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Studium migrace mesenchymálních kmenových buněk v extracelulárním matrix na principu chemotaxe / Study of mesenchymal stem cell migration in the extracellular matrix based on principles of chemotaxisScholasterová, Viktorie January 2021 (has links)
This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.
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Studium migrace mesenchymálních kmenových buněk na principu chemotaxe / Study of mesenchymal stem cell migration based on principles of chemotaxisPošustová, Veronika January 2020 (has links)
The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.
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Evaluation of poly D, L lactic-co-glycolic acid (PLGA) nanoparticle uptake pathways across the nasal mucosaAlbarki, Mohammed Abdulhussein Handooz 01 August 2019 (has links)
The nasal mucosa provides a non-invasive route for drug administration to the systemic circulation and potentially directly to the CNS. Nanoparticles made from biodegradable polymers, including PLGA, are of great interest for use in drug delivery systems due to their relative safety and ease of surface modification. Owing to their small size, nanoparticles may provide enhanced targeting and transport through the nasal mucosa. An improved understanding of the mechanisms and pathways of nanoparticle transfer across the nasal mucosa is needed to design effective new nasal delivery systems. This study focuses on the preparation of PLGA nanoparticles in various diameters and with varying surface characteristics followed by the in vitro investigation of the mechanisms of endocytosis and exocytosis of PLGA nanoparticles in the nasal mucosa.
PLGA nanoparticles (60 nm or 125 nm) containing the lipophilic fluorescent dye, Nile Red, were prepared using a surfactant-free nanoprecipitation method. In one investigation, the inherent negative surface charge of 60 nm PLGA nanoparticles was modified to a positive charge using a 5th generation polyamidoamine dendrimer (PAMAM) during preparation of nanoparticles. In addition, 60 nm PLGA nanoparticle surfaces were coated by adding 5 % (w/v) bovine serum albumin (BSA) to the nanoparticle dispersion and allowing protein adsorption on the particle surface. Nile Red-loaded PLGA nanoparticles were transported into the epithelial layer and reached the sub-mucosal connective tissues, yet only < 5% of the PLGA nanoparticle load was transferred into the nasal mucosa. Total uptake was size dependent, where the uptake of 60 nm unmodified PLGA nanoparticles was significantly higher than uptake of 125 nm nanoparticles. The amount of Nile Red measured in the tissues after expose to the 125 nm nanoparticles was double the amount from the 60 nm nanoparticles due to differences in the carrying capabilities of the 60 and 125 nm PLGA nanoparticles. Modification of the nanoparticle surface with PAMAM or BSA decreased the uptake of 60 nm PLGA nanoparticles into the nasal mucosa.
Endocytic mechanisms involved in the uptake of PLGA nanoparticles were studied using chemical inhibitors. Nanoparticle uptake in the nasal respiratory mucosa involved energy-dependent processes utilizing multiple known mechanisms, including clathrin-mediated endocytosis and macropinocytosis. In the olfactory mucosa, significant energy-independent nanoparticle uptake was also observed.
In order to investigate how nanoparticles exit epithelial cells for further distribution to distant tissues, the exocytosis of 60 nm Nile Red-loaded PLGA nanoparticles was evaluated using three different epithelial cell line models, RPMI-2650 (nasal), Calu-3 (lung) and MDCK-II wild type (kidney) cells. Following a 30 min exposure to a 60 nm PLGA nanoparticles dispersion, nanoparticle exocytosis into a protein-free medium was evaluated for additional 30 or 60 min. Only a limited number of NP (~ 20 % of the endocytosed NP) underwent exocytosis into the medium after 60 min, while the majority of the internalized nanoparticles remained within the cells.
The measurable transfer of PLGA nanoparticles into the nasal mucosal tissues indicates that they may be useful drug carriers for nasal administration. However, the limited exocytosis of 60 nm NP and the resulting potential for intracellular accumulation may raise toxicity concerns and result in potential cellular injury. While PLGA nanoparticles provide promising drug delivery systems for nasal administration, only with careful design of the nanoparticles, including their size and surface characteristics, will efficient and effective, safe drug delivery be accomplished.
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THE ROLE OF ABCG2 IN DRUG ACTIVE TRANSPORT IN MILKWang, Lipeng 01 January 2010 (has links)
Drug active transport into milk is a major concern for breastfeeding mothers and healthcare providers. Studies from the literature indicated that breast cancer resistance protein (ABCG2) plays an important role in drug transfer into milk. There has been limited study on stereoselective interactions with ABCG2. A mechanistic analysis of flux across cell monolayer model is a critical first step toward extrapolating in vitro results for predicting in vivo disposition (including distribution into milk), drug disposition or drug-drug interactions.
The objectives of this thesis were (1) to establish a “Chemical knockout model” in rat for studying drug accumulation into milk, (2) to investigate the impact of stereoselective interaction between ABCG2/Abcg2 and pantoprazole on drug transport in milk, (3) to understand in vitro monolayer flux model using experimental data and a mechanistic mathematical model.
Quantitive PCR, Western blotting and immunohistochemistry results indicated that Abcg2 was up-regulated during lactation and localized on apical side of epithelial cells in mammary gland. In vitro and in vivo experiments confirmed that Abcg2 is responsible for nitrofurantoin active transport in rat milk and GF120918 was established as a chemical knockout model.
Abcg2 interacts stereoselectively with pantoprazole isomers. A significant different apical flux between two pantoprazole isomers was observed in Abcg2-MDCKII cell line. The milk to serum (M/S) ratio of (-)-pantoprazole was almost 3 times as that of (+)-pantoprazole in lactating rats. Administration GF120918 decreased M/S of (-)-pantoprazole (p<0.001) but not (+)-pantoprazole (p>0.05).
A stably transfected ABCG2/Abcg2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships in a monolayer flux model. Based on the profiles of pantoprazole isomer transport, a simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC) and apically efflux ABCG2 (PSA,E) transfection was developed. The mathematical model was developed to more fully understand the interplay of paracellular, passive diffusion, active transport, and flux kinetic parameters (Km, Vmax, IC50 and Ki). This model provided useful insights into the meaning and limitation of parameters obtained from monolayer flux.
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Microporous Membrane-based Co-culture of Human Embryonic Stem CellsAlbert, Kelsey Morgan 01 January 2007 (has links)
Transwell inserts with microporous membranes, available from multiple commercial sources, have been widely used for various mammalian cell culture applications, including the reduction of cell culture mixing. In this study, we examined the feasibility and functionality of using this technology for separating human embryonic stem cells (hESCs) from their respective feeder cells. We found that when hESCs were propagated on transwell inserts positioned directly above feeder cells grown in a separate dish, the hESCs could be maintained in an undifferentiated state for over 10 passages with no change in their basic pluripotent characteristics. In parallel with our transwell insert experiments, we also evaluated the ability of a new defined, xeno-free medium, HEScGRO, to enhance the animal-free characteristics of the transwell insert-based culture system. Results from our studies demonstrate that HEScGRO medium assists in maintaining the pluripotent characteristics of hESCs propagated in the transwell insert- based culture system. These combined results represent a significant development in properly segregating stem cells from their feeders, thus eliminating cell mixing, contamination, and providing the cells with a superior environment for nourishment and controlled self-renewal. Overall, this development in hESC propagation could have wide-reaching applications for self-renewal and differentiation studies within the field of stem cell biology.
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Determining the Effects of CD151 and β<sub>1</sub> on Tumor Cell Adhesion and MigrationEssex, Rachel R. 01 January 2015 (has links)
Previous studies have shown that the upregulation of CD151 and β1 is associated with poor prognosis in many cancers such as breast cancer. Studies have provided evidence that these proteins are associated with the adhesion and migration of tumor cells. In this study, a microfluidic flow chamber was utilized to determine how CD151 and β1 affected the firm and initial adhesion of metastatic breast cancer cells to a planar endothelial monolayer under shear stress. This system mimicked the adhesion of metastatic breast cancer cells to the endothelial cells of the circulatory system. CD151 and β1 increased the firm adhesion of metastatic breast cancer cells onto an endothelial monolayer when subjected to high shear stresses. CD151 and β1 increased the initial adhesion of metastatic breast cancer cells onto an endothelial monolayer. A transwell assay was utilized to determine how CD151 and β1 affected random migration through different matrixes and random transendothelial migration. CD151 and β1 decreased the random migration of metastatic breast cancer cells through matrices. Additionally, background information is provided related to the metastatic cascade, how it can be modeled with microfluidics, and how CD151 and β1 have been known to effect cancer and metastasis.
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Investigation of inhibitors of polysialyltransferase as novel therapeutics for neuroblastoma : development of in vitro assays to assess the functionality and selectivity of novel small-molecule inhibitors of polysialyltransferases for use in neuroblastoma therapySaeed, Rida Fatima January 2015 (has links)
Polysialic acid is a unique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acid is an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma, strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST (ST8SiaIV) and STX (ST8SiaII), with STX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferase could lead to control of tumour dissemination and metastasis. The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferase inhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells (SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acid surface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acid surface expression and invasion. Of the potential polysialyltransferase inhibitors evaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p < 0.05) reduction in cell-surface polysialic acid re-expression and invasion in polysialic acid expressing cells. Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over other members of the wider sialyltransferase family (α-2,3- and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitro cell based assays to discovery more potent derivatives.
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Investigation of inhibitors of polysialyltransferase as novel therapeutics for neuroblastoma. Development of in vitro assays to assess the functionality and selectivity of novel small-molecule inhibitors of polysialyltransferases for use in neuroblastoma therapySaeed, Rida F. January 2015 (has links)
Polysialic acid is aunique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acidis an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma,strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST(ST8SiaIV)and STX(ST8SiaII),withSTX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferasecould lead to control of tumour dissemination and metastasis.The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferaseinhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells(SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acidsurface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acidsurface expression and invasion. Of the potential polysialyltransferase inhibitorsevaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p<0.05)reduction in cell-surface polysialic acidre-expression and invasion in polysialic acid expressing cells.Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over othermembers of the wider sialyltransferase family (α-2,3-and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitrocell based assays to discovery more potent derivatives.
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Characterization of the effect of the membrane on in vitro dissolution profiles for pulmonary drug deliverySimonides, Maral January 2021 (has links)
It has always been a challenge to imitate the lung environment, therefore there is a constant development of standardized in vitro dissolution methods for inhaled products. Dissolution in vitro has been considered as an important parameter, because low solubility determines the bioavailability of inhaled drugs. The in vitro dissolution data generated by the dissolution test experiment can be correlated with in vivo pharmacokinetic data through in vitro-in vivo correlation (IVIVC), because a completed predictive IVIVC model is very useful for drug formulation design and manufacturing changes after approval. The aim of this study was to investigate the effect of the membrane on the dissolution profile of orally inhaled drugs with different solubility, Budesonide (BUD) and Fluticasone propionate (FP) in the different pore sizes of the membrane 8.0 μm, 3.0 μm and 0.4 μm. The method in this study builds on previous dissolution methods, a Transwell® setup to dissolve the drugs with a small amount of dissolution medium, which mimics more the limited lung fluid capacity in vivo. In order to collect the dose from the drugs, Andersen Cascade Impact was used. The dissolution rate of BUD was first in the ranking in all of the pore sizes in the membrane.
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Développement d’un modèle in vitro de la barrière hémato-encéphaliquePuscas, Ina 04 1900 (has links)
La barrière hémato-encéphalique (BHE) est une structure retrouvée au niveau des capillaires cérébraux. Elle représente un véritable obstacle pour les actifs qui doivent se rendre au cerveau pour y exercer un effet pharmacologique. Durant les étapes du développement du médicament, des modèles cellulaires in vitro sont utilisés pour l’évaluation de la perméabilité au cerveau des nouveaux médicaments. Le modèle assemblé avec des cellules endothéliales (CEs) isolées des capillaires des cerveaux de souris présente un intérêt particulier pour la recherche en raison de sa facilité d’obtention et sa pertinence pour le criblage des médicaments. Le but de ce projet a été de construire et de caractériser un modèle monocouche de CEs primaires de souris. En parallèle, un modèle monocouche de la lignée murine b.End3 a été investigué. L’évaluation de ces modèles a été basée sur les valeurs de TEER et de perméabilité aux marqueurs fluorescents, ainsi que sur la présence des protéines spécifiques de la BHE. La validation du modèle a été établie par la corrélation des résultats de perméabilité obtenus avec le modèle développé (in vitro) avec ceux obtenus chez la souris (in vivo). L’intégrité et l’expression des protéines spécifiques de la BHE du modèle primaire se sont montrées supérieures au modèle bEnd.3. La corrélation in vitro/in vivo du modèle primaire a abouti à un r2 = 0,765 comparé au r2 = 0,019 pour le modèle bEnd.3. Ce travail de recherche montre que le modèle primaire monocouche issu de cellules endothéliales cérébrales de souris est un modèle simple et fiable pour la prédiction de la perméabilité des actifs à travers la BHE. / The blood-brain barrier (BBB), a central nervous system structure, is found in the cerebral capillaries. It represents a major obstacle for the drugs that have to reach the brain in order to exercise their pharmacological effect. In the early stages of the drug development, in vitro cell models are used to evaluate the brain permeability of new drugs. Models assembled using primary endothelial cells (ECs) isolated from mouse brain capillaries are of particular interest for research, as for their ease of obtaining and relevance for the drug screening. Thus, the goal of this project was to build and characterize a primary mouse monolayer model. At the same time, a murine b.End3 cell line monolayer model was investigated. The evaluation of these models was based on the TEER and fluorescent marker permeability values, as well as on the presence of the BBB hallmark proteins. The model validation was established by the correlation of the permeability data obtained with the in vitro model and the data obtained in mice (in vivo). As a result, the primary mouse model showed superior monolayer integrity and higher expression of the tight junction and membrane transporter proteins when compared with the bEnd.3 cell line model. The in vitro/in vivo correlation of the primary model resulted in r2 = 0.765 compared to the bEnd.3 model with r2 = 0.019. This research work shows that the primary monolayer mouse model is a simple and reliable model for predicting the drug permeability across the BBB.
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