Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.

Intelligent pipeline control - a simulation study in the automotive sector

Automotive producers are aiming to make their order fulfilment processes more flexible. Opening the pipeline of planned products for dynamic allocation to dealers/ customers is a significant step to be more flexible but the behaviour of such Virtual-Build-To-Order systems are complex to predict and their performance varies significantly as product variety levels change. This study investigates the potential for intelligent control of the pipeline feed, taking into account the current status of inventory (level and mix) and of the volume and mix of unsold products in the planning pipeline, as well as the demand profile. Five ‘intelligent’ methods for selecting the next product to be planned into the production pipeline are analysed using a discrete event simulation model and compared to the unintelligent random feed. The methods are tested under two conditions, firstly when customers must be fulfilled with the exact product they request, and secondly when customers trade-off a shorter waiting time for compromise in specification. The two forms of customer behaviour have a substantial impact on the performance of the methods and there are also significant differences between the methods themselves. When the producer has an accurate model of customer demand, methods that attempt to harmonise the mix in the system to the demand distribution are superior.

Deficiencias minerales en bovinos para carne: diagnóstico, caracterización y control

La demanda de una producción de alimentos cada vez mayor a nivel mundial sumado a la tecnificación y al ritmo acelerado del progreso de las explotaciones agropecuarias actuales hacen que el ganado deba soportar elevadas presiones de producción aumentando los requerimientos de nutrientes. Este es el caso de los minerales considerados actualmente elementos esenciales para los animales, aunque tradicionalmente fueron definidos como los nutrientes pobres de la nutrición y alimentación animal. Actualmente se ha demostrado con evidencia clínica y productiva, el importante rol metabólico de los minerales en el animal sano y productivo, como también se ha definido qué elemento mineral y porcentaje del mismo es requerido para el normal funcionamiento del organismo. Los macro-minerales (calcio, magnesio, fósforo, sodio, potasio, cloro y azufre) y los oligo-minerales (cobre, zinc, hierro, selenio, cobalto, iodo, manganeso, molibdeno y cromo) son elementos esenciales y necesarios para transformar la proteína y la energía de los alimentos en componentes del organismo o en productos animales como leche, carne, crías, piel, lana. Además, ayudan al organismo a combatir las enfermedades, manteniendo al animal en buen estado de salud. Se ha considerado a los minerales como el tercer grupo limitante en la nutrición animal, siendo a su vez, el que mayor potencial y menor costo tiene para incrementar la producción del ganado. Los minerales desempeñan funciones tan importantes como ser constituyentes de la estructura ósea y dental, de tejidos blandos y líquidos corporales. Están involucrados en el funcionamiento celular, siendo activadores de más de trescientas enzimas, constituyentes esenciales de vitaminas, hormonas y pigmentos respiratorios y facilitando la actividad de los microorganismos del rumen. Cuando el aporte de minerales en la ración no es el adecuado en calidad y/o cantidad se originan las deficiencias minerales, encuadradas dentro de las enfermedades metabólicas o enfermedades de la producción. Estas han sido informadas en casi todo el mundo y son responsables de importantes pérdidas económicas en los rodeos de bovinos para carne. Las deficiencias y/o desequilibrios minerales pueden causar los siguientes trastornos en los animales: bajo porcentaje de parición, mayor número de servicios por concepción, abortos, retenciones placentarias, incremento del intervalo entre partos, baja producción de leche, menor peso al nacimiento y al destete, menor porcentaje de destete, menor ganancia de peso, mayor incidencia de enfermedades infecciosas, fracturas espontáneas, diarrea, deformación de huesos y mortandad. Así cobra importancia el diagnóstico mediante el análisis de la sangre de los animales, del pasto y el agua que consumen y la caracterización de estas deficiencias en primarias o secundarias con el objetivo de poder realizar un control de las mismas mediante un adecuado plan de suplementación mineral acorde a las necesidades de los distintos establecimientos agropecuarios.

How to optimize operational shipping processes of non-production materials? The Volkswagen Autoeuropa case study

Field lab: Entrepreneurial and innovative ventures

Branch and Bound algorithms in greenhouse climate control

The horticultural sector has become an increasingly important sector of food production, for which greenhouse climate control plays a vital role in improving its sustainability. One of the methods to control the greenhouse climate is Model Predictive Control, which can be optimized through a branch and bound algorithm. The application of the algorithm in literature is examined and analyzed through small examples, and later extended to greenhouse climate simulation. A comparison is made of various alternative objective functions available in literature. Subsequently, a modidified version of the B&B algorithm is presented, which reduces the number of node evaluations required for optimization. Finally, three alternative algorithms are developed and compared to consider the optimization problem from a discrete to a continuous control space.

Lot production size problem and simulation of urban transport

OBJECTIVES AND STUDY METHOD: There are two subjects in this thesis: “Lot production size for a parallel machine scheduling problem with auxiliary equipment” and “Bus holding for a simulated traffic network”. Although these two themes seem unrelated, the main idea is the optimization of complex systems. The “Lot production size for a parallel machine scheduling problem with auxiliary equipment” deals with a manufacturing setting where sets of pieces form finished products. The aim is to maximize the profit of the finished products. Each piece may be processed in more than one mold. Molds must be mounted on machines with their corresponding installation setup times. The key point of our methodology is to solve the single period lot-sizing decisions for the finished products together with the piece-mold and the mold-machine assignments, relaxing the constraint that a single mold may not be used in two machines at the same time. For the “Bus holding for a simulated traffic network” we deal with One of the most annoying problems in urban bus operations is bus bunching, which happens when two or more buses arrive at a stop nose to tail. Bus bunching reflects an unreliable service that affects transit operations by increasing passenger-waiting times. This work proposes a linear mathematical programming model that establishes bus holding times at certain stops along a transit corridor to avoid bus bunching. Our approach needs real-time input, so we simulate a transit corridor and apply our mathematical model to the data generated. Thus, the inherent variability of a transit system is considered by the simulation, while the optimization model takes into account the key variables and constraints of the bus operation. CONTRIBUTIONS AND CONCLUSIONS: For the “Lot production size for a parallel machine scheduling problem with auxiliary equipment” the relaxation we propose able to find solutions more efficiently, moreover our experimental results show that most of the solutions verify that molds are non-overlapping even if they are installed on several machines. We propose an exact integer linear programming, a Relax&Fix heuristic, and a multistart greedy algorithm to solve this problem. Experimental results on instances based on real-world data show the efficiency of our approaches. The mathematical model and the algorithm for the lot production size problem, showed in this research, can be used for production planners to help in the scheduling of the manufacturing. For the “Bus holding for a simulated traffic network” most of the literature considers quadratic models that minimize passenger-waiting times, but they are harder to solve and therefore difficult to operate by real-time systems. On the other hand, our methodology reduces passenger-waiting times efficiently given our linear programming model, with the characteristic of applying control intervals just every 5 minutes.

Design of a new laboratory for quality control of mussel produced in Sagres

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Evaluación al sistema de control interno del departamento de producción del Centro Gráfico Salesiano de la Editorial Don Bosco

El trabajo que hemos desarrollado es una Evaluación al Sistema de Control Interno del Centro Grafico Salesiano de la Editorial Don Bosco. Esta investigación se desarrolló por la importancia que el Departamento de Producción tiene dentro de la Editorial; esto nos permitirá exponer las falencias, y dar sugerencias para mejorar su funcionamiento. Este estudio se enfoca en los procesos, procedimientos y tareas a cumplir por parte del personal, con productos de calidad, tiempos apropiados, sin desperdicio de materias primas, reducción de tiempos muertos y de costos de producción. En el CAPITULO I, se desarrollaron los conceptos del COSO 1, que serán útiles para nuestro estudio que describe las generalidades de cada uno de sus componentes y métodos de evaluación. En el CAPITULO II, realizamos una breve descripción de los aspectos generales de la empresa, para tener un mayor conocimiento del entorno. En el CAPITULO III realizamos la evaluación, del SCI al departamento productivo, utilizado técnicas de evaluación como las del flujograma, cuestionarios, y con la información obtenida realizamos un diagnóstico y emitimos un informe con conclusiones y recomendaciones. En el CAPITULO IV se finaliza con un planteamiento de conclusiones y recomendaciones que ayuden a mejorar el control de las actividades dentro del departamento de producción. Este estudio no solo servirá como fuente de observación por parte de la Editorial Don Bosco, sino también a empresas del mismo giro comercial, siendo capaz de dar lineamientos de observación de producción.

Microcystin-LR in Brazilian Aquaculture Production Systems

The growth of aquaculture production systems, mostly the sport-fishing kind, coupled with a lack of control, brings about concerns on the quality of water and food produced. The current paper determines which factors may trigger the growth of cyanobacteria, with subsequent concentrations of microcystins in collected water samples, at the surface and in the water column, from 10 aquaculture systems, during the dry and rainy seasons. The above is undertaken by measurements of biotic (counting of Chlorophyceae, cyanobacteria, and microcystin-LR [MC-LR]) and abiotic (total nitrogen and total phosphorus) factors. Because the water from the 10 aquaculture production systems had MC-LR concentrations that were highly correlated with Microcystis aeruginosa (M. aeruginosa) biomass, most MC-LR microcystins were produced by this species. The MC-LR concentrations and M. aeruginosa counting were positively correlated with nitrogen-to-phosphorus ratios and suggest that parameters may affect not only the M. aeruginosa biomass, but also MC-LR concentrations. Water Environ. Res., 82, 240 (2010).

Feeding strategy with alternate fasting and refeeding: effects on farmed pacu production

P>A 36-day trial was conducted to determine the effects of repetitive periods of food restriction and refeeding on growth and energy metabolism in pacu (Piaractus mesopotamicus). A total 264 juvenile fish (36.9 +/- 2.8 g) were fed with the experimental diet for 36 days using three regimes: (i) feeding daily to satiation (FD); (ii) no feed for 3 days, then feeding the same amount offered to the control groups for the next 3 days (NF/R controlled); and (iii) no feed for 3 days, then feeding to apparent satiation for the next 3 days (NF/R at satiation). The treatments were distributed into four tanks each. WG and SGR were higher in FD group. Fish refed showed hyperphagia just up to the second day of refeeding. The worst feed conversion rate and the lowest protein efficiency ratio were found in fish NF/R controlled. The lowest values of visceral fat somatic index were found in both fasted fish groups, particularly in NF/R at satiation. The LL and glycogen concentrations, and the hepatosomatic index were all elevated in both feed restricted fish. Muscle lipid showed a tendency to decrease after the cycle of fasting and refeeding. Plasma free fatty acids and glucose levels were elevated in fish subjected to feeding restrictions while serum triglycerides levels were reduced. Triiodothyronine levels were significantly depressed in fish from the NF/R-controlled group and remained at the same levels as the control fish in fish NF/R at satiation. Results indicated that fish subjected to cyclic periods of 3-day satiation or controlled feeding after 3-days of fasting were unable to achieve the final body weight of fish fed to satiation after 36 days.

The study of agricultural non-point source pollution control policy system

As the agricultural non-point source pollution(ANPSP) has become the most significant threat for water environmental deterioration and lake eutrophication in China, more and more scientists and technologists are focusing on the control countermeasure and pollution mechanism of agricultural non-point source pollution. The unreasonable rural production structure and limited scientific management measures are the main reasons for acute ANSPS problems in China. At present, the problem for pollution control is a lack of specific regulations, which affects the government's management efficiency. According to these characteristics and problems, this paper puts forward some corresponding policies. The status of the agricultural non-point source pollution of China is analyzed, and ANSPS prevention and control model is provided based on governance policy, environmental legislation, technical system and subsidy policy. At last, the case analysis of Qiandao Lake is given, and an economic policy is adopted based on its situation.

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.

Study of nanostructured targets for plasma production via laser ablation

Metal nanowires (NWs) - nanostructures 20-100 nm in diameter and up to tens of micrometers long - behave as waveguides when irradiated with light with wavelength much greater than their diameter. This is due to collective excitations of free electrons (plasmons) in the metal which couple to light and travel on the surface of the nanowire. This effect can be used to efficiently absorb laser pulses to produce dense and hot plasma on special nanostructured targets with metal nanowires vertically aligned on the surface. In this thesis work, nanostructured targets with different parameters (length, diameter, metal and fabrication process) have been irradiated with infrared laser light. X-ray flux emitted by the cooling plasma is measured during irradiation, and the depth of craters formed on the target is measured later. This data is used to choose which target parameters are best for plasma production. Different targets are compared with each other and against a control, non-nanostructured (bulk) target. As will be shown, highly significant (> 5 sigma) differences are found between targets with different nanostructures, and between nanostructured and bulk target. This differences are very difficult to explain whithout accounting for the nanostructures in the targets. Therefore, data collected and analized in this thesis work supports the hypotesys that nanostructured targets perform better than bulk targets for laser plasma production purposes, and provides useful indications for optimization of NWS' parameters.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.

New weapons to fight old enemies: novel strategies for the (bio)control of bacterial biofilms in the food industry

Biofilms are microbial communities characterized by their adhesion to solid surfaces and the production of a matrix of exopolymeric substances, consisting of polysaccharides, proteins, DNA and lipids, which surround the microorganisms lending structural integrity and a unique biochemical profile to the biofilm. Biofilm formation enhances the ability of the producer/s to persist in a given environment. Pathogenic and spoilage bacterial species capable of forming biofilms are a significant problem for the healthcare and food industries, as their biofilm-forming ability protects them from common cleaning processes and allows them to remain in the environment post-sanitation. In the food industry, persistent bacteria colonize the inside of mixing tanks, vats and tubing, compromising food safety and quality. Strategies to overcome bacterial persistence through inhibition of biofilm formation or removal of mature biofilms are therefore necessary. Current biofilm control strategies employed in the food industry (cleaning and disinfection, material selection and surface preconditioning, plasma treatment, ultrasonication, etc.), although effective to a certain point, fall short of biofilm control. Efforts have been explored, mainly with a view to their application in pharmaceutical and healthcare settings, which focus on targeting molecular determinants regulating biofilm formation. Their application to the food industry would greatly aid efforts to eradicate undesirable bacteria from food processing environments and, ultimately, from food products. These approaches, in contrast to bactericidal approaches, exert less selective pressure which in turn would reduce the likelihood of resistance development. A particularly interesting strategy targets quorum sensing systems, which regulate gene expression in response to fluctuations in cell-population density governing essential cellular processes including biofilm formation. This review article discusses the problems associated with bacterial biofilms in the food industry and summarizes the recent strategies explored to inhibit biofilm formation, with special focus on those targeting quorum sensing.