Micoplasmas estão difundidos pela natureza e são caracterizados por um genoma relativamente pequeno, baixo conteúdo GC e ausência de parede celular e de algumas rotas biossintéticas. Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína, capaz de colonizar o trato respiratório e seu crescimento in vitro, comparado com o de outros micoplasmas, é mais lento. Mycoplasma hyorhinis também é encontrado no trato respiratório de suínos. Devido à falta de algumas vias biossintéticas, micoplasmas são incapazes de sintetizar alguns nutrientes e componentes essenciais, sendo forçados a obtê-los do ambiente. Assim, um dos maiores empecilhos enfrentados na pesquisa e diagnóstico de micoplasma tem sido a dificuldade do cultivo in vitro. Portanto, o desenvolvimento de um meio de composição definida que sustente o crescimento celular serviria como uma ferramenta controladamente manipulável, permitindo a definição de suas vias metabólicas assim como análises genéticas. O objetivo do presente estudo foi analisar a viabilidade, taxa de crescimento e regulação gênica de M. hyopneumoniae e M. hyorhinis em diferentes meios de cultivo, assim como em diferentes condições de cultura. Neste trabalho, foi utilizado o meio Friis (1975) como meio complexo, e quatro meios definidos: (i) meio descrito para Mycoplasma pneumoniae por Yus et al. (2009); (ii) meio Yus sem adição de peptona; (iii) meio comercial CMRL e (iv) meio CMRL+ (complementado com lipídeos, aminoácidos e vitaminas). A viabilidade celular foi avaliada em todos os meios definidos e a taxa de crescimento de ambas as espécies nos cinco meios foi avaliada por citometria de fluxo. Os resultados demonstraram que a composição do meio influencia no crescimento da bactéria, uma vez que há diferença entre a concentração celular em cada meio testado. Entretanto, ambas as espécies apresentaram concentração celular semelhante em cada meio. Os resultados demonstram que, dentre os meios definidos testados, o meio CMRL+, desenvolvido no presente estudo, é o meio mais adequado, podendo ser considerado um meio de manutenção para estes microrganismos. Para a avaliação da regulação gênica através de qPCR, M. hyopneumoniae foi cultivado em meio Friis e CMRL+ sendo posteriormente submetido a condições de estresse (choque térmico e estresse oxidativo). Os resultados obtidos sugerem que M. hyopneumoniae altera seus níveis transcricionais mais rapidamente quando cultivado em meio CMRL+, provavelmente devido ao estresse duplo causado pela privação nutricional e estresse oxidativo ou choque térmico. Na condição de choque térmico, o tipo de regulação predominante diferiu entre os dois meios, enquanto que, quando submetido a estresse oxidativo, os genes apresentaram um padrão de regulação semelhante entre Friis e CMRL+. O meio de cultivo definido CMRL+ forneceu uma taxa de crescimento semelhante à do meio complexo Friis, e demonstrou presença de regulação gênica em M. hyopneumoniae em resposta à sua composição e às condições de estresse testadas. Portanto, este meio pode ser usado como uma ferramenta para o avanço da pesquisa com Mycoplasma. / Mycoplasmas are widespread in nature, and are characterized by a relative small genome, low GC content, absence of cell wall and lack of some biosynthetic pathways. Mycoplasma hyopneumoniae is the infective agent of enzootic pneumonia in swine, able to colonize the respiratory tract. Its growth is slower than other porcine mycoplasmas. Mycoplasma hyorhinis f y y . Ty y, ’ f mycoplasma that grows in culture, and its presence can frequently prevent the isolation of other Mycoplasma spp. Due to the lack of some biosynthetic pathways, mycoplasmas are incapable of synthesize some nutrients and essential compounds, being forced to obtain from the environment. Therefore, the major impediment to Mycoplasma research and laboratory diagnosis has been the difficulty of in vitro cultivation. Thus, the development of a defined medium that support mycoplasma growth would provide a tool that can be controllably manipulated to enable the definition of mycoplasmal metabolic pathways as well as genetic analysis. The aim of this work was to analyze viability, growth rate and gene regulation of M. hyopneumoniae and M. hyorhinis in different culture media, and cultivation conditions. In this work, we used Friss broth (1975) as a complex medium, and four different defined media: (i) a medium described for M. pneumoniae by Yus et al. (2009), (ii) defined Yus medium without peptone, (iii) commercial medium CMRL and (iv) CMRL+ medium (supplemented with lipids, amino acids and vitamins). All the defined media were tested towards cell viability and the growth rate of both species in the five media was assessed, through flow cytometry assay comparing between them. The results from flow cytometry assay showed that the composition of the media influences the bacterial growth, once cell concentration in each of the tested media was different. However, both species presented similar cell concentration in each media. The results demonstrate that, amongst the defined media tested, CMRL+ broth, developed in this study, b , g ’ y b . To gene regulation assessment, M. hyopneumoniae was cultivated in Friis and CMRL+ and underwent two stress conditions (heat shock and oxidative stress). Results suggest that M. hyopneumoniae alters the transcriptional levels of some genes more promptly when cultivated in CMRL+ broth, probably due to the dual stress caused by the combination of nutrients deprivation in CMRL+ broth plus heat shock or oxidative stress. In the heat shock condition, the prevailing kind of regulation differed between the two media, while when submitted to oxidative stress, genes presented similar pattern of regulation between Friis and CMRL+. CMRL+ medium provided a growth rate resembling the complex broth and M. hyopneumoniae showed to have gene expression regulation in response to its composition and to the culture conditions tested. Thus, it can be used as a tool that can be controllably manipulated enabling the definition of mycoplasmal nutritional requirements and metabolic pathways as well as genetic analysis, such as gene regulation.

Les bactéries du genre Deinococcus sont des microorganismes qui présentent des propriétés remarquables de résistance aux conditions extrêmes telles que les radiations ionisantes, les stress oxydatifs, la dessiccation ou encore les températures extrêmes. L’exploitation de leurs capacités présente donc un réel intérêt pour les procédés biotechnologiques de production de métabolites d’intérêt, les biocarburants. Néanmoins, la physiologie et le métabolisme de ces microorganismes ont été très peu étudiés à ce jour. L’objectif de ce travail est d’identifier les exigences nutritionnelles d’une souche de déinocoques, Deinococcus geothermalis, dans le but de définir la composition d’un milieu synthétique permettant une croissance sans limitation. L’étude statistique de quarante huit formulations de milieux de culture a mis en évidence la variabilité qualitative et quantitative des compositions des milieux utilisés pour leur culture. Il ressort que l’ajout de facteurs stimulant la croissance, sous forme d’extrait de levure, est indispensable à la croissance non limitée de la souche Deinococcus geothermalis DSM 11300. En bioréacteur à 45 °C, sur un milieu complexe et substrat glucose, une concentration finale de 2,7 gMS.L-1 a été obtenue en six heures avec un taux de croissance égal à 0,65 h 1. Les résultats ont montré une variabilité intra-espèce importante ; parmi les trois souches de Deinococcus geothermalis étudiées, DSM 11300, DSM 11301 et DSM 11302, la souche Deinococcus geothermalis DSM 11302 présente la meilleure croissance en termes de nombre de générations, en milieu défini ou complexe. L’étude de l’influence des étapes de préparation de l’inoculum a permis de standardiser les conditions de préparation de l’inoculum préalablement aux cultures en bioréacteur. La souche Deinococcus geothermalis DSM 11302 présente une croissance exponentielle durant quatre heures en bioréacteur sur un milieu défini avec 10 g.L-1 de glucose ; le taux de croissance de 0,25 h-1 ne se maintient pas sur une durée plus longue. Le rendement de production de biomasse à partir du glucose atteint 0,25 gX.gGlc-1 soit 0,30 CmolX.CmolGlc-1. L’ajout de sources carbonées, sources soufrées, vitamines ou autres facteurs de croissance ne permet pas d’améliorer la croissance de la souche dans ces conditions. Sur ces bases, la quantification de la physiologie de Deinococcus geothermalis DSM 11302 a été étudiée lors d’une culture en mode discontinu alimenté avec une stratégie d’apport de deux substrats, l’extrait de levure et le glucose. Le mode de conduite a permis de révéler que le glucose n’est pas la source de carbone assimilée préférentiellement et que l’extrait de levure peut être consommé comme source azotée organique et/ou comme source carbonée. Les déinocoques sont caractérisés par un métabolisme principalement protéolytique : il a été possible de confirmer que l’extrait de levure est indispensable à la croissance non limitée de Deinococcus geothermalis DSM 11302. Avec cette stratégie d’alimentation co-substrats, 253 g d’extrait de levure et 26 g de glucose ont été ajoutés pour produire 99 gMS de biomasse soit 9,6 gMS.L-1 en six heures. Les vitesses spécifiques maximales de consommation des substrats, extrait de levure et glucose, atteignent respectivement 0,68 et 0,39 Cmol.CmolX-1.h-1. Le taux de croissance exponentiel maximal obtenu est égal à 1.05 h-1, meilleur résultat décrit à ce jour pour un déinocoque. Ces résultats contribuent ainsi à implémenter l’état des connaissances sur la physiologie et les exigences nutritionnelles de Deinococcus geothermalis et donnent accès aux valeurs de paramètres cinétiques et de rendements, données absentes de la littérature / Bacteria belonging to Deinococcaceae family are microorganisms with exceptional resistance properties to extreme environmental conditions such as ionizing radiations, oxidative stress, dehydration or extreme temperatures. These properties make them interesting targets for biotechnological processes for metabolite production like biofuels. Nevertheless, physiological behaviour and metabolism of these microorganisms have been little studied to date. The main objective of this work was to study the nutritional requirements for the growth of a Deinococcaceae strain, Deinococcus geothermalis, in order to define a synthetic medium sufficient for a non-limiting growth of this strain. The statistical study of 48 culture media formulations highlighted a lot of variability between the compositions used for their growth. A limiting growth of the strain Deinococcus geothermalis DSM 11300 was obtained on the defined medium DM chosen for this work, in Erlenmeyer flasks and in bioreactor, unless yeast extract was added to the medium. Cultivated in a bioreactor at 45 °C, on a complex medium with glucose (CMG), the biomass concentration reached 2.7 gMS.L-1 in six hours with a growth rate of 0.65 h-1. The results showed intra-specie variability; the strain Deinococcus geothermalis DSM 11302 exhibits the best number of generation in both complex and defined media. The study of the influence of inoculum preparation steps allowed standardizing the conditions of inoculum preparation before cultures in bioreactor. The strain Deinococcus geothermalis DSM 11302 has a four hours exponential growth when cultured in bioreactor on the defined medium DM at 45 °C, but the maximal growth rate of 0.28 h-1 decreases rapidly. The yield of biomass production on glucose reached 0.25 gX.gGlc-1 (0.30 CmolX.CmolGlc-1). Oxygen uptake and glucose specific uptake rates were 3.3 mmol.L-1.h-1 and 0.57 CmolGlc.CmolX.h-1 respectively. None of the carbon sources, sulfur sources or growth factors added to the defined medium DM could improve growth results of the strain Deinococcus geothermalis DSM 11302. For the quantification of Deinococcus geothermalis DSM 11302 physiology, a fed batch culture strategy with a yeast extract and glucose co-substrate feed was chosen. This strategy revealed that glucose is not the preferential carbon source for biomass production. In addition, yeast extract can be consumed as organic nitrogen source and/or as carbon source. Deinococcaceae are mainly proteolytic microorganisms, yeast extract is an essential element to obtain a non-limiting growth of Deinococcus geothermalis DSM 11302. With this co substrate feed strategy, 253 g of yeast extract and 26 g of glucose were added to produce 99 gMS of biomass or 9.6 gMS.L-1 in six hours. The maximal yeast extract and glucose specific uptake rate reached respectively 0.68 and 0.39 Cmol.CmolX 1.h 1. The maximal exponential growth rate is 1.05 h-1, which is the best result describes to date for a Deinococcaceae strain. These results implement the knowledge on physiology and nutritional requirements of Deinococcus geothermalis and give access to quantitative values for characteristic kinetic parameters and conversion yields

Haemophilus influenzae b (Hib) é uma bactéria patogênica causadora de pneumonia e meningite. Sua cápsula polissacarídica (PRP) é considerada como principal fator de virulência e utilizada como antígeno vacinal. Hib é fastidioso e requer micronutrientes para seu desenvolvimento. A finalidade deste trabalho é estabelecer o meio quimicamente definido para desenvolvimento de Hib e produção de PRP. Inicialmente, definiu-se um meio a partir de dados da literatura. Este meio foi estudado através do delineamento de Plackett-Burman de 44 ensaios, obtendo-se valores máximos de DO540nm de 5,0 UA, e 227,7 mg/L de PRP. A análise estatística revelou que EDTA, NH4Cl, Cys e PVA podem ser removidos do meio sem impactar os parâmetros estudados e que Glm, Hipoxantina, Inosina, Tiamina, Hemina e Tween 80 apresentam efeito significativo positivo para produção de PRP. Analisando os meios estudados, foi possível verificar que a composição do E44 possibilitou produzir o PRP a US$ 16,50/g, sendo considerado o meio quimicamente definido estabelecido neste trabalho. / Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes pneumonia and meningitis. Its capsular polysaccharide (PRP) is considered as a major virulence factor and used as vaccine antigen. Hib is fastidious and requires micronutrients for its development. The purpose of this study is to establish the chemically defined medium for Hib development and PRP production. Initially, a medium was defined based in the literature. This medium was studied by the Plackett-Burman design of 44 trials, achieving maximum values of DO540nm of 5.0 AU and 227.7 mg / L of PRP. Statistical analysis revealed that EDTA, NH4Cl, Cys and PVA can be removed from the medium without impacting the parameters studied and Glm, Hypoxanthine, Inosine, Thiamine, Tween 80 and Hemin exhibit significant positive effect on the PRP production. Analyzing the studied media, it was possible to verify that the composition of E44 enabled to produce PRP to $ 16.50/g, being considered the chemically defined medium established in this work.

In this study, firstly the effects of aspartic acid group amino acids -which were reported to be the potential bottleneck in serine alkaline protease (SAP) synthesis- on SAP production were investigated by substituting at a concentration range of 0-15 mM by using recombinant Bacillus subtilis carrying pHV1434::subC gene. All aspartic acid group amino acids except threonine inhibited SAP activity when CAA&amp / #8805 / 2.5 mM. The highest SAP activities with asparagine, aspartic acid, lysine, threonine, isoleucine and methionine werefound to be 1.89-, 1.87-, 1.61-, 1.48-, 1.4-, and 1.4-fold higher than the reference medium activity, respectively, when the concentration of each amino acid was CAA=0.25 mM. The product and by-product distributions in defined and complex media in SAP production were also analyzed and compared in order to obtain a depth in-sight on functioning of the metabolic reaction network. The highest SAP activity in complex medium was found to be 3&ndash / fold higher than defined medium activity, while, specific SAP production rate was 1.2- fold higher. The highest cell concentration in complex medium (CX= 14.3 g/dm-3) was 8.1-fold higher than that obtained in defined medium (CX= 1.75 g/dm-3). In both media, oxaloacetic acid was observed extracellularly and intracellularly. In complex medium there was also succinic acid in the extracellular medium indicating that the operation of TCA cycle was insufficient. In both media serine, valine and glycine were observed neither in the extracellular nor in the intracellular media indicating that the synthesis of these amino acids can be a secondary rate limiting step. In defined medium asparagine was present neither in the cell nor in fermentation broth whereas, methionine was observed in the cell in high amounts, probably due to the lower flux values towards asparagine. Thus, in defined medium the synthesis of asparagine can also be a potential bottleneck in SAP production in defined medium.

Haemophilus influenzae b (Hib) é uma bactéria patogênica causadora de pneumonia e meningite. Sua cápsula polissacarídica (PRP) é considerada como principal fator de virulência e utilizada como antígeno vacinal. Hib é fastidioso e requer micronutrientes para seu desenvolvimento. A finalidade deste trabalho é estabelecer o meio quimicamente definido para desenvolvimento de Hib e produção de PRP. Inicialmente, definiu-se um meio a partir de dados da literatura. Este meio foi estudado através do delineamento de Plackett-Burman de 44 ensaios, obtendo-se valores máximos de DO540nm de 5,0 UA, e 227,7 mg/L de PRP. A análise estatística revelou que EDTA, NH4Cl, Cys e PVA podem ser removidos do meio sem impactar os parâmetros estudados e que Glm, Hipoxantina, Inosina, Tiamina, Hemina e Tween 80 apresentam efeito significativo positivo para produção de PRP. Analisando os meios estudados, foi possível verificar que a composição do E44 possibilitou produzir o PRP a US$ 16,50/g, sendo considerado o meio quimicamente definido estabelecido neste trabalho. / Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes pneumonia and meningitis. Its capsular polysaccharide (PRP) is considered as a major virulence factor and used as vaccine antigen. Hib is fastidious and requires micronutrients for its development. The purpose of this study is to establish the chemically defined medium for Hib development and PRP production. Initially, a medium was defined based in the literature. This medium was studied by the Plackett-Burman design of 44 trials, achieving maximum values of DO540nm of 5.0 AU and 227.7 mg / L of PRP. Statistical analysis revealed that EDTA, NH4Cl, Cys and PVA can be removed from the medium without impacting the parameters studied and Glm, Hypoxanthine, Inosine, Thiamine, Tween 80 and Hemin exhibit significant positive effect on the PRP production. Analyzing the studied media, it was possible to verify that the composition of E44 enabled to produce PRP to $ 16.50/g, being considered the chemically defined medium established in this work.

The purpose of this research was to study the synthesis and remodeling of extracellular matrix (ECM) by fibroblasts with special emphasis on the culture environment (media composition and initial ECM composition) and the resulting mechanical integrity of the ECM. This was investigated by culturing fibroblasts for 3 weeks in a variety of culture conditions consisting of collagen gels, fibrin gels, or media permissive to the self-production of ECM (Cell-Derived Matrix), and quantifying the mechanics of the resulting ECM. The mechanical characteristics were related to the biochemistry of the resulting ECM, notably in terms of collagen accumulation and collagen fibril diameters. The ultimate tensile strength (UTS) of the collagen gels and fibrin gels at the end of the 3-week period was 168.5 ± 43.1 kPa and 133.2 ± 10.6 kPa, respectively. The ultimate tensile strength of the cell-derived matrices was 223.2 ± 9 kPa, and up to 697.1 ± 36.1 kPa when cultured in a chemically-defined medium that was developed for the rapid growth of matrix in a more defined environment. Normalizing the strength to collagen density resulted in a UTS / Collagen Density in these groups of 6.4 ± 1.9 kPa/mg/cm3, 25.9 ± 2.4 kPa/mg/cm3, 14.5 ± 1.1 kPa/mg/cm3, and 40.0 ± 1.9 kPa/mg/cm3, respectively. Cells were synthetically more active when they produced their own matrix than when they were placed within gels. The resulting matrix was also significantly stronger when it was self-produced than when the cells rearranged the matrix within gels that corresponded to a significantly larger fraction of non-acid and pepsin extractable collagen. These studies indicate that cell-derived matrices have potential both as in vitro wound healing models and as soft connective tissue substitutes.

L’ingénierie du tissu osseux vise à concevoir un substitut tissulaire associant des cellules ostéoprogénitrices à une matrice tridimensionnelle capable de promouvoir la reconstruction osseuse, ouvrant la voie au développement de thérapeutiques substitutives à la pratique de la greffe dont les limitations sont bien connues.Le but de ce travail a été de développer un nouveau produit d’ingénierie tissulaire (PIT) destiné à la régénération osseuse constitué i) d’une matrice tridimensionnelle poreuse constituée de polysaccharides naturels biodégradables, ii) de cellules souches adultes issues du tissu adipeux humain (ADSCs) et d’identifier les conditions de culture optimales permettant le développement d’un produit fonctionnel pour une utilisation clinique. Nos résultats montrent que l’architecture et la composition de la matrice macroporeuse polysaccharidique permet de guider la différenciation ostéoblastique des ADSCs, en l’absence de facteurs ostéogéniques, et notamment en conditions de culture dynamique, grâce à l’organisation cellulaire en agrégats promouvant les interactions cellulaires. Les ADSCs peuvent être marquées à l’aide de nanoparticules superparamagnétiques et suivies in vivo de façon non invasive par imagerie par résonnance magnétique (IRM) au sein des matrices après leur implantation en site sous-cutané chez la souris. Les images IRM montrent que le matériau permet de délivrer une partie des cellules au niveau du site d’implantation participant probablement à un processus de réparation tissulaire. Enfin, en vue d’applications cliniques, un milieu de culture sans sérum répondant aux conditions GMP (Good Manufacturing Practices) pour la différenciation ostéoblastique a été développé par un industriel et validé au cours de ce travail de thèse.En conclusion de ces travaux, l’association d’une matrice macroporeuse composée de polysaccharides avec des ADSCs dans des conditions de culture spécifiques, en conditions dynamiques, semble pertinente et prometteuse pour des applications cliniques en ingénierie du tissu osseux. / Bone tissue engineering may associate osteoprogenitor cells to a tridimensional scaffold that can promote tissue reconstruction in order to replace bone grafting strategies whose limitations are well known. This study aims to develop a new tissue-engineered construct for bone regeneration constituted by i) a tridimensional polysaccharide-based scaffold, ii) adult stem cells extracted from human adipose tissue and identify the best culture conditions needed to develop a functional construct for clinical use. Our results show that this macroporous scaffold offers, without any osteoinductive factors, a suitable architecture and composition for driving osteoblastic differentiation of ADSCs especially when placing the tissue-engineered construct in dynamic conditions, thanks to cell aggregate conformation promoting cell-to-cell interactions. Thanks to ADSCs labeling, the tissue-engineered construct can be tracked in vivo in a non invasive way by magnetic resonance imaging (MRI), after their subcutaneous implantation. Results evidenced that this scaffold behaves as a cell carrier for of holding in its own cell fraction and delivering another fraction to the site of implantation for inducing a better tissue regeneration process. Finally, a serum free medium meeting standards GMPs (Good Manufacturing Practices) has been developed for inducing ADSCs osteoblastic differentiation as a first step towards clinical application.In conclusion, this polysaccharide-based scaffold associated with ADSCs, cultured under low fluid flow in a new bioreactor device, could be a relevant and promising tissue engineered construct for bone tissue engineering applications.