. For the core of the proposed theranostic system, stable aqueous suspensions of superparamagnetic iron oxide nanoparticles with an average diameter of 9 nm were produced. Chitosan-based poly-meric nanoparticles with a hydrodynamic diameter around 150 nm were successfully produced. Incorporation of iron oxide nanoparticles into the polymeric ones increased their hydrodynamic diameter to at least 250 nm. A monoclonal antibody specific for a transmembranar protein (car-bonic anhydrase IX) present in solid tumors was developed by hybridoma technology. Functional hybridomas producing the desired monoclonal antibodies were obtained. The proposed theranostic system functionality was evaluated in separated parts of its components. Uncoated and coated iron oxide nanoparticles with chitosan-based polymers generated heat under the application of an external alternating magnetic field. Uncoated iron oxide nanoparticles sta-bilized with oleic acid were able to enhance contrast in magnetic resonance imaging. Drug deliv-ery studies were conducted in chitosan-based polymeric nanoparticles without and with the in-corporation of iron oxide nanoparticles, demonstrating to be an effective drug delivery platform for doxorubicin. The theranostic system proposed in this PhD thesis is very promising for cancer theranostic, demonstrating to be applicable in solid tumors such as osteosarcoma.

Endemic pemphigus foliaceus (fogo selvagem) and pemphigus vulgaris: immunoglobulin G heterogeneity detected by indirect immunofluorescence

Pemphigus are autoimmune intraepidermal blistering diseases in which immunoglobulin G (IgG) autoantibodies are directed against desmosomal glycoproteins. The aim of this study was to determine the IgG subclass profile of endemic pemphigus foliaceus (fogo selvagem) and pemphigus vulgaris utilizing indirect immunofluorescence. PATIENTS AND METHODS: Twenty-five patients with pemphigus vulgaris, 25 with endemic pemphigus foliaceus (fogo selvagem), and 25 healthy controls were analyzed by indirect immunofluorescence for circulating autoantibodies (total IgG and its subclasses). RESULTS: Our data revealed a significant correlation (P <.05) of disease activity and autoantibody levels in both forms of pemphigus, i.e., negative titers related to clinical remission, whereas positive results related to active disease. Immunoglobulin G subclass analysis in fogo selvagem demonstrated that in patients in remission, 56% showed positive immunoglobulin G4; in active disease, immunoglobulin G4 was the predominant subclass (100% positive in all cases). The IgG subclass profile in pemphigus vulgaris showed that in patients in remission, only 10% were positive for immunoglobulin G4; in active disease, positivity for immunoglobulin G4 was present in 78% to 88% of the cases. CONCLUSION: Subclass characterization of immunoglobulin G autoantibodies is a useful tool for pemphigus follow-up, since immunoglobulin G4 (IgG4) is the subclass that is closely related to recognition of pathogenic epitopes, and consequently with disease activity. Careful monitoring should be performed for fogo selvagem in clinical remission with a homogeneous IgG4 response, since this may indicate more frequent relapses.

Design of bio-inspired ionic liquids for protein stabilisation

Ionic Liquids (ILs) consist in organic salts that are liquid at/or near room temperature. Since ILs are entirely composed of ions, the formation of ion pairs is expected to be one essential feature for describing solvation in ILs. In recent years, protein - ionic liquid (P-IL) interactions have been the subject of intensive studies mainly because of their capability to promote folding/unfolding of proteins. However, the ion pairs and their lifetimes in ILs in P-IL thematic is dismissed, since the action of ILs is therefore the result of a subtle equilibrium between anion-cation interaction, ion-solvent and ion-protein interaction. The work developed in this thesis innovates in this thematic, once the design of ILs for protein stabilisation was bio-inspired in the high concentration of organic charged metabolites found in cell milieu. Although this perception is overlooked, those combined concentrations have been estimated to be ~300 mM among the macromolecules at concentrations exceeding 300 g/L (macromolecular crowding) and transient ion-pair can naturally occur with a potential specific biological role. Hence the main objective of this work is to develop new bio-ILs with a detectable ion-pair and understand its effects on protein structure and stability, under crowding environment, using advanced NMR techniques and calorimetric techniques. The choline-glutamate ([Ch][Glu]) IL was synthesized and characterized. The ion-pair was detected in water solutions using mainly the selective NOE NMR technique. Through the same technique, it was possible to detect a similar ion-pair promotion under synthetic and natural crowding environments. Using NMR spectroscopy (protein diffusion, HSQC experiments, and hydrogen-deuterium exchange) and differential scanning calorimetry (DSC), the model protein GB1 (production and purification in isotopic enrichment media) it was studied in the presence of [Ch][Glu] under macromolecular crowding conditions (PEG, BSA, lysozyme). Under dilute condition, it is possible to assert that the [Ch][Glu] induces a preferential hydration by weak and non-specific interactions, which leads to a significant stabilisation. On the other hand, under crowding environment, the [Ch][Glu] ion pair is promoted, destabilising the protein by favourable weak hydrophobic interactions , which disrupt the hydration layer of the protein. However, this capability can mitigates the effect of protein crowders. Overall, this work explored the ion-pair existence and its consequences on proteins in conditions similar to cell milieu. In this way, the charged metabolites found in cell can be understood as key for protein stabilisation.

COMPORTAMENTO DE LEVEDURAS DO GÊNERO CANDIDA"IN VITRO" A ANTIFÚNGICOS

Estudou-se o comportamento de leveduras do gênero Candida a antifungicos, pela determinação da Concentração Inibitória Mínima (CIM) e da Concentração Fungicida Mínima (CFM) "in vitro"de 30 cepas de Candida frente aos antifungicos: miconazol, cetoconazol e anfotericina B. Empregou-se o método de diluição em meio líquido e os antifungicos foram diluídos visando proporcionar concentrações a partir de 0,06 a 128 μg/mL. O inóculo foi padronizado ajustando-se a suspensão para conter 1X 106 ufc/mL. A concentração para a qual houve maior convergência de cepas foi de 1 μg/mL (26,5%) para anfotericina B. Para miconazol foi de 16 μg/mL (26,0%) e para cetoconazol 32 μg/mL (23,0%). Os valores de CFM foram de 2 μg/mL (23,5%) para anfotericina B, de 16 e 64 μg/mL (26,5%) para miconazol e 32 e 64 μg/mL (30,0%) para cetoconazol. Este imidazólico mostrou os valores de CIM e CFM mais elevados atingindo até 128 μg/mL em algumas espécies. As espécies de Candida mostraram-se mais sensíveis à Anfotericina B, quando comparadas em relação aos antifungicos testados. Sobre o desempenho das espécies de Candida, melhor padrão de comportamento foi verificado para C. albicans com níveis mais baixos de sensibilidade.

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Sistemas aquosos bifásicos uma ferramenta sustentável para a extração de ácido clavulânico a partir de diferentes fontes

O ácido clavulânico (AC) é um inibidor de β-lactamases que tem vindo a ser largamente utilizado na área médica. Embora seja de extrema importância, o desenvolvimento de processos alternativos de produção e purificação é ainda insignificante, sendo fundamental o estudo de técnicas de extração mais biocompatíveis, como os Sistemas Aquosos Bifásicos (SABs). Assim, este trabalho objetivou o estudo de Sistemas Aquosos Bifásicos baseados em polímeros como uma ferramenta alternativa para a extração de AC. Foram testados dois SPAB compostos por Polietileno Glicol (PEG) com massa molecular (M) de 4000 g/mol e Poliacrilato de Sódio de 8000 g/mol, nos quais foi alterado o eletrólito indutor da formação de fases, em particular, sulfato de sódio (Na2SO4,) e cloreto de sódio (NaCl). Ademais, este trabalho visou também avaliar a eficiência de extração do AC, bem como compreender o efeito dos contaminantes no processo de migração. Para tal, foi avaliada a extração do AC a partir de três fontes distintas: solução pura (99,9%); solução comercial (60%); diretamente a partir do sobrenadante de um meio fermentando de Streptomyces clavuligerus. Os resultados obtidos demonstraram que independentemente da fonte inicial do AC, ambos os SABs poliméricos promoveram uma partição preferencial do AC para a fase rica em PEG, sendo o coeficiente de partição maior nos sistemas com Na2SO4 do que com NaCl. Após identificar a grande capacidade de partição de AC, o SAB com PEG/NaPA/Na2SO4 foi também utilizado para avaliar a partição de proteínas presente no meio fermentado, sendo também obtida uma preferencial partição destas para a fase rica em PEG. Assim, apesar da baixa capacidade de purificação de AC frente a proteínas contaminantes, os SABs estudados demonstraram que podem ser uma técnica alternativa sustentável e bastante econômica para uma etapa inicial de clarificação/concentração de bioprodutos a partir de caldos fermentados.