Made available in DSpace on 2018-08-01T21:58:48Z (GMT). No. of bitstreams: 1 tese_10826_DISSERTAÇÃO DE MESTRADO (JONACIR NOVAES DAS VIRGENS (1).pdf: 2898530 bytes, checksum: 48b46f080aa4052ea24556acee922f6c (MD5) Previous issue date: 2017-04-05 / Os denominados biopolímeros, destaque para a quitosana (QUI) e o colágeno (COL), vêm desempenhando um papel importante como biomateriais, esse novo campo de pesquisa na última década tem alcançado avanços notáveis nas áreas de substituição, reconstituição e regeneração tecidual, pois os mesmos apresentam características essenciais como biocompatibilidade, biodegrabilidade e não-citotoxidade. Este trabalho consiste na obtenção e caracterização de membranas de quitosana/colágeno carregadas com nanopartículas de prata obtidas por mistura das soluções poliméricas em diferentes proporções em massa e posterior evaporação do solvente, já as nanopartículas de prata foram obtidas na própria matriz polimérica. As membranas quitosana/colágeno carregadas com nanopartículas de prata foram caracterizadas através das técnicas de infravermelho com transformada de Fourier (FTIR), raios X (DRX), microscopia eletrônica de varredura (MEV), microscopia eletrônica de transmissão (MET) e espectroscopia de energia dispersiva (EDX). Os resultados obtidos demostraram que o método de síntese das membranas por evaporação e a síntese das nanopartículas de prata na matriz polimérica foram realizados com muita eficiência e comprovados pelos resultados obtidos. As blendas de quitosana-colágeno otidas e analisadas por MEV apresentaram uma morfologia formada por estruturas porosas e interconectadas. A proporção em massa de QUI/COL foi importante para a manuntenção da estrutura do colágeno uma vez que na proporção 3:1 (m/m de QUI-COL) observou-se através do espectro vibracional uma diminuição da razão entre os picos de 1235 cm-1 e 1450 cm-1 respectivamente, o que sugere que o aumento da proporção de quitosana acarreta em uma perda da integridade proteíca do colágeno. As nanopartículas de cloreto de prata agregadas a matriz polimérica da quitosana obtidas tinha tamanho médio de 25 nm confirmadas por MET, no difratograma de raios X foram observado picos padrões para nanopartícula de cloreto de prata, no EDX do precipitado obtido da síntese das nanopartículas de prata foi observado além dos elementos prata e cloro a presença dos elementos carbono e oxigênio que confirmam a presença da matriz da quitosana aderida às nanopartículas de cloreto de prata.

Abstract Laminins are basement membrane glycoproteins consisting of three polypeptide chains α, β and γ. Until now 12 members of the protein family have been characterized and all isoforms have an αβγ chain composition, but they assemble in varying combinations of chain variants. The functional properties of laminins include cell adhesion, proliferation, differentiation, growth and migration. Laminin-5 has a chain composition of α3β3γ2 with the distribution mainly restricted to epithelial basement membranes, where its biological functions involve anchorage and locomotion of cells. The importance of this protein for the attachment of basal keratinocytes is clearly demonstrated by the fact that all genes encoding its chains have been shown to be mutated in the severe skin blistering disease Epidermolysis bullosa junctionalis. The present study focused on investigations of the role of the laminin-5 isoform and particularly its γ2 chain in cell adhesion and migration. The role of the short arm of the laminin γ2 chain in the process of epithelial cell attachment is to serve as a kind of a bridging molecule to the extracellular environment, because it does not have any cell binding activity by itself. It was also shown that the newly synthesized γ2 chain participates in the complex process of cell migration, probably as one of the first attachment components for moving cells. Thus, as a migration and differentiation-associated molecule, laminin-5 was considered a potential marker for detection of malignant processes where cell movement plays a role. Subsequently it was shown that the γ2 chain is expressed not only in a restricted manner in human epithelial tissues, but also in a number of human epithelium-derived cancers. In some carcinomas, expression of the γ2 chain appeared to be a characteristic of cancer cells with invasive properties. Examination of over 50 dysplasias and cervical tumors revealed that γ2 chain antibodies were able to distinguish between lesions with or without invasive capacity. This is the first systematic study of epithelial cancers where γ2 chain antibodies have been shown to be a useful marker in the histopathological diagnostics. In addition, this study showed in a mouse tumor model that the γ2 chain of laminin-5 has a potential for being of use for in vivo tumor imaging.

The spatiotemporal organization of membrane proteins is implicated in cellular trafficking, signalling and reception. It was proposed that biological membranes partition into lipid rafts that can promote and control the organization of membrane proteins to localize the mentioned processes. Lipid rafts are thought to be transient (microseconds) and small (nanometers), rendering their detection a challenging task. To circumvent this problem, multi-component artificial membrane systems are deployed to study the segregation of lipids at longer time and length scales. In this thesis, multi-component Droplet Interface Bilayers (DIBs) were imaged using fluorescence and interferometric scattering microscopy. DIBs were used to examine and manipulate microscopic lipid domains and to observe, for the first time, transient nanoscopic lipid domains. The techniques and results described here will have important implications on future research in this field.

Novel composite membrane systems have been devised for use in direct methanol fuel cell (DMFC) with the ultimate aim of improving overall fuel cell performance in terms of achievable power density. The composite membrane system takes the form of a multilayered structure composing of commercial Nafion117 membrane and a novel composite binding layer situated between the anode and the membrane. Within the composite binding layer, inorganic filler particles are evenly dispersed throughout the Nafion matrix presenting a barrier that impedes methanol crossover. Through the current research, three novel membrane electrode assemblies (MEA) have been fabricated, each employing the composite binding layer system with different filler. Mass of filler used is kept constant at 0.5 wt% of Nafion117 membrane. When tested in a DMFC system, the first MEA which utilizes hydrogen form mordenite filler particles yields optimum power density of 60 mW/cm2 with the operation at 90°C, 1M methanol fuel concentration. This represents an improvement of 34.7% compared to the standard MEA which do not include the composite binding layer. Silanefunctionalized hydrogen form mordenite filler is used in the second MEA which yields optimum power density of 64 mW/cm2 at 90°C, 1M methanol, outperforming the standard MEA by 42.5%. The third MEA makes use of TS-1 particles as fillers. This yields an optimum performance of 38 mW/cm2 at 90°C, 1M methanol, a 14.3% reduction in performance compared to the standard. Through the results obtained, it can be deduced that the novel composite binding layer presents a valid approach in reducing methanol crossover, however, the nature of filler particles used exerts a great influence on its performance. Therefore, further research is recommended in exploring new filler materials for use within the composite membrane system.

Abstract Type IV collagen is only found in basement membranes, where it is the major structural component, providing a framework for the binding of other basement membrane components and a substratum for cells. The type IV collagen molecule is triple-helical and composed of three a chains which exist as six distinct forms (α1 - α6). Abnormalities in this basement membrane collagen structure and function are connected to both inherited and acquired diseases. Alport syndrome is a hereditary kidney disease associated with extrarenal complications, such as sensorineural deafness and eye abnormalities. The disease is caused by mutations in the COL4A3, COL4A4 and COL4A5 genes, coding for the type IV collagen α3, α4 and α5 chain genes, respectively. About 85% of the Alport syndrome cases are X-linked dominant, caused by mutations in the COL4A5 gene. In order to develop a basis for automated mutation analysis of the COL4A5 gene, previously unknown intron sequences flanking exons 2 and 37 were determined. Intron sequences flanking the other 49 exons were expanded from 35 to 190, and additionally, two novel 9 bp exons (exons 41A and 41B) were characterized in the large intron 41. In addition to optimization of the PCR amplification and sequencing conditions for all 51 exons and exon flanking sequences, optimization for the 820 bp promoter region and for the two novel exons was performed as well. Mutations were found in 79 unrelated patients of the 107 studied. This gives a high mutation detection rate of almost 75% in comparison with 50%, at its best, in other extensive mutation analyses of the COL4A5 gene using SSCP analysis. None of the mutations involved the promoter region or exons 41A and 41B. Circulating antibodies against basement membrane components have been recognized in some autoimmune diseases. Goodpasture syndrome is a rare autoimmune disease characterized by progressive glomerulonephritis and pulmonary hemorrhage. The target of the antibodies in this disease has been shown to be the noncollagenous NC1 domain of type IV collagen α3 chain. For unknown reasons, a minority of Alport syndrome patients also develops antibodies against α3 and α5 chains after renal transplantation with manifestation of severe anti-GBM disease. In order to investigate the antibodies both in Goodpasture and Alport syndrome, the NC1 domains of all six type IV collagen chains were produced as recombinant proteins in bacterial and mammalian expression systems, and an ELISA method was developed for antibody detection. Antibodies were found in both syndromes, interestingly also in Alport syndrome patients without the anti-GBM disease. The results of this work have a significant clinical value by providing for the first time complete, effective DNA-based analysis of all exon/intron and promoter regions of the COL4A5 gene in Alport syndrome.

Abstract The control and maintenance of the character, number and protein, carbohydrates and lipid composition of intracellular compartments in a changing environment is one of the fundamental features of a living cell. It is effected, to a large measure, by vesicular traffic which connects the various cellular compartments and handles the transportation of cargo between them. Movement of cargo occurs through a transport system in membrane-bounded containers called vesicles. Vesicles originate at the donor membrane from which they are transported to target organelles where they fuse with the acceptor membrane and deliver their cargo. At the donor site, cytosolic coat proteins or 'coats' bind to the donor membrane together with GTP (guanosine 5'-triphosphate)-binding regulatory proteins first to deform a bud, which is then pinched off as a coated vesicle. During budding and targeting events, a number of regulatory proteins interact with the coat components. Currently, several different coat proteins and their adaptor proteins are known. The purpose of this study was to characterize novel components participitating in intracellular vesicle transport. By using computer analysis and EST (expressed sequence tag) database searches, a previously unknown protein was found. Sequencing revealed the presence of a novel protein of 613 amino acids with a calculated molecular mass of 67,149 Da. Based on its structural features, possessing both a VHS domain and an "ear" domain, we named the protein Vear. With its VHS domain in its NH2 terminus, Vear shows similarity to several endocytosis-associated proteins. With the "ear" domain in its C-terminus, it resembles γ-adaptin, a heavy subunit of the AP-1 complex. Vear mRNA showed a widespread distribution in tissues, with high amounts of mRNA in the kidney, skeletal muscle, and cardiac muscle. At the subcellular level, Vear was localized to the Golgi complex in which it colocalized with the trans-Golgi marker γ-adaptin. The preferential membrane-association was demonstrated by subcellular fractionation in which Vear partitioned with the total membrane fraction. Golgi-associated subcellular localization for Vear was sensitive to a treatment with the fungal metabolite brefeldin A, suggesting an ARF (ADP-ribosylation factor)-dependent recruitment onto membranes. In transfection studies, the full-length Vear assembled on and caused structural "compaction" of the Golgi complex, while overexpression of the "ear" domain alone showed diffuse Golgi-localization without "compaction". The VHS domain, on the other hand, was mainly vesicle- and plasma membrane associated and did not show any association with Golgi. In skeletal muscle, Vear was detected preferentially in type I cells by immunohistochemistry and immunofluorescence microscopy. In normal kidney, Vear was exclusively present in glomerular epithelial cells (podocytes) and Vear protein was expressed in a developmentally regulated manner during glomerulogenesis. By immunolabeling electron microscopy, Vear was seen in vesicular and membrane structures adjacent to the Golgi complex. Vear was also abundant in the gastrointestinal tract in cells active in secretion. The results indicate that Vear is a novel vesicle transport-associated protein, detected mainly in the Golgi complex and localized in tissues in a highly cell-type specific manner.

Abstract All neurons and macroglial cells of vertebrates derive from the neuroepithelium. Neuroepithelial (NE) cells first proliferate and, after closure of the neural tube, some cells start generating neurons. It is still unclear what triggers differentiation but apparently there is interplay between extrinsic (secreted or transmembrane signals) and intrinsic factors. Diriving from the embryonic ectoderm, the NE cells inherit epithelial characteristics. It has been shown in other developmental systems that epithelial determinants, such as cell-cell contacts and contact to basal laminar components can guide differentiation. The key epithelial features include cell polarity, and tight junctions. We studied these in the NE at two developmental stages, the neural plate, a proliferative stage and the neural tube, a differentiative stage. The polarity of membrane proteins in NE cells was studied with polarly budding viruses. Mouse embryos were infected with Fowl plague- and vesicular stomatitis viruses and cultured in a whole embryo culture system. Viral envelope proteins (HA and G-protein) were localized by indirect immunofluorescence and immunoelectron microscopy. HA was polarized in the plate stage neuroepithelial cells, whereas in the tube it was not polarized anymore. It is also shown by penetrance of apically injected horseradish peroxidase that in the neural plate, NE cells have functional tight junctions. At this stage, they also express occludin, a transmembrane protein of tight junctions, as shown by indirect immunofluorescence. In the neural tube, the paracellular barrier is lost and there is no occludin expression. In contrast, expression of ZO-1, a cytoplasmic protein binding to occiudin, is upregulated. The downregulation of these epithelial features occurs in all NE cells, irrespective of their mode of division and before any neurons are generated in the NE. The change is initiated already at the plate stage and coincides with the switch from E- to N-cadherin. Later, with birth of neurons, the proliferative cell layer also looses contact to basal lamina. This is probably an important step in the regulation of neurogenesis. Furthermore, lack of apico-basolateral polarity of non-anchored membrane proteins may contribute to the mechanism of rapid neuron generation. Until now, it has been impossible to distinguish a neuroepithelial cell preparing for neuron generation from the surrounding cells that give rise to two precursor cells. In this study, the immediate neuron precursors are shown to express the antiproliferative gene TIS2 1. Using this new marker and ISH in serial sections, we show that the switch to differentiation is initiated in single NE cells.

No description available.

Le traitement des effluents et eaux usées par bioréacteurs à membranes immergées (BAMI) permet d'obtenir une haute qualité de perméat par une dégradation biologique et une séparation physique. Néanmoins, le procédé de filtration est limité par l'influence de facteurs très complexes, en particulier le colmatage de la membrane. Le but du travail est de développer des modèles dynamiques et de simuler le procédé de filtration dans les BAMI. Le développement et la simulation des modèles ont été ciblés sur la description des rapports existants entre les variables les plus importantes du système, comme la pression transmembranaire (PTM), les matières en suspension (MES), les substances polymériques extracellulaires (SPE) et l'influence sur l'évolution du colmatage d'une aération syncopée, injectée à la surface de membrane, et sa synchronisation avec une filtration intermittente. Le modèle et les études d'optimisation du système ont été validé par voie expérimentale. / This thesis studies a submerged membrane bioreactor (MBR) technology that is used to treat effluents. We present in detail the modeling of this process, the validation of developed models, and the results of simulation and optimization carried out with the above mentioned models. The new contributions to scientific knowledge of this work are the following: - A new dynamic model that integrates for such systems, many of the variables and the main phenomena occurring during the process of filtration in MBR wastewater treatment. That constitutes an original contribution to the analysis and development of this technology. – The simulation allows to achieve the quantification of the influence and effect of aeration on the process (membranes fouling) and the influence of the sequencing of the filtration and coarse bubbles aeration cycles. All that takes into account the behavior of biomass, the generation of exopolymeric substances and inlet characteristics. The results provided by the model are validated by comparison with experimental results. – An optimization of MBR operating conditions using the experimental design for simulation, is reported based on the results obtained using the developed models / En la presente tesis se reflejan los estudios realizados en un biorreactor con membrana sumergida, tecnología que se utiliza para el tratamiento de efluentes residuales. Se presentan de forma detallada la modelación de este proceso, la validación de los modelos desarrollados, así como los resultados de la simulación y optimización realizados con los modelos. Entre los nuevos aportes al conocimiento científico del trabajo se encuentran los siguientes: - Un nuevo modelo dinámico que integra por primera vez, para estos sistemas, muchas de las variables y los principales fenómenos que ocurren durante el proceso de filtración y tratamiento de las aguas residuales utilizando los BMS. Lo cual constituye un aporte novedoso para el análisis y desarrollo de esta tecnología. - Se logró por primera vez cuantificar mediante simulación la influencia y el efecto de la aireación sobre el proceso de colmatación de las membranas, así como la influencia de la sincronización de los ciclos de filtración y aireación de burbujas gruesas. Todo esto tomando en cuenta el comportamiento de la biomasa, la generación de sustancias colmatantes y las características de la alimentación. Se validaron los resultados que ofrece el modelo mediante la comparación con resultados experimentales. - Se reporta por primera vez la optimización de las condiciones operacionales de un sistema BMS utilizando el diseño de experimento para la simulación, partiendo de los resultados obtenidos utilizando los modelos desarrollados

Conventional activated sludge-based wastewater treatment is an energy and resource-intensive process. Historically it has been successful at producing safely treated wastewater effluents in the developed world, specifically in places that have the infrastructure and space to support its operation. However, with a growing need for safe and efficient wastewater treatment across the world in both urban and rural settings, a paradigm shift in waste treatment is proving to be necessary. The sustainability of the future of wastewater treatment, in a significant way, hinges on moving towards energy neutrality and wastewater effluent reuse. This potential for reuse is threatened by the recent emergence and study of contaminants that have not been previously taken into consideration, such as antibiotics and other organic micropollutants (OMPs), antibiotic resistance genes, and persistent pathogenic bacteria. This dissertation focuses on investigating the use of anaerobic membrane bioreactor (AnMBR) technology for the sustainable treatment of municipal-type wastewaters. Specifically, a microbial approach to understanding biofouling and methane recovery potential in anaerobic MBR systems has been employed to assess different reactor systems’ efficiency. This dissertation further compares AnMBRs to their more widely used aerobic counterparts. This comparison specifically focuses on the removal and biodegradation of OMPs and antibiotics in both anaerobic and aerobic MBRs, while also investigating their effect on the proliferation of antibiotic resistance genes. Due to rising interest in wastewater effluent reuse and the lack of a comprehensive understanding of MBR systems’ effects on pathogen proliferation, this dissertation also investigates the presence of pathogens in both aerobic and anaerobic MBR effluents by using molecularbased detection methods. The findings of this dissertation demonstrate that membrane-associated anaerobic digestion processes have significant potential to improve the sustainability of wastewater treatment. This is exemplified by attributes of AnMBR systems associated with both increased system efficiency and wastewater reuse potential through methane recovery and lower abundance of effluent microbial contaminants, respectively. Overall, the studies collated in this dissertation have shown that understanding the microbial communities of AnMBRs can play a central role in further improving these attributes and in reducing the risks posed by emerging contaminants and pathogens in wastewater treatment systems.