Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

Foreign Direct Investment in Central and Eastern Europe:post-crisis perspectives

This book examines how foreign direct investment (FDI) inflows to Central and Eastern Europe have changed after the Great Recession. It argues that beyond their cyclical effects, the economic crisis and the changing competitiveness of Central and Eastern European countries have had structural impacts on FDI in the region. FDI has traditionally been viewed as the key driver of national development, but the apparent structural shift means that focusing on cheap labour as a competitive advantage is no longer a viable strategy for the countries in the region. The authors argue that these countries need to move beyond the narrative of upgrading (attracting FDI inflows with increasingly higher value added), and focus on ensuring greater value capture instead. A potential way for doing this is by developing the conditions in which innovative national companies can emerge, thrive and eventually develop into lead firms of global value chains. The book provides readers with a highly informative account of the reasons why this shift is necessary, as well as diverse perspectives and extensive discussions on the dynamics and structural impacts of FDI in post-crisis Central and Eastern Europe.

Engineering design of an integrated sustainable process system for cassava biobased materials

Cassava contributes significantly to biobased material development. Conventional approaches for its bio-derivative-production and application cause significant wastes, tailored material development challenges, with negative environmental impact and application limitations. Transforming cassava into sustainable value-added resources requires redesigning new approaches. Harnessing unexplored material source, and downstream process innovations can mitigate challenges. The ultimate goal proposed an integrated sustainable process system for cassava biomaterial development and potential application. An improved simultaneous release recovery cyanogenesis (SRRC) methodology, incorporating intact bitter cassava, was developed and standardized. Films were formulated, characterised, their mass transport behaviour, simulating real-distribution-chain conditions quantified, and optimised for desirable properties. Integrated process design system, for sustainable waste-elimination and biomaterial development, was developed. Films and bioderivatives for desired MAP, fast-delivery nutraceutical excipients and antifungal active coating applications were demonstrated. SRRC-processed intact bitter cassava produced significantly higher yield safe bio-derivatives than peeled, guaranteeing 16% waste-elimination. Process standardization transformed entire root into higher yield and clarified colour bio-derivatives and efficient material balance at optimal global desirability. Solvent mass through temperature-humidity-stressed films induced structural changes, and influenced water vapour and oxygen permeability. Sevenunit integrated-process design led to cost-effectiveness, energy-efficient and green cassava processing and biomaterials with zero-environment footprints. Desirable optimised bio-derivatives and films demonstrated application in desirable in-package O2/CO2, mouldgrowth inhibition, faster tablet excipient nutraceutical dissolutions and releases, and thymolencapsulated smooth antifungal coatings. Novel material resources, non-root peeling, zero-waste-elimination, and desirable standardised methodology present promising process integration tools for sustainable cassava biobased system development. Emerging design outcomes have potential applications to mitigate cyanide challenges and provide bio-derivative development pathways. Process system leads to zero-waste, with potential to reshape current style one-way processes into circular designs modelled on nature's effective approaches. Indigenous cassava components as natural material reinforcements, and SRRC processing approach has initiated a process with potential wider deployment in broad product research development. This research contributes to scientific knowledge in material science and engineering process design.

Social Tagging and the Next Steps for Indexing: Fordist Relexivity and Intertextuality

Social tagging, as a particular type of indexing, has thrown into question the nature of indexing. Is it a democratic process? Can we all benefit from user-created tags? What about the value added by professionals? Employing an evolving framework analysis, this paper addresses the question: what is next for indexing? Comparing social tagging and subject cataloguing; this paper identifies the points of similarity and difference that obtain between these two kinds of information organization frameworks. The subsequent comparative analysis of the parts of these frameworks points to the nature of indexing as an authored, personal, situational, and referential act, where differences in discursive placement divide these two species. Furthermore, this act is contingent on implicit and explicit understanding of purpose and tools available. This analysis allows us to outline desiderata for the next steps in indexing.

Impact de trois interventions: méthode mère kangourou, massage en incubateur et massage en position kangourou sur la croissance et le développement des enfants prématurés nés à moins de 33 semaines d’âge gestationnel

Cette recherche vise à étudier l’impact d’interventions réalisées par les parents dans l’unité néonatale de soins intensifs. Plus spécifiquement, le premier objectif est de documenter les effets différentiels de la Méthode Mère Kangourou « MMK » accompagnée ou non du Massage en incubateur «MI » ou du Massage en Position Kangourou « MPK » et des Soins Traditionnels «ST » accompagnés ou non du massage dans l’incubateur sur la croissance physique mesurée par le poids, la taille et le périmètre crânien pendant une période de 5 et 15 jours dans l’unité néonatale et l’impact à 40 semaines d’âge gestationnel. Le second objectif est de comparer, chez des enfants qui bénéficient de la « MMK » la valeur ajoutée du « MPK » ou du «MI » sur le neuro-développement à 6 et 12 mois d’âge corrigé de l’enfant. Un échantillon total de 198 enfants et leur famille a été recruté de la façon suivante dans trois hôpitaux de Bogota. Dans chaque hôpital, 66 sujets ont été répartis aléatoirement à deux conditions. Ces hôpitaux ont été choisis afin de tester les effets de diverses conditions expérimentales et de diminuer les bais de sélection. Dans chaque hôpital, deux techniques ont été assignées aléatoirement. Il s’agit, dans le premier, de la « MMK & MPK » vs « MMK & MI ». Dans le second, « MMK sans massage » vs « MMK & MI ». Dans le troisième, « MI » a été comparé aux « ST » ce qui implique une absence de contact physique continu des bébés avec leurs parents. Les résultats rapportés dans le premier article sont à l’effet que, dans le premier hôpital, il y a un effet compensatoire de l’intervention « MMK & MPK » sur la perte physiologique du poids de l’enfant prématuré dans les 15 premiers jours de vie avec un impact sur le poids à 40 semaines d’âge gestationnel, sur la durée du portage kangourou et sur la durée d’hospitalisation totale. Aucun effet sur le périmètre crânien ou la taille n’est apparu. Dans le deuxième hôpital, aucune différence significative n’est rapportée pour le poids sauf quand l’intervention est commencée après le 10ième jours de vie alors que l’enfant « MPK» semble grossir mieux que le «MMK avec MI». Finalement, dans le troisième hôpital il n’y a aucun effet du massage sur les variables anthropométriques, le groupe avec MI grossissant moins vite avec un léger impact sur le poids à 40 semaines. Cela pourrait être dû à la perte de chaleur due à l’ouverture de l’incubateur quand l’enfant est très immature. Dans le second article, les 66 enfants de l’hôpital sont répartis aléatoirement dans le groupe « MMK & MPK» vs le groupe « MMK & MI», ont complété, à 6 et 12 mois d’âge corrigé, un test de neuro-développement, le Griffiths. Les résultats à 6 mois ne montrent aucune différence entre les 2 interventions, mais a 12 mois le IQ semble dépendant du nombre de jours d’hospitalisation de l’enfant, cette durée d’hospitalisation correspond au temps que met l’enfant à se stabiliser physiquement et correspond également au temps que mettent la mère et l’enfant à s’adapter à la méthode kangourou. Une fois, l’adaptation kangourou réussie, la dyade mère enfant sort avec l’enfant toujours en position kangourou. Le temps d’hospitalisation correspond au temps que met l’enfant à être éligible à l’apprentissage de la MMK par la mère. À 12 mois les deux groupes montrent des résultats équivalents, mais des différences positives sont apparues pour le groupe « MMK & MPK» dans les sous échelle Coordination Oculo Manuelle et Audition et Langage du test Griffiths. Dans l’ensemble, les résultats suggèrent que la pratique des deux interventions non traditionnelles peut contribuer à une meilleure croissance physique dans nos cohortes. Le gain de poids du bébé, notamment, est affecté par l’intervention MPK (Hôpital 1) ou sans l’ajout du Massage (Hôpital 2). Par ailleurs, le massage en incubateur n’a pas de différence significative en comparaison aux soins traditionnels, ces interventions ont toutefois un impact mineur (tendances) sur le neuro développement à 6 et 12 mois d’âge corrigé dans cette étude.

Análisis sobre la destrucción y creación de valor dentro del sector de la salud en Bogotá

Una de las maneras que existen para lograr niveles de ingreso mayores y a su vez incrementar las posibilidades de perdurabilidad de las empresas es la generación de valor, el cual permite que las compañías tengan la capacidad de adaptarse a su entorno y así sacar provecho del mismo. Sin embargo, son pocas las herramientas con las que dichas empresas cuentan para determinar de manera efectiva la creación o destrucción de valor que se genera a través del tiempo. El presente proyecto tiene como fin utilizar la herramienta del Valor Económico Agregado (EVA, por sus siglas en inglés) para analizar específicamente la creación de valor en un periodo de cinco años en el sector de la salud en Bogotá, tomando como base de investigación los hospitales pertenecientes al nivel III de atención dentro del total de la red hospitalaria de la capital del país. De igual manera, el análisis permitirá realizar proyecciones acerca de la creación de valor, cuyos resultados serán utilizados como base para evaluar estrategias que permitan a las empresas de este sector mantener dicho valor a través del tiempo o revertir una situación desfavorable en el caso que los resultados arrojados evidencien una destrucción de valor dentro de las instituciones prestadoras de salud. Para llegar a comprender el alcance de estos resultados, este proyecto explicará en qué consiste el valor, cómo se mide en las empresas y la viabilidad y aplicación de estas medidas en el caso específico de las sociedades colombianas pertenecientes al sector de la salud.

Financing innovative start-ups in Portuguese context: what is the role of business angels networks?

Business angels provide both financing and managerial experience, which increase the likelihood of the survival of innovative start-ups. Over the last years, European countries with developing informal venture capital markets have seen governments support the creation of business angels networks (BANs) to increase and consolidate these markets. Using the Portuguese context to carry out the empirical work, this paper provides an assessment of value added provided by angels’ networks. A total of 88 useable responses were received and analysed using non-parametric statistical techniques. This paper demonstrates that is evidence of positive contribution of BANs in terms of bringing together investors and linking them with entrepreneur’s seeking finance. BANs played an important role in financing innovative start-ups also in peripheral regions. Results lead us to conclude that government support BANs would appear to be an effective mechanism to stimulate the angel market in developing informal venture capital markets. The conclusions of this paper are likely to have relevance for countries where there is growing interest in the potential of business angels as a means of financing innovative start-ups.

Non-Equilibrium Atmospheric Plasma As A Novel Route To Nanomaterial Synthesis And Processing For Biomedical Applications

Since last century, the rising interest of value-added and advanced functional materials has spurred a ceaseless development in terms of industrial processes and applications. Among the emerging technologies, thanks to their unique features and versatility in terms of supported processes, non-equilibrium plasma discharges appear as a key solvent-free, high-throughput and cost-efficient technique. Nevertheless, applied research studies are needed with the aim of addressing plasma potentialities optimizing devices and processes for future industrial applications. In this framework, the aim of this dissertation is to report on the activities carried out and the results achieved concerning the development and optimization of plasma techniques for nanomaterial synthesis and processing to be applied in the biomedical field. In the first section, the design and investigation of a plasma assisted process for the production of silver (Ag) nanostructured multilayer coatings exhibiting anti-biofilm and anti-clot properties is described. With the aim on enabling in-situ and on-demand deposition of Ag nanoparticles (NPs), the optimization of a continuous in-flight aerosol process for particle synthesis is reported. The stability and promising biological performances of deposited coatings spurred further investigation through in-vitro and in-vivo tests which results are reported and discussed. With the aim of addressing the unanswered questions and tuning NPs functionalities, the second section concerns the study of silver containing droplet conversion in a flow-through plasma reactor. The presented results, obtained combining different analysis techniques, support a formation mechanism based on droplet to particle conversion driven by plasma induced precursor reduction. Finally, the third section deals with the development of a simulative and experimental approach used to investigate the in-situ droplet evaporation inside the plasma discharge addressing the main contributions to liquid evaporation in the perspective of process industrial scale up.

3D electrocatalysts for the selective glucose oxidation

Nowadays, one of the most important scientific and environmental concern is to reduce global dependence on fossil fuels. The use of lignocellulosic biomass makes it possible to produce important platform molecules such as D-glucose, which is used to synthesize high value-added chemical products such as gluconic acid (GO) and glucaric acid (GA). Moreover, the electrocatalytic oxidation of glucose shows advantages compared to the classical synthesis route, such as the use of non-toxic reactants and milder conditions, making the process greener and more sustainable. In this work, electrocatalysts based on open-cell Ni metal foams were investigated for the glucose electrooxidation. They were used as supplied, oxidized at 500°C and 600°C, and after electrodeposition of Ni(OH)2. The electrocatalysts were characterized by cyclic voltammetry in NaOH solution 0.1M and in a basic D-glucose solution with different glucose concentrations (10mM and 50mM). The effect of the potential applied, the glucose concentration and the reaction time on conversion, selectivity and faradic efficiency were also investigated. 3D Ni electrocatalyst showed promising activity in the conversion of glucose towards gluconic acid, the sample calcined at 500°C showing the best results.

Theoretical and experimental studies of gold catalysts for glucose oxidation

The glucaric acid (GLA) has been identified as a “top value-added chemical from biomass” that can be employed for many uses; for instance, it could be a precursor of adipic acid, a monomer of Nylon-6,6. GLA can be synthetized by the oxidation of glucose (GLU), passing through the intermediate gluconic acid (GLO). In recent years, a new process has been sought to obtain GLA in an economic and environmental sustainable way, in order to replace the current use of HNO3 as a stoichiometric oxidant, or electrocatalysis and biochemical synthesis, which show several disadvantages. Thereby, this work is focused on the study of catalysts based on gold nanoparticles supported on activated carbon for the oxidation reaction of GLU to GLA using O2 as an oxidant agent and NaOH as base. The sol-immobilization method leads us to obtain small and well dispersed nanoparticles, characterized by UV-Vis, XRD and TEM techniques. Repeating the reaction on different batches of catalyst, both the synthesis and the reaction were confirmed to be reproducible. The effect of the reaction time feeding GLO as reagent was studied: the results show that the conversion of GLO increases as the reaction time increases; however, the yields of GLA and others increase up to 1 hour, and then they remain constant. In order to obtain information on the catalytic mechanism at the atomistic level, a computational study based on density functional theory and atomistic modeling of the gold nano-catalyst were performed. Highly symmetric (icosahedral and cubo-octahedral) and distorted Au55 nanoparticles have been optimized along with Au(111) and Au(100) surfaces. Distorted structures were found to be more stable than symmetrical ones due to relativistic effects. On these various models the adsorptions of various species involved in the catalysis have been studied, including OH- species, GLU and GLO. The study carried out aims to provide a method for approaching to the study of nanoparticellary catalytic systems.

Le nozioni autonome di diritto europeo nel sistema dell'imposta sul valore aggiunto

Il presente lavoro intende indagare la formulazione di nozioni autonome di diritto europeo nell’ambito dell’imposta sul valore aggiunto elaborate dalla Corte di giustizia. Più specificamente si intende proporre una tassonomia convenzionale delle nozioni autonome, utile alla loro sistematizzazione e miglior comprensione, fondata sulla distinzione fondamentale tra nozioni autonome e nozioni proprie. A questo scopo il lavoro propone un’ampia analisi delle nozioni nella giurisprudenza europea che vengono distinte a seconda che l’obiettivo finale dell’autonomia sia quello di limitare la discrezionalità applicativa degli Stati relativamente a ipotesi derogatorie, nel caso delle nozioni autonome in senso stretto; oppure che l’obiettivo ultimo sia quello di meglio definire l’ambito di applicazione di nozioni fondamentali nella struttura dell’imposta, nel caso delle nozioni proprie. Lo scopo del lavoro è dimostrare che la definizione funzionalistica risponde a obiettivi diversi, accomunati dal fatto di far valere comunque il primato dell'ordinamento europeo. L’analisi e la sistematizzazione proposte possono essere uno strumento utile nell’attuazione, interpretazione e applicazione delle nozioni. In questo senso, il lavoro esamina anche esempi tratti dalle esperienze nazionali che dimostrino, da un lato, che gli interventi della Corte relativi alle nozioni influenzano gli ordinamenti nazionali; dall’altro, che permangono ancora disallineamenti. L’analisi della giurisprudenza europea e dei regimi nazionali, infine, vuole dimostrare che l’elaborazione di nozioni autonome si inserisce nel processo verso un’armonizzazione sempre più incisiva in ambito IVA, di cui è protagonista la Corte di giustizia. Questo processo, cui l’elaborazione di un “tessuto concettuale europeo” contribuisce, comporta una limitazione della discrezionalità degli Stati, e quindi del principio di attribuzione, in nome dell’efficacia e della primazia del diritto europeo.

Optimization of an innovative T6 heat treatment for the AlSi10Mg alloy processed by Laser-based Powder Bed Fusion: effect on microstructure, mechanical properties, and tribological behavior

Laser-based Powder Bed Fusion (L-PBF) technology is one of the most commonly used metal Additive Manufacturing (AM) techniques to produce highly customized and value-added parts. The AlSi10Mg alloy has received more attention in the L-PBF process due to its good printability, high strength/weight ratio, corrosion resistance, and relatively low cost. However, a deep understanding of the effect of heat treatments on this alloy's metastable microstructure is still required for developing tailored heat treatments for the L-PBF AlSi10Mg alloy to overcome the limits of the as-built condition. Several authors have already investigated the effects of conventional heat treatment on the microstructure and mechanical behavior of the L-PBF AlSi10Mg alloy but often overlooked the peculiarities of the starting supersatured and ultrafine microstructure induced by rapid solidification. For this reason, the effects of innovative T6 heat treatment (T6R) on the microstructure and mechanical behavior of the L-PBF AlSi10Mg alloy were assessed. The short solution soaking time (10 min) and the relatively low temperature (510 °C) reduced the typical porosity growth at high temperatures and led to a homogeneous distribution of fine globular Si particles in the Al matrix. In addition, it increased the amount of Mg and Si in the solid solution available for precipitation hardening during the aging step. The mechanical (at room temperature and 200 °C) and tribological properties of the T6R alloy were evaluated and compared with other solutions, especially with an optimized direct-aged alloy (T5 alloy). Results showed that the innovative T6R alloy exhibits the best mechanical trade-off between strength and ductility, the highest fatigue strength among the analyzed conditions, and interesting tribological behavior. Furthermore, the high-temperature mechanical performances of the heat-treated L-PBF AlSi10Mg alloy make it suitable for structural components operating in mild service conditions at 200 °C.

The green finance apparatus. How ideology and planning steer sustainable capital flows

By investigating the inner working of leading financial institutions, and their dense interconnections, this thesis explores the evolution of traditional financial instruments like bonds to tackle sustainability issues. Building on fieldwork among green financiers, the thesis is based upon participant observation of working groups appointed to define standards for sustainable bonds. Engaging critical theory, one claim is that investors are increasingly recruited or interpellated by an emerging global green ideological apparatus, aimed at ensuring the reproduction of existing social relations. Taking stock of the proliferation of both public and private actors in the definition of green standards and practices, the thesis proposes that this green ideology is becoming hegemonic. Focusing on the case of green bond pricing, it suggests that environmental and climate labels and other financial green signifiers for financial products take on brand-like qualities. Crystallizing imaginaries, meanings, and forms of personhood, they play a fundamental role in what is defined as a dual process of valuation-cum-subjectivation. Identifying themselves as “green”, financiers valuate differently green and brown assets allowing a ‘green’ financial value to slowly come to matter. Yet, alongside their ideological role, green labels have come to be almost exclusively standardized with reference to specific Climate Scenarios (e.g. Net Zero). These scenarios coordinate the optimal path towards achieving a carbon neutral world and represent the quintessential example of socioeconomic planning, crucially undermining neoliberal ideas of ‘the market’ as the ultimate calculative device.

Macroeconomic Conditionality in Cohesion Policy: Added Value or Unnecessary Burden? European Policy Brief No. 13, November 2012

Macroeconomic conditionality has become one of the major elements in discussions on the future of EU cohesion policy. Such conditional-ity would make the cohesion budget dependent on EU economic governance rules. This would have advantages for economic governance and, to a lesser extent, the efficiency of cohesion policy and the EU’s Multiannual Financial Framework negotiations. Yet, conditionality also risks entailing serious disadvantages for the end beneficiaries and cohesion policy itself. If the EU decides to put macroeconomic conditionality in place, it needs to reconsider the design and agree on an ample cohesion budget.