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Showing 41 to 50 of 51 (0.053 seconds)Spelling suggestions: "subject:"invitro model"" "subject:"infvitro model""
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[en] SYNTHESIS OF ARYLATED THIAZOLE DERIVATIVES AND PRELIMINARY EVALUATION OF THEIR TOXICITY AND ANTI T. CRUZI ACTIVITY / [pt] SÍNTESE DE DERIVADOS ARILADOS DE TIAZOL E AVALIAÇÃO PRELIMINAR DA TOXICIDADE E ATIVIDADE ANTI T. CRUZIKELLY LOPES FIGUEIRA 02 December 2021 (has links)
[pt] As doenças negligenciadas são causadas por agentes infecciosos ou parasitas, gerando altas taxas de morbidade e mortalidade em todo o mundo. Os investimentos dos países desenvolvidos em pesquisa, produção de medicamentos e controle dessas enfermidades são muito reduzidos, pois elas afetam, majoritariamente, as populações com baixo Índice de Desenvolvimento Humano. Uma dessas enfermidades é a Doença de Chagas, causada pelo Trypanosoma cruzi. Apresenta-se na forma de uma infecção parasitária crônica e altamente debilitante, acometendo mais de 6 milhões de pessoas na América Latina e este número pode ter um incremento, de aproximadamente 25 por cento nos próximos anos. O fármaco referência para o tratamento da Doença de Chagas disponível no Brasil é o benznidazol, extremamente eficaz na fase aguda da doença, mas sua eficácia na fase crônica é limitada, o que torna essencial a busca por novos compostos que atuem também nesta fase. No presente trabalho, foram descritas rotas sintéticas para uma série de 12 derivados de tiazol, destes 8 são inéditos. Posteriormente, 10 deles foram avaliados quanto a sua atividade tripanocida e toxicidade em modelos in vitro. Os compostos possuindo o anel tiazólico foram estruturalmente planejados, a partir do levantamento bibliográfico, onde verificou-se que certos compostos com um centro tiazólico apresentam propriedade tripanocida. Nas sínteses, a 4-bromoacetofenona (55) foi utilizada como material de partida para a obtenção dos intermediários tiazólicos 4-(4-bromofenil)-2- metiltiazol (56) e 4-(4-bromofenil)-2-aminotiazol (57). A partir desses, diversas modificações foram feitas para se chegar às moléculas alvo. A elucidação estrutural dos compostos foi realizada por RMN de 1H e RMN de 13C. A avaliação preliminar da atividade tripanocida foi realizada na forma amastigota intracelular da Cepa Tulahuen e a toxicidade aguda (LC50) foi feita in vitro em células hospedeiras de mamíferos (linhagem celular L929). Dentre os compostos sintetizados, a maioria apresentou baixa toxicidade, com valores de LC50 maiores que 400 (micro)mol.L-1. O composto tiazólico piridil-substituído 59c apresentou os melhores resultados em termos de atividade tripanocida, tendo reduzido 76 por cento da infecção.
em células hospedeiras em concentração de 20 (micro)mol.L-1 Esse composto servirá como base para otimizações estruturais visando a melhoria da atividade tripanocida. / [en] Neglected diseases are mainly caused by infectious agents or parasites, generating high morbidity and mortality rates worldwide. The investments from developed countries in research, production of drugs/medicines and control of these diseases are small, since they mainly affect populations with low Human Development Index. One of these illnesses is Chagas disease, caused by Trypanosoma cruzi. It presents as a chronic and highly debilitating parasitic infection, affecting more than 6 million people only in Latin America with a perspective of increasing approximately 25 percent in few years. The reference drug for the treatment of Chagas disease in Brazil is benznidazole, which is extremely effective in the acute phase of the disease, but its efficacy in the chronic phase is limited, which makes essential the search for new compounds that also act in this phase. In the present work, synthetic routes were developed for a series of 12 thiazole derivatives, from which eight have been described for the first time. These molecules had their preliminary trypanocidal activity and toxicity evaluated in in vitro models. The compounds with the thiazole ring were structurally planned, based on the bibliographic review, where it was found that certain compounds with this heterocycle display trypanocidal properties. In the synthesis, 1-(4- bromophenyl)ethenone (54) was used as a starting material to obtain the thiazole intermediates 4-(4-bromophenyl)-2-methylthiazole (56) and 4-(4-bromophenyl)thiazol-2- amine (57). Several modifications were performed on these intermediates to produce the target molecules. The structural elucidation of the compounds was performed by 1H NMR and 13C NMR. Preliminary evaluation of trypanocidal activity was performed in the amastigotes intracellular form of the Tulahuen strain and the acute toxicity (LC50) was tested in vitro in mammalian host cells (cell line L929). Among the synthesized compounds, most of them presented low toxicity, with LC50 values greater than 400 (micro)mol/mol.L-1. The thiazole compound pyridil-substituted 59c showed the best results in terms of trypanocidal activity, reducing 76 percent of the infection in host cells in a concentration of 20 (micro)mol/mol.L-1. This thia\ole derivative will be used as a lead compound for structural optimizations aiming at improving trypanocidal activity.
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The Simplified Human Intestinal Microbiota (SIHUMIx) Shows High Structural and Functional Resistance against Changing Transit Times in In Vitro BioreactorsSchäpe, Stephanie Serena, Krause, Jannike Lea, Engelmann, Beatrice, Fritz-Wallace, Katarina, Schattenberg, Florian, Liu, Zishu, Müller, Susann, Jehmlich, Nico, Rolle-Kampczyk, Ulrike, Herberth, Gunda, von Bergen, Martin 11 April 2023 (has links)
Many functions in host–microbiota interactions are potentially influenced by intestinal
transit times, but little is known about the effects of altered transition times on the composition
and functionality of gut microbiota. To analyze these effects, we cultivated the model community
SIHUMIx in bioreactors in order to determine the effects of varying transit times (TT) on the
community structure and function. After five days of continuous cultivation, we investigated the
influence of different medium TT of 12 h, 24 h, and 48 h. For profiling the microbial community,
we applied flow cytometric fingerprinting and revealed changes in the community structure of
SIHUMIx during the change of TT, which were not associated with changes in species abundances.
For pinpointing metabolic alterations, we applied metaproteomics and metabolomics and found,
along with shortening the TT, a slight decrease in glycan biosynthesis, carbohydrate, and amino acid
metabolism and, furthermore, a reduction in butyrate, methyl butyrate, isobutyrate, valerate, and
isovalerate concentrations. Specifically, B. thetaiotaomicron was identified to be affected in terms of
butyrate metabolism. However, communities could recover to the original state afterward. This study
shows that SIHUMIx showed high structural stability when TT changed—even four-fold. Resistance
values remained high, which suggests that TTs did not interfere with the structure of the community
to a certain degree.
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Environmentally Relevant Concentration of Bisphenol S Shows Slight Effects on SIHUMIxSchäpe, Stephanie Serena, Krause, Jannike Lea, Masanetz, Rebecca Katharina, Riesbeck, Sarah, Starke, Robert, Rolle-Kampczyk, Ulrike, Eberlein, Christian, Heipieper, Hermann-Josef, Herberth, Gunda, von Bergen, Martin, Jehmlich, Nico 20 April 2023 (has links)
Bisphenol S (BPS) is an industrial chemical used in the process of polymerization of polycarbonate plastics and epoxy resins and thus can be found in various plastic products and thermal papers. The microbiota disrupting effect of BPS on the community structure of the microbiome has already been reported, but little is known on how BPS affects bacterial activity and function. To analyze these effects, we cultivated the simplified human intestinal microbiota (SIHUMIx) in bioreactors at a concentration of 45 µM BPS. By determining biomass, growth of SIHUMIx was followed but no differences during BPS exposure were observed. To validate if the membrane composition was affected, fatty acid methyl esters (FAMEs) profiles were compared. Changes in the individual membrane fatty acid composition could not been described; however, the saturation level of the membranes slightly increased during BPS exposure. By applying targeted metabolomics to quantify short-chain fatty acids (SCFA), it was shown that the activity of SIHUMIx was unaffected. Metaproteomics revealed temporal effect on the community structure and function, showing that BPS has minor effects on the structure or functionality of SIHUMIx.
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Développement d’un modèle in vitro de Barrière Hémato-Encéphalique humaine pour des études pharmacologiques : Interactions avec les anticoagulants oraux directs / Development of an in vitro model of a human Blood Brain Barrier for pharmacological studies : Interactions with directs oral anticoagulantsPuech, Clémentine 13 December 2018 (has links)
La barrière hémato-encéphalique (BHE) contrôle le passage des médicaments, en partie par la présence d’ATP Binding Cassette (ABC) transporteurs. Dans de nombreuses pathologies cérébrales, la BHE est altérée. Parmi elles, les hémorragies intracérébrales (HIC), qui sont un effet iatrogène des anticoagulants. Des analyses cliniques montrent que les patients sous Anticoagulants Oraux Directs (AODs) présentent moins d’HIC que les patients traités avec les anticoagulants de référence, les anti-vitamine K (AVK), sans que les mécanismes cellulaires soient élucidés. Une des différences entre les AODs et les AVK résident dans leur profil pharmacocinétique, effectivement, les AODs sont des substrats des ABC transporteurs contrairement aux AVKs. Au cours des HIC, la thrombine est activée et entraine une altération de la BHE par clivage et des récepteurs protease activated receptor (PAR). Les objectifs de ce travail de thèse ont été de mettre en place un modèle in vitro de BHE afin d’étudier les interactions des médicaments avec les ABC transporteurs. Ensuite, le modèle est utilisé pour étudier les interactions des AODs en condition pathologique. Le modèle développé est basé sur la lignée HBEC-5i, peu décrite dans la littérature. Les cellules ont été cultivées en monocouche sur insert avec milieu conditionné issu d’astrocytes humains. Le modèle permet l’étude de l’interaction de thérapeutiques avec des ABC transporteurs par des mesures d’efflux ratios. Le modèle a été validé par des études de transport de molécules pharmacologiques. Ensuite, nous avons comparé, sur notre modèle, les effets de l’exposition à la thrombine avec ou sans prétraitement d’anticoagulants (rivaroxaban, dabigatran, apixaban, warfarine et héparine). Les AODs limitent l’ouverture de la BHE induite par la thrombine contrairement aux autres anticoagulants. Nos résultats ont montré que l’altération de la BHE est médiée par le clivage du récepteur PAR-1 par la thrombine. Ce clivage n’est pas le même en fonction de la classe d’anticoagulants utilisée, les AODs minimisant ce clivage. L’ensemble de ce travail de thèse a permis de donner des premières explications cellulaires quant aux mécanismes d’ouverture de la BHE consécutifs aux HIC sous AODs. / The blood-brain barrier (BBB) controls the passage of drugs, in part through the expression of the ATP Binding Cassette (ABC) transporters. In many brain diseases, the BBB is altered. Among them, intracerebral haemorrhages (ICH), which are an iatrogenic effect of anticoagulants. Clinical analyses show that patients with Direct Oral Anticoagulants (DOACs) treatment have less HIC than patients treated with the reference anticoagulants, Vitamin K Antagonist (VKA), without understanding the cellular mechanisms. One of the differences between DOACs and VKA lies in their pharmacokinetic profile, indeed, DOACs are substrates of ABC transporters unlike VKA. During HIC, thrombin, is activated and causes alterations in the BBB by the cleavage of the protease activated receptor (PAR). The objectives of this thesis work were first to set up an in vitro model of the BBB in order to study the passage of drugs and their interactions with ABC transporters. In a second step, the model is used to study the interactions of DOACs in pathological conditions. The model developed is based on the HBEC-5i cell line seldom described in the literature. The cells were cultured in monolayer on insert with conditioned medium from human astrocytes. It allows the study of the interaction of therapeutics with ABC transporters by measuring efflux ratios. The model has been validated by transport studies of pharmacological molecules. In order to meet our second objective, we compared the effect of thrombin exposure with or without pretreatment with anticoagulants (rivaroxaban, dabigatran, apixaban, warfarin and heparin sodium) on our model. DOACs limit the BBB damage induced by the thrombin unlike other anticoagulants. Our results showed that alteration of the BBB is mediated by the cleavage of the PAR-1 receptor by thrombin. This cleavage is not the same depending on the class of anticoagulants used, DOACs minimizing this cleavage. All this thesis work made it possible to provide the first cellular explanations of the opening mechanisms of the BBB following HIC under DOACs.
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Survie et pathogénicité des EHEC dans l'environnement digestif : Interactions avec le microbiote et l'épithélium intestinal. : Influence de l'administration de levures probiotiques. / Survival and Pathogenicity of Enterohemorrhagic Escherichia Coli in Digestive Environment : Interactions with the Microbiota and Intestinal Epithelium. : Influence of the Administration of Probiotic Yeasts.Cordonnier, Charlotte 18 December 2015 (has links)
Les Escherichia coli entérohémorragiques (EHEC) sont des pathogènes majeurs pour l’homme responsables de toxi-infections alimentaires pouvant évoluer vers des complications potentiellement mortelles. La pathogénicité de ces souches est essentiellement due à la production de Shiga-toxines (Stx), même si d’autres facteurs semblent jouer un rôle important dans la virulence, comme des facteurs d’adhésion. La survie et la régulation des facteurs de virulence des EHEC dans l’environnement digestif humain sont des facteurs clés dans la pathogénicité bactérienne, mais restent à ce jour mal décrits, essentiellement en raison d’un manque de modèles d’étude adaptés. De plus, l’absence de traitement spécifique a conduit à s’intéresser à des moyens préventifs et/ou curatifs alternatifs, comme l’utilisation de probiotiques. L’objectif de ce travail de thèse est (i) de mieux comprendre le comportement de la souche de référence EHEC O157:H7 EDL933 dans l’environnement digestif humain simulé, et en particulier ses interactions avec le microbiote résident et l’épithélium intestinal, et (ii) d’évaluer l’effet antagoniste d’une souche de levure probiotique vis-à-vis de la survie, la virulence et l’interaction du pathogène avec l’épithélium intestinal, à l’aide d’approches in vitro et in vivo complémentaires. En modèles digestifs in vitro, la souche EHEC survit dans l’estomac, voire se multiplie dans les parties distales de l’intestin grêle, alors qu’elle ne se maintient pas dans l’environnement colique. Les gènes de virulence codant les Stx et des adhésines majeurs (intimine et « Long Polar Fimbriae » ou Lpf) sont surexprimés dès les parties hautes du tractus digestifs, et ce, même en absence de cellules épithéliales. Les conditions rencontrées dans le tractus digestif supérieur de l’enfant, comparativement à celui de l’adulte, conduisent à une survie et un niveau d’expression des gènes codant les Stx et les Lpf plus élevés chez l’enfant, ce qui peut contribuer à expliquer la grande sensibilité de cette population aux infections à EHEC. Enfin, les Lpf semblent jouent un rôle clé dans le ciblage spécifique des cellules M et le tropisme des EHEC pour les plaques de Peyer, et ce, à la fois in vitro (cellules M en culture) et in vivo (anses iléales murines). Même si elle ne modifie pas la survie du pathogène dans l’environnement colique, la levure probiotique S. cerevisiae CNCM I-3856 a montré des propriétés antagonistes intéressantes vis-à-vis d’EHEC O157:H7 en (i) modulant favorablement l’activité fermentaire du microbiote intestinal, (ii) diminuant significativement l’expression des gènes codant les Stx et (iii) inhibant la translocation bactérienne au travers des plaques de Peyer et les lésions hémorragiques associées. Par ailleurs, l’effet du pathogène et des probiotiques sur le microbiote colique est individu dépendant, confortant l’hypothèse que des facteurs associés à l’hôte, comme le microbiote, pourraient conditionner l’évolution clinique des infections à EHEC et l’efficacité d’une stratégie probiotique.Ce travail de thèse contribue à une meilleure compréhension du comportement des EHEC dans l’environnement digestif humain et confirme l’intérêt d’une stratégie probiotique dans la lutte contre le pathogène. Une étude plus approfondie du transcriptome du pathogène dans l’environnement digestif et une analyse par des méthodes haut débit du microbiote intestinal permettraient de continuer à mieux décrire la physiopathologie des infections à EHEC et comprendre les mécanismes associés à l’effet antagoniste des probiotiques. / The enterohemorrhagic Escherichia coli (EHEC) are major zoonotic pathogens responsible for food-borne infectionwhich leads to life-threatening complications in humans. The main virulence determinant of EHEC is the production of Shigatoxins (Stx), even if other factors seem to play an important role in virulence, such as adhesion factors. Survival and virulenceof EHEC strains in the human digestive environment are a key factor in bacterial pathogenesis but remains unclear owing tolack of relevant model. Moreover, no specific treatment has led to interest in preventative and / or curative alternatives, suchas using probiotics. The objective of this study is to better understand the behavior of the reference strain EHEC O157:H7EDL933 in the entire digestive tract, and in particular its interaction with the resident microbiota and the intestinal epithelium,and to evaluate the antagonistic effect of the probiotic yeast, Saccharomyces cerevisiae CNCM I-3856, using in vitro and in vivo complementary approaches.In vitro, bacterial mortality was noticed in the stomach, whereas bacterial growth resumption was observed in thedistal parts of the small intestine and the pathogen was not able to maintain in the human colonic conditions. Virulence genesencoding Stx and adhesins (intimin and “Long polar fimbriae”) are upregulated in the upper parts of the digestive tract. A ten-time higher amount of cells was found in the ileal effluents of infant compared to adult. stx genes were over-expressed (up to25-fold) in infant conditions compared to the adult ones. This results show that differences in digestive physicochemicalparameters of the upper gastrointestinal tract may partially explain why infants are more susceptible to EHEC infection thanadults. And finally, Lpf seem to play a key role in the interactions of EHEC with murine Peyer’s patches and are needed for anactive translocation of the pathogen across M cells, and both in vitro (M cells culture) and in vivo (murine ileal loops).S. cerevisiae had not effect on EHEC survival in the colonic environment but (i) favorably influenced gut microbiotaactivity through beneficial modulation of short chain fatty acid production, (ii) leading to significantly decrease stx expressionand (iii) significantly reduced EHEC translocation through M cells and inhibited in vivo interactions of the pathogen withPeyer’s patches and the associated hemorrhagic lesions. Probiotic had donor-dependent effect on the gut microbiota strengthenthe hypothesis that host-associated factors such as microbiota could influence the clinical evolution of EHEC infection and theeffectiveness of a probiotic strategy.This work contributes to a better understanding of the behavior of EHEC in the human digestive environment andconfirms the interest of probiotic strategy in controlling EHEC infections. Further transcriptome studies are warranted for thepathogen in the human digestive environment, with or without probiotics for the better understanding of the pathophysiologyof EHEC and so on the mechanisms involved in the antagonistic effect of probiotics.
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Mikrofluidisches in-vitro Modell der Blut-Hirn-Schranke mit aktiver Zellassemblierung mittels DielektrophoreseKießling, Heiko 15 December 2021 (has links)
Neue aussichtsreiche Pharmazeutika scheitern regelmäßig in späten Entwicklungsphasen1 und stehen somit nicht als wertvolle Wirkstoffe zur Verfügung. Ein Grund hierfür ist die komplexe Pharmakinetik und der Mangel an geeigneten in-vitro Modellen.
Daher befasst sich diese Dissertation mit der Entwicklung neuartiger in-vitro Membranmodelle am Beispiel der Blut-Hirn-Schranke (BHS). Zu diesem Zweck wird der aktuelle Stand der Technik vorgestellt und anschließend das Konzept eines Mikrofluidikchips, in welchem mittels Dielektrophorese an eine zuvor erstellte Polyamidmembran CaCo-2-Zellen assembliert wurden. Die Auslegung und Optimierung des Chip-Designs, die Entwicklung der in-situ Membran sowie die Ermittlung der Randbedingungen sind wesentliche Bestandteil dieser Arbeit. Es konnte mittels FEM-Simulationen und Assemblierungsversuchen ein Modell erzeugt werden, mit dem ein Chipdesign entwickelt werden konnte, dass zum einen ein günstigeres Verhältnis von Zellflächen- und Abluminalen Volumen aufweist und zum anderen möglichst wenig Zellen für den Aufbau benötigt. Dieses System bietet somit ein hohes Potenzial für die Herstellung verbesserter in-vitro Modelle. Jedoch konnte auch durch die Charakterisierung mit Rhodamin, Fluorescein und FITC-Dextran aufgezeigt werden, dass dieser Vorteil durch spezifische und unspezifische Bindungen an der größeren Oberfläche z.T. reduziert wird, abhängig vom verwendeten Chipmaterial und untersuchten Wirkstoff.
Als neuartig kann die in-situ Herstellung einer vertikalen Polyamidmembran in einem Polymerchip bezeichnet werden, die im Rahmen dieser Arbeit entwickelt wurde. Für diese wurden die Parameter zur optimale Collagenbeschichtung für die Zelladhäsion ermittelt, sowie der Einfluss auf die Zellvitalität untersucht. Des Weiteren wurde das Medium zur Dielektrophorese und zur Kultivierung der Zellen ohne CO2-Begasung optimiert.:1 Abkürzungsverzeichnis
2 Formelzeichen
3 Einleitung
4 Grundlagen
5 Chipdesign
6 Herstellung und Charakterisierung der Stützmembran
7 Entwicklung des Zellkulturmodells
8 Zusammenfassung
9 Ausblick
10 Anhang
11 Literaturverzeichnis
12 Abbildungsverzeichnis / New promising pharmaceuticals regularly fail at late stages of development1 and thus do not become available as valuable active substances. Two of the main reasons for such failures are the complex pharmacokinetics and the lack of adequate in-vitro models.
Therefore, this dissertation focuses on the development of novel in-vitro membrane models at the example of the blood-brain-barrier (BBB). It starts by presenting current investigations and the state-of-the-art technology and continues with the concept for a microfluidic chip in which CaCo-2 cells were assembled with dielectrophoresis on an in-situ membrane. The essential part of this work was to design and optimize this microfluidic chip, to develop an in-situ membrane to catch the assembled cells and to investigate the required boundary conditions. FEM simulations and assembling experiments conducted to the creation of a model to develop a chip design with a better ratio between cell area and abluminal volume, as well a low number of cells needed for the creation of this model. Such system might have a high potential to establish more sensitive in-vitro models than the current Transwell model. However, it was also demonstrated that this advantage is reduced by specific and nonspecific binding on the larger surface, depending on the chip material and the investigated test substance, shown during the chip characterization by using Rhodamine, Fluorescein and FITC-Dextran.
Furthermore, the creation of a vertical polyamide in-situ membrane in a polymer chip like in this work, can be described as novel. To assemble cells on this supporting membrane, a protocol for a collagen coating as well for the dielectrophoresis medium were developed. Also, a modified culture medium was investigated, to allow the cultivation on standard atmospheric conditions.:1 Abkürzungsverzeichnis
2 Formelzeichen
3 Einleitung
4 Grundlagen
5 Chipdesign
6 Herstellung und Charakterisierung der Stützmembran
7 Entwicklung des Zellkulturmodells
8 Zusammenfassung
9 Ausblick
10 Anhang
11 Literaturverzeichnis
12 Abbildungsverzeichnis
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Studies of the expression and characterization of various transport systems at RBE4 cells, an in vitro model of the blood-brain barrier / Studien zur Expression und Charakterisierung verschiedener Transport Systeme an RBE4 Zellen, einem in vitro Modell der Blut-Hirn SchrankeFriedrich, Anne 05 July 2003 (has links) (PDF)
The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.
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Studies of the expression and characterization of various transport systems at RBE4 cells, an in vitro model of the blood-brain barrierFriedrich, Anne 08 November 2002 (has links)
The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.
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Investigating TRPV4 Signaling in Choroid Plexus Culture ModelsLouise Susannah Hulme (12456711) 12 July 2022 (has links)
<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p>
<p><br></p>
<p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p>
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<p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>
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Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic ProductsMcCanna, David January 2009 (has links)
The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes.
The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.
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