Cette thèse est consacrée à l’étude d’une classe d’équations de réaction-diffusion avec advection non-locale. La motivation vient du mouvement cellulaire avec le phénomène de ségrégation observé dans des expérimentations de co-culture cellulaire. La première partie de la thèse développe principalement le cadre théorique de notre modèle, à savoir le caractère bien posé du problème et le comportement asymptotique des solutions dans les cas d'une ou plusieurs espèces.Dans le Chapitre 1, nous montrons qu'une équation scalaire avec un noyau non-local ayant la forme d'une fonction étagée, peut induire des bifurcations de Turing et de Turing-Hopf avec le nombre d’ondes dominant aussi grand que souhaité. Nous montrons que les propriétés de bifurcation de l'état stable homogène sont intimement liées aux coefficients de Fourier du noyau non-local.Dans le Chapitre 2, nous étudions un modèle d'advection non-local à deux espèces avec inhibition de contact lorsque la viscosité est égale à zéro. En employant la notion de solution intégrée le long des caractéristiques, nous pouvons rigoureusement démontrer le caractère bien posé du problème ainsi que la propriété de ségrégation d'un tel système. Par ailleurs, dans le cadre de la théorie des mesures de Young, nous étudions le comportement asymptotique des solutions. D'un point de vue numérique, nous constatons que sous l'effet de la ségrégation, le modèle d'advection non-locale admet un principe d'exclusion.Dans le dernier Chapitre de la thèse, nous nous intéressons à l'application de nos modèles aux expérimentations de co-culture cellulaire. Pour cela, nous choisissons un modèle hyperbolique de Keller-Segel sur un domaine borné. En utilisant les données expérimentales, nous simulons un processus de croissance cellulaire durant 6 jours dans une boîte de pétri circulaire et nous discutons de l’impact de la propriété de ségrégation et des distributions initiales sur les proportions de la population finale. / This thesis is devoted to the study for a class of reaction-diffusion equations with nonlocal advection. The motivation comes from the cell movement with segregation phenomenon observed in cell co-culture experiments. The first part of the thesis mainly develops the theoretical framework of our model, namely the well-posedness and asymptotic behavior of solutions in both single-species and multi-species cases.In Chapter 1, we show a single scalar equation with a step function kernel may display Turing and Turing-Hopf bifurcations with the dominant wavenumber as large as we want. We find the bifurcation properties of the homogeneous steady state is closed related to the Fourier coefficients of the nonlocal kernel.In Chapter 2, we study a two-species nonlocal advection model with contact inhibition when the viscosity equals zero. By employing the notion of the solution integrated along the characteristics, we rigorously prove the well-posedness and segregation property of such a hyperbolic nonlocal advection system. Besides, under the framework of Young measure theory, we investigate the asymptotic behavior of solutions. From a numerical perspective, we find that under the effect of segregation, the nonlocal advection model admits a competitive exclusion principle.In the last Chapter, we are interested in applying our models to a cell co-culturing experiment. To that aim, we choose a hyperbolic Keller-Segel model on a bounded domain. By utilizing the experimental data, we simulate a 6-day process of cell growth in a circular petri dish and discuss the impact of both the segregation property and initial distributions on the finial population proportions.

Le cancer du poumon non-à-petites-cellules (CPNPC) représente 85% des cas de cancer du poumon. Cependant, les modèles utilisés de culture cellulaire en deux dimensions (2D) représentent partiellement les caractéristiques physiopathologiques du CPNPC. Notre objectif est de réaliser un modèle in vitro plus représentatif des caractéristiques de ce type de cancer. La culture tridimensionnelle (3D) dans laquelle les cellules forment un sphéroïde, est considérée comme un modèle plus fidèle aux tumeurs retrouvées chez les patients grâce à la structure et aux interactions intercellulaires du modèle. Pour mieux représenter le microenvironnement tumoral, nous intégrons une lignée cellulaire de fibroblastes dans le sphéroïde de CPNPC, afin d’imiter l’interaction entre cellules cancéreuses et cellules stromales. En effet, bien que les fibroblastes représentent un faible pourcentage des cellules au sein des tumeurs de CPNPC, elles jouent un rôle prépondérant dans la biologie tumorale et la réponse aux médicaments. Nous avons testé différentes lignées de fibroblastes et différents ratios de co-culture afin de déterminer les conditions optimales de notre modèle de co-culture 3D. Après 7 jours de co-culture, les cellules cancéreuses démontrent un potentiel migratoire plus élevé lorsque mesuré dans des chambres de Boydens. Cet effet n’est dépendant ni de la prolifération, ni d’un changement de phase dans le cycle cellulaire. Nous avons caractérisé la localisation des fibroblastes au sein du sphéroïde par des expériences de microscopie à fluorescence. L’expression de la protéine α-SMA du cytosquelette a aussi été déterminée par immunofluorescence. Nous avons par la suite établi un modèle de co-culture sur 24 jours afin de maximiser la communication entre les cellules cancéreuses et les fibroblastes. Ce modèle de co-culture long terme a par la suite été analysé selon son contenu en cytokines, chimiokines et métabolites. Enfin, nous avons réalisé un criblage de médicaments au jour 24 de notre co-culture long terme afin d’évaluer une réponse thérapeutique des cellules cancéreuses, dans des conditions plus semblables au microenvironnement tumoral. / Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. However, the two-dimensional (2D) cell culture models used represent partially the pathophysiological characteristics of NSCLC. Our goal is to develop an in vitro model that is more representative of the characteristics of this type of cancer. The three-dimensional (3D) culture, in which the cells form a spheroid, is considered to be a model more faithful to the tumors found in patients due to the structure and intercellular interactions of the model. In order to better represent the tumor microenvironment, we are integrating a fibroblast cell line into the spheroid of NSCLC to mimic the interaction between cancer cells and stromal cells. Although fibroblasts represent a small percentage of cells in NSCLC tumors, they play a major role in tumor biology and drug response. We tested different fibroblast cell lines and co-culture ratios to determine the optimal conditions of our 3D co-culture model. After 7 days of co-culture, the cancer cells show a higher migratory potential when measured in Boydens chambers. This effect is not dependent on proliferation or change of phase in the cell cycle. We characterized the localization of fibroblasts within the spheroid by fluorescence microscopy experiments. The expression and localization of the cytoskeletal protein α-SMA was also determined by immunofluorescence. We then established a 24-day co-culture model to maximize communication between cancer cells and fibroblasts. This long-term co-culture model was subsequently analyzed for cytokine, chemokine and metabolite content. Finally, we performed drug screening on day 24 of our long-term co-culture to evaluate a therapeutic response of cancer cells under conditions more similar to the tumor microenvironment.

No description available.

Intimal hyperplasia (IH) is defined as the abnormal migration and proliferation of smooth muscle cells with associated deposition of extracellular matrix in the intimal layer. It is a natural response to endovascular injury induced by procedures such as angioplasty, stent implantation, or atherectomy. Research on the molecular pathways and mediators has led to the discovery of a variety of substances aimed to interrupt or attenuate IH. Heparin, low-molecular-weight heparin, aspirin, corticoids, angiotensin-converting enzyme inhibitors, cyclosporin, vascular endothelial growth factor (VEGF) and other agents have been investigated. However, none of these agents has been used with marked success at the clinical level. Therapeutic angiogenesis studies have demonstrated the potential of heparin binding angiogenic growth factors such as VEGF and basic fibroblast growth factor (bFGF/FGF-2) to treat ischemic heart diseases. Studies on endothelial nitric oxide synthase (eNOS) gene transfer in vivo showed attenuated IH caused by constitutive generation of nitric oxide (NO) via the NOS pathway. FGF-2 increased VEGF mRNA levels in single-cultures of rabbit smooth muscle cells (SMC) and also promoted NO production from endothelial cells (EC). Therefore, we hypothesize that FGF-2 mediates SMC inhibition through the NOS pathway. In order to elucidate the influence of these growth factors, we employed an appropriate SMC-EC co-culture system.Studies on SMC-EC interactions have been established in various in vitro co-culture systems. However, there exist only few co-culture systems in which the structure of a vessel wall is imitated. A direct co-culture model was used in this study to determine the effect of growth factors on the SMC and EC proliferation. In the following study we investigate the effects of VEGF, FGF-2 and FGF-2+VEGF on porcine aorta smooth muscle and endothelial cells. Addition of the higher concentrations (>10 ng/ml) of FGF-2 to the SMC-EC direct co-culture greatly reduced smooth muscle cell numbers and cell cycle S-phase, as judged by propidium iodide DNA analysis using flow cytometry. We also observed that coadministration of FGF-2 with VEGF did not show any difference on SMC proliferation compared to control. These data demonstrate the potent regulatory capabilities of FGF-2 on smooth muscle cell inhibition. Nitric oxide which is generated by the enzyme NOS is hindered by the addition of NOS inhibitor, NG-Methyl-L-arginine acetate (L-NMMA). Utilizing L-NMMA we found that FGF-2 mediated smooth muscle cell inhibition does not follow the NOS pathway. This study is intended to understand the interactions of combination of therapeutic growth factors on vascular cells. The current study is a first step towards an overall goal of setting up an in vivo porcine model for clinical treatment of IH using FGF-2.

Bacteriocinas produzidas por bactérias láticas (BAL) apresentam um importante potencial de aplicação na bioconservação de alimentos, por sua ação antimicrobiana contra algumas espécies de microrganismos patogênicos de relevância, como Listeria monocytogenes. Este estudo analisou o efeito da interação entre cepas selecionadas de BAL produtoras de bacteriocinas com outras BAL viáveis ou não viáveis (bacteriocinogênicas ou não) na indução da produção de bacteriocinas. O efeito dos metabólitos produzidos por estas cepas na indução da bacteriocinogênese também foi avaliado. As cepas produtoras de bacteriocinas selecionadas para o estudo foram Lactobacillus sakei MBSa1, produtora de sakacina A e Pediococcus acidilactici ET34, produtora de pediocina, isoladas de salame e salmão defumado, respectivamente. A produção de pediocina por P. acidilactici ET34 foi avaliada também em leite em pó desnatado reconstituído, além de meio de cultura (caldo MRS). Os resultados indicaram que, quando em co-cultura com Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 ou Listeria monocytogenes (cepas 104, 711 e 637), ou na presença do sobrenadante livre de células (SLC) dessas culturas, nenhuma das duas cepas testadas produziu maior quantidade de bacteriocina do que a produzida quando em monocultura ou na ausência do SLC. A bacteriocina produzida por P. acidilactici ET34 apresentou um efeito bacteriostático contra L. monocytogenes 104 no leite em pó desnatado reconstituído nas 12 h analisadas, com extensão da fase lag, de forma dose-dependente. Os resultados indicaram, também, que P. acidilactici ET34 não foi capaz de produzir pediocina no leite em pó desnatado reconstituído quando em monocultura ou em co-cultura, ao contrário do observado para o caldo MRS. Mais investigação é necessária para esclarecer os efeitos de possíveis interações entre as BAL presentes em um alimento, bem como o efeito dos componentes dos alimentos na produção das bacteriocinas pelas BAL bacteriocinogênicas. / Bacteriocins produced by lactic acid bacteria (LAB) present an important application potential in food biopreservation, by their antimicrobial activity against some species of pathogenic microorganisms of relevance, such as Listeria monocytogenes. This study analyzed the effect of the interaction between selected strains of bacteriocin-producing LAB with other viable or non-viable LAB (bacteriocinogenic or not) in the induction of bacteriocin production. The effect of the metabolites produced by these strains on the induction of bacteriocinogenesis was also evaluated. The bacteriocin-producing strains selected for the study were Lactobacillus sakei MBSa1, producer of sakacin A and Pediococcus acidilactici ET34, producer of pediocin, isolated from salami and smoked salmon, respectively. The production of pediocin by P. acidilactici ET34 was also evaluated in reconstituted skimmed milk powder as well as culture medium (MRS broth). The results indicated that when co-cultivated with Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 or Listeria monocytogenes (strains 104, 711 and 637), or in the presence of the cell free supernatant (SLC) of these cultures, neither of the two strains tested produced greater amount of bacteriocin than that produced in monoculture or in the absence of SLC. The bacteriocin produced by P. acidilactici ET34 presented a bacteriostatic effect against L. monocytogenes 104 in skimmed milk powder reconstituted in 12h, with extension of lag phase, in a dose-dependent manner. The results also indicated that P. acidilactici ET34 was not able to produce pediocin in the reconstituted skimmed milk powder when in monoculture or in co-culture, unlike that observed for the MRS broth. More research is needed to clarify the effects of possible interactions between BAL present in a food and the effect of food components on bacteriocin production by bacteriocinogenic BAL.

A endometriose é caracterizada pela presença de tecido endometrial fora do útero. No estudo, utilizou-se um sistema de co-cultivo 3D contendo células do estroma e endotélio endometrial. O sistema foi exposto ao soro de mulheres saudáveis ou com endometriose, que fazem ou não o uso de anticoncepcional. Posteriormente, foi avaliada a resposta do sistema no que diz respeito ao perfil de citocinas. Nossos resultados mostraram que o perfil sérico das mulheres com endometriose sem anticoncepcional apresentaram elevados níveis séricos de IL2 e IL10 com relação às saudáveis. Enquanto que as mulheres com endometriose, que fazem uso de anticoncepcional, apresentaram altos níveis de IL6 e IL8. O perfil de citocinas encontrado no homogenato das mulheres com endometriose induziu a produção exacerbada de IL2 e IL10 além de IFN-γ, TNF-α, IL6. Quando as células eram incubadas com soro de mulheres com endometriose que fazem tratamento, os níveis de IL6 e IL8 aumentaram. Os achados sugerem que o uso de co-cultivos 3D de células estromais e endoteliais endometriais é um bom modelo para novos estudos. / Endometriosis is characterized by the presence of endometrial tissue outside the uterus. In this study, we used a 3D co-culture system with stromal and endothelium endometrial cells. The system was exposed to serum of healthy women or with endometriosis do or not use contraceptive. Subsequently, he studied the response of endometrial cells with respect to the cytokine profile. Our results showed that the serum profile of women with endometriosis without contraceptive had high serum levels of IL2 and IL10 regarding healthy women. While women with endometriosis who use contraceptive, showed high levels of IL6 and IL8. The cytokine profile seen in homogenate of women with endometriosis induced high production of IL2 and IL10, in addition IFN-γ, TNF-α, IL6. When cells were incubated with serum from women with endometriosis under treatment, the levels of IL6 and IL8 increased. The findings suggest that the use of co-cultivation 3D stromal cells and endometrial endothelial is a good model for further studies.

Insetos como formigas cortadeiras, cupins e alguns besouros têm sido descritos como agricultores de fungos mutualistas. No entanto, apenas recentemente esse fenômeno foi descrito em abelhas. O objetivo desse trabalho foi estudar os micro-­organismos associados à abelha sem ferrão Scaptotrigona depilis, a primeira abelha agricultora descrita. Foram isolados 149 micro-­organismos a partir de diferentes materiais coletados da colônia, e destes, 28% apresentaram atividade antimicrobiana frente a patógenos humanos. Os micro-­organismos Bacillus sp. SDLI1, Candida sp. SDCP2, Zygosaccharomyces sp. SDBC30G1 e Monascus ruber SDCP1, isolados do favo de cria de S. depilis, foram selecionados para estudo químico e biológico. Foi demonstrado que o fungo-­alimento de S. depilis é Zygosaccharomyces sp., que produz grande quantidade de adipossomos. Os lipídeos e esteroides estocados nessas organelas citoplasmáticas podem ajudar na nutrição larval, uma vez que Zygosaccharomyces sp. é requerido para o desenvolvimento das larvas de S. depilis. Em culturas in vitro de ovos dessa abelha, verificou-­se que ergosterol depempenha papel semelhante ao de Zygosaccharomyces sp. para a metamorfose de S. depilis, indicando que esse mutualista serve como fonte de esteroides para as larvas. Verificou-­se que compostos voláteis produzidos por Candida sp. SDCP2 estimulam o crescimento de Zygosaccharomyces sp. SDBC30G1. Análises revelaram que os compostos voláteis majoritários produzidos por Candida sp. SDCP2 foram etanol (C1) e álcool isoamílico (C2), e essas substâncias também foram encontradas nas células de cria dessa abelha. Já o fungo M. ruber SDCP1 produz lovastatina (M6), um produto natural reconhecido pela inibição da enzima HMG-­CoA redutase, essencial para biossíntese de esteroides, e M6 pode modular o desenvolvimento de Zygosaccharomyces sp. SDBC30G1. Candida sp. SDCP2 estimula a produção de monascinol (M2) e monascina (M3) pelo fungo M. ruber SDCP1 em co-­cultura. Monascina (M3) é ativa frente Candida sp. SDCP2, podendo controlar o crescimento dessa levedura, sendo a produção de M3 aumentada em 57 vezes após sete dias de cocultivo. A investigação química de Bacillus sp. SDLI1 revelou que essa bactéria produz sete surfactinas (B1-­B7) e bacillomicina D (B8), que apresentam atividade antifúngica. O cromossomo circular de Bacillus sp. SDLI1 possui oito clursters biossintéticos para a produção de diferentes classes de antimicrobianos. Bacillus sp. SDLI1 também produz o fago SDLI1-­1, ativo contra Paenibacillus larvae, patógeno causador da cria pútrida americana em Apis mellifera. Cultivos de larvas in vitro revelaram que S. depilis apresenta resistência contra os entomopatógenos Beauveria bassiana e Metarhizium anisopliae, sendo que Bacillus sp. SDLI1 pode desempenhar um papel protetivo para as larvas de S. depilis contra o patógeno P. larvae. Os resultados sugerem que o ambiente encontrado nas colônias de S. depilis favorece o desenvolvimento de uma microbiota especializada. Esses micro-­ organismos interagem e beneficiam a abelha hospedeira, estabelecendo relações de simbiose que devem ser preservadas para a sobrevivência desse polinizador / Some ants, termites and beetles have established mutualistic relationship with fungi that they culture for food. However, just recently a fungus agricultural behavior has been described for bees. The main goal of this study was to investigate the microbiota associated with the stingless bee Scaptotrigona depilis, the first fungus-­ farming bee described. Different materials from S. depilis colony were used to isolate 149 microbial strains, and among these microorganisms, 28% showed antimicrobial activity against human pathogens. The microorganisms Bacillus sp. SDLI1, Candida sp. SDCP2, Zygosaccharomyces sp. SDBC30G1 and Monascus ruber SDCP1, isolated from brood cells of S. depilis, were selected for further studies. Using different approaches, it was confirmed that Zygosaccharomyces sp. SDBC30G1 is the fungus required for S. depilis larval development. This fungus accumulates cytoplasmic lipid droplets that can be a source of sterols and lipids to the larvae. Using in vitro eggs culturing, it was verified that ergosterol, the major sterol produced by Zygosaccharomyces sp. SDBC30G1, can stimulates larval metamorphosis, confirming this yeast as an important sterol source. Co-­cultures provided information about Candida sp. SDCP2 volatile organic compounds (VOCs) production, which stimulates Zygosaccharomyces sp. SDBC30G1 growth. The majoritarian VOCs produced by Candida sp. SDCP2 were ethanol (C1) and isoamyl alcohol (C2), which were also detected in S. depilis brood cells. The fungus M. ruber SDCP1 produces lovastatin (M6), an inhibitor of HMG-­CoA reductase, which can modulate Zygosaccharomyces sp. SDBC30G1 pellicle formation and lipids accumulation. Candida sp. SDCP2 stimulates the production of monascinol (M2) and monascin (M3) by M. ruber SDCP1 in co-­culture. Monascin (M3) is active against Candida sp. SDCP2 and can control the growth of this yeast, once M3 biosynthesis increased ~ 57 folds after seven days of co-­culturing. Bacillus sp. SDLI1 produces seven surfactins (B1-­B7) and bacillomycin D (B8) that presented antifungal activity against pathogenic fungi. This bacterium had its whole-­genome sequenced and the circular chromosome harbors biosynthetic gene clusters to produce eight classes of antimicrobial compounds. The phage SDLI1-­1, which presented activity against Paenibacillus larvae, a pathogen causative of American Foulbrood Disease, was also produced by Bacillus sp. SDLI1. S. depilis larvae were resistant against Beauveria bassiana and Metarhizium anisopliae infections, and Bacillus sp. SDLI1 was capable to increase larval survival during in vitro culturing with P. larvae. The results suggest that the environmental conditions found in S. depilis colonies favor the development of a specialized microbiota. These microorganisms interact and produce beneficial factors to its host. These stablished symbiotic relationships contribute with the homeostasis inside the colony and they must be preserved to safeguard S. depilis survival

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.