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Lipid bilayers form the basis of the membranes that serve as a barrier between a cell and its physiological environment. Their physical properties make them ideally suited for this role: they are extremely soft with respect to bending but essentially incompressible under lateral tension, and they are quite permeable to water but essentially impermeable to ions which allows the rapid establishment of the osmotic gradients. The function of membrane proteins, which are vital for tasks ranging from signal transduction to energy conversion, depends on their interactions with the lipid environment. Because of the complexity of natural membranes, model systems consisting of simpler lipid mixtures have become indispensable tools in the study of membrane biophysics. The objective of the work reported here is to develop a deeper understanding of the underlying physics of lipid bilayers through nanoscale measurements of the mechanical properties of mixed lipid systems including cholesterol, a key ingredient of cell membranes. Atomic force microscopy (AFM) has been used extensively to measure the topographical and elastic properties of supported lipid bilayers displaying complex phase behaviour and containing mixtures of important PC, PE lipids and cholesterol. Phase transformations have been investigated varying the membrane temperature, and the effects of cholesterol in controlling membrane fluidity, phase, and energetics have been studied. Elastic modulus measurements were correlated with phase behaviour observations. To aid in the nanoscale probing of lipid bilayers, AFM probes with a high aspect ratio and tip radii of $sim$4~nm were fabricated and characterised. These probes were used to investigate the phase boundary in binary and ternary lipid systems, leading to the discovery of a raised region at the boundary which has implications for the localisation of reconstituted proteins as well as the role of natural domains or lipid rafts. The electrical properties of the probes were examined to assess their potential application for combined structural and electrical measurements in liquid. A novel technique was developed to aid in the study of the physical properties of lipid bilayers. Membrane budding was induced above microfabricated substrates through osmotic pressure. Modification of the adhesion energy of the bilayer through biotin-avidin linking was successful in modulating budding behaviour of liquid disordered bilayers. The free energy of the system was modelled to allow quantitative information to be extracted from the data.

Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Barbora Šmídová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Supervisor - specialist: RNDr. Mgr. Alena Tichá, PhD. Title of diploma thesis: CHANGES OF LIPID SPECTRUM DURING BODY MASS REDUCTION IN PATIENTS WITH DIABETES MELLITUS The thesis deals with the determination of lipid parameters (plasma total fatty acids, plasma total cholesterol, lipoprotein cholesterol, plasma triglycerides, plasma cholesterol precursors (lathosterol and squalene) and markers of cholesterol absorption (β-sitosterol and campesterol)) in obese patiens with diabetes mellitus type 1 and type 2 who were tested with a seven-day fasting, followed by low-calorie diabetic diet. It is assumed that weight loss should improve insulin resistence. The aim of this work is to evaluate the lipid parameters during the body weight reduction in obese patients suffering with diabetes mellitus. Lipids were determined before and after a seven-day fasting and after one month from the beginning of fasting (in patiens with diabetes mellitus type 1 also after one year). Gas chromatography was used for the determination of fatty acids and non-cholesterol sterols and squalene. Cholesterol and triglycerides were determined by a routine...

Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes

Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).

Integrins are large heterodimeric (αβ) cell surface receptors that play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are ‘activated’ to a high affinity state by the formation of an intracellular complex between the membrane, the integrin β-subunit tail and talin, a process known as ‘inside-out activation’. The head domain of talin, a FERM domain homologue, plays a vital role in the formation of this complex. Recent studies also suggest that kindlins act in synergy with talin to induce integrin activation. Despite much available structural and functional data, details of how talin activates integrins remain elusive. In this thesis a multiscale simulation approach (using a combination of coarse-grained and atomistic molecular dynamics simulations) together with NMR experiments were employed to study talin-mediated integrin inside-out activation. A number of novel insights emerged from these studies including: (i) the crucial role of negatively charged lipids in talin/membrane association; (ii) a novel V-shape conformation of the talin head domain which optimizes its interactions with negatively charged lipids; (iii) that interactions of talin with negatively charged moieties in the membrane orient talin to optimize interactions with the β cytoplasmic tail; (iv) that binding of talin to the β cytoplasmic tail promotes rearrangement of the integrin TM helices and weakens the integrin α/β association; and (v) that an increase in the tilt angle of the β integrin TM helix initiates a scissoring movement of the two integrin TM helices. These results, combined with experimental data, provide new insights into the mechanism of integrin inside-out activation. The same multiscale approach was used to demonstrate the crucial role of the Gx3G motif in the packing of the integrin transmembrane helices. Using recent structural data the integrin/talin complex was modelled and inserted in bilayers which resemble the biological plasma membrane. The results demonstrate the dynamic nature of the integrin receptor and suggest that the integrin/talin complex alters the lipid organization and motion in the outer and inner bilayer leaflets in an asymmetric way and that diffusion of lipids in the vicinity of the protein is slowed down. The work in this thesis demonstrates that multiscale simulations have considerable strengths when applied to investigations of structure/function relationships in membrane proteins.

Master of Science / Department of Plant Pathology / William W. Bockus / The effects of tan spot on lipid profiles in wheat leaves were quantified by mass spectrometry. Inoculation with Pyrenophora tritici-repentis significantly reduced the amount of many lipids, including the major lipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in leaves over time. These two lipids accounted for 89% of the mass spectral signal of detected lipids in wheat leaves. Reductions in amounts of lipids were at much higher rates over time for susceptible cultivars compared with resistant cultivars. Furthermore, data show that cultivars resistant to tan spot have different lipid profiles when compared with susceptible cultivars. Resistant cultivars had more MGDG and DGDG than susceptible ones, even in non-inoculated leaves. Using linear models that were fit to data, non-inoculated cultivars with a rating of 1 (highly resistant to tan spot) were calculated to have 66.1% more MGDG and 52.7% more DGDG signal than cultivars with a rating of 9 (highly susceptible). These latter findings are indirect evidence that the amounts of some lipids in wheat leaves may be determining factors in the resistance response of cultivars to tan spot.

Carbohydrate molecules are essential parts of living cells. They are used as energy storage and signal substances, and they can be found incorporated in the cell membranes as attachments to glycoproteins and glycolipids, but also as free molecules. In this thesis the effect of carbohydrate molecules on phospholipid model membranes have been investigated by the means of Molecular Dynamics (MD) computer simulations. The most abundant glycolipid in nature is the non-bilayer forming monogalactosyldiacylglycerol (MGDG). It is known to be important for the membrane stacking typical for the thylakoid membranes in plants, and has also been found essential for processes related to photosynthesis. In Paper I, MD simulations were used to characterize structural and dynamical changes in a lipid bilayer when MGDG is present. The simulations were validated by direct comparisons between dipolar couplings calculated from the MD trajectories, and those determined from NMR experiments on similar systems. We could show that most structural changes of the bilayer were a consequence of lipid packing and the molecular shape of MGDG. In certain plants and organisms, the enrichment of small sugars such as sucrose and trehalose close to the membrane interfaces, are known to be one of the strategies to survive freezing and dehydration. The cryoprotecting abilities of these sugar molecules are long known, but the mechanisms at the molecular level are still debated. In Papers II–IV, the interactions of trehalose with a lipid bilayer were investigated. Calculations of structural and dynamical properties, together with free energy calculations, were used to characterize the effect of trehalose on bilayer properties. We could show that the binding of trehalose to the lipid bilayer follows a simple two state binding model, in agreement with recent experimental investigations, and confirm some of the proposed hypotheses for membrane–sugar interactions. The simulations were validated by dipolar couplings from our NMR investigations of TRH in a dilute liquid crystal (bicelles). Furthermore, the assumption about molecular structure being equal in the ordered and isotropic phases was tested and verified. This assumption is central for the interpretation of experimentally determined dipolar couplings in weakly ordered systems. In addition, a coarse grain model was used to tackle some of the problems with slow dynamics that were encountered for trehalose in interaction with the bilayer. It was found that further developments of the interaction models are needed to properly describe the membrane–sugar interactions. Lastly, from investigations of trehalose curvature sensing, we concluded that it preferably interacts in bilayer regions with high negative curvature. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Objetivos: Etapa 1 - avaliar características organolépticas e físico-químicas de formulações tópicas contendo nanopartículas lipídicas sólidas encapsulando curcuminoides; Etapa 2 - avaliar a citotoxicidade das nanopartículas lipídicas sólidas e dos curcuminoides encapsulados em nanopartículas lipídicas sólidas ou não em cultura de células de fibroblastos e queratinócitos com ou sem serem submetidos a RI; avaliar a atividade alergênica dos curcuminoides encapsulados em nanopartículas lipídicas sólidas ou não encapsulados; Etapa 3 - avaliar a toxicidade aguda de uma formulação contendo curcuminoides encapsulados em nanopartículas lipídicas sólidas na pele e em órgãos-alvo de camundongos BALB após 21 dias de aplicação tópica; induzir radiodermite em camundongos BALB; avaliar o efeito de formulações tópicas contendo curcuminoides encapsulados em nanopartículas lipídicas sólidas na prevenção e no tratamento de radiodermite, em camundongos BALB. Método: Etapa 1 - inspeção visual, cor, sensibilidade ao tato e odor, quantificação do pH, análise do comportamento reológico e quantificação de ativo (curcuminoides) das formulações; Etapa 2 - avaliação da viabilidade celular de queratinócitos, fibroblastos e mastócitos, quantificação da % de liberação da enzima ?-hexosaminidade em mastócitos; Etapa 3 - avaliação do peso, consumo alimentar e comportamento dos animais; quantificação de marcadores sanguíneos de toxicidade renal (ureia e creatinina) e hepática (TGP, FA e proteínas totais); análises histológicas qualitativas do coração, fígado, rim, pulmão e pele; quantificação de colágeno na pele; analisar a probabilidade de desenvolvimento de radiodermite, em grupos independentes, após crescentes doses de radiação ionizante; quantificação de citocinas (IL-1?, IL-6, IL-10, KC e FNT-?) avaliação do grau de radiodermite. Resultados: Etapa 1 - as formulações permaneceram estáveis por três meses, sem alteração de características organolépticas e físico-químicas; aos seis meses a formulação mais concentrada ficou mais opaca e observou-se aumento do pH e da perda de curcumina e curcuminoides totais; Etapa 2 - os resultados mostraram que as nanopartículas lipídicas sólidas são mais citotóxicas para os queratinócitos do que para os fibroblastos nas doses avaliadas, apesar de serem menos citotóxicas que os curcuminoides não encapsulados; não se observou diferença na viabilidade destas células sem ou após radiação ionizante (2 Gy); as nanopartículas lipídicas sólidas contendo curcuminoides e os curcuminoides não encapsulados não estimularam a liberação de ?-hexosaminidase em mastócitos, sugerindo que a formulação com nanopartículas não possui propriedades alergênicas. Etapa 3 - a formulação tópica contendo nanopartículas lipídicas sólidas com 30 mg de curcuminoides não conferiu toxicidade local e nem aos órgãos-alvo; os animais não apresentaram perda ponderal, alterações comportamentais ou mudanças na ingestão alimentar ou hídrica; foi possível a indução de radiodermite a partir de 25 Gy, sendo 30 Gy a que apresentou maior probabilidade de desenvolvimento; os animais não apresentaram perda ponderal, redução do consumo alimentar e hídrico significativos; o painel de citocinas avaliado evidenciou que a pele responde ao tratamento de forma diferente do controle da radiação; não foi observado infiltrado inflamatório; a quantificação do colágeno mostrou grande variabilidade. Conclusões: Devido a uma toxicidade mínima do sistema nanocarreador desenvolvido, somos encorajados a realizar testes futuros para aplicações clínicas em dermocosmética e na prevenção e tratamento de lesões cutâneas ou osteoarticulares / Aims: Step 1 - to evaluate organoleptic and physicochemical characteristics of topical formulations containing solid lipid nanoparticles encapsulating curcuminoids; Step 2 - to evaluate the cytotoxicity of solid lipid nanoparticles and curcuminoids encapsulated in solid lipid nanoparticles or not in culture of fibroblast and keratinocytes cells without and after being submitted to IR; to evaluate the allergenic activity of encapsulated curcuminoids in solid or non-encapsulated lipid nanoparticles; Step 3 - to evaluate the acute toxicity of a formulation containing encapsulated curcuminoids in solid lipid nanoparticles on the skin and target organs of BALB mice after 21 days of topical application; to induce radiodermatitis in BALB mice; to evaluate the effect of topical formulations containing encapsulated curcuminoids in solid lipid nanoparticles in the prevention and treatment of radiodermatitis in BALB mice. Method: Step 1 - visual inspection, color, sensitivity to touch and odor, pH quantification, rheological behavior analysis and quantification of active (curcuminoids) of the formulations; Step 2 - evaluation of the cellular viability of keratinocytes, fibroblasts and mast cells, quantification of the release % of ?-hexosaminase enzyme in mast cells; Step 3 - evaluation of weight, food consumption and behavior of the animals, quantification of markers of renal toxicity (urea and creatinine) and hepatic (TGP, FA and total proteins), qualitative histological analysis of the skin, heart, liver, kidney and lung and quantitative analysis of collagen in the skin; analysis of the probability to developing radiodermatitis in independent groups after increasing doses of ionizing radiation; quantification of the cytokines (IL-1?, IL-6, IL-10, KC and TNF-?) and collagen, qualitative histological analyzis of the skin, and the degree of radiodermatitis. Results: Step 1 - the formulations remained stable for three months, without alteration of organoleptic and physicochemical characteristics; at six months the more concentrated formulation became more opaque and there was an increase in pH and loss of curcumin and total curcuminoids; Step 2 - the results showed that solid lipid nanoparticles are more cytotoxic for keratinocytes than for fibroblasts at the doses evaluated, although they were less cytotoxic than non-encapsulated curcuminoids; no significant difference was observed in the viability of these cells without or after ionizing radiation (2 Gy); the solid lipid nanoparticles containing curcuminoids and non-encapsulated curcuminoids did not estimulated ?-hexosaminidase release in mast cells, suggesting that the nanoparticle formulation does not have allergenic properties. Stage 3 - the topical formulation containing solid lipid nanoparticles with 30 mg curcuminoids did not confer local toxicity and nor to the target organs the animals did not showed any weight loss, behavioral changes or changes in food or water ingestion; the radiodermatitis induction was possible from 25 Gy, with the dose of 30 Gy being the most likely to develop; the animals had no significant weight loss, nor reduction in food and water ingestion, the cytokine panel evaluated showed that skin responds to treatment differently from radiation control, no inflammatory infiltrate was evident and the quantification of collagen showed great variability. Conclusions: Due to the minimal toxicity of the developed nanocarrier system, we are encouraged to perform future tests for clinical applications in dermocosmetic and in the prevention and treatment of cutaneous or osteoarticular lesions