The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale. This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction. A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB). For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF). / <p>QC 20101108</p>

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

<p>Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.</p><p>Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.</p><p>Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.</p>

Apesar da existência de diversos estudos que validem o uso de implantes de diâmetro estreito, a maioria é baseada em ligas de titânio puro. Há pouca evidência clínica em relação ao sucesso do implantes de diâmetro estreito de titânio-zircônia (TiZr NDIs), no que diz respeito à taxa de sobrevida (SR) e perda óssea marginal (MLB). O objetivo desta revisão sistemática foi analisar sistematicamente SR, assim como MBL de TiZr NDIs quando comparados a implantes de diâmetro estreito de titânio comercialmente puro (cpTi NDIs). A busca foi conduzida nas bases Medline/Pubmed, Cochrane, Scopus e Embase (do ano 2000 a novembro de 2016). Foram incluídos estudos clínicos do tipo coorte e randomizados. Dos 3453 artigos inicialmente identificados, foram incluídos seis estudos clínicos. Não houve diferença estatisticamente significante em taxa de sobrevida quando comparados os grupos TiZr NDIs e cpTi NDIs, em um ano de acompanhamento (p=0,5), além disso, também não houve diferença na comparação entre região de implantação (anterior ou posterior) no grupo TiZr NDIs. Não houve diferença entre os grupos anterior e posterior em relação à taxa de sobrevida em um ano: -0,01 (95% CI, -0,05 a 0,03) e perda óssea marginal: -0,01 mm (95% CI: -0,14 a 0,12). Pode-se concluir que TiZr NDIs apresentam taxas de sucesso e reabsorção óssea peri-implantar similares às de cpTi NDis. / Despite the existence of several studies validating the use of narrow diameter implants, most of them are based on pure Ti alloys. There is few clinical evidence of the success of TiZr narrow diameter implants (TiZr NDIs) regarding survival rate and marginal bone loss. The aim of this review was to systematically assess survival rate, as well as marginal bone loss of TiZr NDIs compared to commercially pure titanium narrow diameter implants (cpTi NDIs). Material and Methods: The search was conducted in Medline/PubMed, Cochrane, Scopus and Embase databases (year 2000 to November 2016). Cohort studies and randomized trials were included. Results: Six clinical studies from the 3453 articles initially identified met the inclusion criteria. There were no statistically significant differences in survival rate when TiZr NDIs and cpTi NDIs were compared in the 1-year follow up (p=0.5), or when comparing TiZr NDIs placed in posterior and anterior regions. There was no difference between groups regarding 1-year SR: -0.01 (95% CI, -0.05 to 0.03) and MLB: -0.01 mm (95% CI: -0.14 to 0.12). Conclusion: It can be concluded that TiZr NDIs present similar success rates and peri-implant bone resorption to cpTi NDIs.

Orientador: Idelmo Rangel Garcia Júnior / Banca: Roberta Okamoto / Banca: Élio Hitoshi Shinohara / Banca: Oswaldo Bergamaschi / Banca: Paulo Sérgio Perri de Carvalho / Resumo: O presente estudo teve por finalidade analisar as respostas teciduais do tecido subcutâneo na presença de transplante homógeno de células de medula óssea, por meio dos estudos histológico e imunoistoquímico das proteínas Cbfa1/Runx2 e osteocalcina. A metodologia utilizada nesse estudo envolve processamento em tecido descalcificado e neste caso, possibilitando cortes com menor espessura, para análise das características morfológicas das células presentes no tecido formado ao redor do enxerto e mesmo a resposta inflamatória do tecido subcutâneo ao material enxertado. Como complementação realizou-se o processamento em tecidos calcificados, após a injeção de fluorocromos, a calceína aos 10 dias e a alizarina aos 20 dias possibilitando a análise do processo de deposição de cálcio ao redor do material enxertado no tecido subcutâneo. Com a análise histológica, verificou-se que o enxerto de medula óssea isoladamente foi incorporado ao tecido subcutâneo, sendo que no período de 20 dias são observadas algumas estruturas semelhantes a tecido ósseo, pois apresentam-se morfologicamente, como tecido calcificado. Com o objetivo de caracterizar o tecido medular implantado no tecido conjuntivo subcutâneo, foi observada imunomarcação positiva contra Cbfa1/Runx2 e contra osteoclacina. Os resultados sugerem que a presença da medula óssea junto ao osso cortical, quando transplantada em seres de mesma espécie, em tecido subcutâneo, favoreceu a osteogênese pelo processo de osteoindução. / Abstract: The purpose of this study was to analyze the cellular response to the subcutaneous tissue in a presence of autolougous bone marrow cells transplantation, through the histological and imunohistochemic study of Cbfa1/Runx2 and osteocalcin proteins. The methodology uses the decalcified tissue processing, and in this case, provides thinnest slices cuts for the morphological analyses and inflammatory responses of the cells around the graft in the subcutaneous tissue. As an extra study, was evaluated the calcified tissues after the fluorochroms injection, calcein in the tenth day and alizarin in the twentieth day, given the possibility to analyze the calcium deposition process around the graft in the subcutaneous tissue. Through the histological analyses it was possible to verify that the bone marrow was well accepted by the connective tissue, at the twentieth day it was possible to see some structures similar to bone, because it was morphologically similar to calcified tissue. With the objective to characterize the bone marrow implanted in the conjunctive subcutaneous tissue, positive results against Cbfa1/Runx2 and osteocalcin was observed. The results suggest that the presence of the bone marrow next to the cortical bone, when grafted in beings of same species, in subcutaneous tissue, it favored osteogneses for the osteoinduction process. / Doutor

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-14T15:41:38Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoPrecisaoGuias.pdf: 4828751 bytes, checksum: 4f67a722f8937eec4e565f5418e64246 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-30T13:45:33Z (GMT) No. of bitstreams: 2 Dissertacao_AvaliacaoPrecisaoGuias.pdf: 4828751 bytes, checksum: 4f67a722f8937eec4e565f5418e64246 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-30T13:45:33Z (GMT). No. of bitstreams: 2 Dissertacao_AvaliacaoPrecisaoGuias.pdf: 4828751 bytes, checksum: 4f67a722f8937eec4e565f5418e64246 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2008 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O planejamento implantodôntico exige precisão e confiabilidade. A prototipagem rápida oferece a possibilidade de transferir o planejamento para o campo operatório, sendo este um importante aliado no tratamento cirúrgico. O presente estudo tem por objetivo avaliar a precisão de guias cirúrgicos obtidos através de tomografia computadorizada, planejamento em software para implantodontia (Implantviwer – Anne Solutions ltda.) e prototipagem rápida, avaliando a angulação e a posição horizontal dos implantes e ainda comparar guias com apoio ósseo e guias com apoio gengival. Foram selecionadas cinco mandíbulas humanas secas, que foram fixadas a um crânio e realizadas as tomografias computadorizadas, o planejamento dos implantes e os guias confeccionados apartir de prototipagem. Após a instalação de vinte implantes foram realizadas novas tomografias e comparação das medidas referentes a angulação e posicionamento horizontal dos implantes através da sobreposição das imagens pré e pós operatórias. Tais resultados foram submetidos a análise estatística pelo teste T para amostras dependentes e análise de variância de Friedman com índice de significância de 95%. Observou-se uma discrepância média de angulação de 2,60º (DP = 3,20º) e de 0.40 mm (DP = 0,51 mm) para o posicionamento horizontal nos guias com apoio sobre gengiva artificial e 5,30º (DP = 3,88º) e 0,70 mm (DP = 0,82 mm) para os guias com apoio ósseo . Os guias com apoio ósseo e gengival apresentaram diferença estatística entre si apenas quanto a angulação. Os guias cirúrgicos para implantes osteointegrados construídos apartir de prototipagem rápida oferecem precisão na transferência do planejamento para o campo operatório e sua utilização poderá aumentar a previsibilidade dos resultados, assim como facilitar a cirurgia e minimizar a intervenção cirúrgica. / The implantodontic planning demands precision and trustworthiness, the rapid prototyping offers the possibility to transfer the planning to the surgical field, being this an important ally in the surgical treatment. The present study it has for objective to evaluate the precision of surgical guides constructed through computerized tomography, planning in software for implantodontics and fast prototyping and compare bone supported guides to gingival tissue supported guides. Five dry human mandibulas had been selected, who had been fixed to an ideal skull and carried through the computerized tomograph, the implant planning and the prototyped guides. After the installation of the twenty implants, had been carried through new tomographs and comparison of the referring measures the axis and horizontal positioning of the implantations through the overlapping of the images before and after surgery, such results forms submitted to the statistic analysis for test T for dependent samples and Friedman´s variance analysis, with index of significance of 95%. An average discrepancy in axis of 2,60º (SD = 3,36º) and of 0,40 mm (SD = 0,68mm) for the horizontal positioning was observed to the gingival tissue supported guides and 5,30º (SD = 3,88º) and 0,70 mm (SD = 0,82mm) to bone supported guides, bone supported guides and gingival tissue supported guides did not demonstrated statistical differences. The surgical guides for osteointegrated dental implants constructed rapid prototyping offer precision in the transference of the planning for the surgical field and its use will be able to increase the previsibility of the results, as well as facilitating the surgery and minimizing the intervention.

Orientador: Eduardo Piza Pellizzer / Banca: Dalva Cruz Laganá / Banca: Eduardo Passos Rocha / Resumo: A influência do diâmetro e do comprimento de implantes osseointegrados em associação com próteses parciais removíveis de extremidade livre (PPRELs) ainda não foi relatada na literatura. Dessa forma, este trabalho visou avaliar a esta influência, utilizando o método dos elementos finitos (MEF) bi-dimensional, associando implantes osseointegrados do sistema Branemark sob a base protética de uma PPREL, funcionando apenas como suporte, e analisando mapas de deslocamento e de tensão de von Mises. Para o estudo foram confeccionados oito modelos representativos de uma hemi-arcada mandibular com a presença apenas dos dentes 33 e 34, com as seguintes características: A - sem PPR; B - com PPR sem implante; C - com PPR e implante de 3,75 x 7,00 mm; D - com PPR e implante de 3,75 x 10,00 mm; E - com PPR e implante de 3,75 x 13,00 mm; F - com PPR e implante de 5,00 x 7,00 mm; G - com PPR e implante de 5,00 x 10,00 mm; H - com PPR e implante de 5,00 x 13,00 mm. As forças aplicadas foram estritamente verticais e de 50 N em cada ponta de cúspide. Pelos resultados notou-se que a presença da PPR sobrecarregou o dente suporte, bem como as demais estruturas de suporte da PPR quando comparadas com o modelo A. A presença do implante diminuiu as tensões, principalmente nas extremidades do rebordo desdentado, quando comparados o modelo B com os modelos que possuíam implantes associados. Tanto o comprimento quanto o diâmetro do implante tenderam a diminuir as tensões à medida que aumentavam suas dimensões. Pelos resultados foi possível concluir que: o modelo B foi o que apresentou a maior tendência a deslocamento; quanto maior o comprimento do implante, menores foram os valores do mapa de deslocamento; o modelo B apresentou níveis de tensão de von Mises superiores ao modelo A; os modelos com implantes associados apresentaram níveis menores de tensão de von Mises que o modelo B nas estruturas de suporte da PPR. / Abstract: The influence of the diameter and length of osseointegrated implants in association with removable partial prostheses of distal-extension (DERPDs) it was not still reported in the literature. In that way, this study evaluated this influence, associating osseointegrated implants of the Branemark system under the prosthetic base of a DERPD, just working as support, and analyzing displacement and von Mises tension maps, using bi-dimensional finite element analysis (FEA). For this study, eight representative models of a lower hemi-arcade were made with the presence of only the teeth 33 and 34, with the following characteristics: A - without RPD; B - with RPD and without implants; C - with RPD and implant of 3,75 x 7,00 mm; D - with RPD and implant of 3,75 x 10,00 mm; E - with RPD and implant of 3,75 x 13,00 mm; F - with RPD and implant of 5,00 x 7,00 mm; G - with RPD and implant of 5,00 x 10,00 mm; H - with RPD and implant of 5,00 x 13,00 mm. The applied forces were strictly vertical and of 50 N in each edge tip. The results shown that the presence of RPD overloaded the support tooth, as well as the other structures of support of RPD when compared with the model A. The presence of the implants reduced the tensions, mainly in the extremities of the toothless edge, when compared model B with the models that possessed implant associated. So much the length as the diameter of the implants tended to reduce the tensions as increased your dimensions. For the results it was possible to conclude that: the model B was what presented the largest tendency to displacement; as larger the length of the it implants, smaller they were the values of the displacement map; the model B presented levels of von Mises tension superiors to the model A; the models with implant associated presented smaller levels of von Mises tension that the model B in the structures of support of RPD; the increase of the length and of the diameter of the implants. / Mestre

To address the concern of classical guidance and control designs (where guidance and control loops are designed separately in an “outer loop – inner loop” structure), integrated guidance and control (IGC) ideas have been proposed in the recent literature. An important limitation of the existing IGC algorithms, however, is that they do not explicitly exploit the inherent time scale separation that exist in aerospace vehicles between rotational and translational motions, and hence, can be ineffective unless the engagement geometry is close to the collision triangle. To address this, a time scale separated partial integrated guidance and control (PIGC) structure has been proposed in this thesis. In this two-loop design, the commanded pitch and yaw rates are directly generated from an outer loop optimal control formulation, which is solved in a computationally efficient manner using the recently-developed model predictive static programming (MPSP) and Model Predictive Spread Control (MPSC) techniques. The necessary roll-rate command is generated from a roll-stabilization loop. The inner loop then tracks the outer loop commands using the nonlinear dynamic inversion philosophy. However, unlike classical guidance and control designs, in both the loops the Six-DOF interceptor model is used directly. This intelligent manipulation preserves the inherent time scale separation property between the translational and rotational dynamics, and hence overcomes the deficiency of current IGC designs, while preserving the benefits of the IGC philosophy. The new approach has been applied in the terminal phase of an endo-atmospheric interceptor for engaging incoming high speed ballistic missile targets. Six–DOF simulation results will be presented accounting for a 3-D engagement geometry to demonstrate the usefulness of this method. It offers two important advantages: (i) it leads to very small (near-zero) miss distance, resulting in a “hit-to-kill” scenario and (ii) it also leads to lesser and smoother body-rate demands, relaxing the demand on actuators as well as enlarging the ‘capture region’ (which relaxes the demand on mid-course guidance). Next, to address the problem of modeling inaccuracy that is inherent in aerospace vehicles (mainly because of the inaccuracy of aerodynamic model generated from wind-tunnel testing), a neuro-adaptive design is augmented to dynamic inversion technique in the inner loop. In this design the unmodelled dynamics is adaptively captured using three neural networks in the roll, pitch and yaw channels. Training of the neural networks is carried out online using the Lyapunov stability theory, which results in stability of the inner-loop error dynamics as well as boundedness of network weights. This adaptive body rate tracking loop augmented with the sub-optimal feedback guidance loop results in substantial enhancement of interception performance in presence of realistic (i.e. fairly large) modeling uncertainties of the interceptor. The results have also been validated with representative seeker noise.

Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus. / QC 20100802

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections. / QC 20100902