Ce travail apporte une contribution au développement de la stratégie PAT pour les procédés de culture de cellules animales. Il propose l'amélioration de la compréhension et de la maîtrise de la culture de cellules Vero, dédiées à la production de vaccins viraux, et cultivées sur microporteurs dans un milieu sans sérum. Une première partie a permis de cribler les effets de certains groupes de composés du milieu de culture par le suivi de la croissance en microplaques. Puis, des études cinétiques et métaboliques plus approfondies, réalisées en spinners, ont montré que le métabolisme carboné des cellules Vero est saturé par l'accumulation intracellulaire de pyruvate et qu’il est peu orienté vers la croissance. Alors que le renouvellement du milieu ou l'ajout ponctuel de glutamine amélioraient la croissance cellulaire sans ré-équilibrer le métabolisme, la substitution du glucose et de la glutamine a permis de réduire l’apoptose et d'améliorer les performances métaboliques et de croissance. Par ailleurs, les spectroscopies diélectrique et proche-infrarouge ont été évaluées pour le contrôle en-ligne du procédé, en prenant en compte les particularités des cellules adhérentes. Nous avons montré leur capacité à évaluer les concentrations de cellules, de composés du milieu, et à détecter l'apoptose. Enfin, les principales améliorations par substitution de la glutamine ont été appliquées en bioréacteurs, à la production d'un vaccin prototype contre la dengue, dans des conditions proches de celles d'un procédé industriel. Ceci a permis de limiter les renouvellements de milieu pendant l’expansion cellulaire, sans compromettre la production de particules virales infectieuses / This work contributes to the development of the PAT strategy for animal cell culture processes. The aim of this study was to improve the understanding and the control of Vero cell culture, dedicated to the production of viral vaccines, and grown on microcarriers in serum-free medium. An initial study was performed to screen the effects of certain groups of compounds of the culture medium, by the cell growth monitoring in microplates. Then, kinetic and metabolic studies conducted in spinners flasks allowed to go further and to show that the Vero cell metabolism is saturated through the pyruvate intracellular accumulation and that it is not oriented toward growth. While media renewal or punctual addition of glutamine improve the cell growth without improving the metabolism balance, the substitution of glucose and glutamine allowed to reduce apoptosis and to improve growth and metabolic performances.Furthermore, dielectric and near-infrared spectroscopies have been evaluated for the in-line process monitoring, taking into account the particularities of adherent cells. We have demonstrated their ability to quantify cell concentrations, medium component concentrations, and to detect apoptosis. Finally, major improvements by substitution of glutamine have been applied to bioreactor culture to produce a dengue vaccine prototype, with culture conditions close to industrial process. In these cases, medium renewal during the cell expansion was removed without compromising the production of infectious viral particles

Aujourd’hui, la biologie des systèmes est confrontée aux défis de l’analyse de l’énorme quantité de données biologiques et à la taille des réseaux métaboliques pour des analyses à grande échelle. Bien que plusieurs méthodes aient été développées au cours des dernières années pour résoudre ce problème, ce sujet reste un domaine de recherche en plein essor. Cette thèse se concentre sur l’analyse des propriétés structurales, le calcul des modes élémentaires de flux et la détermination d’ensembles de coupe minimales du graphe formé par ces réseaux. Dans notre recherche, nous avons collaboré avec des biologistes pour reconstruire un réseau métabolique de taille moyenne du métabolisme cellulaire de la plante, environ 90 noeuds et 150 arêtes. En premier lieu, nous avons fait l’analyse des propriétés structurelles du réseau dans le but de trouver son organisation. Les réactions points centraux de ce réseau trouvés dans cette étape n’expliquent pas clairement la structure du réseau. Les mesures classiques de propriétés des graphes ne donnent pas plus d’informations utiles. En deuxième lieu, nous avons calculé les modes élémentaires de flux qui permettent de trouver les chemins uniques et minimaux dans un réseau métabolique, cette méthode donne un grand nombre de solutions, autour des centaines de milliers de voies métaboliques possibles qu’il est difficile de gérer manuellement. Enfin, les coupes minimales de graphe, ont été utilisés pour énumérer tous les ensembles minimaux et uniques des réactions qui stoppent les voies possibles trouvées à la précédente étape. Le nombre de coupes minimales a une tendance à ne pas croître exponentiellement avec la taille du réseau a contrario des modes élémentaires de flux. Nous avons combiné l’analyse de ces modes et les ensembles de coupe pour améliorer l’analyse du réseau. Les résultats montrent l’importance d’ensembles de coupe pour la recherche de la structure hiérarchique du réseau à travers modes de flux élémentaires. Nous avons étudié un cas particulier : qu’arrive-t-il si on stoppe l’entrée de glucose ? En utilisant les coupes minimales de taille deux, huit réactions ont toujours été trouvés dans les modes élémentaires qui permettent la production des différents sucres et métabolites d’intérêt au cas où le glucose est arrêté. Ces huit réactions jouent le rôle du squelette / coeur de notre réseau. En élargissant notre analyse aux coupes minimales de taille 3, nous avons identifié cinq réactions comme point de branchement entre différent modes. Ces 13 réactions créent une classification hiérarchique des modes de flux élémentaires fixés et nous ont permis de réduire considérablement le nombre de cas à étudier (approximativement divisé par 10) dans l’analyse des chemins réalisables dans le réseau métabolique. La combinaison de ces deux outils nous a permis d’approcher plus efficacement l’étude de la production des différents métabolites d’intérêt par la cellule de plante hétérotrophique. / Nowadays, systems biology are facing the challenges of analysing the huge amount of biological data and large-scale metabolic networks. Although several methods have been developed in recent years to solve this problem, it is existing hardness in studying these data and interpreting the obtained results comprehensively. This thesis focuses on analysis of structural properties, computation of elementary flux modes and determination of minimal cut sets of the heterotrophic plant cellmetabolic network. In our research, we have collaborated with biologists to reconstructa mid-size metabolic network of this heterotrophic plant cell. This network contains about 90 nodes and 150 edges. First step, we have done the analysis of structural properties by using graph theory measures, with the aim of finding its owned organisation. The central points orhub reactions found in this step do not explain clearly the network structure. The small-world or scale-free attributes have been investigated, but they do not give more useful information. In the second step, one of the promising analysis methods, named elementary flux modes, givesa large number of solutions, around hundreds of thousands of feasible metabolic pathways that is difficult to handle them manually. In the third step, minimal cut sets computation, a dual approach of elementary flux modes, has been used to enumerate all minimal and unique sets of reactions stopping the feasible pathways found in the previous step. The number of minimal cut sets has a decreasing trend in large-scale networks in the case of growing the network size. We have also combined elementary flux modes analysis and minimal cut sets computation to find the relationship among the two sets of results. The findings reveal the importance of minimal cut sets in use of seeking the hierarchical structure of this network through elementary flux modes. We have set up the circumstance that what will be happened if glucose entry is absent. Bi analysis of small minimal cut sets we have been able to found set of reactions which has to be present to produce the different sugars or metabolites of interest in absence of glucose entry. Minimal cut sets of size 2 have been used to identify 8 reactions which play the role of the skeleton/core of our network. In addition to these first results, by using minimal cut sets of size 3, we have pointed out five reactions as the starting point of creating a new branch in creationof feasible pathways. These 13 reactions create a hierarchical classification of elementary flux modes set. It helps us understanding more clearly the production of metabolites of interest inside the plant cell metabolism.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic organ that responds to its external environment. Cell signaling cascades are initiated within bone cells when changes in mechanical loading occur. To describe these molecular signaling networks that sense a mechanical signal and convert it into a transcriptional response, we proposed the mechanosome model. “GO” and “STOP” mechansomes contain an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. “GO” mechanosomes functions to promote the anabolic response of bone to mechanical loading, while “STOP” mechanosomes function to suppress the anabolic response of bone to mechanical loading. While much work has been done to describe the molecular mechanisms that enhance the anabolic response of bone to loading, less is known about the signaling mechanisms that suppress bone’s response to loading. We studied two adhesion-associated proteins, Src and Pyk2, which may function as “STOP” mechanosomes. Src kinase is involved in a number of signaling pathways that respond to changes in external loads on bone. An inhibition of Src causes an increase in the expression of the anabolic bone gene osteocalcin. Additionally, mechanical stimulation of osteoblasts and osteocytes by fluid shear stress further enhanced expression of osteocalcin when Src activity was inhibited. Importantly, fluid shear stress stimulated an increase in nuclear Src activation and activity. The mechanism by which Src participates in attenuating anabolic gene transcription remains unknown. The studies described here suggest Src and Pyk2 increase their association in response to fluid shear stress. Pyk2, a protein-tyrosine kinase, exhibits nucleocytoplasmic shuttling, increased association with methyl-CpG-binding protein 2 (MBD2), and suppression of osteopontin expression in response to fluid shear stress. MBD2, known to be involved in DNA methylation and interpretation of DNA methylation patterns, may aid in fluid shear stress-induced suppression of anabolic bone genes. We conclude that both Src and Pyk2 play a role in regulating bone mass, possibly through a complex with MBD2, and function to limit the anabolic response of bone cells to fluid shear stress through the suppression of anabolic bone gene expression. Taken together, these data support the hypothesis that “STOP” mechanosomes exist and their activity is simulated in response to fluid shear stress.

Indiana University-Purdue University Indianapolis (IUPUI) / Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.

<p dir="ltr">Skeletal muscle accounts for 40% of total body weight and the homeostasis of muscle tissue is critical in maintaining proper body function. Skeletal muscle develops during the embryonic stages from the muscle progenitor cells derived from the dermomyotome structure. The myogenic progenitor cells contribute to the primary myogenesis by forming the primary myotubes which are the founding structures that the secondary myogenesis continues to build on. A portion of the myogenic progenitor cells makes up the adult muscle stem cells residing in homeostatic muscle tissue. The adult muscle stem cells contribute substantially for the adult muscle regeneration. Due to the significance of the muscle tissue and the importance of muscle stem cells, dysregulation of the muscle homeostasis or the muscle stem cell homeostasis will result in severe pathological conditions such as myopathy.</p><p dir="ltr">Mitochondria are cellular organelles that are responsible for generating energy needed for cellular processes, especially for muscle tissue where muscle contraction requires the presence of ATP. On the other hand, mitochondria also serve as signaling molecules and provide macromolecules for the biosynthesis. FAM210A (Family With Sequence Similarity 210 Member A) protein was shown to impact the lean mass of human subjects yet a detailed study on the effect of FAM210A in skeletal muscle was not performed, nor has the molecular mechanisms through which FAM210A function been elucidated. Therefore, I take on the task to unveil the function of FAM210A in muscle development, muscle homeostasis and muscle stem cell behavior by using a combination of mouse models with different myogenic promoters to target <i>Fam210a</i> at different developmental stages.</p><p dir="ltr">In the first part of the thesis, I investigated the role of FAM210A in post differentiation myofibers. Using the <i>Myl1</i>^<em>Cre</em> driven deletion of <i>Fam210a</i>, I found that <i>Fam210a</i>^<em>MKO</em> had normal development before 3 weeks of age, but the growth was stagnant from 4 weeks on, and the mice did not survive past 8 weeks of age. I found that the assembly of the ribosomes in the <i>Fam210a</i>^<em>MKO</em> was defective, leading to impaired translation which attenuated the muscle atrophy phenotype. I identified through proteomics that the mitochondrial autophagy and proteostatic control pathways were significantly induced yet mitochondrial organization and energetic proteins were downregulated. Metabolomics analysis showed that the signaling metabolite acetyl-CoA was increased in the <i>Fam210a</i>^<em>MKO</em> which led to increased protein acetylation, specifically, we showed that the ribosomal proteins were hyperacetylated, and that the acetylation increase was elicited by the <i>Fam210a</i>-null mitochondria.</p><p dir="ltr">In the second part of the thesis, I investigated the function of FAM210A in muscle progenitor cells. In the <i>FamMKO</i> mice, I found that deletion of <i>Fam210a</i> from embryonic myogenic progenitor cells led to developmental arrest and postnatal death at day 6. In the <i>FamPKO</i> mice, I found that <i>Fam210a</i> is needed for adult muscle stem cell to contribute to regeneration. Loss of <i>Fam210a</i> leads to the regenerative defects when the muscle was exposed to injury cues. We further showed that <i>Fam210a</i> deletion in muscle stem cells resulted in disruption of the proteostatic control over muscle stem cell activation, thereby forbidding the translational increase necessary to facilitate activation and proliferation. Furthermore, I showed that <i>Fam210a</i> deletion leads to excessive OPA1 cleavage, which contributes to the regenerative failure of muscle stem cells as fusion is required for the mitochondrial network remodeling during regeneration. Therefore, <i>Fam210a</i> safeguards the mitochondrial network and proteostasis during regeneration.</p><p dir="ltr">In summary, my studies characterized the functional contribution of FAM210A during embryonic muscle development, muscle mass maintenance and adult muscle stem cell homeostasis. The regulation of FAM210A in these three processes impinge on the translational regulation. My studies further demonstrated the importance of mitochondrial regulated protein translation in skeletal muscle and muscle stem cells.</p>

<p dir="ltr">Newborn animals undergo a lot of early-life stress that heavily impact on their long-term growth, performance, and welfare. Typically, the stress would indirectly interfere with the capacity of these neonates to utilize dietary nutrients and consequently impact tissue growth and development. In piglets, weaning is a stressful situation characterized by disruption of intestinal epithelial cell development which causes poor digestion of solid feed and a negative impact on absorption of nutrients especially in the post-gastric region. In addition, weaning in piglets could cause an increase in cellular assault by reactive oxygen species thereby potentially causing gut leakiness and paracellular loss of nutrients along the intestinal tract. In broiler chickens, access to feed may take up to 72 h following hatching which may affect their gut development as well as their gut microbiota. After the first feed ingestion, there is a sharp increase in the gut microbiota which triggers an increase in the development of the immune system as well as the gut. There is continuous attention on the strategies and nutritional interventions to mitigate or ameliorate the adverse effects of early life stressors in these food animals, especially in broiler chickens and piglets. In the studies described in this dissertation, myo-inositol (purely supplemented or phytase-induced) and luteolin were tested as nutritional strategies to mitigate the effects of early-life stressors on growth and the potential mechanisms by which myo-inositol and luteolin regulate growth were investigated.</p><p dir="ltr">In study I, the effect of myo-inositol on growth in 128 postweaning piglets fed protein-deficient corn-soy diets was tested. There were 4 dietary treatments in a randomized complete block design with body weight as the blocking factor. The treatments consisted of 1 positive control (PC) diet formulated to meet all the nutrient requirements of the piglets with a 20% crude protein (CP); the remaining 3 diets were the negative control (NC) diets with a 3% reduction in CP, a 2 g/kg myo-inositol supplemented negative control diet (NC+INO), and phytase (3,000 FTU/kg) supplemented negative control (NC+PHY) diet. The results showed that phytase enhanced the apparent total tract digestibility (ATTD) of P in the weanling pigs. Myo-inositol supplementation in a protein-deficient diet improved (P < 0.05) porcine plasma myo-inositol concentration while an in vitro myo-inositol incubation with intestinal epithelial cells increased the expression of genes that encode for Claudin-1, Claudin-3, Claudin-4, ZO-1, NaPiIIb, GLUT2, and SLC7A2. The in vitro analysis of tight junction integrity in the IPEC-J2 cells indicated by the transepithelial electrical resistance and FITC-Dextran permeability showed an enhancement in response to myo-inositol treatment. Although the in vivo study found that myo-inositol did not improve growth performance or ATTD, the in vitro myo-inositol enhanced markers of gut health and function.</p><p dir="ltr">In study II, the effect of myo-inositol on the growth of broiler chickens was tested. In this study, there were 6 experimental treatments based on two dietary protein levels (PC and NC) and three supplement types (BASAL, INO, and PHY) resulting in a 2 x 3 factorial arrangement in a completely randomized design. A total of 384 broiler chickens comprising 6 treatments with eight replicates per treatment and 8 birds per replicate were used. The birds were fed a common starter diet for the initial 7 days after they arrived at the poultry unit followed by a 14-day trial. The protein-deficient diet decreased the feed efficiency of the birds. Phytase addition increased (P < 0.05) the apparent ileal digestibility (AID) and ATTD of P and Ca in both PC and NC groups. The jejunal gut morphology was enhanced by supplemental phytase as indicated by an increase in villus height and the ratio of the villus height-to-crypt depth, coupled with an increase in serum myo-inositol concentration caused by both myo-inositol and phytase. In conclusion, myo-inositol showed a differential influence on growth performance, nutrient digestibility, and gut morphology.</p><p dir="ltr">In study III, the effects of luteolin on weanling pigs and IPEC-J2 cells were examined. A total of 48 piglets were randomly allotted to two dietary treatments consisting of a control group and a luteolin (LUT)-supplemented dietary group for a 4-week trial. A weekly assessment of the growth performance and expression of specific proteins in the jejunal mucosa was performed. In each dietary group, 8 piglets were slaughtered at weeks 1, 2, and 4 postweaning to collect blood, jejunal and ileal mucosa, and tissues. Luteolin supplementation numerically improved the ADG and G:F of the pigs. Luteolin feeding altered the jejunal and ileal gut morphology with increased villi height (P < 0.05) and villus height-to-crypt depth ratio (VCR, P < 0.05) in the jejunum and decreased crypt depth in the ileum. The effect of luteolin on IPEC-J2 global proteome and phosphor-proteome showed that luteolin could potentially improve intestinal barrier integrity by enhancing the abundance of proteins important in cell growth and survival. </p><p dir="ltr">In summary, dietary supplementation with myo-inositol and luteolin could regulate growth and nutrient digestibility in broiler chickens and weanling pigs by enhancing the integrity of intestinal cells and facilitating the expression of nutrient transporters that are significant in the uptake of nutrients across the lining of the gastrointestinal tract. Phytase supplementation improves the P release from phytate in the diets thereby alleviating its loss.</p>

La sclérose en plaques est une maladie inflammatoire et démyélinisante caractérisée par la formation de lésions multifocales à travers le système nerveux central. Les lymphocytes pro- inflammatoires CD4 Th1 et Th17 sont considérés pathogéniques en sclérose en plaques et dans son modèle animal, l’encéphalomyélite auto-immune expérimentale. L’obésité et la composition du microbiome intestinal sont associées à une exacerbation de l’inflammation et à la progression de la sclérose en plaques, faisant des approches thérapeutiques métaboliques une méthode de choix afin d’améliorer l’évolution de cette maladie. La méthionine est un acide aminé essentiel métabolisé en S-adénosylméthionine (SAM), considéré comme le donneur universel de groupements méthyl, influençant l’expression génique. La restriction en méthionine est quant à elle associée à une diminution de la prise de poids et de l’inflammation, et in vitro, permet de réduire l’expansion des cellules Th17. Notre hypothèse est donc que diminuer l’activité du cycle de la méthionine permettrait de limiter la progression de l’encéphalomyélite autoimmune expérimentale en modulant le métabolisme des cellules T, la barrière hémo-encéphalique et le microbiome intestinal. Pour ce faire, l’encéphalomyélite autoimmune expérimentale est induite chez des souris mâles et femelles exposées à une diète réduite en méthionine ou contrôle afin d’effectuer une évaluation clinique quotidienne. Des expériences de cytométrie en flux, d’immunofluorescence et de séquençage d’ADN ribosomal 16S sont effectuées afin de déterminer l’activité des cellules immunitaires, la perméabilité de la barrière hémo-encéphalique et la composition du microbiome intestinal. Nos résultats montrent que la diète réduite en méthionine est associée à un délai significatif de l’apparition des symptômes neurologiques, avec une évolution clinique différente entre les mâles et les femelles. Ceci est associé à une diminution du nombre de cellules immunitaires infiltrant le système nerveux central, en plus d’influencer la composition du microbiome intestinal, suggérant des changements vers un profil plutôt anti-inflammatoire. La diète réduite en méthionine semble donc améliorer la progression de l’encéphalomyélite autoimmune expérimentale de manière différente selon les sexes et pourrait représenter une nouvelle voie thérapeutique contre la sclérose en plaques. / Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system with a sex bias towards women. Proinflammatory Th1 and Th17 cells are considered pathogenic in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Obesity, western diet and gut dysbiosis increase inflammation and influence the course of this disease. Contrarily, dietary methionine restriction is associated with lower weight gain and reduced inflammation. In vitro, T cells upregulate components of methionine metabolism, including S- adenosylmethionine, upon activation and proliferation, while methionine restriction in the milieu reduces the expansion of Th17 cells. Thus, we hypothesized that limiting the activity of the methionine cycle will improve experimental autoimmune encephalomyelitis course by modulating Th17 cells through sex-specific epigenetic mechanisms and modification of the gut microbiome. To test this hypothesis, active experimental autoimmune encephalomyelitis is induced in male and female mice exposed to a methionine restricted or control diet. Clinical scores, flow cytometry, immunofluorescence and 16S rRNA sequencing are used to characterize the properties of immune cells, the blood-brain barrier, and the gut microbiome. Our results show that dietary methionine restriction is associated with a significantly delayed onset of neurological symptoms, with clinical differences in disease evolution between males and females. This is associated with a reduced number of immune cells and pathogenic proinflammatory T cells in periphery, then in the central nervous system. Methionine restricted diet is moreover associated with modification of the gut microbiome, suggesting a shift towards an anti-inflammatory profile. Finally, methionine restriction also influences the expression of adherence molecules such as VCAM-1, present in the blood-brain barrier, suggesting a neuro-protective effect. Our data reveal that methionine restricted diet ameliorates the clinical course and neuroinflammatory processes in experimental autoimmune encephalomyelitis in a sex-dependent manner and could represent a new therapeutic avenue to improve multiple sclerosis course.

No description available.