Doctoral projects

PhD thesis projects in Biophysics (Department of Biophysics and Center for Interdisciplinary Biosciences) for the academic year 2019/2020:

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1. Mechanism of cell responses to oxidative stress: time-resolved imaging of cell

Supervisor: RNDr. Veronika Huntošová, PhD. – CIB TIP-P. J. Safarik University

Annotation: The main aim of the Thesis is to apply time-resolved fluorescence microscopy for monitoring of cellular responses to oxidative stress. Classical fluorescence microscopy is intensity-based technique. The interaction between molecule-molecule, molecule- biomolecule and molecule-cell structures required to apply fluorescence/phosphorescence lifetime measurements (FLIM/PLIM). From the fluorescence lifetime values of fluorescence probes, we will be able to estimate the localization and the environment of the used fluorescence probe. FLIM/PLIM will be used mostly to monitor important signaling pathways agents. These agents will be fluorescence immune-stained. Their involvement to cell survival will be assumed according to the changes in fluorescence lifetimes of immune-probe.

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2. Mechanism of cell resistance and apoptosis in 3D spheroids: cross-correlation of protein kinase C and ABC transporters functions

Supervisor: RNDr. Veronika Huntošová, PhD. – CIB TIP-P. J. Safarik University

Annotation: The main aim of the Thesis is to investigate cross-correlation of protein kinase C and ABC transporters during apoptosis and generally, during cell response after stimulation with different drugs. Preferentially, we will pay attention to cancer. Tumors are spherical systems. For this reason the study will be performed in 3D spheroids. The results will be compared with cell monolayers approach. Protein kinase C (PKC) phosphorylates different proteins and enzymes in cells that are active in apoptotic signaling pathways. Drug resistance is one of the main problem in cancer treatment. We will focus our study to investigate mechanism of PKC interactions with ABC transporters and other multi-drug resistance proteins. Mechanisms will be studied by the techniques available at Center for Interdisciplinary Biosciences and Department of Biophysics: steady-state and time-resolved microscopy, spectroscopy, western blotting, different isolation technique for protein expression determination in cells.

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3. Chemical nature of ferryl intermediates of the respiratory oxidases

Supervisor: RNDr. Marian Fabian, CSc. – CIB TIP-P. J. Safarik University
Co-supervisor: doc. Mgr. Daniel Jancura, PhD. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University

Annotation: In aerobic organisms, mitochondria are the principal source of the metabolic energy. The mitochondrial respiratory chain performs the primary transformation of the energy to the metabolically usable form. Cytochrome oxidase (CcO), terminal component of the respiratory chain, is a membrane protein that catalyzes reduction of oxygen to water. This reaction is coupled to the proton pumping through the inner mitochondrial membrane. Two ferryl intermediates of the catalytic cycle, P (peroxy) and F (ferryl), generated during the catalytic cycle of CcO, are direct participants in the proton pumping. In spite the crucial role of P and F in the proton transfer, the chemical nature of the catalytic center of these two forms is not established. Aim of this work is a determination of the chemical state of both intermediates and the nature of the transition between these two forms. For this goal both biochemical and biophysical (UV-Vis spectroscopy, isothermal titration calorimetry, magnetic susceptibility measurements) methods will be employed. Studies will be performed on CcO purified from beef heart mitochondria.

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4. Development and characterization of lipoprotein nanoparticles for targeted drug delivery to cancer tissues

Supervisor: doc. Mgr. Daniel Jancura, PhD. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University
Co-supervisor: RNDr. Veronika Huntošová, PhD. – CIB TIP-P. J. Safarik University

Annotation: Naturally occurring low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are adequate vehicles for drug delivery and targeting to cancer tissues. The capacity of both types of the lipoproteins to bind hydrophobic drugs and their functionality as drug carriers has been examined in several studies. However, a difficult isolation of the lipoproteins in large quantity from a biological organism as well as a variability of the composition and size of these molecules makes practical application of LDL and HDL as drug delivery systems quite complicated. Synthetic LDL and HDL and large unilamellar vesicles (LUV) are potentially candidates to substitute the native lipoproteins for targeted and effective drug delivery. This main goal of this thesis is to develop and characterize several types of synthetic lipid-based nano-particles (sLNP) and large unilamellar vesicles (LUV) containing various amount of cholesterol. The complex physico-chemical characterization of these systems: chemical composition, size, zeta potential, stability and the mechanisms of their interactions with hydrophobic/amphiphilic molecules with potential to be applied in cancer treatment, will be thoroughly studied. Further, cellular uptake of the constructed lipoprotein/drug complexes by several cell types will be also investigated.

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5. Molecular mechanism of colloidal stability of immunoglobulins

Supervisor: RNDr. Gabriel Žoldák, PhD. – CIB TIP-P. J. Safarik University
Co-supervisor: doc. RNDr. Erik Sedlák, PhD. – CIB-P. J. Safarik University

Annotation: Stability of immunoglobulins is one of the most critical properties affecting the success of the clinical application of immunotherapeutics. Determination of correct parameters which describe the stability and aggregation of IgG is a very ambitious goal mostly due to the complexity of their structures. In this thesis, we will research how exogenous (e.g., pH, ionic strength, and osmolytes) and endogenous (glycosylation, mutagenesis, fragments) factors determine the kinetic and colloidal stability of selected IgGs. Methods developed by our group will help us to understand the molecular basis of the factors. Obtained experimental data will help us to drive the conceptual development of approaches focusing on the prediction of IgG colloidal stability.

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6. Utilization of ribosome display in development of enzymes

Supervisor: doc. RNDr. Erik Sedlák, PhD. – CIB TIP-P. J. Safarik University
Co-supervisor: RNDr. Gabriel Žoldák, PhD. – CIB TIP-P. J. Safarik University

Annotation: Techniques of protein evolution offer an efficient tool in modulation of properties of proteins and enzymes such as for example stability, solubility, affinity and specificity of binding towards selected ligand and catalytic activity. Improving of catalytic activity of enzymes belong to complex problems, which are practically unsolvable by rational design approach. In this work therefore, to “improve” properties of selected enzyme from the family of dehalogenases we apply the technique of ribosome display that we have recently established in laboratories of Center of Interdisciplinary Biosciences.

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7. Drug tansport in biological systems

Supervisor: doc. Mgr. Gregor Bánó, PhD. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University

Annotation: Drug treatment optimization requires the knowledge of the pharmacokinetic properties of the used drug molecules. The concentration time course of active substances in the organism is greatly influenced by their transport in the blood stream, in tissue, and inside cells. The passage of substances across biological membranes plays an important role here. Membrane transport can be monitored using model systems (vesicles or various artificial lipid bilayers) or directly on live cell cultures. The project is focused on experimental study of drug transport in solutions and membranes using a modular micro-Raman apparatus in combination with optical tweezers. The project comprises development of new experimental techniques for monitoring drug movement dynamics in biological systems.

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8. Photobiomodulation effect on damaged neuronal cells

Supervisor: doc. RNDr. Katarína Štroffeková, CSc. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University
Co-supervisor: doc. Mgr. Gregor Bánó, PhD. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University

Annotation: The civilization diseases common denominator, including cardiovascular and neurodegenerative ones, is increased oxidative stress and mitochondrial dysfunction, which result into cell dysfunction or apoptosis. Traumatic brain and spinal cord injury also result in increased ROS production, Ca2+ overload and mitochondrial dysfunctionNear-infrared (NiR) photobiomodulation (PBM) has the potential to fulfill protective and regenerative functions for cells. The emphasis will be to study PBM effects and their optimization at the molecular and cellular level. We will use interdisciplinary approach of metabolic fluxes measurements, confocal fluorescent microscopy, molecular biology and spectroscopy.

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9. Hypericin as potential BH3 sensitizer and its role in apoptosis and autophagy

Supervisor: doc. RNDr. Katarína Štroffeková, CSc. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University
Co-supervisor: RNDr. Gabriela Fabriciová, PhD. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University

Annotation: Members of the Bcl2 family of proteins are key regulators of apoptosis. The intricate network of protein-protein interactions between multi BH domain anti- and pro-apoptotic Bcl2 proteins, and/or BH3-only proteins control cell survival or death via regulation of mitochondria function and fission/fusion processes. The BH3-only proteins has been shown to fulfill role of either sensitizer or direct activator of pro-apoptotic Bax and Bak. The importance of interaction between pro-survival Bcl2 proteins and BH3 motifs of either pro-apoptotic or BH3 only proteins for cell death or survival decisions makes this interaction an appealing target for cancer therapy and at the present, more than 20 small molecule inhibitors of pro-survival Bcl2 proteins, termed as BH3 mimetics, were explored. We have shown evidence that Hypericin (Hyp) may be another naturally occurring BH3 mimetic. The goal of this study will focus on Hyp interaction with anti-apoptotic Bcl2, BclXL and Mcl1 in apoptosis and autophagy pathways in cancer cells. We will use interdisciplinary approach of confocal fluorescent microscopy, molecular biology and spectroscopy.

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10. Nové prístupy k rekonštrukcii 2.5 a 3D dát zo single-shot zobrazovacích experimentov X-ray s využitím apriórnej informácie

Supervisor: doc. RNDr. Jozef Uličný, CSc. – Dpt. of biophysics, Faculty of Science, P. J. Safarik University

Annotation: Structural dynamics of biomedical and technological objects can be revealed using short intense X-ray pulses. Within few tens of femtoseconds, the sample is completely destroyed, so that only few simultaneous projections are available for 2.5D or even 3D reconstruction. For such underdetermined system, the reconstruction is possible only for systems with fair amount of structural a priori information. The task of PhD student will be to design 3D models of fibrillar structures with optimized placement of sequence-specific fiducial markers for imaging experiment at European XFEL. Subsequently, to design and implement reconstruction algorithms for obtaining structural information with gradually increasing realism of experiment. The PhD work assumes active participation on experiments utilising 3rd and 4th generation coherent X-ray sources in international collaboration comprising of scientists from Institute of Physics of Czech Academy of Sciences, University of Lund, European XFEL and CFEL – Center for free electron laser science.

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11. Formation and inhibition of self-assembly of protein amyloid structures – impact on amyloid diseases

Supervisor: doc. RNDr. Zuzana Gažová, CSc. – IEP SAS
Co-supervisor: RNDr. Zuzana Bednáriková, PhD. – IEP SAS

Annotation: Amyloidoses represent an important group of human diseases associated with formation of different protein amyloid structures in the human body. Conversion of the native protein molecules into supramolecular amyloid aggregates and their accumulation in different tissues are the main pathological features of diseases such as Alzheimer´s and Parkinson´s diseases and diabetes mellitus. The main aims of PhD project will be identification of factors inducing protein structural and functional changes leading to amyloid formation. Moreover, the project will be focused on search for compounds with ability to reduce the amount of amyloid aggregates with aim to find effective therapy for these diseases. Various physico-chemical methods, namely spectroscopic and calorimetric techniques, atomic force microscopy as well as various cytotoxicity assays will be used.

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12. Evaluation of the Importance of Hydrophobic Interactions in Functional and Structural Integrity of Supramolecular Protein Assemblies

Supervisor: RNDr. Ing. Katarína Šipošová, PhD. – IEP SAS
Co-supervisor: MUDr. Andrej Musatov, DrSc. – IEP SAS

Annotation: The self-assembly of proteins resulting in the formation of amyloid fibrillar aggregates has emerged as a subject of fundamental importance in biomedical research as well as in protein chemistry due to its involvement in a number of neurodegenerative disorders. The overall aim of this thesis is better understanding of the protein/peptide mechanism of aggregation with the focus on the role of hydrophobic interactions in amyloid aggregation pathways implicated in diseases. Specific aims are: i) to examine the molecular/kinetic mechanisms involved in the formation of the different amyloid structures; and ii) to assess the effectiveness of either, natural or synthesized hydrophobic chemical compounds to interfere with amyloid fibril formation. Therefore, this work will clarify the ways that may lead to plaque prevention and/or removal, based on the understanding of neurotoxic structures assembles.

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13. Regulation of the ryanodine receptor from the rat brain under physiological and pathophysiological conditions

Supervisor: Mgr. Marta Gaburjáková, PhD. – IMPG SAS
Co-supervisor: Mgr. Jana Gaburjáková, PhD. – IMPG SAS

Annotation: Ca2+ signaling in neuronal cells plays a key role in memory formation and learning processes. Dysregulation of this signaling pathway has been revealed to make an important contribution to neurodegeneration (e.g. Alzheimer’s disease). The ryanodine receptor (RYR) is an intracellular Ca2+ channel that belongs to the most important components of Ca2+ signaling in muscle cells. Its role in neuronal cells has not been very well characterized. The aim of the thesis is to give a detail understanding of the molecular mechanisms of RYR regulation in the rat brain under physiological and pathophysiological conditions. The main experimental approach will be the reconstitution of an ion channel into a planar lipid membrane in combination with biochemical and bioinformatics methods.

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14. Structure-function relationships of dyadic complexes in cardiac myocytes during systole and diastole

Supervisor: Ing. Alexandra Zahradníková, DrSc. – IMPG SAS
Co-supervisor: RNDr. Ivan Zahradník, CSc. – IMPG SAS

Annotation: Excitation-contraction coupling takes place in specialized structures of cardiac myocytes – dyads – in which voltage sensitive calcium channels of the surface membrane and ryanodine receptors of the sarcoplasmic reticulum are juxtaposed. The structure of the dyad is highly variable within a single myocyte, differs between species, and undergoes changes in response to physiological and pathological stimuli. The aim of the work is to study, using mathematical modelling, the effect of dyad structure and of the properties of its ion channels on myocyte function – i.e., on excitation-contraction coupling during systole and on the formation of calcium waves in diastole.

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Supervisor: RNDr. Alexandra Zahradníková ml., PhD. – Biomedical Research Center SAS
Co-supervisor: RNDr. Ivan Zahradník, CSc. – IMPG SAS

Annotation: Sarcolemma of cardiac myocytes is important for their excitability. Changes in its electrical potential cause influx of calcium ions to the cell that triggers processes leading to the contraction of myocytes. Development of the heart, or changing physiological load, or pathological states, lead to adaptation of the myocardium. In these processes, the sarcolemma changes its electrical and structural characteristics dynamically to adapt to changing function of myocytes. Electrical properties of sarcolemma are determined by its surface area and relative content of membrane lipids, proteins, transporters and ion channels. The aim of the work is to study the membrane mechanisms of changes in passive and active electrical properties of sarcolemma of isolated cardiac myocytes by methods of cell electrophysiology and laser fluorescence confocal microscopy.

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