|
|
Vliv povrchových modifikací lipidových nanočástic na jejich vlastnosti
Mašková, Vendula
The aim of the thesis was to study the effect of surface modifications of liposomal nanoparticles on their physicochemical properties and to evaluate their uptake by cells in vitro. For this purpose, two types of liposomal nanoparticles (LNPs) that differed in their sensitivity to pH changes, i.e. pH-sensitive LNPs containing the lipid CPA (cholesteryl-PEG350-aminoxylipid) in their composition, were prepared for comparison, to which PEG (polyethylene glycol propionaldehyde) was attached by an oxime bond, and pH-insensitive LNPs containing DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol). The siRNAs were encapsulated into these nanoparticles and the storage stability, pH sensitivity, cleavage of pH-sensitive linkage, protein corona formation and their uptake by HepG2 cells were studied. The pH sensitivity of the linkages between PEG and CPA, or between PEG and DSPE, was verified using MALDI-TOF mass spectrometry at decreasing pH. Nanoparticle size, PDI and zeta potential were also monitored during the pH sensitivity study. According to the results, pH insensitive LNPs indeed appeared to be inert to pH change and pH sensitive LNPs showed changes in parameters at pH 6.0. Furthermore, the stability of both types of LNPs during long-term storage under different temperature conditions was assessed based on the change in size and PDI. The prepared LNPs appeared to be size uniform and stable throughout the experiment even at different temperatures. The exceptions were the fluorescently labelled LNPs, which exhibited poorer stability within the parameters studied. Finally, the effect of surface modifications on the reduction of protein corona formation was confirmed. The prepared LNPs also efficiently entered the cells, and within 24 hours there was a release of siRNA into the cytoplasm from pH-sensitive LNPs and a probable accumulation of siRNA in endosomes in the case of pH-insensitive LNPs. Based on the results of this work, it can be concluded that the prepared pH-sensitive LNPs exhibit desirable behavior in biological systems, and thus have potential as a suitable carrier for siRNA transport in biological applications.
|
|
|
Effect of amyloid β on the function of endosomes and lysosomes
Tmějová, Monika ; Rudajev, Vladimír (advisor) ; Čočková, Zuzana (referee)
Alzheimer's disease is progressive neurodegenerative disease characterized by accumulation of amyloid β aggregates in brain tissue. Understanding the mechanisms of amyloid β pathogenesis and neuronal cell destruction is still not clear. The most toxic form of amyloid β are 42 aminoacids long oligomers that tends to cumulate and speed up disease progression. Membrane dynamics which affect protein degradation and recycling within the cell plays a criticale role in maintaining homeostasis. Vesicular trafficking plays fundamental role in balancing physiological level of amyloid β. Disruption of endolysosomal complex leads to cycle of disruptions within the cell which results in neuronal cell death. The main aim of this thesis was to look through different ways how amyloid β42 affects endolysosomal compartment. Results of our work confirmed toxic effect of amyloid on SH-SY5Y cell line and its ability to damage functions of lysosomes. We were not able to confirm amyloid β toxicity on endosomal function. Key words: amyloid β, Alzheimer disease, oligomers, plasma membrane, endocytosis, endosome, lysosome, neurotoxicity
|
|
|
The role of phosphoinositides in macropinocytosis
Hájek, Tomáš ; Doubravská, Lenka (advisor) ; Španielová, Hana (referee)
Macropinocytosis is non-selective actin-dependent type of endocytosis. It is important for the normal physiology of some cell types. However, it is used by intracellular parasites which internalise themselves into host cells in this way and also play a role in the nutritional supply in some type of cancer cells. During macropinocytosis a self-organized subdomain of plasma membrane is separated by a diffusion barrier named macropinocytic cup. RAC1-driven actin polymerization is required for membrane protrusion at the cup periphery, where a narrow ring of the actin nucleating factors is present. In contrast, actin dissociation occurs at the base of the cup due to RAS-controlled formation of phosphatidylinositol trisphosphates (PIP3). During cup closure sequential breakdown of PIP3 to phosphatidylinositol and acquisition of the endosomal identity of the newly formed vesicle is necessary. As a result of tubulation in the early stages of macropinocytosome maturation the vesicle decreases in diameter and stabilizes. At late stages the macropinocytic vesicle may fuse with the lysosome, allowing internalized material to enter this degradative organelle. Throughout the process specific types of phosphatidylinositols are part of the membrane providing signal transduction and membrane identity. These phospholipids...
|
|
|
The role of CUP-4 protein in Wnt signalling
Žídek, Radim ; Macůrková, Marie (advisor) ; Pospíchalová, Vendula (referee)
Wnt signalling is indispensible for proper development of organisms and maintaining of adult tissue homeostasis. Its disruption often leads to disease. In nematode Caenorhabditis elegans, Wnt signalling governs vast array of developmental processes, among others also migration of the Q neuroblasts and their descendants. The sole Wnt acting in this process, EGL-20, triggers the canonical β-catenin Wnt signal transduction pathway in QL but not in QR which leads to QL remaining in the posterior while the QR migrates anteriorly. This represents a useful tool for studying Wnt signalling. Recently, mutation of gene cup-4 was found to disrupt migration of the QL neuroblast in a small proportion of the mutant population. cup-4 encodes a ligand-gated ion channel family homologue and it was shown to participate in endocytosis by coelomocytes, specialized phagocytic cells in the C. elegans body cavity. Here, I present the results of my effort to determine the place of CUP-4 action in Wnt signalling and to elucidate the mechanism of its function. I found that CUP-4 acts upstream of PRY- 1/Axin, which is involved in signal transduction in signal receiving cells, and most probably downstream of adaptin AP2, which is important for recycling of Wnt cargo receptor Wntless (Wls) in Wnt producing cell. cup-4 also...
|
|
|
Functional analysis of syntaxin 16 phosphorylation using yeast as a model
Volfová, Barbora ; Entlicher, Gustav (advisor) ; Dráber, Petr (referee)
4 Abstract Mechanism of fusion of intracellular membranes in eukaryotic cells involves several protein families including soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins and Sec1/Munc-18 related proteins (SM proteins). It is known that the transport is evolutionary conserved from yeast to man. Therefore for facilitating of the research, we can use simple eukaryotes Saccharomyces cerevisiae. Mammalian SNARE protein syntaxin 16 has a yeast homologue Tlg2p which is used in this study as a model for studying affects of phosphorylation to the syntaxin 16 function. Also their binding partners, SM proteins mVps45p (mammalian) and yeast Vps45p are homologous. Phosphorylation of SNARE proteins is known as a possible way of regulation of membrane fusion. Abolishment of one of the putative phosphorylation sites in Tlg2p protein, serine 90 leads to dominant effects on the exocytic and endocytic pathways. The work presented in this study shows some phenotypes of mutants based on this phosphorylation site of protein Tlg2p. Those mutants are S90A (cannot be phosphorylated) and S90D (phosphomimetic - acid carboxyl group mimics phosphate group). It was revealed that the phosphorylation of Tlg2p protein at serine 90 or the mutation Tlg2p-S90D may play some role in protecting Tlg2p...
|
|
|
Cytoplasmic membrane and tonoplast dynamics during closing and opening of stomata
Röder, Matěj ; Žárský, Viktor (advisor) ; Albrechtová, Jana (referee)
Stomata are epidermal structures mediating regulated contact of plant apoplast with surrounding environment via stomatal opening. Change of turgor plays crucial part in initiation of stomatal opening or closure. During stomatal movement, guard cell undergo considerable and repetitive changes in cell volume and consequently surface area over a period of minutes. Alteration in the surface of membrane must occurs due to limited stretching capability of the plasma membrane. It can be achieved through membrane invaginations and endocytosis of small vesicles. Microscopy and electrophysiology techniques have proven that both processes are happening in guard cells. These processes are controlled and managed by complex web of signal pathways in which actin and microtubular cytoskeletons, SNARE proteins, ion channels and others molecules have crucial parts. The aim of this work is to summarize current knowledge on the processes and mechanisms of these membranes surface changes and their molecular principle.
|
|
|
Application on nanoparticles in bio-medicine.
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Beranová, Jana (referee)
During last few years nanoparticles of different origin have been used in biomedicine. Their interactions with cells are however a big point of concern and so further research is needed to be done regarding their properties, internalization into cells and their fate within the cell. All of this is needed for correct selection of proper nanoparticles for bioimaging and also for future use in human medicine. One of the main parts of research is deep understanding of interactions of nanodiamonds and silicon based nanoparticles with cells, because of their highly unique properties (autofluorescence), biocompatibility and in case of silicon nanoparticles also their degradability.
|
|
|
Mechanisms of establishment and maintenance of PIN polarity in Arabidopsis
Glanc, Matouš ; Friml, Jiří (advisor) ; Grebe, Markus (referee) ; Luschnig, Christian (referee)
Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...
|
|
|
Effect of amyloid β on the function of endosomes and lysosomes
Tmějová, Monika ; Rudajev, Vladimír (advisor) ; Búran, Peter (referee)
Amyloid β peptide is produced by proteolytic processing of amyloid precursor protein. Accumulation of toxic Aβ in lysosomes and endosomes is considered to be one of the earliest signs of Alzheimer disease. Alzherimer disease was first described in 1907 by doctor Alois Alzheimer. This disease is most common in elderly people over the age of 65 and it is currently the most common cause of dementia. Although significant progress has been made in recent years, the key mechanism of formation of senile plaques and neurofibrillary tangles still remains unclear. Vesicular trafficking plays fundamental role in regulation of APP and generation of Aβ. This thesis summarizes molecular mechanisms of pathological effect of Aβ on the endolysosomal complex.
|
|
|
Mechanisms regulating the function of adaptor protein 2 complex during endocytosis
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Pleskot, Roman (referee)
Adaptor protein complex 2 (AP2) mediates the interaction of clathrin with the plasma membrane and thus enables the clathrin-coated vesicle formation. AP2 is also responsible for cargo recognition and it recognizes cargo either directly using endocytic motifs YxxΦ or [DE]xxxL[LI] in the cytosolic domains of cargo or indirectly via additional adaptor proteins from which β-arrestin and ARH are the best-known. The binding sites for endocytic motifs are located in the core of AP2 complex and, similarly to the clathrin-binding site, they are blocked by autoinhibitory mechanism in the inactive cytosolic form of AP2. Therefore, binding of endocytic motifs and clathrin must be preceded by conformational change of AP2 complex which is triggered by membrane-bound phosphatidylinositol-4,5-bisphosphates and greatly facilitated by phosphorylation at Thr156 by AAK1 kinase. AP2 is also important for later stages of endocytosis during which it recruits proteins responsible for membrane curvature, fission, and eventual disassembly of clathrin coat. Repeated association of AP2 with the plasma membrane is prevented by the protein NECAP, but the mechanism of inactivation is still poorly understood.
|
guest ::
