A importância do estudo das celulases não se limita à aquisição de conhecimento científico, mas também ao grande potencial biotecnológico que elas representam. Isso se deve ao fato da celulose ser a molécula mais abundante presente na natureza e prover uma vasta gama de produtos e processos sustentáveis. Muitas famílias de celulases já foram bem caracterizadas, enquanto outras permanecem ainda desconhecidas. Dentre estas últimas, a família 45 das hidrolases de glicosídeos é a família de celulases fúngicas menos caracterizada tanto estruturalmente quanto funcionalmente. Recentemente foi proposta a divisão dessa família em três subfamílias e, até agora, apenas membros da subfamília A tiveram enzimas estruturalmente elucidadas. Nesse trabalho reportamos a estrutura cristalográfica da proteína recombinante endoglucanase de Phanerochaete chrysosporium (PcCel45A), a primeira das hidrolases de glicosídeos da subfamília C, e seu complexo com celobiose a 1,4 Å e 1,7 Å de resolução, respectivamente. A PcCel45A é uma enzima de domínio único, com uma estrutura em β-barril e seu empacotamento geral remete ao formato de âncora. O sítio ativo da enzima forma um longo sulco na superfície da estrutura, sendo que o seu centro catalítico é diferente das outras enzimas publicadas dessa família e o aspartato catalítico, que atua como aceptor de próton na reação de inversão, (Asp10) não é conservado. Adicionalmente, a estrutura cristalográfica dessa enzima apresenta mais similaridades com as β-expansinas (proteínas de plantas) e transglicosilases líticas (proteínas que clivam o peptidoglicano de bactérias) do que com as outras representantes da família 45, o que a torna ainda mais singular. Para entendermos melhor seu funcionamento foram realizadas mutações sítio-dirigidas nos principais resíduos do sítio ativo. O Asp121, conhecido por participar da reação de inversão das outras enzimas da família como doador de próton, mostrou-se essencial para a atividade da enzima, enquanto que outros resíduos conservados como a Tyr25, o Trp161 e o Asp92 afetaram, mas não aniquilaram a atividade da enzima, apresentando aproximadamente 20%, 50% e 10% da atividade da enzima nativa, respectivamente. / The importance of the study of the cellulases is not limited to generating significant scientific knowledge, since these enzymes represents an enormous potential in biotechnology. This is partly because cellulose is the most abundant molecule in nature and provides a wide range of products and sustainable process. Many cellulases families have been well characterized, while others still remain unknown. Among them, the glycoside hydrolase family 45 is the least well characterized both structurally and functionally, between fungal cellulases. It was recently proposed the subdivision of this family into three subfamilies, with structural information available only for subfamily A. In this work, we report the chrystallographic structure of the recombinant endoglucanase from Phanerochaete chrysosporium (PcCel45A), the first GH45 subfamily C and its complex with cellobiose at 1.4 Å and 1.7 Å respectively. The PcCel45A is a single domain enzyme, which has a β-barrel structure with the overall shape resembling an anchor. The active site of the enzyme has a long cleft on the surface, being remarkably different from those members of subfamily A, and the catalytic aspartate responsible for acting as proton acceptor (Asp10) is not present. Additionally, the chrystallographic structure of this enzyme has shown more similarity with β -expansins (plant proteins) and lytic transglycosylase (proteins that cleave the peptidoglycan of bacteria) than others representants of family 45, which makes it more singular. For a better understanding of its function, we perform pontual mutations in the main residues from active site. The Asp121, known for acting as proton acceptor in the inversion reaction of others enzymes, proved to be essential for the enzyme activity, while others conserved residues as Tyr25, Trp161 and Asp92 affected but not annihilated the enzyme activity, leaving approximately 20%, 50% and 10% of the native enzyme activity.

A existência de uma maior variedade de aminoácidos codificados pelo código genético tem estimado estudos sobre os mecanismos de síntese, reconhecimento e incorporação desses resíduos nas cadeias polipeptídicas nascentes. Um exemplo é a via de incorporação de selenocisteína evento cotraducional dirigido pelo códon UGA. Em bactérias, essa via conta com uma complexa maquinaria molecular composta por: Selenocisteína Sintase (SelA), Fator de Elongação Específico de Reconhecimento (SelB), Selenofosfato Sintetase (SelD), tRNA específico (SelC ou tRNAsec), sequência específica no mRNA (Sequência de Inserção de Selenocisteínas - SECIS) e Aminoacil tRNA Sintetase (aaRS). Pelo fato do selênio ter uma toxicidade elevada em ambientes celulares, é fundamental a compreensão do mecanismo catalítico e razão estequiométrica na formação dos complexos da via na etapa de incorporação junto ao tRNAsec, bem como sua caracterização estrutural foram os objetivos deste trabalho. A proteína SelA foi expressa e purificada para utilização em análises envolvendo microscopia de força atômica, microscopia eletrônica de transmissão com contraste negativo e em gelo vítreo foram realizadas nos complexos SelA e SelA-tRNAsec, visando obter um modelo estrutural e a razão estequiométrica dos complexos. A fim de compreender o mecanismo de passagem do selênio, ensaios de anisotropia de fluorescência e de microcalorimetria, corroborados pelas análises de troca de hidrogênio-deutério acoplado a espectrometria de massa e espectroscopia de infravermelho, elucidaram a formação e estequiometria do complexo ternário SelAtRNA sec-SelD. Tentativas de cristalização e análises cristalográficas também foram realizadas, no entanto, sem sucesso. Com os resultados obtidos foi possível propor que o reconhecimento de SelD e, consequentemente, a entrega do selenofosfato, seja uma etapa crucial da via de incorporação de selenocisteínas. / The existence of a greate variety of amino acids encoded by the genetic code has stimulated the study of the mechanisms of synthesis, recognition and incorporation of these residues in the nascent polypeptide chains. An example of genetic code expansion is the selenocysteine incorporation pathway an event cotraducional by the UGA codon. In bacteria, this pathway has a complex molecular machinery comprised: Selenocysteine Synthase (SelA), Specific Elongation Factor (SelB), Selenophosphate Synthetase (SelD), tRNA-specific (SelC or tRNAsec), Specific mRNA Sequence (SElenocysteine Insertion Sequence - SECIS) and Aminoacyl tRNA Synthetase (aaRS). Because selenium has high toxicity in cellular environments; it is essential for cell survival the association of this compound with proteins, in this case, selenoprotens and the associated proteins involved in the selenocysteine synthesis. Therfore the understanding of the catalytic mechanism, stoichiometric ratio, protein complex formation with the tRNAsec, and its structural characterization were the objectives of this work. The SelA protein was expressed and purified to used in analyzes involving atomic force microscopy, transmission electron microscopy with negative stain and in vitreous ice were performed in the complex SelA and SelA-tRNAsec in order to obtain a structural model of the complex and the stoichiometric ratio of its components. To study the selenium association with protein of the synthesis pathway, fluorescence anisotropy assays and isothermal titration calorimetry corroborated by the analysis hydrogen-deuterium exchange coupled to mass spectrometry and infrared spectroscopy were employed.Crystallization attempts were made and preliminary crystallographic analyzes were also performed, however, so far unsuccessfuly. The results obtained were possible to develop the hypothesis about the SelD recognition and, consenquently, the selenophosphate delivery, a crucial stage of the selenocysteine incorporation pathway.

No description available.

Orientadores: Chang Tien Kiang, Elias Hage Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-20T05:26:20Z (GMT). No. of bitstreams: 1 Cardoso_Giselia_M.pdf: 2679256 bytes, checksum: 20c2cfedadeeb83bd8a03e60f9c2c1a4 (MD5) Previous issue date: 1994 / Resumo: Misturas poliméricas binárias de poliamida 6,6 com aditivo macromolecular ¿ poliamida 6 modificada, policarbonato e poli(metacrilato de metila) ¿ em quantidades de 1 a 10% em massa, foram preparados por fusão e moldadas por injeção através do Mini Max Molder de fabricação da Custom Scientific Instruments. Os produtos obtidos foram caracterizados por microscopia ótica (MO), calorimetria diferencial de varredura (DSC) e análise dinâmico-mecânica (DMA). A análise em microscopia ótica foi realizada de forma qualitativa na observação da influência da natureza química e da quantidade de aditivo macromolecular na modificação da microestrutura da poliamida 6,6. as análises de DSC foram realizadas de duas maneiras: varredura de temperatura, na verificação de existência de miscibilidade nas e determinação. Pela equação de Nishi ¿ Wang, do parâmetro de interação de Flory ('X IND. 12¿); e isotérmica, na avaliação do efeito da presença dos aditivos macromoleculares na velocidade de cristalização da poliamida 6,6. A existência de separação de fase no estado sólido das misturas foi verificada através da análise do comportamento dinâmico-mecânico. O estudo conclui que a adição de pequena quantidade de macromoléculas retarda o aparecimento e reduz a taxa de crescimento dos esferulitos na poliamida 6,6 / Abstract: Binary polymeric blends of polyamide 6,6 with macromolecular additive ¿ modified polyamide 6, polycarbonate and poly(methyl methacrylate) ¿ in amounts varying from 1% to 10% in mass, were prepared by melting and injection ¿ molded a Mini Max Molder ¿ Custom Scientific Instruments. The obtained products were characterized by optical microscopy (OM), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The optical microscopy analysis was performed as a qualitative observation of the influence of chemical nature and quantity of macromolecular additive in the modification of microstructure of polyamide 6,6. the DSC analysis were done in two ways: temperature scanning, where it was verified the miscibility of mixtures and the Flory¿s interaction parameter 'X IND. 12¿ was determined by Nishi ¿ Wang equation; and isothermally, where the presence of macromolecular additives in the rate of crystallization of polyamide 6,6 was evaluated. DMA of rod samples were performed to evaluate the presence of phase separation in the solid state. It is concluded that blending small amounts of macromolecular additives can delay and reduce the spherulitic crystallization growth in the polyamide 6,6 / Mestrado / Ciencia e Tecnologia de Materiais / Mestre em Engenharia Química

Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.

Adhesives are widespread. They hold together the furniture, cars and electronics that we use on a daily basis. The majority of commercially available glues are sourced from petroleum-based monomers and are not degradable in any practical way. The permanent nature of these adhesive materials makes disassembly for recycling difficult. Current bio-based glues such as hide and starch glue are not strong enough to compete with commercial glues. Inspiration from nature is helping us to tackle this problem. Marine mussels achieve strong bonding to underwater surfaces through the use of adhesive plaques containing the uncommon amino acid 3,4-dihydroxyphenylalanine. Incorporating this chemistry into a degradable polylactic acid backbone allows for the development of strong bonding biodegradable glue. Throughout this work, the synthesis of these materials is discussed as well as methods to improve the bonding of these materials to compete with commercial glues. The degradation of these materials as well as their cytocompatibility is discussed.

This dissertation investigates non-covalent macromolecular chemistry using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) based techniques. The included studies reveal the impact of molecular structure on conformation and binding energetics. Supramolecules that might be too heavy to be dissociated in single collision-induced dissociation (CID) were dissociated using sustained off-resonance collision induced dissociation (SORI-CID) techniques. Relative binding energies and thresholds were evaluated for various macromolecular host-guest systems. Besides the non-covalent binding energies, conformation characterization was undertaken by a novel method to determine collision cross sectional areas using FTICR (CRAFTI, and multi-CRAFTI), initially developed by the Dearden lab. The systems chosen for further understanding of macromolecular interactions include calixarene-alkali metal complexes, cucurbit[5]uril-alkali halide complexes and cryptand-alkali metal complexes. The results were found to be consistent with expected behavior, and strongly correlated with predictions from computations. Size- and shape selectivity, as well as host-guest polarizability, are the main factors that govern the non-covalent macromolecular interactions that control complex conformation and dissociation. The results demonstrate the ability of FTICR to simultaneously determine binding energy, structure and conformation, which are the most important aspects for determination of comprehensive molecular characterization.

Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.

Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.