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Individuals living with depression and anxiety show systemic increases of the bacterial endotoxin lipopolysaccharide (LPS), which induces an inflammatory cascade, resulting in negative effects across the gut-brain axis (GBA). LPS administration in mice has previously been used as a rodent model of depression/anxiety. Flaxseed (FS) contains key bioactives, including an omega-3 fatty-acid, dietary fibre, and a poly-phenolic compound which all may attenuate the effects of LPS through modulation of the GBA. The objectives of this thesis were to examine the effects of LPS on the GBA in C57Bl/6 mice and to determine if dietary supplementation with FS and/or FS oil (FO) provided protection against the LPS challenge. The LPS-induced negative effects across the GBA were partially attenuated by dietary supplementation with FS, but not FO, through changes in microbiota composition/function and systemic-/neuro-inflammation. Therefore, the potential benefits of FS are independent of the oil or are synergistic of all bioactives.

In the past two decades, molecular cell biology has transitioned from a traditional descriptive science into a quantitative science that systematically measures cellular dynamics on different levels of genome, transcriptome and proteome. Along with this transition emerges the interdisciplinary field of systems biology, which aims to unravel complex interactions in biological systems through integrating experimental data into qualitative or quantitative models and computer simulations. In this dissertation, we applied various systems biology tools to investigate two important problems with respect to cellular activation dynamics and reprograming. Specifically, in the first section of the dissertation, we focused on lipopolysaccharide (LPS)-mediated priming and tolerance: a reprogramming in cytokine production in macrophages pretreated with specific doses of LPS. Though both priming and tolerance are important in the immune system's response to pathogens, the molecular mechanisms still remain unclear. We computationally investigated all network topologies and dynamics that are able to generate priming or tolerance in a generic three-node model. Accordingly, we found three basic priming mechanisms and one tolerance mechanism. Existing experimental evidence support these in silico found mechanisms. In the second part of the dissertation, we applied stochastic modeling and simulations to investigate the phenotypic transition of bacteria E.coli between normally-growing cells and persister cells (growth-arrested phenotype), and how this process can contribute to drug resistance. We built up a complex computational model capturing the molecular mechanism on both single cell level and population level. The paper also proposed a novel way to accelerate the phenotypic transition from persister cells to normally growing cell under resonance activation. The general picture of phenotypic transitions should be applicable to a broader context of biological systems, such as T cell differentiation and stem cell reprogramming. / Ph. D.

Mathematical models have long been used to describe complex biological interactions with the aim of predicting mechanistic interactions hard to distinguish from data. This dissertation uses modeling, mathematical analyses, and data fitting techniques to provide hypotheses on the mechanisms of immune response formation and function. The immune system, comprised of the innate and adaptive immune responses, is responsible for protecting the body against invading pathogens, with disease or vaccine induced immune memory leading to fast responses to subsequent infections. While there is some agreement about the underlying mechanisms of adaptive immune memory, innate immune memory is poorly understood. Stimulation with lipopolysaccharide induces differential phenotypes in innate immune cells depending on the strength of the stimulus, such that a secondary lipopolysaccharide encounter of a constant dose results in either strong or weak inflammatory cytokine expression. We model the biochemical kinetics of three molecules involved in macrophages responses to lipopolysaccharide and find that once a macrophage is programed to show a weak inflammatory response this cannot be reverted. Contrarily, a secondary lipopolysaccharide stimulus of a very high dose or applied prior to waning of the effects of the primary stimulus can induce a phenotype switch in macrophages initially programed to show strong inflammatory responses. Some pathogens, such as the hepatitis B virus, have developed strategies that hinder an efficient innate immune response. Hepatitis B virus infection is a worldwide pandemic with approximately 257 million chronically infected people. One beneficial event in disease progression is the seroclearance of hepatitis B e antigen often in combination with hepatitis B antibody formation. We propose mathematical models of within-host interactions and use them to predict that hepatitis B e antibody formation causes hepatitis B e antigen seroclearance and the subsequent reactivation of cytotoxic T cell immune responses. We use the model to quantify the time between antibody formation and antigen clearance and the average monthly hepatocyte turnover during that time. We further expand the study of hepatitis B infection, by investigating the kinetics of the virus under an experimental drug administered during a clinical trial. Available drugs usually fail to induce hepatitis B s antigen clearance, defined as the functional cure point of chronic hepatitis B infections. Drug therapy clinical trials that combined RNA interference drug ARC-520 with entecavir have shown promising results in reducing hepatitis B s antigen titers. We develop pharmacokinetic-pharmacodynamic models describing the mechanistic interactions of the drugs, hepatitis B virus DNA, and virus proteins. We fit the model to clinical trial data and predict that ARC-520 alone is responsible for the reduction of hepatitis B s and e antigens, while entecavir is the driving force behind viral reduction. This work was supported by Simons Foundation, Grant No. 427115, and National Science Foundation, Grant No. 1813011. / Doctor of Philosophy / Mathematical models have long been used to describe complex biological interactions with the aim of predicting interactions that explain observed data and informing new experiments. This dissertation uses modeling, mathematical analyses, and data fitting techniques to provide hypotheses on the mechanisms of immune response formation and function. The immune system, comprised of the innate and adaptive immune responses, is responsible for protecting the body against invading pathogens, such as viruses, bacteria, or fungi. If an immune response to a secondary pathogen encounter differs from the response when the body first encounters the specific pathogen, this is called immune memory. The mechanisms underlying the memory of immune responses are well understood in the context of adaptive immune responses, but less so for innate immune responses. Stimulation with lipopolysaccharide, a cell wall component of many bacteria, programs innate immune cells, such as macrophages, to be in one of two states, called phenotypes, depending on the strength of the stimulus. Based on their phenotype the macrophages show either a weak or strong inflammatory response upon a secondary lipopolysaccharide encounter of a constant dose. We model the biochemical kinetics of three molecules involved in macrophages responses to lipopolysaccharide. We find that once a macrophage is programed to show a weak inflammatory response this cannot be reverted. Contrarily, a secondary lipopolysaccharide stimulus that is either of a very high dose or applied before the effects of the primary stimulus have waned, can induce a phenotype switch in macrophages initially programed to show strong inflammatory responses. Some pathogens, such as the hepatitis B virus, have developed strategies that hinder an efficient innate immune response. Hepatitis B virus infection is a worldwide pandemic with approximately 257 million chronically infected people. Hepatitis B e antigen is a protein that infected liver cells release into blood and that impairs adaptive immune responses. It is considered a beneficial event in disease progression, and called hepatitis B e antigen clearance, when hepatitis B e antigen becomes indetectable in a patient's blood. We propose mathematical models of interactions between liver cells, the virus, hepatitis B e antigens and hepatitis B e antibodies, which neutralize the antigens. We predict that antibody formation causes antigen clearance and a reactivation of immune responses. We furthermore use the model to quantify the time between antibody formation and antigen clearance and the average number of liver cells killed during that time. We further expand the study of hepatitis B infection, by investigating the kinetics of the virus under an experimental drug administered during a clinical trial. Available drugs rarely induce hepatitis B s antigen clearance, but clinical trials that combined a novel drug, called ARC-520, with the commonly used drug entecavir have shown promising results in reducing hepatitis B s antigen titers in the blood of infected patients. Following the clearance of hepatitis B s antigen, a protein that is released by infected cells and impairs adaptive immunity, the body usually has the capability to control the infection without medication. We develop mathematical models describing the interactions of the drugs, hepatitis B virus, and virus proteins. We fit the model to clinical trial data and predict that ARC-520 alone is responsible for the reduction of hepatitis B s and e antigens, while entecavir is the driving force behind viral reduction.

Die Sepsis ist ein gefürchtetes Krankheitsbild, das in hochentwickelten Industrienationen mit einer hohen Mortalität verknüpft ist und damit zu den häufigsten Todesursachen gehört. Die Pathomechanismen dieses komplexen und heterogenen Krankheitsbildes zu entdecken, gehört momentan zu den Hauptinteressengebieten der Sepsisforschung. Da die Interpretation klinischer Studien aufgrund der Heterogenität des Patientenguts schwierig ist, kommt der Entwicklung adäquater Tiermodelle eine entscheidende Bedeutung zu. Die hierbei gängigen Tiermodelle in Mäusen weisen jedoch Unzulänglichkeiten auf, die die Übertragung der in Tierexperimenten gewonnen Daten auf den klinischen Kontext nur teilweise ermöglichen. Eine Brücke kann hierbei das Tiermodell der humanisierten Maus schlagen, in der, durch Transplantation mit humanen hämatopoetischen Stammzellen, ein humanes Immunsystem reift. Die vorliegende Arbeit beschäftigt sich mit der Fragestellung, inwieweit die humanen Immunzellen in der humanisierten Maus in der Lage sind, auf LPS als Stimmulus zu reagieren. Darüberhinaus wird die Nutzung der Endotoxämie in der humanisierten Maus als alternatives Sepsismodell im Bezug zum klinischen Kontext untersucht. Hierbei ergab sich eine mögliche Nutzung des Endotoxämiemodells in der humanisierten Maus zur genaueren Erforschung des Zytokinmilieus, sowie neuer Surrogatmarker wie Pentraxin 3. Bezüglich der Reaktion einzelner immunologischer Subpopulationen und deren Bedeutung für die Klinik scheint eine Untersuchung an Modellen, die eine B- und T-Zell-Reifung nachvollziehen können und in der murine Residualzellen möglichst gering vorhanden sind, als sinnvoll.

L’état fébrile et le vieillissement normal sont deux processus physiologiques conduisant à un déséquilibre hydrominéral de l'organisme. Ce déséquilibre se traduit par une déshydratation sévère qui peut être aggravée par des conditions climatiques comme nous l'avons vu durant l'été 2003. Dans les deux cas, fièvre et vieillissement, l'organisme répond par une stimulation du système hypothalamo-neurohypophysaire conduisant à l’augmentation de la libération de vasopressine ou hormone antidiurétique, qui pourrait prévenir une déshydratation possiblement critique. Cependant, les modalités d’activation des neurones vasopressinergiques (AVP) dans ces conditions restent inconnues.Le but des recherches réalisées dans cette thèse, a été de déterminer les mécanismes cellulaires et moléculaires responsables de l’activation des neurones vasopressinergiques (AVP) lors d’une réponse inflammatoire et au cours du vieillissement.Nous avons pu démontrer dans la première partie de ce travail que lors d’un épisode inflammatoire (mimé par une injection de lypopolysaccharide LPS) l’activité des neurones AVP est rapidement augmentée et cette activation est soutenue pendant plus de six heures. De plus, cette activation n’est pas due à un effet potentiel secondaire du LPS sur l'osmolarité plasmatique ou la pression artérielle. L’activation précoce des neurones AVP par le LPS semble être soutenue par l’IL-6 (qui mime les effets du LPS), puisque l’activation par le LPS est bloquée par une injection préalable d’anticorps anti-IL-6.Dans la seconde partie de ce travail, nous avons pu montrer le traitement chronique d’IGF-I chez le rat âgé permet de restaurer une fonction urinaire comparable à celle observée chez l’adulte, en agissant vraisemblablement directement sur les neurones AVP puisque le taux plasmatique d’AVP chez les rats âgés traités par l’IGF-I revient à des valeurs normales, i.e., équivalente à celle de rats adultes. Cette hypothèse est confortée par le fait que (i) les neurones AVP expriment le récepteur de l’IGF-I et qu’il n’y a pas de différence dans l’expression de ces récepteurs entre rats âgés et adultes, et (ii) les neurones AVP sont inhibés par l’IGF-I.Enfin, dans la dernière partie de ce travail, nous avons pu montrer que lors du vieillissement, les neurones AVP sont activés, ce qui se traduit par un taux plasmatique d’AVP élevé et un taux d’apeline très faible. De même, les astrocytes sont activés et ne présentent plus de plasticité morphofonctionelle. La microglie, en état d’alerte, ne semble pas jouer un rôle prépondérant dans cette suractivation neuronale et astrocytaire. De plus, cette suractivation neuronale est palliée par un traitement central par un anticorps anti-IL-6 ou un inhibiteur non sélectif des canaux TRPV. Cependant, un traitement central par un anticorps anti-IL-6 n’affecte pas l’expression des TRPV2 dans le noyau supra-optique (NSO). En conclusion générale, il apparait que :1/ L’IL-1 n’est pas le chef d’orchestre de tous les processus inflammatoires. En effet, dans le NSO, l’activation des neurones AVP est soutenue par l’IL-62/ La balance pro- / anti-inflammatoire est un élément importante du dysfonctionnement neuronal. Cependant, le facteur critique du dysfonctionnement des neurones AVP n’est pas la production excessive de facteurs inflammatoires mais l’insuffisante production compensatoire de facteurs anti-inflammatoires.3/ lors du vieillissement, la neuroinflammation responsable du dysfonctionnement des neurones AVP peut être qualifiée de type « chronique à bas bruit », processus dans lequel (i) la microglie, en alerte, voit sa réactivité décuplée lors d'une sollicitation inflammatoire supplémentaire; (ii) le cross-talk astrocytes-neurones est figé dans une configuration d'hyperactivité, semblable à celle observée à l'âge adulte en condition de stimulation physiologique soutenue (comme lors d'une déshydratation), mais qui empêche toute réponse appropriée du réseau à toute demande physiologique supplémentaire, quelle soit transitoire (comme la réponse à une injection aigüe de LPS ou de NaCl 9%) ou soutenue (déshydratation de 48h).Cependant, les données de la littérature montrent le rôle majeur de la microglie dans d'autres types de neuroinflammation dites à « haut bruit », et dont les effets délétères - qui vont du dysfonctionnement neuronal à la neuro-dégénérescence – trouvent leur origine dans la surexpression de molécules microgliales telles l'IL-1 ou le TNF. Pour tenter de comprendre les mécanismes cellulaires et moléculaires impliqués dans un tel dysfonctionnement et pour caractériser la nature du dysfonctionnement neuronal, nous avons mis au point un modèle pharmacologique de neuroinflammation à haut bruit, en injectant directement dans les NSO de l'IL-1. Nos données préliminaires montrent que le dysfonctionnement neuronal ainsi que les mécanismes cellulaires et moléculaires à l’origine de ce dysfonctionnement diffèrent de ceux observés lors du vieillissement : la microglie est activée et surexprime de nombreuses molécules inflammatoires, probablement à l’origine du dysfonctionnement neuronal (absence de pattern phasique, même lors d’une stimulation osmotique), puisque les astrocytes ne semblent pas être affectés. L’absence de pattern phasique à l’origine du faible taux d’AVP plasmatique traduit une perturbation des propriétés électrophysiologiques intrinsèques sous-tendant ce pattern phasique (récepteurs ; canaux ioniques) et/ou des afférences excitatrices (Glu ; ACh ; Na) ou inhibitrices (GABA) modulant cette activité phasique. / The fever and normal aging are two physiological processes leading to water and mineral imbalance in the body. This imbalance results in severe dehydration which can be aggravated by climatic conditions as we saw during the summer of 2003. In both cases, fever and age, the body responds by stimulating the hypothalamic-neurohypophysial system leading to increased release of vasopressin or antidiuretic hormone, which could possibly prevent dehydration criticism. However, the modalities of activation of vasopressinergic neurons (AVP) in these conditions remain unknown. The aim of the research done in this thesis was to determine the cellular and molecular mechanisms responsible for the activation of vasopressinergic neurons (AVP) during an inflammatory response and during aging. We showed ,in the first part of this work, that during an inflammatory episode (mimicked by an injection of lypopolysaccharide LPS) the activity of AVP neurons is rapidly increased and this activation is sustained for more than six hours. Moreover, this activation is not due to a potential secondary effect of LPS on plasma osmolarity and blood pressure. The early activation of AVP neurons by LPS seems to be supported by IL-6 (which mimics the effects of LPS), since activation by LPS is blocked by prior injection of anti-IL-6. In the second part of this work, we showed chronic treatment of IGF-I in old rats can restore bladder function similar to that observed in adults, presumably by acting directly on neurons AVP as the rate plasma AVP in aged rats treated with IGF-I returned to normal values, ie, equivalent to that of adult rats. This hypothesis is supported by the fact that (i) AVP neurons express the receptor for IGF-I and there is no difference in the expression of these receptors between adult and aged rats, and (ii) AVP neurons are inhibited by IGF-I. Finally, in the latter part of this work, we showed that during aging, the AVP neurons are activated, which results in increased serum AVP level and a very low rate of apelin. Similarly, astrocytes are activated and show more morphofunctional plasticity. Microglia does not seem to play a role in neuronal and astrocytic overactivation. Moreover, this neuronal overactivation is overcome by a central processing by an anti-IL-6 or a nonselective TRPV channels. However, an icv treatment by an anti-IL-6 does not affect the expression of TRPV2 in the supraoptic nucleus (SON). In general conclusion, it appears that: 1 / IL-1  is not the conductor of all inflammatory processes. Indeed, in the NSO, the activation of AVP neurons is sustained by IL-6 2 / the balance of pro-/ anti-inflammatory is significant in neuronal dysfunction. However, the critical factor in the dysfunction of AVP neurons is not the excessive production of inflammatory factors, but the insufficient production of compensatory anti-inflammatory factors. 3 / during aging, neuroinflammation responsible for the dysfunction of AVP neurons can be classified as type "chronic and low-grade" process in which (i) microglia, in alert, saw its reactivity increased tenfold during inflammatory additional solicitation; (ii) cross-talk astrocyte-neuron is stuck in a pattern of hyperactivity, similar to that observed in adulthood under conditions of sustained physiological arousal (such as in dehydration), but that would prevent the proper response network to any additional physiological demand, which is transient (as the response to acute injection of LPS or NaCl 9%) or sustained (48 h dehydration). However, literature data show the important role of microglia in other types of neuroinflammation called "high grade", and whose deleterious effects - ranging from neuronal dysfunction to neurodegeneration - are rooted in Microglial overexpression of molecules such as IL-1 or TNF  . In an attempt to understand the cellular and molecular mechanisms involved in such dysfunction and to characterize the nature of neuronal dysfunction, we have developed a pharmacological model of neuroinflammation high grade by injecting IL-1  directly into the SON. Our preliminary data show that neuronal dysfunction and the cellular and molecular mechanisms behind this dysfunction differ from those observed during aging: activated microglia overexpressing many inflammatory molecules, probably at the origin of neuronal dysfunction ( absence of phasic pattern, even during osmotic stimulation), since astrocytes do not appear to be affected. The absence of phasic pattern causing the low plasma AVP reflects a disturbance of intrinsic electrophysiological properties underlying the phasic pattern (receptors, ion channels) and / or afferent excitatory (Glu, ACh, Na) or inhibitory (GABA) modulating the phasic activity.

Les bactéries Pectobacterium sont classées parmi les agents pathogènes les plus importants économiquement pour la culture de la pomme de terre. Au cours des dernières années, une augmentation des maladies dues à ces bactéries a pu être observée. Le but de ce travail de thèse était d’analyser certains aspects de la diversité liés aux Pectobacterium sp à savoir : (i) la diversité génétique liée aux régulateurs du pouvoir pathogène de la bactérie (ii) la diversité de virulence et d’agressivité des souches de Pectobacterium vis-à-vis de leurs hôtes et (iii). Le rôle et la diversité des effets induits par le lipopolysaccharides (LPS), composants de la surface bactérienne de bactéries phytopathogène ou non phytopathogène. Ce travail de thèse souligne également le rôle potentiel que pourrait jouer ces molécules LPS dans le biocontrôledes Pectobacterium sp. Différentes expérimentations cellulaires et moléculaires allant de l’identification de la bactérie à la compréhension des voies de signalisation ont été utilisées. Les résultats obtenus nous ont permis de montré, en premier lieu que, le séquençage du gène pmrA, gène connu pour être impliqué dans la régulation du pouvoir pathogène desPectobacterium, est un outil moléculaire complémentaire d’identification de sous espèces de Pcc et pourrait être aussi un moyen efficace d’évaluation de la diversité génétique intraspécifique. Dans un second temps, nous avons montré que les cultures cellulaires d’Arabidopsis thaliana pourraient être un modèle végétal d’évaluation de l’agressivité des Pectobacterium. Ceci a été obtenu par quantification des cinétiques de trois paramètres associés à la pathogénie de ces bactéries à savoir : l’activité des pectate-lyases, déterminant majeur du pouvoir pathogène des Pcc, la fuite des électrolytes, considérée comme un marqueur précoce de la mort cellulaire, et la mort cellulaire des cultures elle-même. Enfin nous avons également montré que les effets induits par les LPS chez les cellules d’Arabidopsis thaliana sont dépendant du type bactérien. En effet Les résultats obtenus nous ont permis de mettre en évidence trois types de réponses différentes aux LPS en fonction de leur origine: les réponses identiques (régulation des flux d’ions), des réponses communes mais présentant des intensités et de cinétiques différentes (production de ROS, induction de gènes de défense) et des réponses spécifiques (induction d’une PCD, alcalinisation du milieu). Ces résultats indiquent que différentes voies de signalisation pourraient être activées chez Arabidopsis thaliana. Ils nous ont permis également de mieux comprendre l’implication des LPS dans le biocontrôle contre les Pectobacterium sp. / Pectobacterium are classified among the most economically important pathogens of culture of potato. Recently, an increase in diseases caused by these bacteria was observed. The work presented in this thesis has allowed highlighting some aspects on diversity associated with Pectobacterium sp namely: (i) genetic diversity related with the pathogenicity of thebacterium (ii) the diversity in virulence and aggressiveness of Pectobacterium strains on its hosts and (iii) the role and the diversity of effects induced by lipopolysaccharide (LPS), bacterial surface components of phytopathogenic and non- phytopathogenic bacteria. First, we have demonstrated that sequencing the pmrA gene, known to be involved in the regulation of pathogenicity of Pectobacterium, is an additional molecular tool for identification of Pectobacterium subspecies. Moreover, pmrA gene could be an effective tool for evaluation of genetic diversity within species. In a second time, we have showed that cell cultures of Arabidopsis thaliana, could be used as an alternative system to evaluate rapidly and efficiently the virulence of different Pectobacterium strains. This was achieved by quantification of different parameters associated to the pathogenesis of these bacteria namely, the activity of pectate lyases, major determinant of the pathogenicity of Pcc, the electrolyte leakage, considered an early marker of cell death and the cell death itself. Finally, our data further suggest the effects induced by LPS from different origin on Arabidopsis thaliana cells, could be different. Indeed, three different types of responses to LPS have been shown: the identical responses (regulation of ion flux), the common responses but having different intensities and kinetics (ROS production, induction defense genes) and specific responses (induction of PCD, alkalinization of the medium). These results indicate that different signaling pathways could be involved in Arabidopsis thaliana in response to LPS and allowed us to highlight the potential role of LPS in the biocontrol of Pectobacterium sp.

Foi investigada a utilização de Sistema Micelar de Duas Fases Aquosas (SMDFA) para remoção de lipolissacarídeos (LPS) de preparações contendo proteínas recombinantes de interesse farmacêutico, como a proteína verde fluorescente (GFPuv). Os SMDFA são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, que possibilitam seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, permitindo a remoção de LPS contaminante. Neste trabalho, foi realizada a implementação do método para a quantificação de LPS em amostras contaminadas e a obtenção de LPS e GFPuv puros a partir de cultivo de E. coli recombinante. Além disso, foi estudada a influência do Triton X-114 na metodologia de quantificação de LPS, e a adição de MgSO4, CaCl2, KI e (NH4)2SO4 na partição de GFPuv e LPS puros em SMDFA. E ainda, realizou-se um planejamento experimental (22) para avaliar os maiores KGFPuv e %RECGFPuv. O homogeneizado celular de E. coli foi testado nas melhores condições obtidas com o planejamento experimental. E finalmente, o processo por cromatografia de afinidade por íons metálicos (IMAC) foi empregado para investigar a adsorção de LPS em matriz IDA-Ca2+. Conforme os resultados obtidos, o TX-114 causou elevada interferência no método cinético cromogênico, em função da similaridade desta molécula com os LPS. Os LPS apresentaram partição preferencial para a fase concentrada em micelas, com altos valores de remoção, %REMLPS>98,0%. Ao contrário, a GFPuv foi recuperada preferencialmente na fase diluída, na qual existe maior volume disponível, resultando em valores de KGFPuv>1. A adição de sais ocasionou diminuição nos valores KGFPuv, provavelmente por causa da carga negativa que GFPuv adquiriu nas condições avaliadas. Os resultados do planejamento experimental mostraram que a melhor condição de partição obtida foi na região do ponto central, 4,0% (p/p) a 60,0°C, com KGFPuv>10. O processo por IMAC apresentou as maiores capacidades de adsorção de LPS-IDA-Ca+2 nas condições de menor pH e maior força iônica 4,0 e 1,0 mol/L, respectivamente. O processo de purificação por SMDFA empregado para a remoção de LPS contaminante presente em meio fermentado contendo GFPuv, demonstrou ser eficiente em recuperar a biomolécula-alvo na fase diluída e separar o principal contaminante na fase rica em micelas. Portanto, pode ser empregado como primeira etapa para a remoção de altas concentrações de LPS na purificação de proteínas hidrofílicas como a GFPuv. / The Aqueous Two-Phase Micellar System (ATPMS) was investigated for endotoxin (LPS) removal from preparations containing recombinant proteins of pharmaceutical interest, such as the green fluorescent protein (GFPuv). These systems usually consist of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the biomolecules partitioning according to its hydrophobicity. In this work, the implementation of the method for LPS quantification in contaminated samples was accomplished, as well as the obtaining of pure LPS and GFPuv from recombinant E. coli. Furthermore, the influence of Triton X-114 in the methodology for LPS quantification was studied, as the addition of MgSO4, CaCl2, KI, and (NH4)2SO4 into the partition of pure GFPuv and LPS in ATPMS. In addition, a statistical design (22) was carried out to evaluate the highest KGFPuv and %RECGFPuv. The E. coli cell lysate was tested under optimum conditions obtained with the statistical design. And, finally, the process by ionmetal affinity chromatography (IMAC) was used to investigate the adsorption of LPS in IDA-Ca2+ matrix. The results showed that the TX-114 caused high interference in the kinetic chromogenic method, according to the similarity of this molecule to LPS. The LPS showed preferential partitioning to the micellerich phase, with high values of removal, %REMLPS>98.0%. In the other hand, the GFPuv was preferentially recovered in the micelle-poor phase, in which there is greater volume available resulting in values of KGFPuv>1. The addition of salts caused a reduction in the values KGFPuv, probably because of the negative charge that the GFPuv acquired at the conditions evaluated. The results of the statistical design showed that the best partitioning condition obtained was in the central point region, 4.0% (wt/wt) at 60.0°C, with KGFPuv>10. The process by IMAC showed the highest adsorption of LPS-IDA-Ca+2 capacities at the conditions of lower pH and higher ionic strength 4.0 and 1.0 mol/L, respectively. The purification process for the LPS contaminant removal from E. coli fermentation containing GFPuv by ATPMS proved to be efficient in the recovering of target biomolecule in the micelle-poor phase, and separating the main contaminant in the micelle-rich phase. Furthermore, this system can be exploited as the first step for removal of higher LPS concentrations from hydrophilics protein purification.