Die Semaphorine Sema3A und Sema3C sowie Netrin-1 und deren Rezeptoren, die Neuropiline und DCC wurden in der vorliegenden Arbeit hinsichtlich ihrer Expression und auf ihre funktionellen Eigenschaften bezüglich des Auswachsens von Axonen, die die intrinsischen und afferenten hippokampalen Projektionen bilden, untersucht. Während die Expressionsmuster von Sema3A schon gut bekannt waren, wurde in der hier vorliegenden Arbeit die Expression des Rezeptors von Sema3A, Neuropilin-1 (NP-1), untersucht. NP-1 wird von Embryonaltag E17 an im entorhinalen Kortex, dem Subiculum und der hippokampalen Anlage exprimiert. Es konnte eine starke postnatale Expression von NP-1 in der CA3-Region und eine schwächere Expression in der CA1-Region, dem Gyrus dentatus und dem entorhinalen Kortex gezeigt werden. Außerdem wurden in dieser Arbeit die Expressionsmuster von Sema3C und Neuropilin-2 (NP-2) genauer analysiert. Etwa zum Zeitpunkt der Geburt (P0) wurde Sema3C im Gyrus dentatus und in der Cornu ammonis Region exprimiert. Der Sema3C-Rezeptor Neuropilin-2 wurde zu diesem Zeitpunkt ebenso im Gyrus dentatus und CA3-Region, schwächer auch in der CA1-Region exprimiert. Es wurde keine Expression dieser beiden Faktoren im entorhinalen Kortex detektiert. In Kokulturstudien zwischen mit Sema3A bzw. Sema3C transfizierten Zellaggregaten und Explantaten aus den hippokampalen Subregionen wurden für spezifische Explantate ein funktioneller Zusammenhang zwischen der Sekretion der Semaphorine und dem Auswachsen der jeweiligen Explantate in einer drei-dimensionalen Kollagenmatrix deutlich. Sema3A besitzt repulsive Eigenschaften auf Explantate vom Gyrus dentatus, der CA1- und der CA3-Region sowie dem entorhinalen Kortex. Die Interaktion zwischen Sema3A und NP1 beeinflußt das Einwachsen bzw. die Terminierung entorhinaler Fasern in der Molekularschicht des Gyrus dentatus, indem Sema3A eine repulsive Barriere für einwachsende Fasern und Moosfasern, die in Richtung der CA3-Region auswachsen, darstellt. Sema3C besitzt repulsive Eigenschaften auf Fasern des medialen Septums und beeinflußt dadurch das Einwachsen dieser Fasern entlang der Cornu ammonis Region in den Hippokampus. Weiterhin wurden in dieser Arbeit die Expressionsmuster von Netrin-1 und DCC im Hippokampus sowie die funktionellen Eigenschaften von Netrin-1 untersucht. Netrin-1 wird bereits zum Zeitpunkt E17 im Neokortex exprimiert, konnte im Hippokampus jedoch erst ab dem postnatalen Entwicklungsstadium P1 detektiert werden. Während im Gyrus dentatus nur ein schwaches und im entorhinalen Kortex kein Signal gefunden werden konnte, wird Netrin-1 stark in der Cornu ammonis Region exprimiert. Der Rezeptor DCC wird dagegen schon früher in der Embryonalentwicklung, ab E15, diffus in der hippokampalen Anlage exprimiert. Ab P1 lassen sich diese Signale im Gyrus dentatus und in den CA1-CA3-Regionen unterscheiden. Von den untersuchten Explantaten der hippokampalen Region zeigte Netrin-1 nur auf die Fasern von Gyrus dentatus und CA3, welche die hippokampale Kommissur bilden, einen attraktiven Wachstumseffekt. Dies bestätigt Befunde aus Netrin-1- und DCC-defizienten Tieren, in denen die hippokampale Kommissur aufgrund des fehlenden axonalen Leitmoleküls bzw. seines Rezeptors nicht ausgebildet wird. / In this work, the semaphorins Sema3A and Sema3C as well as Netrin-1 and their receptors, the neuropilins and DCC, were investigated regarding their expression and functional properties on outgrowing axons, which are forming the intrinsic and afferent hippocampal projections. Because of the already well known expression patterns of Sema3A, this work focused on the expression of the receptor of Sema3A, NP-1. From embryonic stage E17 on, NP-1 is expressed in the entorhinal cortex, the subiculum and the hippocampal Anlage. A strong postnatal expression of NP-1 in the CA3-region could be detected, while the expression pattern in the CA1-region, the dentate gyrus and the entorhinal cortex was weaker. Additionally, the expression patterns of Sema3C and NP-2 were investigated in greater detail. At birth (P0), Sema3C was expressed in the dentate gyrus and the cornu ammonis region. The expression of its receptor NP-2 could be detected at the same timepoint P0 in the dentate gyrus and the CA3-region and, less pronounced, in the CA1-region.There could not be detected any expression of Sema3C or NP-2 in the entorhinal cortex. In functional coculture studies between with Sema3A or Sema3C transfected cell clusters and neuronal explants from subregions of the hippocampal formation, these factors were investigated for their influence on axonal outgrowth within a three-dimensional collagen gel matrix. Sema3A has repulsive properties on explants from the dentate gyrus, the CA1- and CA3- regions and the entorhinal cortex. I the resulting model, the interaction between Sema3A and NP-1 influences the ingrowth and/or the termination of entorhinal fibers into the molecular layer of the dentate gyrus by a repulsive barrier formed by Sema3A. The same barrier also acts on mossy fibers to allow them to grow only in direction of the CA3-region. Sema3C has repulsive properties on fibers from the medial septum and shapes the ingrowth of these fibers along the cornu ammonis region into the hippocampus. Additionally, the expression patterns of Netrin-1 and DCC and their functional properties in the hippocampus were investigated. Netrin-1 is already expressed in the cortex at E17, although the onset of expression in the hippocampus is at P1. In the dentate gyrus, a weak signal could be detected, but no signal was found in the entorhinal cortex. In the cornu ammonis region, however, Netrin-1 showed a strong expression signal. The Netrin-1 receptor DCC could be detected as early as E15 with a diffuse distribution in the hippocampal Anlage. From P1 on, these signals could be distinguished in the dentate gyrus and the CA1-CA3-regions. Netrin-1 showed attractive properties only on fibers from explants of the dentate gyrus and the CA3-region, which form the hippocampal commissure. These results confirm previous findings from Netrin-1 and DCC deficient animals in which the absence of the hippocampal commissure was described.

O ambiente marinho apresenta uma rica biodiversidade e, por ser ainda pouco explorado configura-se como uma fonte potencial de novos organismos da flora e fauna marinhas, o que possibilita a descoberta de estruturas distintas e biologicamente ativas. Microrganismos marinhos como cianobactérias e fungos possuem um metabolismo secundário rico, o qual produz substâncias bioativas. Em virtude do exposto, um dos objetivos deste trabalho foi avaliar química e biologicamente as linhagens de cianobactérias marinhas Cyanobium sp. CENA178, Cyanobium sp. CENA181 e Oxynema sp. CENA135 provenientes de manguezais da Ilha do Cardoso, do Estado de São Paulo. Foram identificados os aminoácidos alanina, treonina e valina presentes na fração ACN:H2O (40:60) da linhagem Oxynema sp. CENA135. Por meio das análises realizadas em CG-EM, a fração n-hexano/acetato de etila (9:1) da linhagem Cyanobium sp. CENA178 apresentou os componentes: 2,4-bis (1,1-dimetiletil) fenol, (Z)-7-hexadeceno 6,10,14-trimetil-2-pentadecanona e eicosano; a fração n-hexano da linhagem Cyanobium sp. CENA181 apresentou como componente majoritário o neoftadieno, o qual também foi encontrado na fração acetato de etila da linhagem Oxynema sp. CENA135, juntamente com os componentes majoritários: heptadecano, [R-[R*,R*-(E)]]- 3,7,11,15-tetrametil-2-hexadeceno e octadecino. Com relação à triagem biológica, as linhagens Cyanobium sp. CENA178 e Cyanobium sp. CENA181, apresentaram atividade anticolinesterásica moderada e fraca, respectivamente. Concomitantemente foi realizada uma co-cultura entre a linhagem de cianobactéria Cyanobium sp. CENA181 e o fungo endofítico marinho Penicillium decaturense, onde foi isolada do extrato bruto do fungo P. decaturense a substância 10,11-deidrocurvularina e como produto da co-cultura foi obtida a substância curvularina. Deste modo, foi demonstrado que houve interação química entre os microrganismos devido a produção de diferentes metabólitos secundários. Adicionalmente, foram investigadas nove linhagens de cianobactérias do gênero Symploca quanto à análise química e biológica, provenientes do Panamá, Samoa Americana e uma da coleção de cultura de Pasteur (PCC8002). Por meio da análise de redes moleculares (molecular networking) foram identificadas três substâncias conhecidas: apratoxina A; palmiramida A e curacina D. Das frações A2143 E e F foi isolada uma substância inédita denominada de caracolamida A. Da fração G foi isolada uma substância conhecida pertencente à classe dos polimetoxi-alquenos isotáticos denominada de 4,6,8,10,12,14,16,18,20,22-decametóxi-heptacos-1-eno. A substância inédita caracolamida A foi comparada com o padrão feniletilamina e ambas demonstraram ter atividade neuromoduladora em concentrações subnanomolares, possuindo efeitos similares na oscilação e frequência dos canais de Ca2+. Os resultados apresentados neste trabalho acrescentam dados químicos e biológicos às espécies estudadas e enriquecem a área de Produtos Naturais Marinhos. / The marine environment contains a rich biodiversity and, since it is relatively underexplored, is a great source for finding new organisms including marine flora and fauna. This enables the discovery of chemicals with distinct and structures and biological activity. Marine microorganisms, such as cyanobacteria and fungi, have rich secondary metabolism, which yield biologically active molecules. Accordingly, one aim of this work was to evaluate the chemical profiles and biological activities of the compounds isolated from marine cyanobacteria strains Cyanobium sp. CENA178, Cyanobium sp. CENA181 and Oxynema sp. CENA135 from Ilha do Cardoso mangrove, State of São Paulo. The results indicate that it would help to adopt different laboratory cultivation methods for growing cyanobacteria strains to mimic the natural habitat and increase the opportunity to obtain new secondary metabolites. Aminoacids alanine, threonine and valine were identified of the fraction ACN:H2O (40:60) of the strain Oxynema sp. CENA135. Through the analyzes performed in CG-MS, the hexane/ethyl acetate (9:1) fraction of the strain Cyanobium sp. CENA178 showed the components: 2,4-bis (1,1-dimethylethyl) phenol, (Z)-7-hexadecene, 6,10,14-trimethyl-2-pentadecanone and eicosane. From the hexane fraction of Cyanobium sp. CENA181, neophytadiene was observed as the major component, and this was also found in the ethyl acetate fraction of Oxynema sp. CENA135 strain along with heptadecane, 2-hexadecene, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]] and 1-octadecyne. Cyanobium sp. CENA178 and Cyanobium sp. CENA181 showed moderate and weak anticholinesterase activity, respectively. Simultaneously, a co-culture was performed using Cyanobium sp. CENA181 and the marine endophytic fungus, Penicillium decaturense. The compound 10,11-dehydrocurvularin was isolated from the crude extract of P. decaturense, and curvularin from only the co-culture. Therefore, it was clear that the microorganisms exhibited an interaction leading to the different production of secondary metabolites. Nine species of cyanobacteria of the genus Symploca from Panama, American Samoa and one from Pasteur culture collection (PCC8002) were investigated to yield new natural products. Through molecular networking analysis three known compounds were identified: apratoxin A; palmiramide A and curacin D. Caracolamide A is a new compound isolated from fractions A2143 E and F. A known compound isolated from the same organism is called 4,6,8,10,12,14,16,18,20,22-decamethoxy-heptacos-1-ene, and it is an isotactic polymethoxy-1-alkene. The new compound caracolamide A demonstrated neuromodulatory activity at subnanomolar concentrations, and displayed similar effects as a phenylethylamine standard on the oscillation amplitude and frequency in Ca2+ channels. The results presented in this work provide chemical and biological information about the species studied, and enrich marine natural products research

The aortic valve maintains unidirectional blood flow between the left ventricle and the systemic circulation. When diseased, the valve is replaced either by a mechanical or a bioprosthetic heart valve, that carry issues such as thrombogenesis, long term structural failure, and calcification, necessitating the development of more structurally and biologically sufficient long-term replacements. Tissue engineering provides a possible avenue for development, combining cells, scaffolds, and biochemical factors to regenerate tissue. The overall goal of this dissertation was to create a foundation for the rational design of a tissue engineered aortic valve. The novel approach taken in this thesis research was to view each of the three leaflets as a laminate structure. The first three aims consider the leaflet as a laminate structure comprising of layers of collagen, elastin, and glycosaminoglycans (GAGs). In the first aim, the effect of GAGs on the tensile properties and stress relaxation in the leaflet was investigated, by removing GAGs through increasing amounts of hyaluronidase. A decrease in GAGs led to significantly higher elastic moduli, maximum stresses, and hysteresis in the leaflet. In the second aim, the 3D elastic fiber network of the leaflet was characterized using immunohistochemistry and scanning electron microscopy. This structure was found to have regionally varying thicknesses and patterns. In the third aim, a novel hydrogel-fiber composite design was proposed to match the anisotropy of the leaflet. This composite composed of aligned electrospun poly(ε-caprolactone) (PCL) within a poly(ethylene glycol) diacrylate (PEGDA) matrix. Surface modification and embedding of the PCL did not significantly alter the anisotropy or strength of the underlying PCL scaffold, providing the basis for an anisotropic, biocompatible scaffold. In the last aim, a novel co-culture model was designed using magnetic levitation as a layered structure of valvular endothelial cells and interstitial cells. This technique was used to create co-culture models within hours, while maintaining cell phenotype and function, and inducing extracellular matrix formation, as shown by immunohistochemical stains and their gene expression profiling. The overall result of this dissertation is a clearer understanding of the layered structure-function relationship of the aortic valve, and its application towards heart valve tissue engineering.

No description available.

Na literatura encontram-se referências de estudos envolvendo micro-organismos endofíticos, e mais recentemente estudos que avaliam a interação entre micro-organismos o que resulta na modificação, no tipo ou quantidade dos compostos que são produzidos. Neste trabalho foram realizados cultivos simples e mistos do fungo Colletotrichum boninense FLe 8.1 e da actinobacteria Streptomyces albospinus RLe 7, endófiticos isolados de Lychnophora ericoides que pertencem à coleção do Laboratório de Química de Microorganismos (LQMo) da FCFRP-USP, com o objetivo de aumentar suas capacidades de produção de novos compostos com atividade biológica. O cultivo misto, ou co-cultivo, é uma estratégia que tem sido usada para o acesso aos produtos naturais de origem microbiana. Existem poucos relatos de compostos com atividade biológica isolados a partir de S. albospinus e não há relatos de metabólitos secundários obtidos a partir de C. boninense. Nenhum desses micro-organismos tem sido descrito como endofítico na literatura e não existem relatos sobre co-cultivos envolvendo qualquer um deles na busca de compostos bioativos. Juntando as informações geradas através das diferentes técnicas de detecção utilizadas, como TLC, HPLC-DAD, GC-MS, ESI-MS, RMN e depois da análise correspondente, foi possível identificar e atribuir as estruturas de algumas substâncias conhecidas de origem microbiana como a mevalonolactona, o tirosol, a fisostigmina, a desferrioxamina E. ESI-MS foi utilizada para análises dos extratos brutos originados dos cultivos em meio arroz parbolizado permitindo visualizar compostos produzidos em baixas quantidades pelos micro-organismos na cultura simples quanto na co-cultura. Além disso, foram obtidos os perfis metabólicos desses micro-organismos a partir de cultivos em placa de Petri, possibilitando a detecção dos metabólitos em regiões específicas da interação microbiana, o que conduziu à identificação da fisostigmina e o seu análogo N-etilcarbamato produzidos pela actinobactéria, na interação com o fungo. Análises posteriores de MS sequencial permitiram obter perfis de fragmentação, os quais, juntamente com os dados dos íons detectados nos extratos, foram comparados com a informação disponível em bases de dados como DNP, METLIN e MassBank. Tais informações geradas a partir dessas análises permitiram sugerir possíveis compostos envolvidos na interação dos micro-organismos endofíticos mencionados. Foi sugerido que a fisostigmina, substância produzida pela actinobactéria S. albospinus RLe 7 e isolada nesse trabalho, poderia ter algum papel na inibição do crescimento do fungo C. boninense FLe 8.1 já que a inibição só foi observada quando cultivados ambos os micro-organismos na mesma placa de Petri. Porém, bioensaios com fisostigmina pura demonstraram que essa substância não possui atividade antifúngica per se, mas é possível que exista uma sinergia com outras substancias produzidas pela actinobactéria. Os resultados apresentados nesse trabalho demonstram a importância do uso de técnicas de detecção muito sensíveis, como a ESI-MS, na identificação de substâncias envolvidas na troca metabólica e permitiram gerar informações que serão utilizadas em estudos futuros utilizando esses micro-organismos endofíticos. / There are several studies involving endophytic microorganisms, and more recently some studies evaluate microbial interaction resulting in metabolic profiles modifications. In this study, simple and mixed cultures of the fungus Colletotrichum boninense FLe 8.1 and the actinobacteria Streptomyces albospinus RLe 7 were carried out. These endophytic microorganisms, isolated from Lychnophora ericoides, belong to the collection of the Laboratory of Microbial Chemistry (LQMo) of the FCFRP-USP. The main goal of this work was to increase the bioactive compounds production capacities. Co-culture, or mixed culture, is a recent strategy for accessing microbial natural products. There are few reports of bioactive metabolites from S. albospinus RLe 7 and there are no one about secondary metabolites obtained from C. boninense FLe 8.1. Neither the actinobacteria nor the fungus have been described as endophytic microorganisms and there are no co-culture studies involving these microorganisms in order to obtain natural products. Joining and analyzing all together the generated information from several detection techniques, such as TLC, HPLCDAD, GC-MS, ESI-MS, NMR, it was possible to identify and attribute the chemical structures of some known natural, such as mevalonolactone, tyrosol, physostigmine and deferrioxamine E. ESI-MS was used for extracts analysis from cultures in parboiled rice medium, allowing visualization of compounds produced in very low yields. Besides that, it was possible to obtain metabolic profiles from cultures in Petri dishes, leading to the detection of metabolites in specific regions of the microbial interaction. Physostigmine and its N-ethylcarbamate analogue were identified as actinobacteria metabolites in the interaction against the fungus. Posterior analysis by MS/MS allowed to obtain fragmentation profiles, which together with the ions detected from the extracts analysis, were compared by searching in databases, such as DNP, METLIN and MassBank. Such information allowed to suggest possible candidates for the compounds involved in the microbial metabolic exchange. It was hypothesized that physostigmine, isolated from the actinobacteria in this work, had a role in the fungal growth inhibition observed when both microorganisms were co-cultured. However, this substance did not show considerable antifungal activity when tested against C. boninense FLe 8.1, but it is possible that other compounds produced by this actinobacteria are acting in a synergistic way. The results showed here demonstrated the importance of very sensitive techniques, as ESI-MS, in the identification of molecules involved in the microbial metabolic exchange and will help in the generation of information for future studies involving those endophytic microorganisms.

As técnicas de cultivo de folículos e oócitos in vitro, com o objetivo de se obter oócitos maduros para procedimentos de Reprodução Assistida (RA), têm sido aplicadas em diferentes contextos. O sucesso destes procedimentos está diretamente relacionado ao sistema de cultivo utilizado. A utilização de células da granulosa (CG) humanas cultivadas in vitro como um suporte para o co-cultivo destes oócitos imaturos e folículos tem sido descrita por alguns autores. A criopreservação destas células, considerando-se o contexto de sua obtenção em procedimentos de RA, permitiria a viabilização da aplicação destas células na prática clínica diária. Sendo assim, o objetivo deste estudo foi padronizar o congelamento de células da granulosa (CG) humanas para aplicação em sistemas de co-cultivos de folículos e oócitos imaturos. Foram obtidas CG de 20 voluntárias em tratamento de reprodução assistida, células de 10 voluntárias foram cultivadas em meio ?-MEM suplementado para interrupção da luteinização e congeladas após 48 horas em container \"Cryostep\" (grupo 2C- 2 cultivos) (etapa 2) e células de 10 voluntárias foram congeladas em container \"Cryostep\" sem cultivo prévio (grupo CD- congelamento direto) (etapa 3). Após o descongelamento estas células foram (re)cultivadas por 144 horas, com troca de meio em 48, 96 e 144 horas para avaliações da produção de estradiol (E2) e progesterona (P4) (ng/mL). Verificamos redução na contagem celular e na viabilidade celular tanto no método de congelamento direto (CD) quanto no método com dois cultivos (2C) após o descongelamento (p<0,05), e isso se refletiu na produção de estradiol e progesterona que foi maior nas culturas de células frescas em relação às células criopreservadas (p<0,05). Porém, a relação de E2/célula foi mantida após o descongelamento, sugerindo que esta redução na produção se deve à redução no número de células, as que sobrevivem se mantém normofuncionantes (p=0,23).O CD foi mais eficiente pois permitiu uma maior recuperação celular e uma melhor viabilidade quando comparado ao grupo 2C. A relação estradiol/progesterona foi mantida em todos os tempos de cultivo, fresco, CD e 2C (p>0,05), indicando que a característica funcional destas células foi preservada após o descongelamento. Concluímos que a criopreservação de CG humanas obtidas durante a captação de oócitos compromete a contagem celular e a viabilidade geral da cultura, entretanto, a capacidade funcional e a característica destas células se mantêm preservadas (manutenção das relações E2/célula e E2/P4) / Follicle and oocyte in vitro culture techniques, aiming to obtain mature oocytes for Assisted Reproductive Treatments (ART), have been applied to different contexts. The success of these procedures depends on the culture system used. The use of human granulosa cells (GC) in co-culture systems for follicle and oocyte maturation have been described by some authors. The cryopreservation of these cells, considering the context in which they are obtained during ART, would enable the usage of these cells in such procedures in daily clinical practice. Thus, the objective of this study was to standardize the freezing protocol for human granulosa cells (GC) for future applications in co-culture systems for follicle and oocyte maturation. Twenty volunteers submitted to ART donated their granulosa cells after oocyte retrieval, 10 were cultivated previously in order to interrupt the luteinization process and then frozen \"Cryostep\" container (group 2C- two cultures) (step 2) and 10 were directly frozen with no previous culture in the \"Cryostep\" container (group DF- direct freeze) (step 3). After thawing these cells were (re)cultured for 144 hours, with medium exchange at 48, 96 and 144 hours to evaluate the estradiol (E2) and progesterone (P4) production (ng/mL). After thawing, there was a reduction in the cell number (p<0,05) and cell viability in both methods, the direct freezing (DF) and the two cultures (2C) (p<0,05); this had an impact in the production of estradiol and progesterone, which were higher in fresh cultures than in the frozen ones (p<0,05). However, the E2/cell ratio was maintained after thawing (p=0.23), suggesting that this impairment in steroid production was probably due to the reduction in the cell count. The cells that survive remain functionally normal. The DF was more efficient since it allowed greater cell recovery and better viability when compared to 2C. The estradiol/progesterone ratio was maintained in all culture times, in the fresh, DF or 2C groups (p>0.05), indicating that the functional characteristic of these cells was preserved post-thawing. We conclude that cryopreservation of human GC obtained during oocyte retrieval compromises the cell count and the overall viability of the culture; however, the functional capacity and the characteristic of these cells are preserved (maintenance of E2/cell and E2/P4 relations)

Aujourd’hui, la transplantation est le seul traitement efficace proposé aux patients souffrant d’une insuffisance hépatique fulminante. La nécessité de disposer d’un système de suppléance hépatique transitoire apparaît donc indispensable. C’est dans cet axe que se sont développés les systèmes qualifiés de foies bio artificiels (BAL). Leur principale caractéristique est d’incorporer un bioréacteur hébergeant des cellules pouvant restaurer l’activité hépatiques dans son ensemble. A l’heure actuelle, les hépatocytes primaire humains (HEP) issus de foies de donneurs non transplantables sont considérées comme le meilleur choix. Cependant, leur utilisation reste limitée par leur faible disponibilité et la difficulté à les maintenir différenciés en culture in vitro. Pour remédier à ce dernier point, l’approche la plus prometteuse semble être une co-culture des hépatocytes avec les cellules non parenchymateuses afin de recréer un environnement proche des sinusoïdes hépatiques. Ce travail de thèse repose sur la mise en place d’une nouvelle approche de co-culture tridimensionnelle sous la forme de sphéroïdes, d’HEP primaires avec les principaux types de cellules non-parenchymateuses (les cellules de Kupffer, les cellules endothéliales et les cellules étoilées) selon des proportions spécifiques. Puis de leurs encapsulations dans des billes d’alginate et leurs cultures au sein d’un bioréacteur à lit fluidisé. Ce modèle s’est révélé pertinent et approprié à maintenir les fonctions hépatiques dans le temps. Bien que beaucoup d’optimisation reste à définir, ce travail exploratoire témoigne de l’intérêt de cette approche intéressante pour le progrès des systèmes BAL. / Liver shortage makes transplantation inapplicable to all acute liver failure patients. Bioartificial Iiver (BAL) devices represent a temporary solution for these patients which are thereby bridged tilt Iiver transplantation or regeneration BAL treatment offers blood purification and substitution of metabolic functions through the activity of hepatocytes (HEPs), which are integrated in the device within acclimating containers, so-called bioreactors. Primary human hepatocytes are the ideal cell type to use in BAL, but they are scarcely available and difficult to maintain in vitro. Co-culture of HEPs with supporting cells has been proposed as the most promising strategy for preserving HEP behaviors in in vitro conditions. In fact, assisting cells types hold their ability to influence functional responses of the HEPs by providing them with cues of the native organ.This PhD work proposed a novel approach of co-culture for the functional sustain and preservation of the HEPs in the environment of the fluidized bed bioreactor (designed in our Iaboratory). Definition of this model took inspiration from the cellular organization in the organ; therefore, it employed three major sinusoidal non-parenchymal cell populations (liver sinusoidal, Kupffer, and hepatic stellate cells) which, together with HEPs, were cultured with three-dimensional arrangement (spheroids) and according to specific proportions. The resulting model was characterized in terms of functional benefits for the HEPs, and then applied in the microenvironment of alginate beads, which provide cells with immunological and mechanical protection in the fluidized bed bioreactor. This spheroidal multi-cultured model revealed its potentiality in sustaining in vitro HEP behaviors over time. Although much remains to be refined, this model may represent an interesting approach for the progress of BAL

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Ziel dieser Studie war die Etablierung einer Kokultur aus equinen endometrialen Epithel- und Stromazellen. Nach der erfolgreichen Umsetzung des Kokulturmodells sollte im weiteren Versuchsablauf durch die Zugabe von 17β-Östradiol (E2) und/oder Progesteron (P4) zum Nährmedium der Einfluss der Hormone auf die Zellen untersucht werden. Neben einer lichtmikroskopischen Auswertung der zytomorphologischen Charakteristika beider Zellarten sollte die Expression der Steroidhormonrezeptoren Östrogenrezeptor α und Progesteronre-zeptor sowie der uterinen Proteine Uteroglobin und CalbindinD9k immunzytologisch überprüft werden. Für die Etablierung der Kokultur wurden Endometriumproben von lebenden (n = 5) sowie frischtoten (n = 4) Stuten gewonnen. Eine jeweils parallel entnommene Gewebeprobe von jedem Tier wurde in Formalin fixiert und diente als Referenzmaterial (in situ). Auf die Zelliso-lierung (mechanisch und enzymatisch) folgte die Separation von Epithel- und Stromazellen (EZ/SZ) mittels Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. An-schließend wurden die EZ auf die Außenseite von Millicell®-Membraneinsätzen aufgebracht. Nach zwei Tagen erfolgte das Einsäen der bis zu diesem Zeitpunkt separat kultivierten SZ auf die Innenseite der Membranen. Als Nährmedium diente ein Gemisch aus DMEM und Ham’s F-12, wobei diesem 2,5 % fötales Kälberserum sowie verschiedene Additive zugesetzt wurden. Ab Kulturtag 4 wurden dem Medium definierte Konzentrationen und Kombinationen von E2 und P4 zugesetzt. Die Kultivierung erfolgte bei einem CO2-Partialdruck von 5 % in 37 °C warmer wasserdampfgesättigter Raumluft. Mit der polarisationsmikroskopisch er-fassbaren Ausbildung durchgehender Zellrasen („scheinbare Konfluenz“) wurden die Kokul-turen in Formalin fixiert und für die Lichtmikroskopie aufgearbeitet. Das Ausgangsgewebe zeigte mehrheitlich eine sekretorische Funktionsmorphologie (n = 6). Einzelne Endometrien befanden sich in einem Übergangsstadium von der Sekretions- zur Proliferationsphase (n = 1), bzw. vice versa (n = 1) oder wiesen eine irregulär proliferative Differenzierung (n = 1) auf. Im Rahmen der Kokultivierung bildeten die EZ innerhalb der Schnittebene vier und die SZ drei verschiedene morphologische Zelltypen aus. Dabei traten rundovale bis polygonale EZ (Typ 1) selten bis gelegentlich, spindelförmige EZ (Typ 2) gelegentlich bis häufig und iso-prismatische (Typ 3) sowie mehrschichtig wachsende EZ (Typ M) jeweils selten auf. Die SZ zeigten innerhalb der Schnittebene selten eine rundovale bis polygonale Zellform (Typ 1), sehr häufig eine spindelförmige Morphologie (Typ 2) und selten ein mehrschichtiges Wachstum (Typ M). Ein Zusammenhang zwischen der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung oder dem Hormonzusatz und der Häufigkeitsverteilung der Zell-typen sowie der Wachstumsgeschwindigkeit der kultivierten Zellen war nicht offensichtlich. Zytokeratin 19 wurde stets von EZ exprimiert, während es auf Seiten der SZ nur sporadisch in maximal 5 % der Zellen im Bereich mehrschichtig wachsender Zellrasen auftrat. Die Stero-idhormonrezeptoren konnten lediglich in einzelnen Kokulturen aus sekretorisch differenzier-tem Ausgangsgewebe detektiert werden. Uteroglobin wurde in vitro mit einer variablen Häufigkeit in den EZ-Typen exprimiert. Während ein übergreifender Zusammenhang zur hormonellen Supplementierung nicht abgeleitet werden konnte, wurde jedoch ersichtlich, dass im Bereich einschichtig wachsender EZ in Ansätzen aus sekretorisch differenzierten Endometrien unter niedrigen Hormondosen (Zusatz von entweder nur E2 oder nur P4) im Median häufiger Uteroglobin exprimiert wurde. Mit zunehmender Hormonkonzentration im Medium nahm der Anteil immunopositiver Zellen (Typen 1, 2 und 3) deutlich ab. Innerhalb der Stromazellpopulation wurde Uteroglobin selten und ausschließlich in Zellen aus sekretorisch differenziertem Ausgangsmaterial nachgewiesen. CalbindinD9k wurde in vitro vornehmlich intrazytoplasmatisch und sehr vereinzelt intranukleär exprimiert. Insgesamt konnte das Protein in vitro stets in wenigen Typ-1-EZ, sehr selten in Typ-2-EZ und in einer geringen bis mäßigen Anzahl von Typ-3- und Typ-M-EZ beobachtet werden. Innerhalb der Stromazellpo-pulation trat CalbindinD9k ausschließlich in einer geringen (Endometrien aus dem Östrus) bis mäßigen (Endometrien aus dem Interöstrus) Anzahl der Typ-2- und wenigen Typ-M-SZ auf. Insgesamt wurden keine deutlichen Einflüsse der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung und/oder der hormonellen Supplementierung in vitro auf die im-munzytologischen Charakteristika der kokultivierten Zellen ersichtlich. Abschließend betrachtet, konnte ein Kokultursystem equiner endometrialer Epithel- und Stromazellen erfolgreich etabliert und charakterisiert werden. Es bietet dabei, trotz der z. T. fehlenden Kongruenz zu den Gegebenheiten in situ, Ansätze für potenzielle Folgearbeiten, insbesondere hinsichtlich der Erfassung interzellulärer Wechselwirkungen sowie bezüglich der Vermittlung und Wirkung hormoneller Einflüsse auf zellulärer Ebene. / The aim of the present study was the establishment of a coculture system of equine endome-trial epithelial and stromal cells. Subsequent to the successful development of the coculture model the culture medium should be supplemented with 17β-estradiol (E2) and/or progester-one (P4) in order to study the influence of the hormones on the cellular level. In addition to the examination of cytomorphological characteristics of both cell types via light microscopy, the expression of the steroid hormone receptors (estrogen receptor α and progesterone receptor) as well as of the uterine proteins Uteroglobin and CalbindinD9k was investigated. For the establishment of the coculture system endometrial samples were obtained from living (n = 5) as well as freshly deceased mares (n = 4). A simultaneously taken tissue specimen of each animal was fixed in formalin and served as in situ reference material. After an initial mechanical and enzymatical isolation the epithelial and stromal cells (EC/SC) were separat-ed via filtration, density gradient centrifugation and differential adhesion. Subsequently, the EC were applied to the outer surface of Millicell® inserts. The SC were cultivated separately for 2 days before they were seeded onto the inner surface of the same insert. The culture medium used was comprised of a DMEM and Ham‘s F-12 basis as well as 2.5 % foetal calf serum and different additives. Starting on day 4 of cultivation the standardised medium was supplemented with different concentrations and combinations of E2 and P4. Throughout the study the cultures were kept in a humidified atmosphere of 37°C and a 5 % partial pressure of carbon dioxide. Once the cocultures formed continuous cell layers, as determined via a polarisation microscope (“apparent confluency”), the membranes were fixed in formalin and routinely processed for light microscopical evaluation. The initial tissue samples predominantly showed a secretory functional morphology (n = 6), while single specimens were obtained during the transition from the secretory to the prolifera-tive phase (n = 1) or vice versa (n = 1). One endometrial sample exhibited an irregular proli-ferative differentiation. In the course of cocultivation the EC formed 4 and the SC 3 different cellular morphologies within the section plane. EC with a round-oval to polygonal cell form (type 1) were rarely to occasionally encountered, while spindle-shaped EC (type 2) were occasionally to frequently seen and EC with a cuboidal morphology (type 3) as well as such cells growing in stratified layers (type M) were only infrequently detected. The SC only rarely showed a round-oval to polygonal cell form (type 1) or areas of a stratified cell growth (type M), whereas spindle-shaped SC (type 2) were observed very often. A correlation of the endometrial functional morphology at the time of cell isolation or the hormonal supplementation and the frequency distribution of the cell types as well as the growth rate of the cultivated cells was not evident. The EC always expressed Cytokeratin 19, while on the side of the SC only up to 5 % of the cells in areas of stratified cell growth exhibited this filament. Solely in individual cocultures from secretory differentiated endometrial tissue the steroid hormone receptors could be de-tected. Uteroglobin was expressed in vitro in EC with a variable frequency. An overall corre-lation of the hormonal supplementation and the Uteroglobin expression could not be derived. However, under low hormone doses (only E2 or only P4 supplement) Uteroglobin was detect-ed in EC in areas of single-layered cell growth more often (median value). With an increase in hormone concentration the amount of immunopositive cells (types 1, 2 and 3) diminished noticeably. In SC the protein could only rarely be seen and exclusively in cells from endome-tria with a secretory functional morphology. In vitro CalbindinD9k was predominantly detected intracytoplasmatically, while single cells showed an additional intranuclear expression. Alto-gether, CalbindinD9k could always be observed in a few type-1-EC, rarely in type-2-EC and with a variable frequency in small to moderate numbers of type-3- and type-M-EC. In SC the protein was exclusively expressed in a small (endometrial samples form the oestrous phase) to moderate (endometrial tissue from the interoestrous phase) number of type-2-SC and a few type-M-SC. Generally, no distinct influence of the endometrial functional morphology at the time of tissue sampling and/or of the hormonal supplementation in vitro on the immuno-cytochemical characteristics of the cocultured cells could be observed. In summary, a coculture system of primary equine endometrial epithelial and stromal cells was successfully established and characterised. Despite of the partly absent congruence to the in situ conditions/prerequisites, the present study offers a basic approach and scaffold for further investigations, particularly regarding the ascertainment of intercellular dependencies or the mediation and effectiveness of hormonal influences on the cellular level.

Microbes typically form highly complex and diverse communities that account for a significant portion of life's genetic diversity. Analysis of living systems, e.g. bacterial or cell population, plays a significant role in detecting and identifying pathogens, testing antibiotic susceptibility, and the fundamental research of population diversity and evolution. This work focuses on the analysis of bacterial communities using droplets based millifluidics. To monitor the bacteria growth, we designed an optofluidic system, combining the encapsulation of bacteria in numerous emulsion droplets to monitor their long-term behavior and relationship in a co-culture environment using fluorescent signals. In the first part of this work, we co-encapsulated and cultured two isogenic strains of Escherichia coli (E. coli) in numerous emulsion droplets to reveal their competition and cooperation relationship. Since two strains of E. coli express blue and yellow fluorescent proteins (BFP and YFP, respectively), we quantified their growth by integrating a fluorescence detection system. We analyzed the following parameters: doubling time, population yield, final biomass ratio, correlation map of doubling time and competition coefficient to characterize and compare the bacterial growth kinetics and behavior in mono and co-cultures. In addition, the experimental observations were compared with the predictions from a single growth model. Finally, we employed the millifluidic device to verify the appearance of cross-protection between antibiotic-sensitive bacteria and antibiotic-resistant bacteria. It is one of the mechanisms by which different bacteria, sharing the same environment, protect each other to survive in the presence of antibiotics. For this purpose, the E.coli YFP strain was chosen as an antibiotic-sensitive group. Simultaneously, the E.coli BFP strain with β-lactam and its mutations were selected as resistant strains. Combining the millifluidic droplet reactor method with other detection strategies, e.g. fluorescence microscopy, fluorescence flow cytometry, and plate reader, we proved the appearance of cross-protection by detecting the filamentary cells, the fluorescence of cell-free media, viable cell rates, cell shape and size, as well as β-lactamase activity. All these results obtained by millifluidic devices proved that this strategy could be used in a high-throughput bacterial coexistence study. In addition, the research of these general fields, such as bacterial community and antibiotic impact, can help us to reveal the interaction between microbial species and determine the right dose of antibiotics to inhibit bacterial growth in a co-existent environment efficiently.