| 1. |
- Bauer, Brigitte, 1978
(författare)
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Controlling Chemistry and Membrane Proteome Composition in Nanotube-Vesicle Networks
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents a combination of experimental and theoretical techniques to elucidate the dynamics of chemical reactions in confined biological systems and bridges the gap between biomimetic and biological systems by using cell membranes as simplified biomimetic devices. These are in turn used and analyzed to draw conclusions about the cellular membrane makeup.Cells constantly undergo changes in shape and volume while biochemical reactions occur inside. In these confined spaces, the influence of geometry and structural dynamics on reaction behavior is an important factor which has to be taken into account to get a more complete understanding of dynamic cellular processes. The use of biomimetic nanotube-vesicle networks (NVN) have generated important knowledge about membrane behavior, and reaction and transport phenomena in small-scale systems. In this work, they are a helpful tool to study the reaction-diffusion behavior of enzymatic reactions. Investigations of reactions in different static network geometries imply that the geometry in which a reaction takes place can control the behavior of a catalytic reaction. Also, a high sensitivity of a reaction-diffusion system to changes in network topology is shown, implying that chemical reactions can be readily induced or boosted in certain nodes as a function of connectivity. Such changes in connectivity are related to the dynamic tube formations found inside Golgi stacks, for example. The relationship between enzymatic reaction rate, and volume fluctuations is shown by demonstrating that reactions can be turned on and off just by changing compartment volume.In order to add functionality to NVNs, a method to construct NVNs from the cell plasma membrane (PM) has been employed. The membrane is taken from unilamellar PM protrusions and possesses the native composition of membrane proteins (MP) and lipids from the PM. This enables functional studies of plasma membrane constituents, e.g. transport activity of MPs, but also reaction-diffusion behavior in a cell-like environment. In order to perform such studies, but also in the view of drug discovery, it is crucial to know which MPs reside in the plasma membrane. Cells are able to release the PM in form of micron-sized vesicles for which a purification protocol was developed. The membrane protein content was analyzed by exposing plasma membrane vesicles (PMVs) to proteolytic digestion of the embedded membrane proteins and analysis of peptides by mass spectrometry. More than 90% of the identified proteins are annotated to the PM which presents an unprecedented degree of purity in PM proteome analysis. PMVs originate solely from the PM, providing a platform for proteomic and functional studies, where the possibility to control MP composition via e.g. recombinant or overexpression of proteins is especially exciting. PMVs can be regarded as a versatile simplistic cell model, enabling studies of more complex cellular processes.
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| 2. |
- Bauer, Brigitte, 1978, et al.
(författare)
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Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 22:22, s. 9329-9332
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.
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| 3. |
- Bauer, Brigitte, 1978, et al.
(författare)
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Metal nanoparticles amplify photodynamic effect on skin cells in vitro
- 2011
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Ingår i: Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Optical Interactions with Tissue and Cells XXII; San Francisco, CA; 24-26 January 2011. - : SPIE. - 1605-7422. - 9780819484345 ; 7897
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- We report on an investigation aimed to increase the efficiency of photodynamic therapy (PDT) through the influence of localized surface plasmon resonances (LSPR's) in metal nanoparticles. PDT is based on photosensitizers that generate singlet oxygen at the tumour site upon exposure to visible light. Although PDT is a well-established treatment for skin cancer, a major drawback is the low quantum yield for singlet-oxygen production. This motivates the development of novel methods that enhance singlet oxygen generation during treatment. In this context, we study the photodynamic effect on cultured human skin cells in the presence or absence of gold nanoparticles with well established LSPR and field-enhancement properties. The cultured skin cells were exposed to protoporphyrin IX and gold nanoparticles and subsequently illuminated with red light. We investigated the differences in cell viability by tuning different parameters, such as incubation time and light dose. In order to find optimal parameters for specific targeting of tumour cells, we compared normal human epidermal keratinocytes with a human squamous skin cancer cell line. The study indicates significantly enhanced cell death in the presence of nanoparticles and important differences in treatment efficiency between normal and tumour cells. These results are thus promising and clearly motivate further development of nanoparticle enhanced clinical PDT treatment.
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| 4. |
- Bauer, Brigitte, 1978, et al.
(författare)
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Modification and expulsion of keratins by human epidermal keratinocytes upon hapten exposure in vitro.
- 2011
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Ingår i: Chemical research in toxicology. - : American Chemical Society (ACS). - 1520-5010 .- 0893-228X. ; 24:5, s. 737-43
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Tidskriftsartikel (refereegranskat)abstract
- Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten-peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations.
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| 5. |
- Bauer, Brigitte, 1978, et al.
(författare)
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Proteomic Analysis of Plasma Membrane Vesicles
- 2009
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Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 48:9, s. 1656-1659
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Tidskriftsartikel (refereegranskat)abstract
- (Figure Presented) A simple and scalable method is presented for harvesting, purification, and on-chip processing of mammalian plasma membrane vesicles (PMVs) optimized for downstream proteome analysis. After immobilization on a microfluidic flow-cell of PMVs, the embedded membrane proteins are proteolytically digested, and the peptides harvested and analyzed by LC-MS/MS. Over 93% of the detected proteins are plasma-membrane-derived. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
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| 6. |
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| 7. |
- Kirejev, Vladimir, 1984, et al.
(författare)
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Novel nanocarriers for topical drug delivery: investigating delivery efficiency and distribution in skin using two-photon microscopy
- 2011
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Ingår i: Proc. SPIE, Multiphoton Microscopy in the Biomedical Sciences XI, editors: Ammasi Periasamy, Karsten König, Peter T. C. So, 23 January 2011. - : SPIE. ; 7903:1
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Konferensbidrag (refereegranskat)abstract
- The complex structure of skin represents an effective barrier against external environmental factors, as for example, different chemical and biochemical compounds, yeast, bacterial and viral infections. However, this impermeability prevents efficient transdermal drug delivery which limits the number of drugs that are able to penetrate the skin efficiently. Current trends in drug application through skin focus on the design and use of nanocarriers for transport of active compounds. The transport systems applied so far have several drawbacks, as they often have low payload, high toxicity, a limited variability of inclusion molecules, or long degradation times. The aim of these current studies is to investigate novel topical drug delivery systems, e.g. nanocarriers based on cyclic oligosaccharides - cyclodextrins (CD) or iron (III)-based metal-organic frameworks (MOF). Earlier studies on cell cultures imply that these drug nanocarriers show promising characteristics compared to other drug delivery systems. In our studies, we use two-photon microscopy to investigate the ability of the nanocarriers to deliver compounds through ex-vivo skin samples. Using near infrared light for excitation in the so called optical window of skin allows deep-tissue visualization of drug distribution and localization. In addition, it is possible to employ two-photon based fluorescence correlation spectroscopy for quantitative analysis of drug distribution and concentrations in different cell layers.
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| 8. |
- Lizana, Ludvig, 1977, et al.
(författare)
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Controlling Chemistry by Geometry in Nanoscale Systems
- 2009
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Ingår i: Annual Review of Physical Chemistry. - : Annual Reviews. - 1545-1593 .- 0066-426X. ; 60, s. 449-468
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Tidskriftsartikel (refereegranskat)abstract
- Scientific literature dealing with the rates, mechanisms,and thermodynamic properties of chemical reactions in condensed media almost exclusively assumes that reactions take place in volumes that do not change over time. The reaction volumes are compact (such as a sphere, a cube, or a cylinder) and do not vary in shape. In this review article, we discuss two important systems at small length scales (similar to 10 nm to 5 mu m), in which these basic assumptions are violated. The first system exists in cell biology and is represented by the tiniest functional components (i.e., single cells, organelles, and other physically delineated cellular microenvironments). The second system comprises nanofluidic devices, in particular devices made from soft-matter materials such as lipid nanotube-vesicle networks. In these two systems, transport, mixing, and shape changes can be achieved it or very close to thermal energy levels. In further contrast to macroscopic systems, mixing by diffusion is extremely efficient, and kinetics can be controlled by shape and volume changes.
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| 9. |
- Lizana, Ludvig, 1977, et al.
(författare)
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Controlling the rates of biochemical reactions and signaling networks by shape and volume changes
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:11, s. 4099-4104
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Tidskriftsartikel (refereegranskat)abstract
- In biological systems, chemical activity takes place in micrometerand nanometer-sized compartments that constantly change in shape and volume. These ever-changing cellular compartments embed chemical reactions, and we demonstrate that the rates of such incorporated reactions are directly affected by the ongoing shape reconfigurations. First, we show that the rate of product formation in an enzymatic reaction can be regulated by simple volume contraction-dilation transitions. The results suggest that mitochondria may regulate the dynamics of interior reaction pathways (e.g., the Krebs cycle) by volume changes. We then show the effect of shape changes on reactions occurring in more complex and structured systems by using biomimetic networks composed of micrometer-sized compartments joined together by nanotubes. Chemical activity was measured by implementing an enzymatic reaction-diffusion system. During ongoing reactions, the network connectivity is changed suddenly (similar to the dynamic tube formations found inside Golgi stacks, for example), and the effect on the reaction is registered. We show that spatiotemporal properties of the reaction-diffusion system are extremely sensitive to sudden changes in network topology and that chemical reactions can be initiated, or boosted, in certain nodes as a function of connectivity. © 2008 by The National Academy of Sciences of the USA.
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| 10. |
- Rago, Gianluca, et al.
(författare)
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Uptake of gold nanoparticles in healthy and tumor cells visualized by nonlinear optical microscopy
- 2011
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 115:17, s. 5008-16
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the mechanism underlying the interactions between inorganic nanostructures and biological systems is crucial for several rapidly growing fields that rely on nano-bio interactions. In particular, the further development of cell-targeted drug delivery using metallic nanoparticles (NP) requires new tools for understanding the mechanisms triggered by the contact of NPs with membranes in different cells at the subcellular level. Here we present a novel concept of multimodal microscopy, enabling three-dimensional imaging of the distribution of gold NPs in living, unlabeled cells. Our approach combines multiphoton induced luminescence (MIL) with coherent anti-Stokes Raman scattering (CARS) microscopy. Comparison with transmission electron microscopy (TEM) reveals in vivo sensitivity down to the single nanostructure. By monitoring the incorporation of NPs in human healthy epidermal keratinocytes and squamous carcinoma cells (SCC), we address the feasibility of noninvasive delivery of NPs for therapeutic purposes. While neutralizing PEG coating was confirmed to prevent NP integration in SCCs, an unexpectedly efficient integration of NPs into keratinocytes was observed. These results, independently validated using TEM, demonstrate the need for advanced surface modification protocols to obtain tumor selectivity for NP delivery. The CARS/MIL microscopy platform presented here is thus a promising tool for noninvasive study of the interaction between NPs and cell.
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