| 1. |
- Unnerståle, Sofia
(författare)
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NMR Investigations of Peptide-Membrane Interactions, Modulation of Peptide-Lipid Interaction as a Switch in Signaling across the Lipid Bilayer
- 2010
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.
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| 2. |
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| 3. |
- Wallin, Patric, 1985
(författare)
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Creating cell microenvironments in vitro
- 2012
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Stem cells have a great potential to bring about advancements in fields like developmental biology, drug discovery, cancer biology and tissue engineering. In order to be able to use stem cells to their full potential, it is important to have control over their behavior. In vivo cellular fate processes are controlled by the microenvironment around them and the many different factors in it. Cells communicate with their surrounding environment and shape it actively via cell-cell, cell-matrix and cell-liquid interactions. These interactions often happen on the cellular and subcellular length scale in defined time dependent sequences. Consequently, it is important to have systems that can provide different molecular cues with a high spatial and temporal resolution, in order to mimic cell microenvironments in vitro and study cells under controlled conditions. This thesis focuses on cell-matrix and cell-liquid interactions and different ways to create cell niches in cell culture systems. The focus is on designing and characterizing microfluidic cell culture platforms and, in particular, systems that are capable of forming molecular gradients. Flow-based and diffusion-based microfluidic gradient generators were combined with substrates coated with biofunctionalized gold nano dots, chemical active molecules, or electrospun microfibers. Thus, it was possible to provide cells with topographical cues and a defined surface chemistry, as well as soluble molecular cues in a gradient manner, simultaneously. COMSOL Multiphysics simulations were used to assist the design process and characterization of the microfluidic systems, and also to study cell receptor binding interactions in great detail. The developed toolbox of COMSOL modeling, a liquid handling system, a variety of microfluidic networks, surface modification techniques and molecular gradients allows the formation of multifactorial microenvironments to now study induction of cellular fate process of different cell types in vitro.
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| 4. |
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| 5. |
- Akpe, Victor
(författare)
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Photophysical and Chemical Approaches to Cellular Biophysics
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The central theme in this thesis is reversibility. Two main attempts has been made to approach reversibility in cellular systems from both chemical and physical points of view. Reversibility of immunolabeling of proteins on the cell surface has been adressed by development of new fluorescent substances optimized for CALI (Chromophore-Assisted Laser Inactivation of protein). Aluminum phthalocyanine (AlPc) is here identified to be a good candidate for a new generation of fluorophores for efficient hydroxyl radical generation. It is shown that cells can be reversibly labeled with antibody-AlPc conjugates. In experiments on living cells the AlPcs were not only active as classic fluorophores but also as photocatalytic substances with destaining properties. Reversibility of cell immobilization is also reported, where cells cultured in microstructures were immobilized and 3D supported using hydrogels. Hydrogel formulation and application was optimized to achieve a system where both viability and ease of use was satisfied. Gel reversibility was actualized with pH and enzyme treatment. The developped method offers the possibility of stop flow culturing cells in controlled and reusable 3D environments.
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| 6. |
- Björnerås, Johannes, 1982-
(författare)
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Dynorphin A – Interactions with receptors and the membrane bilayer
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.
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| 7. |
- Liebau, Jobst, 1985-
(författare)
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Membrane interactions of glycosyltransferases
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work.To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells.A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain.Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.
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| 8. |
- Olsson, Thomas, 1977
(författare)
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Characterization of artificial and biological lipid vesicles using TIRF and SPR
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Synthetic lipid vesicles serve as important mimics of cells and the natural membranes that they are enclosed by. As such they are frequently used as simplified models of the highly complex cell membrane to aid in-depth physicochemical and biological characterization of this essential biological structure. Lipid vesicles also fulfill fundamental biological functions, for example in intercellular signaling via extracellular vesicles and also as signal-substance containing intercellular secretory vesicles in the synapses of neurons and in secretory cells of the endocrine glands, which release their cargo to the extracellular space in response to external cues. Lipid-based nanoparticles are also of increasing importance as drug carriers, both for targeted release at specific tissues and for improved cellular uptake. There are today many techniques available to probe a multitude of lipid vesicle properties, including size, structure, content, molecular composition etc. In this thesis work, we have contributed improved means to quantify vesicle size using fluorescence microscopy by using total internal reflection fluorescence (TIRF) microscopy to correlate the measured distribution in fluorescence intensity of individual vesicles to their size, as measured by nanoparticle particle tracking analysis (NTA). A similar approach has been used before by others, but the formalism used that have won prevalence contains a mathematical error, which motivated the introduction of an improved expression for converting total vesicle intensity to vesicle size. We present the difference between the former and the latter formalism, as well as the possible negative impact of the former when used to draw conclusions for larger sized vesicles in a number of studies. One example when this type of analysis is crucial, is in studies were a certain vesicle property is correlated with vesicle size. One such example is studies of membrane protein function, which is often dependent of membrane curvature. In the second work, we used surface plasmon resonance (SPR) and amperometry as quantitative methods to investigate whether secretory dense core vesicles, isolated from bovine chromaffin cells from the medulla of adrenal glands, are able to maintain their high loading of catecholamine molecules after vesicle isolation and purification and how the vesicle catecholamine content is affected by vesicle exposure to osmotic stress. We found, as also previously reported by intracellular amperometry measurements in live cells, that dense core vesicles release part of the vesicle catecholamine content when exerted to a hyperosmotic shock, and also that this release occurs very rapidly in response to the applied osmotic stress. This work demonstrates the strength of using different complementary label-free measurements to account for the total number of catecholamine molecules in such vesicles and for monitoring molecule release from vesicle compartments in real-time. Further, by using knowledge gained about chromaffin vesicle size from TEM together with changes in refractive index as probed with SPR using suspensions based on ordinary and heavy water, we could estimate the hydration level of the dense protein core of the chromaffin vesicles.
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| 9. |
- Carlred, Louise M, 1985
(författare)
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Imaging of lipids and proteins in Alzheimer's disease using Time-of-Flight Secondary Ion Mass Spectrometry
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the formation of senile plaques. These plaques, which consist of aggregations of a peptide called amyloid-β, are deposited in-between the nerve cells in the brain, where they disrupt the signaling processes. The reason for the generation of these plaques is not completely known, but one has found that the regulation of lipids, such as cholesterol, in the cell membrane is one of many factors involved in the process.One method used to study the generation of AD is imaging of brain tissue samples with fluorescence microscopy. To be able to study individual types of molecules in the tissue, immunohistochemistry is often applied, in which antibodies are used to target the molecule of interest. In this way, several different proteins can be visualized simultaneously, while lipids often remain unseen. Another method that can be used to image molecules in tissue samples, and especially lipids, is time-of-flight secondary ion mass spectrometry (ToF-SIMS). However, this method cannot detect intact molecules over ~2 kDa, thereby excluding most peptides and proteins from being identified.In this work, the capability of ToF-SIMS to detect lipids is utilized for targeting proteins in tissue samples using antibody-coupled lipid vesicles, so called liposomes. The antibody-coupled liposomes were specifically bound to amyloid-β deposits in transgenic AD mouse brains, enabling ToF-SIMS imaging of both amyloid-β and, at the same time, surrounding lipids, such as cholesterol, in the tissue. The specificity of the liposome binding was investigated by analyzing their interaction with a model surface using quartz crystal microbalance with dissipation monitoring (QCM-D). Furthermore, the binding of the antibody-coupled liposomes to amyloid-β deposits in tissue sections was analyzed with fluorescence microscopy, confirming specific binding. To unravel possible artifacts in the tissue sample due to the demanding sample preparation required for ToF-SIMS imaging, the effects of the tissue preparation protocol were using ToF-SIMS and scanning electron microcopy (SEM), revealing no severe spatial redistribution of the native lipids or any major disruption of the surface morphology. This method may thus provide an important complement to traditional tissue imaging approaches for the investigation of the interaction between lipids and proteins, which may result in important clues about the generation of different diseases, such as AD.
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| 10. |
- Norling, Karin, 1988
(författare)
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Studying the influence of the physicochemical properties of lipid nanoparticles for mucosal vaccine delivery
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipid-based nanoparticles have attracted attention as promising pharmaceutical carriers. Reports of them having inherent adjuvant properties make them particularly interesting as vaccine vectors; however, the physicochemical profile of an ideal nanoparticle for mucosal vaccine delivery remains unknown. The aim of this thesis work is to contribute a better understanding of the connection between physicochemical properties of lipid nanoparticles used as vaccine carriers and the activation of the immune response at several different levels of complexity. As combined antigen and adjuvant, we used a novel fusion protein comprising the Cholera toxin A1 subunit, combined with either the M2e or Ealpha peptide and a dimer of the D subunit of Staphylococcus aureus protein A. This fusion protein was coupled to liposomes and lipodisks with systematically varied poly(ethylene glycol) (PEG) content, protein load, rigidity and size/shape. Firstly, a detailed characterization of the biological response in vitro and in vivo, in a mouse model, to two types of fusion protein-carrying lipid particles was performed. Compared with the free fusion protein, which is in itself already an effective vaccination compound, the result showed that the non-PEGylated liposomes more efficiently induce both cell- and antibody-mediated immune responses as well as protection against a lethal virus challenge than both free fusion protein and the PEGylated liposomes. Secondly, an in vitro study was performed, focusing on elucidating the effect of the physicochemical properties of the carrier particle on processing, in particular the antigen presentation in major histocompatibility complex class II (MHC II), by dendritic cells. Out of 6 different formulations, which varied with respect to PEGylation, fusion protein load, membrane rigidity, size and shape it was found that only the DSPC-based liposome formulation, the only liposome formulation in gel phase, was able to increase antigen presentation compared to free fusion protein. Additionally, this formulation lead to an increased amount of surface-bound MHC II, indicating that the liposomes themselves might have an immunostimulatory effect, making them a promising candidate for further evaluation as a vaccine carrier with inherent adjuvant properties.
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