Academic literature on the topic 'Cellic ctec2'

The performance of cellulase in the enzymatic saccharification of lignocellulose depends on the characteristics of lignocellulosic biomass feedstocks and the pretreatment method used. Efficient hydrolysis of specifically pretreated lignocellulose necessitates the knowledge of the characteristics of the optimal commercial cellulases. In this study, commercial cellulase preparations (Accellerase™ 1000, Accellerase® 1500, and Spezyme® CP from DuPont and Cellic® CTec2 from Novozymes) were evaluated for their hydrolysis efficiency of hydrothermally pretreated empty fruit bunches (EFBs). The highest glucose yields of 91.3% and 84.7% were achieved for 30 FPU of Cellic® CTec2/g glucan with and without Cellic® HTec2, respectively. Of the four cellulases tested, Cellic® CTec2, which showed the highest cellobiohydrolase, xylanase, and β-glucosidase activities, showed the highest glucose yield in the enzymatic hydrolysis of hydrothermally pretreated EFBs. The results of this study are valuable for those who plan to enzymatically hydrolyze hydrothermally pretreated EFBs.

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 50 °C. Cellulose obtained by the Kürschner-Hoffer method from the wood of 3-year-old poplar (Populus trichocarpa) was subjected to enzymatic hydrolysis. Cellic® CTec2 enzymes (Novozymes, Denmark) were used. The enzymatic hydrolysis was tested within the conditions recommended by the manufacturer and the literature. The process was carried out at 50 °C at various pH – 4.8, 5.0, 5.5 and enzymes doses - 25, 50 and 100 mg per 100 mg of the dry mass of cellulose. The process was ended after 24 h. The hydrolysates were analysed by high-performance liquid chromatography (HPLC) to determine the glucose content, and then the highest glucose yield. The highest glucose yield was obtained for pH 4.8 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 72 %.

Estudou-se a hidrólise enzimática da palha da cana para a obtenção de açúcares fermentescíveis. A palha passou por processos físicos (moagem, lavagem e secagem), foi submetida à uma deslignificação com H2O2 em meio alcalino e hidrólise. Após caracterização da biomassa lignocelulósica in natura e deslignificada observou-se remoção de 50% da lignina. Foi quantificada a atividade enzimática da celulase de T. reesei e comercial Cellic® CTec2. Foram estudados pH da reação, concentração de enzima e tempo de reação, obtendo-se pH ótimo 4,0 e 5,5 para a T. reesei e Cellic® CTec2, respectivamente. O maior rendimento em termos de concentração de enzima foi de 10 FPU/g de palha para ambas as enzimas e para o tempo, ambas enzimas apresentaram queda de conversão após 24 horas de hidrólise, possivelmente por contaminação microbiana. Por fim realizou-se o balanço material para a celulase comercial, projetando-se um rendimento do processo de hidrólise enzimática da palha de 23,8%.

BackgroundAspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However,A. nigernaturally secretes low levels of CBH. Hence, recombinant production ofA. nigerCBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH fromA. niger.MethodsIn this study, the gene encoding a cellobiohydrolase B (cbhB) fromA. nigerATCC 10574 was cloned and expressed in the methylotrophic yeastPichia pastorisX-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic®CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.ResultsRecombinant CBHB was over-expressed as a hyperglycosylated protein attached toN-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC),p-nitrophenyl-cellobioside (pNPC) andp-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel®and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic®CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iαand Iβ in the treated OPEFB samples.DiscussionSince CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic®CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic®CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence,A. nigerCBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 45 °C. The enzymatic hydrolysis with the use of industrial enzymes Cellic® CTec2 (Novozymes, Denmark) was carried out within the conditions recommended by the manufacturer and literature. Cellulose obtained by the Kürschner-Hoffer method from a wood of 3-year-old poplar (Populus trichocarpa) was used for the study. Three pH values of 4.8, 5.0 and 5.5 were applied. Also, three amounts of enzymes were used: 25, 50 and 100 mg per 100 mg of the dry mass of cellulose for each pH used. The temperature was 45 °C. Samples were taken after 24 h and subjected to chromatographic analysis to determine the glucose content in the hydrolysates, and then the process parameters allowing for the highest glucose yield after the enzymatic hydrolysis process. The highest glucose yield was obtained for pH 5.0 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 79 %.

Abstract Cheap, renewable lignocellulosic materials are relevant to the future of biofuel production. Wood and agricultural wastes (e.g. straw, corn stover) provide a raw material source that cannot be used for human consumption, thus biofuels from such sources do not threaten the food supply. The aim of the work was to carry out the pre-treatment and hydrolysis of lignocellulosic material in the same ionic liquid solvent (1-n-butyl-3- methyl-imidazolium-chloride, [Bmim]Cl), using ground wheat straw and a mixture of corn (Zea mays) leaf and stover, as substrates. Our measurements show that it is possible to achieve an acceptable glucose content from the cellulose by applying Cellic® CTec2 and Cellic® HTec2 enzyme complexes.

Improved cost-effective bioethanol production using inexpensive enzymes preparation was investigated. Three types of waste lignocellulosic materials were converted—for the production of enzyme preparation, a mixture of sugar beet pulp and wheat bran, while the source of sugars in hydrolysates was sweet sorghum biomass. A novel enzyme cocktail of Trichoderma citrinoviride C1 is presented. The one-step ultrafiltration process of crude enzyme extract resulted in a threefold increase of cellulolytic and xylanolytic activities. The effectiveness of enzyme preparation, compared to Cellic® CTec2, was tested in an optimized enzymatic hydrolysis process. Depending on the test conditions, hydrolysates with different glucose concentrations were obtained—from 6.3 g L−1 to 14.6 g L−1 (representing from 90% to 79% of the CTec2 enzyme yield, respectively). Furthermore, ethanol production by Saccharomyces cerevisiae SIHA Active Yeast 6 strain DF 639 in optimal conditions reached about 120 mL kg d.m.−1 (75% compared with the CTec2 process). The achieved yields suggested that the produced enzyme cocktail C1 could be potentially used to reduce the cost of bioethanol production from sweet sorghum biomass.

The effect of binding between the lignin isolates from an alkali (NaOH)– and an acid (H2SO4)– pretreated Miscanthus and cellulolytic enzymes in Cellic® CTec2 was investigated. Additonally, cellobiose and Avicel were enzymatically hydrolyzed with and without lignin isolates to study how enzyme binding onto lignin affects its conversion to glucose. Three carbohydrate–lignin loadings (0.5:0.25, 0.5:0.5, and 0.5:1.0% (w/v)) were employed. The results indicated that β-glucosidase (BG) had a strong tendency to bind to all lignin isolates. The overall tendency of enzyme binding onto lignin isolate was similar regardless of pretreatment chemical concentration. Though enzyme binding onto lignin isolates was observed, hydrolysis in the presence of these isolates did not have a significant (p > 0.05) impact on glucose production from cellobiose and Avicel. Cellobiose to glucose conversion of 99% was achieved via hydrolysis at both 5 and 10 FPU/g carbohydrate. Hydrolysis of Avicel with 5 and 10 FPU/g CTec2 resulted in 29.3 and 47.7% conversion to glucose, respectively.

The demand for ethanol in Brazil is growing. However, although the country is one of the largest producers of this fuel, it is still necessary to diversify the production matrix. In that regard, studies with different raw materials are needed, mainly the use of low cost and high available wastes such as lignocellulosic residues from agriculture. Therefore, this study aimed to analyze the bioethanol production from corn stover. An alkaline pretreatment (CaO) was carried out, followed by enzymatic hydrolysis (Cellic Ctec2 and Cellic Htec2) to obtain fermentable sugars. The best experimental condition for the pretreatment and hydrolysis steps resulted in a solution with 0.31 gsugar∙gbiomass-1. Then, the fermentation was performed by the industrial strain of Saccharomyces cerevisiae (PE-2) and by the wild yeast strain Wickerhamomyces sp. (UFFS-CE-3.1.2). The yield obtained was 0.38 gethanol∙gdry biomass-1 was, demonstrating the potential of this process for bioethanol production.

Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten different fucoidan samples extracted enzymatically from Laminaria digitata (LD), Saccharina latissima (SL) and Fucus distichus subsp. evanescens (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using different enzymes and SL fucoidans were further separated into three fractions (SL_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL_F2 and SL_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective effects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic effects. In OMM-1 SL_F2 and several FE fucoidans were protective. LD_SiAT2 (Cellic®CTec2 + Sigma-Aldrich alginate lyase), FE_SiAT3 (Cellic® CTec3 + Sigma-Aldrich alginate lyase), SL_F2 and SL_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL_F2 and SL_F3 are very promising for beneficial AMD relevant biological activities.

Bio-refining of renewable resources such as waste biomass into value added products has increased rapidly over the last decades. The aim is to find new environmentally friendly yet economically feasible ways of replacing current utilization of non renewable resources. It can be expected that a commercially viable next generation bio-ethanol will be produced from lignocellulosic feedstock in the near future.This master thesis set out to look at a specific Canadian company in depth (Pure Lignin Environmental Technology Ltd), known as PLET henceforth. PLET has a new approach in pre-treatment of biomass, with a recently patented environmentally friendly technology, which could be extremely lucrative if applied in the right context. It is desirable to look at the possibility of using the master thesis as a take-off platform to launch a consulting bio-tech company directly after completion.There are several different ways to process biomass into higher value added products. In this master thesis focus is laid upon biomass waste, but the same principles could be applied on biomass processing in general. That is the separation and purification of products. In order to put the technology of PLET into a context, other company approaches are used as a comparison, such as SEKAB E-Technology, Lignoboost and Borregaard Lignotech.It is however hard to find suitable examples equivalent to the PLET approach since the technology of PLET considers all the three constituents of wood as raw material for production of three stand alone commercial products; pure lignin, pure cellulose and hemi-cellulose. These three products are separated and purified in the pre-treatment process at high yield and practically no formation of by-products. Process parameters are only moderately adjusted depending on what raw material is used.The catalytic reactor process (=CRP), is performed by PLET as a pre-treatment in a continuous and batch system, in which lignocellulosic material (= wood chips, saw dust or other waste biomass) undergo acid catalyzed hydrolysis by dilute nitric acid. The impregnated wood chips are partially depolymerised from the lignin matrix when heated in the low pressure catalytic reactor. After that, follows distillation, condensation and almost complete recovery of dilute nitric acid. After CRP, the cellulose pulp is separated from the black liquor by an alkaline solution (=NaOH). The obtained black liquor is pumped to a separation tank to precipitate lignin and sweet liquor through filtration. The final step is to dry the lignin, while the sweet liquor is utilized by Torula yeast (=Candida utilis) in order to produce unicellular protein.It would be interesting to see if the pre-treatment that PLET provides, could be exploited in a context of production of next generation of cellulosic ethanol from the cellulosic part of waste biomass. In this master thesis, an evaluation of producing next generation ethanol from dilute-nitric acid pre-treated cellulose rich softwood was performed and dilute sulphuric acid pre-treated hemi-cellulose rich hardwood birch pulp was used as a comparison.The technical part of this master thesis was conducted on a laboratory scale, using samples from two companies, referred to as PLET and SEKAB E-technology (Sweden). The strategy of PLET is currently to find a commercial platform to produce value added products from waste biomass generated by saw mills and pulping industries, while SEKAB E-Technology mainly works with Swedish softwood as a raw material.ivExperiments of this master thesis includes a series of fermentation trials using either SSF (= Simultaneous Saccharification and Fermentation) or SHF (= Separate Hydrolysis and Fermentation). Yields of the enzymatic hydrolysis and subsequent fermentations were estimated with HPLC measurements complemented with dry weight measurements.Obtained experimental data support the potential to make lignocellulosic ethanol out of either softwood or hardwood. Ethanol yield from dry material from SEKAB E-Technology hardwood birch slurry was 0.43 (g ethanol / g dry raw material), whereas samples supplied by PLET achieved the following results. The ethanol yield from dry material from softwood pine washed cellulose was 0.33 g (ethanol / g cellulose). The ethanol yield from dry material obtained for softwood pine unwashed cellulose in the three SHF was 0.47; 0.34 and 0.19 (g ethanol / g cellulose) respectively, while the yield in the two SSF was 0.45 and 0.46 (g ethanol / g glucose).The economical part of this master thesis includes the use of Business Model Canvas and the NABC- model (= Need, Approach, Benefit and Competition) to define the basic topics and tasks that need to be addressed in the start up phase of a small consulting bio-tech company.At least 6 million US dollars is needed as a minimum starting capital to be able to construct a small commercially viable pilot plant, according to PLET. This investment would cover the basic requirements and is scalable. The construction could preferably be done in Sweden in the vicinity of an existing saw mill or paper pulp industry or elsewhere, where a steady supply of cheap raw material could be assured. The size of the operation envisioned, depends greatly on location and adjacent businesses. Large pulp mills in the range of 400-500 MWh would be preferred, the largest pulp mills in Canada process 5000 ton per day which is equivalent to a capacity of 1 GW wood per day (GW = giga watt), but the concept of the technology of PLET must first prove its value on a smaller scale.According to PLET, a future full scale production plant could be designed for processing 56 tons of wood chips per day of wood chips would generate annual revenues of ≈ 7500000 US dollars. The annual profit would be ≈ 2500000 US dollars, and capital cost (equipments) ≈ 5000000 US dollars, while the payback time on investment would be 2-3 years. According to PLET, the products are estimated to have the following values; 25 tons/day of pure cellulose with a market value of 400-500 US dollars/ton, 10 tons/day of pure lignin with a market value of 1000-1200 US dollars/ton and finally 21 tons/day of sweet liquor with a market value of 50-100 US dollars/ton.
Program: Masterutbildning i Energi- och materialåtervinning - industriell bioteknik

The thymus is the primary lymphoid organ responsible for the development and maturation of T lymphocytes (aka T-cells) in vertebrates. The complex architecture of the thymic microenvironment orchestrates the formation of a diverse and self-tolerant T-cell repertoire capable of supporting the development and maintenance of a functional immune system. The main component of this microenvironment, the thymic epithelium, is crucially required to direct thymus organogenesis and homeostasis, and to mediate T-cell repertoire development and selection. The thymic epithelial progenitor cells (TEPCs) from which the mature thymus develops originate from the endoderm of the 3rd pharyngeal pouch by embryonic day 9 in mouse development (or early week 6 in human embryos). Expression of the transcription factor FOXN1 is required to drive TEPCs differentiation in each thymic epithelial lineage (TEC), while the absence of functional FOXN1 causes athymia. Moreover, forced expression of Foxn1 in mouse embryonic fibroblasts (MEFs) converts these MEFs into TECs that can support the development of a normal thymic system. Despite the great therapeutic potential that TEPCs present in regenerative medicine, there is currently no detailed model describing regulation of the TEPC state and its differentiation into cortical (c) and medullary (m) TECs, or explaining the dominant role of FOXN1 in the thymic epithelial system. Comparative transcriptomics analysis in conjunction with pathway enrichment analysis of the developing TEPCs could reveal the signalling pathways that regulate the early TEPC state and progression into differentiation. Additionally, integrative bioinformatics analysis of transcriptomics and genomics datasets could identify the functional networks that are directly regulated by FOXN1 during early TEC progression. In this thesis I provide, for the first time, an in silico model explaining fetal TEPC differentiation into the functionally distinct TEC lineages, in the cellular, molecular and signalling contexts of thymus early development. Furthermore, I present evidence which suggests that FOXN1 could be a pioneer factor, capable of fully establishing the transcriptional programme that underpins thymic epithelial cell identity and function. Finally, in this thesis, I introduce the development of an interactive thymic-specific database that provides a platform for easy access, analysis and integration of curated bioinformatics datasets.

Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

Dissertação de Mestrado Integrado em Engenharia Química apresentada à Faculdade de Ciências e Tecnologia
A presente dissertação de mestrado tem como objetivo principal a dissolução da matriz fibrosa presente no papel de impressão e escrita, com o intuito de separar o material inorgânico sem que estas partículas sofram degradação. A matriz fibrosa é constituída praticamente por fibras de celulose e hemicelulose e quantidades residuais de lenhina. A celulose, é um biopolímero de cadeia linear, com regiões altamente cristalinas, que estabelece fortes ligações de hidrogénio, que dificulta em grande medida o acesso dos solventes. Por esta razão e porque é um polímero insolúvel em água e na maioria dos solventes orgânicos conhecidos, a sua dissolução é um processo difícil e exigente. No caso da presente tese, a dissolução do material orgânico foi tentada com recurso a três reagentes químicos distintos: Cuproetilenodiamina, N,N-Dimetilacetamida/Cloreto de Lítio e Hidróxido de Sódio/Ureia. Como estes agentes de dissolução de celulose são bastante dispendiosos e extremamente agressivos para o ambiente, testou-se também a solubilização com recurso ao complexo enzimático Cellic CTec2 da Novozymes. Todos estes agentes foram aplicados a dois materiais distintos: i) duas folhas de papel de impressão e escrita (material com cerca de aproximadamente 70% (w/w) de fibras) e ii) um substrato composto quase exclusivamente por fibras de celulose e hemicelulose, o papel de filtro Whatman no1. Uma vez que a dissolução de celulose presente nos papéis de impressão e escrita ainda não se encontra reportada na literatura, foi necessário otimizar os procedimentos utilizados, de modo a obter elevada eficiência de dissolução sem que ocorra uma severa degradação das partículas minerais. Os resultados obtidos através destes procedimentos foram sempre comparados com a metodologia utilizada atualmente para a separação de cargas minerais, a calcinação. À exceção do procedimento de dissolução de celulose com DMAc/LiCl, nos restantes procedimentos foi utilizada uma etapa preliminar que consistiu no intumescimento das fibras de celulose em água ultrapura a 40 ºC. Este pré-tratamento físico facilitou, na etapa seguinte, a impregnação de solvente na matriz de celulose, contribuindo positivamente para a etapa de dissolução. Alguns estudos afirmam mesmo que esta etapa permite a diminuição do grau de cristalinidade, o que leva a um aumento da solubilidade da celulose nos diferentes solventes testados. O material não dissolvido foi caracterizado quanto à morfologia, tamanho de partícula, carga superficial, composição química e estabilidade térmica. Os resultados foram sempre que possível comparados com o material de partida, nomeadamente papéis de impressão e escrita e carbonato de cálcio. Através das análises realizadas é possível concluir que nem todos os procedimentos de dissolução se mostraram eficientes. O procedimento com solvente CED permitiu atingir o objetivo proposto. Através da calcinação e de análise termogravimétrica é notório a elevada eficiência do procedimento, pois a percentagem de matéria orgânica no resíduo final ronda os 9% (w/w). Com a técnica de FTIR e FSEM é notório que o resíduo é, na sua grande maioria, constituído por carbonato de cálcio na forma escalenoédrica, semelhante ao presente no papel de impressão e escrita. Este procedimento foi também aplicado ao papel de filtro onde é notória a total degradação da matéria orgânica. Com as metodologias DMAc/LiCl e NaOH/Ureia, a dissolução do papel de filtro não se mostrou eficiente, tendo-se verificado o mesmo no caso dos dois papéis de impressão e escrita. Com recurso a FESEM e AE é possível concluir que não ocorreu significativa dissolução da componente orgânica, sendo também visível a impregnação deste material com os solventes químicos utilizados. Por fim, a aplicação do complexo enzimático revelou-se também como um procedimento não totalmente positivo, isto porque apesar de se verificar a dissolução praticamente total do material orgânico, ocorre severa decomposição do material inorgânico, o que se torna uma enorme limitação. A dissolução do material orgânico é provada pela degradação total do papel de filtro com a aplicação deste complexo enzimático. Quando o material de partida é papel de impressão e escrita, composto essencialmente por fibras de celulose e cargas minerais o procedimento torna-se ineficaz para o presente objetivo. De facto, como este complexo enzimático requer que o pH do meio seja ácido, a adição da solução de tampão citrato 1M promove a reação química deste tampão com o carbonato de cálcio, levando a formação de outros sais como citrato de cálcio e de sódio. Assim, no final do procedimento de hidrólise enzimática não resta carbonato de cálcio, mas sim o produto da reação química que ocorre. Uma vez obtidos estes resultados é importante referir novamente que o solvente CED cumpre totalmente o objetivo da presente tese e o complexo enzimático também consegue hidrolisar praticamente toda a celulose, embora provoque degradação das cargas minerais.
This thesis aims at the deconstruction of the fibrous matrix present in commercial printing and writing papers, for a non-degrading separation of the inorganic particles. The fibrous matrix is mainly composed of cellulose and hemicellulose fibers with residual amounts of lignin. Cellulose is a natural polymer of linear chain with regions of high crystallinity. The strong intra and inter hydrogen bonds makes its dissolution difficult and demanding: it is insoluble in water and most organic solvents. Three distinct reactants for the dissolution of organic material have been used in this work: Cupriethylenediamine, N,N-Dimethylacetamide/Lithium Chloride system and Sodium Hydroxide/Urea aqueous system. Since the reactants are expansive and environmental pollutants. The enzymatic complex Cellic CTec2, Novozymes was also employed as dissolving agent. All these methods were applied to two distinct materials: printing and writing papers (approximately 70 % wt of fibers) and filter paper (Whatman no1, almost entirely composed of cellulose and hemicellulose fibers). To our knowledge the dissolution of the cellulose fibers present in printing and writing filler containing papers is not yet reported in the literature and therefore, the optimization of the procedures used in order to maximize the dissolution process was mandatory. The results obtained with the different tested methodologies were compared with the currently used technique for the fillers recovery, calcination. A preliminary stage, consisting in the swelling the fibers in water at 40 ºC, was applied, except for the DMAc/LiCl. This treatment improves the impregnation of the solvents through the fibrous matrix improving the fibers dissolution, mainly due to the decrease in then crystallinity as well reported in the literature. The residues obtained were characterized in terms of morphology, particle size, superficial charge, chemical composition and thermal stability. The results were compared with those of the starting materials: printing and writing paper and calcium carbonate. The characterization carried out revealed that not all of the methodologies were efficient. By calcination and TGA it was conclude that CED was highly efficient for the separation, with only approximately 9% wt of organic material in the residue. Furthermore, FTIR and FESEM showed that the residue is mainly composed of scalenohedral calcium carbonate, similar to the one found in the printing and writing paper. The filter paper was completely degraded by CED. The DMAc/LiCl and NaOH/Urea based methodologies were not efficient in dissolving both the writing and printing papers and the filter papers. In fact, FESEM and EA showed that no significant degradation of the organic matter occurred. The enzymatic complex did not give satisfactory results because, in spite of achieving a complete dissolution of the organic fibers, the inorganic materials were severely degraded. The dissolution of the organic material was confirmed by the complete degradation of the filter paper. The acidic media required by the enzymes, achieved by adding citrate buffer, promotes the reaction of the acid with the calcium carbonate and leads to the formation of other calcium and sodium salts distinct from calcium carbonate. In conclusion, the CED procedure fully allowed to achieve the goal of this work. The enzyme treatment allows a near full dissolution of the cellulose fibers but it profoundly changes the mineral fillers.

Risk stratification, prognostication and longitudinal monitoring of therapeutic efficacy in lung cancer patients remains highly challenging. It is imperative to establish robust surrogate biomarkers for identifying eligible patients, predicting and effectively monitoring clinical response as well as timely detecting emerging resistance to therapeutic regimens. Circulating tumor biomarkers, analyzed by liquid biopsy, are primarily composed of nucleic acid-based circulating tumor DNA (ctDNA) and an aneuploid cell-based category of circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs). Unlike ctDNA, cancer cells are the origin of all categories of various tumor biomarkers. Involvement of aneuploid CTCs and CTECs in tumorigenesis, neoangiogenesis, tumor progression, cancer metastasis and post-therapeutic recurrence has been substantially investigated. Both CTCs and CTECs possessing an active interplay and crosstalk constitute a unique category of cellular circulating tumor biomarkers. These cells concurrently harbor the intact cancer-related genetic signatures and full tumor marker expression profiles in sync with disease progression and therapeutic process. Recent progress in clinical implementation of non-invasive liquid biopsy has made it feasible to frequently carry out ctDNA analysis and unbiased detection of a full spectrum of non-hematologic circulating rare cells including CTCs and CTECs in lung cancer patients, regardless of variation in heterogeneous cell size and cancer cell surface anchor protein expression. In situ phenotypic and karyotypic comprehensive characterization of aneuploid CTCs and CTECs, in combination with single cell-based genotyping and improved ctDNA analyses, will facilitate and benefit multidisciplinary management of lung cancer.