Neuronal cell lipidomics and role of cholesterol in α-synuclein binding and aggregation

α-Synuclein is an intrinsically disordered protein whose fold and multimeric state is strongly affected by its environment. The monomers of α-synuclein are enriched in the presynaptic terminals of dopaminergic neurons and seem to have a role in synaptic vesicles recycling and transmitter release. Specific cellular conditions can promote α-synuclein oligomerization which, in the end, results in neuronal death. Lipids seem to be one of these conditions as they are involved in both the native and pathogenic role of α-synuclein. This thesis aims to explore α-synuclein:lipid interaction and how binding to certain lipids may trigger or suppress oligomerization. Furthermore, it aims to establish and use lipidomic techniques for improving the state of knowledge of the lipid environment that might affect α-synuclein function and dysfunction. In Paper I we investigated how cholesterol, the most abundant lipid in mammals, affect α-synuclein affinity towards the lipid bilayer and its oligomerization properties. For this work we tested and optimized the preparation of SMA lipid nanodiscs containing cholesterol. Then, we used these nanodiscs to investigate lipid-mediated changes in fibrillation. We also determined individual amino acid affinities towards the lipid bilayer within the α-synuclein primary sequence. The results suggest the existence of two binding modes: the N-terminal and NAC binding mode. The N-terminal mode seems to be preferred in the presence of anionic lipids (like PG), and this binding leads to a delay in fibrillation onset. The NAC-binding mode seems to be promoted in the presence of lipid bilayers containing cholesterol and speed up α-synuclein oligomerization significantly. In Paper II, we focused on lipidomics methods. We established and optimized a method for lipid isolation, followed by phospholipid identification and quantification using 31P NMR. These methods were first tested on populations of a prokaryotic organism, L. innocua, which were arrested at the C/D boundary of its cell cycle. We discovered that L. innocua modulates its lipid composition, and both cardiolipin and phosphatidylethanolamine fall significantly between the B period and the C/D boundary. When we investigated how these lipid changes could affect the physical properties models of the cell membrane, we observed that the decrease of PE content seems to be compensated for by an increase of PG. In the absence of other factors, this would result in decreasing stored curvature stress, while maintaining membrane fluidity. In Paper III, we used methods established in Paper II for the lipidomic analysis of the SH-SY5Y cell line, which is a commonly used neuronal cell model for Parkinson’s disease research. We also developed an automated script for analysis of the fatty acid chain distribution of phospholipids by LC-MS/MS. We observed several deviations of lipid content from commonly used lipid models and lipid content of brain matter. The most interesting ones are a high content of PC, low content of SM and PS and absence of longer fatty acid chains, including 22:6 PS

Replacing a Mold With a 3D Plastic Model to Create and Validate a Coordinate Machine Measuring Program

Diplomová práce se zabývá možnostmi spojení a 3D tiskem modelu výlisku rozděleného na více částí. Úvodní část diplomové práce představuje nejpoužívanější technologie aditivní výroby a nejpoužívanější materiály pro technologii FDM. Experimentální část se již zabývá návrhem možných spojení, která jsou následně aplikována přímo do modelu výlisku. Po vytisknutí jednotlivých částí modelu a jejich úspěšném spojení probíhají měření na souřadnicovém měřícím stroji. Na základě tohoto měření je vyhodnocena vhodnost jednotlivých výrobků pro vytvoření měřícího programu. Závěr práce popisuje použitelné druhy spojení a obsahuje seznam doporučených 3D tiskáren.The master thesis deals with the possibilities of connection and 3D printing model mold divided into several parts. The introductory part of the thesis presents the most used technologies of additive production and the most used materials for FDM technology. The experimental part deals with the design of possible connections, which are then applied directly to the molding model. After the individual parts of the model have been printed and connected successfully, measurements are taken on the coordinate measuring machine. On the basis of this measurement, the suitability of individual products for the creation of a measuring program is evaluated. The conclusion of the thesis describes usable types of connections and contains a list of recommended 3D printers.346 - Katedra obrábění, montáže a strojírenské metrologievýborn

Free Form Surface Milling

Bakalářská práce obsahuje návrh a následné otestování frézovacích strategií při programování v CAM softwaru Mastercam. Výsledkem je realizace výroby tvárníku na 5osém frézovacím centru DMU 50. Navržené strategie a řezné nástroje byly otestovány při výrobě na tvarových plochách tvárníku. Následně probíhají měření daných bodů na dvou různých strojích. Pro měření na obráběcím centru DMU je v bakalářské práci vytvořen i NC program, který lze také využít pro měření jakékoli jiné součásti. Výstupem práce je tvárník v různých fázích obrobení s informačními kartami, které informují o daných fázích obrábění.Bachelor thesis includes the suggestion and subsequently testing of milling strategies when programming in CAM software Mastercam. The result is the realization of mansory production at the 5axis milling center DMU 50. The proposed strategies and cutting tools have been tested during production on the form surfaces of the mansory. Subsequently, the points are measured on two different machines. For measurement at the DMU machining center is in this bachelor’s thesis created NC program, which can be also used for measuring any other component. The output of the work is a product in various stages of machining with information cards, which inform about given stage of machining.346 - Katedra obrábění, montáže a strojírenské metrologievýborn

Detection of misfolded protein aggregates from a clinical perspective

Neurodegenerative Protein Misfolding Diseases (PMDs), such as Alzheimer’s (AD), Parkinson’s (PD) and prion diseases, are generally difficult to diagnose before irreversible damage to the central nervous system damage has occurred. Detection of the misfolded proteins that ultimately lead to these conditions offers a means for providing early detection and diagnosis of this class of disease. In this review, we discuss recent developments surrounding protein misfolding diseases with emphasis on the cytotoxic oligomers implicated in their aetiology. We also discuss the relationship of misfolded proteins with biological membranes. Finally, we discuss how far techniques for providing early diagnoses for PMDs have advanced and describe promising clinical approaches. We conclude that antibodies with specificity towards oligomeric species of AD and PD and lectins with specificity for particular glycosylation, show promise. However, it is not clear which approach may yield a reliable clinical test first. Relevance for patients: Individuals suffering from protein misfolding diseases will likely benefit form earlier, less- or even non-invasive diagnosis techniques. The current state and possible future directions for these are subject of this review.publishedVersio

Data and Query Adaptation Using DaemonX

The most common applications of the today's IT world are information systems. The problems related to their design and implementation have sufficiently been solved. However, the true problems occur when an IS is already deployed and user requirements change. In this paper we introduce DaemonX - an evolution management framework which enables to manage evolution of complex applications efficiently and correctly. Using the idea of plug-ins, it enables to model almost any kind of a data format (currently XML, UML, ER, and BPMN). Since it preserves also mapping among modeled constructs of modeled formats via a common platform-independent model, it naturally supports propagation of changes to all related and affected parts

Contrasting the phospholipid profiles of two neoplastic cell lines reveal a high PC:PE ratio for SH-SY5Y cells relative to A431 cells

Lipids have been implicated in Parkinson's Disease (PD). We therefore studied the lipid profile of the neuroblastoma SH-SY5Y cell line, which is used extensively in PD research and compared it to that of the A431 epithelial cancer cell line. We have isolated whole cell extracts (WC) and plasma membrane (PM) fractions of both cell lines. The isolates were analyzed with 31P NMR. We observed a significant higher abundance of phosphatidylcholine (PC) for SH-SY5Y cells for both WC (55 ± 4.1%) and PM (63.3 ± 3.1%) compared to WC (40.5 ± 2.2%) and PM (43.4 ± 1.3%) of A431. Moreover, a higher abundance of phosphatidylethanolamine was detected for the WC of A431 compared to the SH-SY5Y. Using LC-MS/MS, we also determined the relative abundance of fatty acid (FA) moieties for each phospholipid class, finding that SH-SY5Y had high polyunsaturated FA levels, including arachidonic acid compared to A431 cells. When comparing our results to reported compositions of brain and neural tissues, we note the much higher PC levels, as well as very low levels of docosahexaenoic acid. However, relative levels of arachidonic acid and other polyunsaturated fatty acids were elevated, in line with what is desirable for a neural model system

Evidence that Listeria innocua modulates its membrane's stored curvature elastic stress, but not fluidity, through the cell cycle.

This paper reports that the abundances of endogenous cardiolipin and phosphatidylethanolamine halve during elongation of the Gram-positive bacterium Listeria innocua. The lyotropic phase behaviour of model lipid systems that describe these modulations in lipid composition indicate that the average stored curvature elastic stress of the membrane is reduced on elongation of the cell, while the fluidity appears to be maintained. These findings suggest that phospholipid metabolism is linked to the cell cycle and that changes in membrane composition can facilitate passage to the succeding stage of the cell cycle. This therefore suggests a means by which bacteria can manage the physical properties of their membranes through the cell cycle

WIND-PVPA: Water/Ion NMR Detected PVPA to assess lipid barrier integrity in vitro through quantification of passive water- and ion transport

Water/Ion NMR Detected – Phospholipid Vesicle Permeability Assay (WIND-PVPA), is presented as a novel, straightforward and automatable method to assess lipid barrier integrity in vitro. The apparent permeability constants of water- and ions across the PVPA barriers are determined in a one-pot experiment under the influence of membrane-active guest molecules. NMR spectroscopy is used to quantify the water directly (D2O) and the ions indirectly (complexed with EDTA) as a function of time. WIND-PVPA is demonstrated using four anti-microbial peptides, to show that membrane active molecules can be differentiated by their disruptive influence on the PVPA system. The results obtained are compared with explicit molecular dynamics simulations of lipid bilayers, AMPs, water and salt, where the motions of all individual water molecules relative to the lipid bilayer are monitored over the course of the simulations, allowing the calculation of theoretical apparent permeability constants of the corresponding single bilayer systems. Proof-of-principle is presented that WIND-PVPA can be used to evaluate the lipid barrier destabilizing effect of active guest molecules by measuring changes in passive water- and ion permeabilities upon exposure. The method is highly flexible in terms of barrier composition, choice of probes and membrane active compounds

Graphene Acid for Lithium‐Ion Batteries—Carboxylation Boosts Storage Capacity in Graphene

Environmentally sustainable, low-cost, flexible, and lightweight energy storage technologies require advancement in materials design in order to obtain more efficient organic metal-ion batteries. Synthetically tailored organic molecules, which react reversibly with lithium, may address the need for cost-effective and eco-friendly anodes used for organic/lithium battery technologies. Among them, carboxylic group-bearing molecules act as high-energy content anodes. Although organic molecules offer rich chemistry, allowing a high content of carboxyl groups to be installed on aromatic rings, they suffer from low conductivity and leakage to the electrolytes, which restricts their actual capacity, the charging/discharging rate, and eventually their application potential. Here, a densely carboxylated but conducting graphene derivative (graphene acid (GA)) is designed to circumvent these critical limitations, enabling effective operation without compromising the mechanical or chemical stability of the electrode. Experiments including operando Raman measurements and theoretical calculations reveal the excellent charge transport, redox activity, and lithium intercalation properties of the GA anode at the single-layer level, outperforming all reported organic anodes, including commercial monolayer graphene and graphene nanoplatelets. The practical capacity and rate capability of 800 mAh g−1 at 0.05 A g−1 and 174 mAh g−1 at 2.0 A g−1 demonstrate the true potential of GA anodes in advanced lithium-ion batteries

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.publishedVersio