Ce projet a pour objectif l'amélioration de souches châssis méthylotrophes capables de convertir le méthanol fourni comme source de carbone et d'énergie en biomasse et, à terme, en composés chimiques dans les conditions de production industrielle. Le méthanol est une alternative aux glucides en tant que matière première pour les fermentations industrielles, car son utilisation n'entre pas en concurrence avec les denrées alimentaires et il peut être produit par réduction chimique du CO2.Une condition préalable à la production efficace à grande échelle de composés d'intérêt est la stabilité de la souche méthylotrophe productrice dans des concentrations élevées de méthanol. À cette fin, deux souches méthylotrophes apparentées, Methylobacterium extorquens AM1 et TK 0001, qui croissent de façon optimale avec 1% de méthanol, ont été adaptées en culture continue à la prolifération en présence de concentrations de méthanol allant jusqu'à 10%. Ces adaptations ont été réalisées à l'aide des automates de culture GM3, qui permettent de maintenir des populations de microorganismes en croissance à long terme dans des conditions contrôlées.Les courbes de croissance enregistrées pour des isolats issus de populations évoluées ont montré une prolifération accrue en présence de 5 % de méthanol en comparaison avec les cellules non évoluées. Les isolats croissent de façon comparable mais non identique, ce qui suggère une hétérogénéité des cellules adaptées dans une même population.Le séquençage génomique de clones isolés à différentes étapes de l'évolution en concentration croissante de méthanol a révélé des profils mutationnels variés. En revanche, le gène metY codant pour la O-acétyl-L-homosérine sulfhydrylase est muté dans tous les isolats. L'enzyme codée catalyse une réaction secondaire en présence de méthanol produisant la méthoxinine, analogue de la méthionine, connue pour sa toxicité par son incorporation dans les protéines. Les résultats des tests enzymatiques réalisés sur les protéines MetY mutées montrent une perte de fonction presque complète avec le méthanol ainsi qu'avec les substrats naturels, suggérant la participation de MetY dans la toxicité du méthanol.Une analyse transcriptomique a été entreprise afin d'étudier l'expression génique de la souche évoluée G4105 de M. extorquens TK 0001 en réponse à une exposition brève ou prolongée à une concentration élevée de méthanol en comparaison avec la réponse de la souche sauvage. Les gènes impliqués dans la division cellulaire, la structure des ribosomes et des flagelles, la stabilité des protéines et l'absorption du fer montrent une différence d'expression entre les deux souches.Les variantes de M. extorquens TK 0001 adaptées à une concentration élevée de méthanol produisent plus de biomasse à partir de méthanol que les cellules de type sauvage. On peut supposer qu'un composé synthétisé via une voie dérivant du métabolisme central par des cellules adaptées pourrait être produit à partir de méthanol avec un rendement supérieur à celui atteint par des cellules de type sauvage. La production de D-lactate a été testée dans les souches sauvage et évoluée sur-exprimant la lactate déshydrogénase endogène. Les cellules évoluées produisent plus de lactate que la souche contrôle, confirmant ainsi l'intérêt de cette adaptation au méthanol. / The objective of this project was the development of enhanced methylotrophic chassis strains capable of converting methanol as carbon and energy source into biomass and ultimately into commodity chemicals under industrial conditions. Methanol is an alternative to carbohydrates as feedstock in industrial biotechnology as its use does not interfere with food supply and its production can start from CO2.A prerequisite for an efficient and large scale industrial fermentation is stable growth of the methylotrophic producer strain on high methanol concentrations. For this purpose, two closely related methylotrophic strains, Methylobacterium extorquens AM1 and TK 0001, which both have a growth optimum at about 1% methanol, were adapted in continuous culture to proliferate stably in the presence of methanol of up to 10% (v/v). The adaptations were conducted using GM3 devices enabling automated long term cultivation of microorganisms.Growth curves recorded for isolates obtained from evolved populations showed enhanced proliferation in the presence of methanol at 5% as compared with wild type cells. The isolates showed comparable albeit not identical growth pointing to heterogeneity among the adapted cells in the population.Genomic sequencing of isolated clones at different steps of the adaptation revealed differences in their mutation profiles. The gene metY coding for O-acetyl-L-homoserine sulfhydrylase was found to be mutated in all isolates. This enzyme undergoes a side reaction with methanol leading to the production of the methionine analogue methoxinine known to be toxic through incorporation into proteins.Enzymatic tests conducted with these mutants showed an almost complete loss of activity even with their natural substrates, validating the involvement of MetY in methanol toxicity.Transcriptomic analysis was performed to study the gene expression response of an evolved derivative of M. extorquens TK 0001 to short and long term exposure to high methanol and compared with the response of the ancestor strain. Genes implicated in cell division, ribosomal and flagellar structures, protein stability and iron uptake showed differences in expression patterns between the strains.The M. extorquens TK 0001 cells adapted to high methanol produced more biomass from methanol than the wildtype cells. This suggests that a compound synthesized through a pathway branching from the central metabolism would be produced in higher yield from methanol by the adapted cells compared to the wildtype cells. The production of D-lactate was tested for wildtype and evolved cells both overexpressing native lactate dehydrogenase. The evolved cells produced more lactate than the control cells, confirming the interest of this methanol adaptation.

<p dir="ltr">Corn stover is a promising resource for biofuels, but its high viscosity makes it challenging to convert into a pumpable slurry. This thesis covers the scale-up, techno-economic assessment, and life cycle assessment to ensure profitable and sustainable biofuel production. In this study, a slurry was created using corn stover pellets, enzymes, and deionized water with a moisture content of 16%. The viscosity of the slurry was measured at different concentrations and shear rates using an Anton Parr rheometer. Important parameters such as the consistency index and flow behavior index were obtained through linear regression, which were then used to predict energy usage by correlating them with slurry concentration. Steady and unsteady state mass and energy balances were conducted to determine operation time, verify power consumption, and provide values for techno-economic and life cycle assessments.</p><p dir="ltr">Our study found that slurry can be pumped up to 300g/L concentrations with a viscosity of less than 300 Pa*s at shear rate close to 3Hz. We established a correlation between consistency and flow behavior index with solids content, predicting power consumption in the mixing tank. Our findings enabled us to calculate the production cost of $0.0155 per pound of the pumpable slurry, which is 38% less than the sale price to biorefineries.</p><p dir="ltr">This study confirms that it is economically viable to convert corn stover pellets into pumpable slurry for biofuel production without pretreatment. Although the process faces challenges with viscosity, it ensures a steady supply of feed for the biorefinery and results in profitability. The research takes a comprehensive approach that includes benchtop scale-up and assessments, establishing the practicality and cost-effectiveness of slurry production. Experimental findings and correlation analysis provide accurate predictions of power consumption, while steady-state energy balance results closely align with predictions. Techno-economic and life cycle assessments further support the competitiveness of slurry conversion. In essence, this research offers a sustainable solution to challenges in energy generation.</p>

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by "leaky" expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR. / Master of Science

Mise en oeuvre de microréseaux de lectines naturelles et recombinantes dédiés au suivi de production des glycoprotéines d’intérêt thérapeutiqueLes anticorps thérapeutiques, biomédicaments dont le marché est en pleine expansion, sont des glycoprotéines obtenues en bioproduction dans des cellules eucaryotes. Leur N-glycosylation, cruciale pour la modulation des fonctions effectrices des anticorps, confère un effet pro-inflammatoire ou anti-inflammatoire aux anticorps. Les anticorps thérapeutiques recombinants actuellement commercialisés présentent des glycosylations dites tronquées (GlcNAc terminaux) ou non humaines (α-Gal, NeuGc) rendant les anticorps immunogènes et, par conséquent, réduisant notablement leur efficacité. Les Agences de Santé exigent de réduire cet effet secondaire. La société SiaMed’Xpress a développé un savoir-faire pour obtenir une glycosylation complète, incluant la sialylation terminale des N-glycanes par ingénierie des cellules eucaryotes. Il est nécessaire de disposer d’outils d’analyse permettant de suivre la qualité de la N-glycosylation au cours de la production.La modification du milieu de culture par ajout de suppléments nutritionnels permet une modulation supplémentaire de la glycosylation. L’utilisation de suppléments nutritionnels commerciaux augmentent le taux de production mais bloquent la glycosylation au stade G0(F) (GlcNAc terminaux) alors que l’utilisation d’un supplément propre à SiaMed’Xpress permet de poursuivre jusqu’à la galactosylation et la sialylation, mettant ainsi en évidence un carrefour métabolique clé dans la glycosylation. Pour suivre les modifications de glycosylation dans différentes conditions de production, un test utilisant des lectines a été développé. Un jeu de treize lectines naturelles et recombinantes a été sélectionné par analyse bio-informatique et intégré dans un glycotest. L’utilisation de ce glycotest pour l’analyse de la glycosylation de trois anticorps produits en présence de différents suppléments nutritionnels permet d’obtenir de façon rapide, fiable et reproductible les profils de glycosylation des anticorps. Ainsi, le glycotest développé est fonctionnel et permet de caractériser la glycosylation des anticorps thérapeutiques recombinants. / Development of natural and recombinant lectin microarrays for monitoring the production of glycosylated protein drugsTherapeutic antibodies, biologic drugs whose market is growing, are glycoproteins obtained though bioproduction in eukaryotic cells. Their N-glycosylation, which is crucial for the modulation of effector functions, confers a pro-inflammatory or anti-inflammatory effect to antibodies. Currently, marketed therapeutic recombinant antibodies have truncated (terminal GlcNAc) or non human (α-Gal, NeuGc) glycosylation, inducing immunologic reaction and, consequently, reducing their effectiveness. Health agencies demand to reduce such secondary effects. SiaMed’Xpress developped a knowledge to obtained a complete glycosylation, including terminal sialylation of N-glycans by engineering eukaryotic cells for production. It is now necessary to develop analysis tools to monitor the quality of the N-glycosylation during bioproduction.Modification of culture media with feeds allows to further modulate glycosylation. The use of marketed feeds increase the production rate but block the glycosylation process in the G0(F) state (terminal GlcNAc) whereas SiaMed’Xpress feed allows for galactosylation and sialylation, highlighting a metabolic key point during the glycosylation. To follow the glycosylation modifications under different conditions of production, an assay using lectins has been developed. A panel of 13 lectins, recombinants and naturals, has been determined by bio-informatic analysis and integrated into a glycotest. The use of this glycotest to analyse glycosylation of 3 antibodies produced with different feeds allows fast, reliable and reproductible glycosylation profils of these antibodies. So, this developped glycotest is functionnal and this using permits to monitore therapeutic recombinant antibody glycosylation during bioproduction.

Dans un monde où la course à l’innovation est de plus en plus rapide, il est important pour une entreprise innovante ou un laboratoire de recherche public de mettre en place une stratégie de protection et de valorisation de ses inventions qui soit performante. La protection des résultats par des brevets revêt une importance capitale pour le développement industriel des biotechnologies qui forment un secteur innovant et prometteur, et où la R&D exige des investissements financiers considérables. Au delà de cet intérêt fondamental, les brevets sont aussi une source de premier plan en matière d'informations technologiques, juridiques et stratégiques, pouvant être exploitées à travers des paysages brevets. Ces études constituent un outil privilégié d'aide à la décision en matière de stratégie de R&D puisqu’elles permettent de définir les axes de recherche des concurrents et les niches de développement technologique libres de droits de Propriété Intellectuelle. / In a world where the innovation race is increasing fast, it is of economic importance for an innovative company or a public research laboratory to develop a strategy for the protection and enhancement of its inventions is efficient Protection of results through patents is critical for the industrial development of biotechnology which are an innovative and promising sector where R&D requires considerable financial investments. Beyond this fundamental interest, patents are also a source of information on technological, legal and strategic, which can be exploited through patent landscapes. These studies are a key tool for decision supportin R&D since they allow to identify research strategies of competitors and technological niches free from of Intellectual Property rights.

La biologie synthétique est le domaine de la bioingénierie permettant de concevoir, de construire et de tester de nouveaux systèmes biologiques en réécrire le code génétique. Les circuits biologiques synthétiques sont des outils sophistiqués permettant de construire des réseaux biologiques pour des applications médicales, industrielles et environnementales. Cette thèse de doctorat porte sur le développement de voies métaboliques synthétiques conçues à l'aide d'outils informatiques. Ces voies métaboliques sont intégrées à la couche de régulation transcriptionnelle pour développer des biocircuits pour la bioproduction, la biodétection et la biocalcul dans des systèmes cellulaires et acellulaires. Les résultats obtenus durant cette thèse de doctorat révèlent le nouveau potentiel des voies métaboliques dans l'établissement de biocircuits synthétiques. Le volet bioproduction-biodétection de la thèse vise à développer un nouveau biocapteur pour un sucre rare utilisé pour améliorer l'activité catalytique d’enzyme dans la cellule (in vivo). Ce biocapteur a ensuite été implémenté dans un système acellulaire (in vitro) pour découvrir et optimiser le comportement de biocapteurs à base de répresseurs. Une fois optimisé en système acellulaire, notre biocapteur a été utilisé pour surveiller la production enzymatique de sucre rare. Le développement de biocapteurs procaryotes acellulaires, qui reposent principalement sur des répresseurs, permet d'accélérer et de rendre plus efficace le cycle “design-build-test” dans le prototypage des voies métaboliques dans les systèmes acellulaires. L'application de la biodétection des circuits métaboliques pour le diagnostic est la mise en œuvre et l'optimisation des transducteurs métaboliques dans le système acellulaire. Les transducteurs sont des voies métaboliques composées d'au moins une enzyme catalysant un métabolite indétectable en un inducteur transcriptionnel, augmentant ainsi le nombre de petites molécules biologiquement détectables. En tant que nouvelle approche pour effectuer des biocalculs, des circuits métaboliques ont été appliqués pour construire des additionneurs métaboliques et des perceptrons métaboliques. Dans la cellule, trois transducteurs métaboliques et un additionneur métabolique ont été construits et caractérisés. Les systèmes acellulaires permettent d’accélérer la caractérisation de circuits biologiques, de finement régler le niveau d’expression d’un ou plusieurs gènes et facilite l’expression de plusieurs plasmides simultanément. Ceci a permis de construire de multiples transducteurs pondérés et des additionneurs métaboliques. Le modèle basé sur des données expérimentales a permis de concevoir un perceptron métabolique pour construire des classificateurs binaires à quatre entrées. Les additionneurs, perceptrons et classificateurs peuvent être utilisés dans des applications avancées telles que la détection de précision et dans le développement de souches pour le génie métabolique ou la thérapeutique intelligente. / Synthetic biology is the field of engineerable life science and technology to design-build-test novel biological systems through reprogramming the code of DNA. Synthetic biocircuits are sophisticated tools to reconstruct biological networks for medical, industrial, and environmental applications. This doctoral thesis focuses on the development of synthetic metabolic pathways designed by computer-aided tools integrated with the transcriptional regulatory layer enabling bioproduction, biosensing, and biocomputation in whole-cell and cell-free systems. The achievements of this doctoral thesis bring attention to new potentials of metabolic pathways in the development of synthetic biocircuits. The bioproduction-biosensing section of the thesis is to build a novel sensor for a rare sugar used to improve the catalytic activity of its producing enzyme in the whole-cell system (in vivo). This sensor was then implemented in a TX-TL cell-free system (in vitro) as a proof of concept of a repressor based biosensor to discover and optimize the behavior of repressor based biosensors in the cell-free system that suffer from low fold repression. The optimized cell-free biosensor was then used to monitor the enzymatic production of the rare sugar. The development of cell-free prokaryotic biosensors which are mostly relying on repressors enables faster and more efficient design-build-test cycle in metabolic pathways prototyping in cell-free systems. The biosensing application of the metabolic circuits for diagnosis is the implementation and optimization of cell-free metabolic transducers. The transducers are metabolic pathways composed of at least one enzyme catalyzing an undetectable metabolite to a transcriptional inducer, hence expanding the number of biologically detectable small molecules in cell-free systems. Finally, as a radical approach to perform biocomputation, metabolic circuits were applied to build metabolic adders and metabolic perceptrons. In whole-cell system, three metabolic transducers and a metabolic adder (multiple transducers receiving multiple input metabolites and transform them into a common metabolite) were built and characterized. By taking advantage of cell-free systems in rapid characterization, high tunability, and the possibility of using tightly controlled multiple DNA parts, multiple weighted transducers and metabolic adders were implemented. The integrated model trained on the experimental data enabled the designing of a metabolic perceptron for building four-input binary classifiers. The adders, perceptrons and classifiers can be applied in advanced applications such as multiplex detection/precision medicine and in the development of designer strains for metabolic engineering or smart therapeutics.

Esta dissertação é o resultado de uma pesquisa sobre a categoria trabalho que principiou pelas obras escritas em conjunto por Antonio Negri e Michael Hardt, em especial a trilogia Império, Multidão e Commonwealth e se estendeu posteriormente para suas obras individuais e os autores que dialogam com Negri apoiando e criticando suas ideias e inovações. A motivação fundamental foi encontrar caminhos de mudança das relações assimétricas de poder e recompensa que reifica o trabalhador e sua vida cada vez mais levando sofrimento à maior parte dos seres humanos ao viverem uma vida menor considerando o potencial de aprender, produzir e ser sempre fonte e destino de mais vida. A pesquisa levou também à crítica construtiva dirigida sobretudo à ausência, na obra de Negri, do saber psicanalítico como instrumental para compreender a gênese do sujeito considerando quão fundamentais são as questões sobre trabalho e produção de subjetividade levantadas por Negri. / This dissertation is the result of a research over the category of labour initially focused on the work developed together by Antonio Negri and Michael Hardt, especially the trilogy Empire, Multitude and Commonwealth and was later extended to their individual work and to those authors that has stablished a dialogue with Negri as supporters and critics of his ideas an innovations. The primary motivation was to find ways to change asymmetrical relations of power and welfare which reifies the worker and his life more and more implying suffering for most of mankind as they live a minor life considering the potential of each one to learn, create and become a source and a destination of more life. The research has led to a constructive critique oriented overall to the absence of psychoanalytical knowledge as an instrument to understand the genesis of the subject, especially considering how essential the questions about labour and production of subjectivity raised by Negri are.

Esta dissertação é o resultado de uma pesquisa sobre a categoria trabalho que principiou pelas obras escritas em conjunto por Antonio Negri e Michael Hardt, em especial a trilogia Império, Multidão e Commonwealth e se estendeu posteriormente para suas obras individuais e os autores que dialogam com Negri apoiando e criticando suas ideias e inovações. A motivação fundamental foi encontrar caminhos de mudança das relações assimétricas de poder e recompensa que reifica o trabalhador e sua vida cada vez mais levando sofrimento à maior parte dos seres humanos ao viverem uma vida menor considerando o potencial de aprender, produzir e ser sempre fonte e destino de mais vida. A pesquisa levou também à crítica construtiva dirigida sobretudo à ausência, na obra de Negri, do saber psicanalítico como instrumental para compreender a gênese do sujeito considerando quão fundamentais são as questões sobre trabalho e produção de subjetividade levantadas por Negri. / This dissertation is the result of a research over the category of labour initially focused on the work developed together by Antonio Negri and Michael Hardt, especially the trilogy Empire, Multitude and Commonwealth and was later extended to their individual work and to those authors that has stablished a dialogue with Negri as supporters and critics of his ideas an innovations. The primary motivation was to find ways to change asymmetrical relations of power and welfare which reifies the worker and his life more and more implying suffering for most of mankind as they live a minor life considering the potential of each one to learn, create and become a source and a destination of more life. The research has led to a constructive critique oriented overall to the absence of psychoanalytical knowledge as an instrument to understand the genesis of the subject, especially considering how essential the questions about labour and production of subjectivity raised by Negri are.

The aim of this dissertation is to summarize the current ecological situation in the Czechia. A very important aspect of this alternative agriculture is its regional differentiation. Czechia has a lot of organic farms, the mian aim of this thesis is to map their location and determine the location is correct or not. NUTS 4 units will be used in mapping the distribution of ecological farms as they are the suitable units for making comparisons. The source of data on which this research is based is the Ministry of Agriculture and Control organization KEZ, o.p.s. The results of this research are displayed in the form of tables and pictures. Cartograms and cart-diagrams are used to display the geographical distribution of the organic subjects. The commentaries on the cartograms and cart-diagrams are the key part of this research. This dissertation also briefly looks comparison Czechia with other member states of the EU member states in organic farming and own research which aims to offer organic products in stores. C lick to buy N O W ! PDF-XCHANGE w w w .docu-track.c o m C lick to buy N O W ! PDF-XCHANGE w w w .docu-track.c o m

Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12. / Ph. D.