Modern drug development is an astronomically expensive and time consuming undertaking. Because of this, studying the pharmacokinetic properties of drugs in vitro has become an integral step early in the process of drug development, with the goal of preventing costly failures late in the process, and dangerous side effects. Artificial phospholipid bilayers known as droplet interface bilayers (DIBs) have the potential to be used for these pharmacokinetics assays, combining the low cost of cell-free assays with the ability to more closely mimic structures found in life than current cell-free in vitro techniques. Combined with the reproducibility, ease of use, and low reagent consumption found with microfluidic methods, disruptive new low cost techniques for assessing pharmacokinetics in drug development may be possible using DIBs as an artificial cell membrane model. In this work, I establish the potential of DIBs to be used as a pharmacokinetics modelling platform, and advance the use of microfluidic methods for carrying out pharmacokinetics assays in drug discovery. I first developed a new microfluidic platform for the formation of DIBs, which sought to solve some of the shortcomings of current microfluidic methods for DIB formation (Chapter 2). This device is the first that can be used to form DIB networks from dissimilar droplets in parallel, without use of active controls, and with droplet contact gentle enough to enable use of biomimetic lipid mixtures. I examine for the first time the behaviour of phospholipids on microfluidic devices, and characterise the interaction that they have with a common material used to construct microfluidic devices (Chapter 3). Not only has this interaction never been studied before, but my unexpected findings indicate a new area requiring further study in order to advance the adoption of DIBs on microfluidic devices. In collaboration with my colleague Jaime Korner, I use my newly developed microfluidic platform to carry out an on-chip permeation assay for the first time using biomimetic lipid formulations and bespoke compartments modelled after the human intestine. We demonstrate that this on-chip assay has predictive accuracy greater than that of a current widely used cell-free technique (Chapter 4). Finally, I demonstrate that a DIB based microfluidic platform enables, and is critical for, characterising the effect of structural features such as membrane asymmetry on drug permeation. With this, I find measurable, previously unknown effects of membrane asymmetry on the absorption of the chemotherapy drug doxorubicin, highlighting a possible contributing factor to chemoresistance in some cancers (Chapter 5). I find, and demonstrate throughout the body of this work that microfluidic methods and DIBs can not only provide alternatives to current cell-free in vitro pharmacokinetics assays, but that they can exceed the performance of existing assays, and be used for entirely new ways of examining pharmacokinetics. Through building bespoke artificial cell membranes from the ground up, I hope to demonstrate herein the great potential of these powerful new cell-free methods. / Graduate / 2022-09-12

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This diploma thesis is focused on determination of aggregation behavior of selected phospholipids (lecithin; 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) in water and on the effect of native hyaluronan addition of various molecular weights and concentrations on this behavior. The behavior has been investigated with fluorescence spectroscopy using pyrene and perylene as fluorescence probes being able to penetrate into hydrophobic cavities of formed aggregates. Critical aggregation concentration and the concentration at which lecithin begins to aggregate have been determined. Regarding 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, it was possible to determine only the beginning of aggregation value. The values of this parameter for lecithin and for 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine correspond in the order. It has been investigated, that the addition of native hyaluronan has only in some systems slight effect on the aggregate behavior of selected phospholipids.

Le lipidome désigne la sous-fraction lipidique du métabolome d’une cellule, d’un tissu ou d’un organisme. Cependant, bien qu’appartenant au cadre général de la métabolomique, la lipidomique représente un champ d’investigation particulier en raison des spécificités fonctionnelles et métaboliques des lipides de même que de leurs propriétés physico-chimiques. L’analyse lipidomique représente un changement majeur d’échelle par rapport à l’analyse des lipides telle que pratiquée classiquement. L’objectif est ici d’atteindre l’échelle de l’espèce moléculaire lipidique en termes d’identification et de quantification afin de décrire l’ensemble des perturbations d’un réseau métabolique liées à un état physiologique ou pathologique. L’identification des classes et espèces repose sur principalement sur l’utilisation du couplage de la chromatographie liquide couplée à la spectrométrie de masse. La quantification peut faire appel à la spectrométrie de masse quand la spécificité et des faibles limites de détection sont requises ou à l’utilisation de détecteurs « universels » ou détecteurs par nébulisation que sont le détecteur évaporatif à diffusion de la lumière (DEDL) ou le détecteur d’aérosol chargé (Corona®-CAD) quand l’objectif est de quantifier l’ensemble de la classe lipidique. / The lipidome means the lipid subfraction of a metabolome, of a cell, a tissue or an organism. Lipidomics represents a particular field of investigation because of the functional and metabolic specificities of lipids as well as their physico-chemical properties. Lipidomics analysis represents a major change of scale with respect to lipid analysis as practiced conventionally. The aim of this work is to reach across the lipid molecular species in terms of identification and quantification in order to describe all the perturbations of a metabolic network related to a physiological or pathological state. The identification of the classes and the specie is based mainly on the use of liquid chromatography coupled to mass spectrometry. Quantification may use mass spectrometry when the specificity and low detection limits are required or the use of "universal" detectors that are nebulized detectors:evaporative detector light scattering (ELSD) or charged aerosol detector (CAD-Corona®) when the aim is to quantify the total lipid classes.

La Structuration des molécules d’eau à l’interface eau/vapeur d’eau fait l’objet de l’intérêt scientifique depuis des années. La plupart des études sont focalisées sur le bulk d’eau mais des études plus détaillées sur l’eau de surface sont nécessaires. De plus, les interfaces avec les alcanes sont intéressantes d’un point de vue biologique et industriel. Puisque pour des applications biologiques et industrielles les interfaces eau/air et eau/huile possèdent des médiateurs amphiphiles, l’influence d’une monocouche de tensioactif sur la structuration de la surface de l’eau mérite aussi une attention particulière.Dans cette thèse, plusieurs modèles atomistiques d’eau ont été sélectionnés. Des simulations de dynamique moléculaire classique ont été réalisées à 298K pour le bulk d’eau, des systèmes eau/vapeur d’eau et eau/alcane (C5-C9), ainsi que des systèmes eau/DLPC/vapeur d’eau et eau/DLPC/octane (DLPC : dilauroyl phosphatidylcholine). Plusieurs propriétés structurelles, ainsi que les moments dipolaires, la tension de surface et les liaisons hydrogène, du bulk d’eau et des couches de surface d’eau ont été examinées grâce à la fonction de distribution radiale et les diagrammes de Voronoi. L’objectif a été d’estimer l’impact de l’incorporation de la polarisabilité sur les propriétés de l’eau et de sélectionner un modèle optimal (qualité/temps de calcul) pour leur description, ainsi que d’enrichir les données existantes sur la structuration de l’eau à l’interface.Cette étude aborde la structuration de l’eau du bulk et de la surface à l’interface avec la vapeur d’eau ou des alcanes. Un des objectifs de ce travail a été d’évaluer la reproductibilité des données expérimentales en utilisant différents modèles et de s’assurer pour quelles propriétés l’utilisation d’un modèle polarisable est critique. De simples modèles polarisables basés sur les oscillateurs de Drude ont été testés afin de limiter le temps de calcul. Pour le bulk d’eau et les systèmes eau/vapeur d’eau, les modèles TIP4P, SWM4-NDP et COS/G2 ont été les plus performants. Dans la mesure où le modèle TIP4P produit des résultats commensurables avec les modèles polarisables, il a été utilisé pour la simulation des interfaces eau/alcanes (C5-C9). Les molécules à la surface sont organisées de manière plus compacte et moins ordonnée. Cette diminution de l’ordre est principalement due aux liaisons hydrogène qui sont deux fois plus nombreuses dans le bulk qu’en surface. Les analyses de Voronoi ont montré que la coordination tétraédrique n’est pas si claire et que des polyèdres plus complexes sont formés. Les couches de surfaces trouvées s’avèrent être formées de 2 sous-couches, interne et externe, avec des polarités inégales orientées de manières opposées, définissant des zones de charges résiduelles à l’interface.En plus des systèmes avec un contact direct entre l’eau et le fluide apolaire, des interfaces comportant des monocouches de lipides ont été modélisées. La compacité de l’eau de surface, déjà renforcée par la présence d’alcanes, a été augmentée davantage par l’introduction de lipide. Néanmoins, l’orientation de l’eau a été changée et la polarité de la surface inversée, équilibrée par les têtes lipidiques au lieu des sous-couches externes diffuses.Les résultats principaux de cette thèse de doctorat sont les suivants:1. Il a été montré que l’utilisation de modèles d’eau polarisable n’est pas nécessaire pour une évaluation correcte d’un certain nombre de propriétés, mais elle est critique pour les moments dipolaires et la tension de surface.2. Pour la première fois une analyse structurelle a été réalisée en utilisant les diagrammes de Voronoi et un ensemble de modèles d’eau démontrant la différence entre propriétés de l’eau liquide en bulk et en surface.3. Considérant le nombre limité de données existantes, l’étude d’une monocouche de DLPC solide condensée à l’interface eau/vapeur et eau/octane en utilisant différents modèles d’eau a été une contribution originale. / The structuring of water molecules at the water/vapour interface is an object of scientific interest for decades. Most of the existing theoretical studies are focused on bulk water but there is still need of a more detailed research on surface water. In addition, interfaces with alkanes are interesting as being instructive from both biological and industrial perspectives. Since in both bio- and industrial applications water/air and water/oil interfaces are mediated by amphiphiles, the role of a surfactant monolayer on surface water structuring deserves more attention as well.In the present Ph. D. thesis several atomistic water models were chosen and classical molecular dynamics simulations were carried out on bulk water, water/vapour and water/alkane (from pentane to nonane) systems, as well as on water/DLPC/vapour and water/DLPC/octane models, DLPC being dilauroyl phosphatidylcholine. In all cases the temperature was kept at 298 K. Several structural properties of bulk and surface water layers were examined by means of radial distribution functions and Voronoi diagrams. Dipole moments, surface tension and hydrogen bonding were tackled too. The objective was to estimate the impact of accounting for polarisability on the water properties of interest and to select a cost-efficient water model for describing them, as well as to add new data to the existing knowledge about interfacial water structuring.The study addresses the water structuring in bulk and surfacial water at the interface with vapour or alkanes of different chain length. One of the aims of the work was to assess the reproducibility of experimental data using an assortment of polarisable and non-polarisable water models and to check for which properties the utilisation of polarisable models is critical. Simple polarisable models based on Drude oscillators were tested in order to keep the computational costs low. For bulk water and water/vapour systems the models TIP4P, SWM4-NDP and COS/G2 performed the best. Since the TIP4P model produced results commeasurable with the polarisable ones, it was used predominantly further on to simulate water/alkane (C5-C9) interface and to quantify the structural parameters of water obtained from RDFs and Voronoi analyses. The molecules in this layer are organised in a more compact and less ordered manner. The ordering is owed mainly to hydrogen bonds which are twice as many in the bulk compared to the surface. The analysis of the Voronoi diagrams showed that the tetrahedral coordination was blurred and more complex polyhedra were formed. The surface layer was found to consist of two sublayers, inner and outer, with oppositely oriented unequal polarity, defining areas of residual charges at the interface.In addition to the systems with direct contact between water and non-polar fluids, interfaces mediated by lipid monolayers were modelled. The monolayer was meant to seam together the two phases. The compactness of the surfacial water, which was enhanced by the presence of alkanes, was tightened further by the lipid introduction. However, the water orientation was changed and the surfacial polarity was inverted, balanced by the lipid heads instead of the diffuse outer sublayer.The main contributions of the Ph.D. thesis are as follows:1. It is shown that the usage of a polarisable water model is not necessary for correct evaluation of a number of properties, but is critical for characteristics such as dipole moments and surface tension.2. For the first time a structural analysis has been made using Voronoi diagrams and an assortment of water models which demonstrates the difference between bulk and surfacial characteristics of liquid water.3. An original contribution is the study of a solid-condensed DLPC monolayer at the water/vapour interface utilising different water models and at the interface of water/octane, considering the limited experimental data available.

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen.

Background: Idiopathic inflammatory bowel disease (IBD) and food-responsive diarrhea (FRD) are common categories of chronic inflammatory enteropathy (CIE) in dogs. The intestinal microbiota is considered a major contributing factor to the pathogenesis of IBD. Intestinal dysbiosis has been identified in dogs with IBD, but only little information is available on differences in the mucosal microbiota of dogs diagnosed with IBD and FRD. In humans, dyslipidemia has also been described in patients with IBD. However, studies on the lipid profile in dogs with IBD or FRD are currently lacking. Objectives: This is the first study to (1) compare the intestinal mucosal microbiota of dogs with IBD and FRD in the duodenum and the colon, (2) evaluate differences in the mucosal microbiota of each dog before and after the induction phase of treatment, and (3) evaluate the systemic phospholipid profile of dogs with IBD or FRD also both prior to and after the induction phase of treatment. Materials and Methods: Duodenal and colonic mucosal biopsies from 24 dogs with CIE (15 FRD, 9 IBD) and EDTA-plasma and whole blood from 32 dogs (16 FRD, 16 IBD) were retrieved from a former study on canine CIE. All client-owned dogs were prospectively enrolled in the study. All dogs received a standardized diagnostic work-up and treatment including a dietary trial. Dogs that responded to the elimination diet within 14 days were classified as FRD; the remaining dogs requiring additional immunosuppressant treatment were classified as IBD. Biopsy specimens of duodenum and colon were obtained endoscopically both before and after standard therapy. DNA was extracted from these biopsies and the intestinal mucosal microbiota of the duodenum and colon were evaluated by Illumina sequencing of the bacterial 16S rRNA gene. The phospholipids in whole blood and EDTA plasma, collected both before and after treatment, were analyzed by hydrophilic interaction liquid chromatography (HILIC). Differences in the composition were statistically assessed by alpha diversity indices, principal coordinate analysis, analysis of similarities (ANOSIM) and linear discriminant (LDA) analysis effect size (LEfSe) for the microbiota and by principal component analysis (PCA), analysis of variance (ANOVA) and random forest analysis for the phospholipid profile. Results: All differences in the microbial composition and phospholipid profile described below are statistically confirmed with significance set at p-value < 0.05 or LDA score > 2.0. No difference in the global bacterial composition was identified neither between the two disease groups of dogs nor with the treatment status. However, abundances of several bacterial taxa varied between disease groups and also with the treatment status. When comparing disease groups, an unclassified genus of Neisseriaceae was more abundant in the duodenum in the IBD group, whereas Bilophila spp. occurred more frequently in the duodenum of the FRD group. Comparison before and after treatment revealed Enterococcus spp., Corynebacterium spp. and Proteobacteria to be enriched in the duodenum of FRD dogs before treatment. Bacteroides spp. was more abundant in the colon in the FRD group post-treatment. In the IBD dogs, Unclassified_Neisseriaceae was more abundant in the duodenum and mainly Proteobacteria (Burkholderia spp., Citrobacter spp.) in the colon prior to treatment. Bacteroides spp. was significantly more abundant in the colon after treatment. The phospholipidome differed dependent on the type of specimen (whole blood vs. plasma). In addition, treatment and disease severity presented the most significant factors determining the variance of the phospholipid profile. An increase in lysolipids and a significant shift of the phosphatidylcholine (PC) species from PC 38:4 (18:0 / 20:4) to mainly lysophosphatidylcholine 18:0 was observed after treatment. Conclusions: Some differences in individual bacterial taxa were identified both between disease groups of dogs and with regard to treatment status. The role of these bacterial groups in the pathogenesis of IBD and FRD is still unknown. However, Bacteroides spp. might be of importance, and this species could potentially serve as marker of treatment response. Furthermore, significant variances were identified in the phospholipid profiles of dogs with IBD and FRD, which were particularly associated with the type of specimen used, disease severity, and treatment status. These alterations in the phospholipid profile could potentially aid in monitoring the response to treatment. Subsequently, specific modulation of the intestinal microbiota and the phospholipid profile might also present novel therapeutic strategies in dogs with CIE.

Obesity, one of the most serious health problems of the 21st century, often occurs as a result of an imbalance between energy intake and energy expenditure. Dietary lipids play an important role in the development of obesity, partly because they represent the richest source of energy amongst all macronutrients. It is, however, not only the amount of consumed lipids, but also the composition of fatty acids, which strongly influences health effects of a particular diet. Saturated fatty acids (SFA) are generally considered as unhealthy due to their pro-inflammatory and lipotoxic properties, while monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) represent a healthier alternative, as they are more readily oxidized and do not disrupt biochemical properties of cellular membranes. Amongst PUFA, PUFA of n-3 series (Omega-3) represent an utterly unique class of lipids that have been documented to protect against cardiovascular disease and dyslipidemia in men and improve insulin sensitivity and glucose tolerance primarily in animal models of obesity. Some molecular mechanisms of Omega-3 action have been already uncovered, such as the modification of biological membranes composition, activation of various transcription factors and membrane receptors, and their role as precursors for...

Bovine milk fat globules naturally vary from less than 0.2 µm to 15 µm in diameter. Milk has at least two distinct distributions of fat globules. While the majority (~90%) of globules in milk are of the smaller distribution (average diameter of 0.4 µm), virtually all the fat is carried in the larger globules (average diameter 3.5 µm). This distribution suggests some compositional and/or functional significance might exist between the two populations of fat globules, which may be related to origin of these globules in the lactating cell. Milk fat globules have a unique structure, composed of a core droplet of non polar lipids (triacylglycerol) surrounded by a lipid bilayer membrane known as milk fat globule membrane (MFGM). Other than MFGM, there is another source of membrane that has been identified in skim milk. It has been hypothesized that this skim milk membrane (SMM) is derived from MFGM, but little data are available to support this idea, and the membrane may also have alternate origins. In this study, different aggregates of lipids (small and large fat globules, SMM, skim milk) from milk were isolated and characterized for their lipid contents. Isolation of small and large fat globules fractions was verified by laser diffraction particle size analysis. The lipids were extracted from isolated different lipid aggregates and individual classes were separated using thin layer chromatography. Lipids were transesterified to fatty acid methyl esters and analyzed by gas chromatography-mass spectrometry. The results indicate that there are some compositional differences between native milk fat globule membranes of different sizes. For example, the total phospholipid fraction of small fat globules (SFG) contained significantly more unsaturated C18:1n9 and C18:2n6 than large fat globules (LFG). Conversely, sphingomyelin composition of SFG contained less C18:1n9 and C18:2n6cc, but more long chain fatty acids C22:0, C23:0, and C24:0. Phosphatidylethanolamine composition of SMM contained more C17:1 than SFG and LFG. The composition of C18:1n9 in triacylglycerol increased with fat globule size. Clear differences were also found in lipid profile of SMM and small and large fat globules from milk. Composition differences between SMM and native milk fat globules of different sizes suggest that origin of this membrane material in skim milk might have some different source than that of MFGM.

Indiana University-Purdue University Indianapolis (IUPUI) / High choline uptake and increased choline kinase activity have been reported in many cancers. This has motivated the use of choline as a biomarker for tumor imaging. Tumors in general are heterogeneous in nature with respect to oxygen tension. There are regions of hypoxia and normoxia that are expected to have different metabolism but regulation of choline metabolism under hypoxia is poorly understood. It is important to clarify the status of choline metabolism in hypoxic microenvironment as it will have an impact on potential of choline as a cancer biomarker. The primary goal was to determine the status of choline phosphorylation in hypoxic cancer cells and its effect on uptake of choline. This was examined by tracer studies in cancer cells exposed to hypoxia. It was observed that hypoxia universally inhibits choline uptake /phosphorylation in cancer cells. Decreased choline phosphorylation resulted in transient uptake of choline radiotracers in cultured cancer cells and 9L tumors suggesting potential problem in using choline as a biomarker for cancers in hypoxic microenvironment. To investigate the mechanism behind decrease in choline phosphorylation, steady state levels of choline metabolites were measured and choline kinase catalyzed choline phosphorylation step was found to be rate-limiting in PC-3 cells. This suggested that modulation in choline kinase levels can alter choline metabolism in hypoxic cancer cells. Expression and activity assays for choline kinase revealed that choline kinase expression is down-regulated in hypoxia. This regulation involved transcriptional level mediation by HIF1 at the conserved HRE7 site in choline kinase promoter. To further understand the importance of down-regulation of choline kinase in hypoxia, stable prostate cancer cell lines over-expressing choline kinase were generated. Effect of over-expression of choline kinase in hypoxia was evaluated in terms of malignant phenotypes like proliferation rate, anchorage independent growth and invasion potential. Both over-expression of choline kinase and hypoxia had a pronounced effect on malignant phenotypes of prostate cancer cells. Further study showed that increased choline kinase activity and hypoxic tumor microenvironment are important for progression of early-stage, androgen-dependent LNCaP prostate cancer cells but confer little survival advantage in undifferentiated, androgen-independent PC-3 prostate cancer cells.