Our laboratory has long been involved in the use of AFM microscopy in the study of cellular and molecular structure and mechanics. We are now equipped with new modules (FluidFM, MEA) for further operations with cells and the acquisition of additional parameters (adhesiveness, cell potential). These methods are described below, the student's goal would be to characterize biomechanical properties of various cells, such as cardiomyocytes. However, also adhesivity of epithelial cells to the new types of implant materials. Combined characterization of mechano-electrical feedback of electrically active cells may bring new insight into the diseases, such as CPVT. FluidFM, micro-fluidic force microscopy, is a relatively new (2009) and very interesting application of atomic force microscopy (AFM) based on a microchannel inside the tip and probe for localized injection or aspiration of very small volumes of fluids. It is very well suited for work with living cells - pico-injection into cells, isolation of molecules or organelles from single cells, the study of cell adhesion, etc. Microelectrode array (MEA) is an electrophysiological method using a region with tens to hundreds of small electrodes (electrode size is on the order of tens of micrometers in a region of the order of units of millimeters) to monitor the cell potential of cardiac and nerve cells. The main use is to test new drugs, but also to describe cellular processes in electrically active cells.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.cz/centralni-laborator-nanobiotechnologie/cf104

Supervisor

Mgr. Jan Přibyl, Ph.D.

Antimicrobial peptides (AMPs) can disrupt the membrane barrier function and kill bacteria, including resistant bacteria recognized by the UN and WHO as one of the biggest threats to global health. AMPs are part of the host-defense system of many organisms and promising candidates to become a new class of therapeutics. Particularly advantageous are peptides that kill bacteria by the formation of leaky pores in their membranes because bacteria have difficulties to develop resistance against such peptides. However, the peptide sequence motives responsible for pore formation remain elusive. This project is focused on evaluation and design of sequence motives able to stabilize pores by reducing the membrane line tension associated with the pore. The main tool will be Molecular dynamics simulations with free energy calculations using Gromacs program package and coarse-grained force field. This project will be complemented by experiments providing verification and feedback of membrane disruption. The findings are expected to lead to the design of new antimicrobial peptides.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor or/and fill in the registration form on the web page of the CEITEC PhD School:

https://www.ceitec.eu/phd-school-call-registration-form/t10587

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://vacha.ceitec.cz/

Supervisor

doc. RNDr. Robert Vácha, PhD.

Cell penetrating peptides (CPPs) are peptides that can spontaneously translocate cell membranes. CPPs can thus carry drugs into the cell or act as drug themselves. However, it remains unclear what conditions enables peptides to translocate and how it is affected by membrane composition. The aim of this project is to investigate the translocation of selected peptides across phospholipid membranes under non-equilibrium conditions, which correspond to experiments. The main tool will be Molecular dynamics simulations with free energy calculations using Gromacs program package and coarse-grained force field. This project will be complemented by experiments providing verification and feedback of membrane disruption. The findings could be used in the design of new antimicrobial and cell-penetrating peptides.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor or/and fill in the registration form on the web page of the CEITEC PhD School:

https://www.ceitec.eu/phd-school-call-registration-form/t10587

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://vacha.ceitec.cz/

Supervisor

doc. RNDr. Robert Vácha, PhD.

DNA guanine quadruplexes (G4) belong to the most important noncanonical nucleic acid structures. They regulate gene expression, are potential pharmacological targets, and are often used as a basis for DNA supramolecular assemblies and nanostructures. G4 molecules have intriguing folding mechanisms, which appear to be unique in the biomolecular world. Understanding of the basic principles of folding of biomolecules is an important scientific question per se.

The aim of the project is to study folding mechanisms of G4s and some other types of noncanonical nucleic acids using state of the art atomistic molecular dynamics (MD) simulations. The computational research will be conducted in one of the world-leading laboratories in the field of nucleic acids simulations, in a close collaboration with experimental laboratories, leading to clear interrelation between theory and experiment. The main techniques will be standard and diverse enhanced-sampling MD simulations; part of the research may be even development of cutting-edge methods (force fields and enhanced sampling protocols) for studies of biomolecular folding.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/non-coding-genome/rg109

Supervisor

prof. RNDr. Jiří Šponer, DrSc.

DNA guanine quadruplexes (G4) belong to the most important noncanonical nucleic acid structures. They regulate gene expression, are potential pharmacological targets, and are often used as a basis for DNA supramolecular assemblies and nanostructures. G4 molecules have intriguing folding mechanisms, which appear to be unique in the biomolecular world. Understanding of the basic principles of folding of biomolecules is an important scientific question per se.

The aim of the project is to study folding mechanisms of G4s and some other types of noncanonical nucleic acids using state of the art atomistic molecular dynamics (MD) simulations. The computational research will be conducted in one of the world-leading laboratories in the field of nucleic acids simulations, in a close collaboration with experimental laboratories, leading to clear interrelation between theory and experiment. The main techniques will be standard and diverse enhanced-sampling MD simulations; part of the research may be even development of cutting-edge methods (force fields and enhanced sampling protocols) for studies of biomolecular folding.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/non-coding-genome/rg109

Supervisor

doc. Mgr. Lukáš Trantírek, Ph.D.

Correlative Light Electron Microscopy (CLEM) uses a combination of an optical (fluorescence) microscope and a cryo-electron microscope. Two images of the sample are taken simultaneously – one with the optical light, the other with the electron beam. This technology allows to capture not only dynamic changes but also the molecular ultrastructure of living systems. New developments in accurate positional referencing of specimens on mounting grids, advances in the instrumentation, and the availability of software packages for cross-platform data correlation allow to image the ultrastructure of nucleolar sub-compartments and to track specific proteins found in phase-separated organelles. In this project, we will implement the CLEM technology to investigate and visualize phase-separated organelles involved in transcription by RNA polymerase II and investigate their regulatory mechanism during transcription. This biophysically focused project will also involve other imaging approaches, including single-particle reconstruction cryo-electron microscopy and cryo-electron tomography, which will help to obtain an overall picture of condensate-based transcription at different resolutions. The project is linked to the CEITEC's teaming project CORMIC – bridging academia and industry in correlative microscopy.

Supervisor

prof. Mgr. Richard Štefl, Ph.D.

The novel forms of nucleotides will be incorporated in silico in oligomers with sequences relevant for biosystems. The biocompatibility of artificial building blocks will be evaluated using advanced methods of quantum chemistry that provide also analytical tools for investigation of crucial noncovalent interactions. Available candidates of modified nucleobases and sugars will be studied experimentally by using NMR spectroscopy in solution.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/structure-of-biosystems-and-molecular-materials/rg108

Supervisor

prof. RNDr. Radek Marek, Ph.D.

CDK11 is ubiquitously expressed in all tissues and the CDK11 null mouse is lethal at an early stage of development indicating an important role for Cdk11 in the adult as well as during development. CDK11 is believed to play a role in RNAPII-directed transcription and co-transcriptional mRNA-processing, particularly alternative splicing and 3'end processing. However, its genome-wide function in regulating the human transcriptome is unknown. Notably, several recent studies identified CDK11 as a candidate essential gene for growth of several cancers therefore, understanding the molecular mechanism(s) of CDK11-dependent gene expression would be also of significant clinical interest. In this research we will use various techniques of molecular biology and biochemistry to characterize genome-wide role of CDK11 in regulation of gene expression and tumorigenesis.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/inherited-diseases-transcriptional-regulation/rg38/

Supervisor

Mgr. Dalibor Blažek, Ph.D.

Plants, unlike animals, possess a unique developmental plasticity that enables them to develop new organs throughout their entire life. This ability requires the presence of regulators that trigger specific spatiotemporal changes in developmental programs. In de novo shoot regeneration, cytokinins are the prime candidates for the signal that determines the shoot identity. The process is accompanied by changes in the metabolism, signalling and by differential regulation of gene expression.

The project will aim to elucidate the role and the impact of cytokinins during de novo shoot regeneration. A wide range of molecular biology methods will be used, mainly CRISPR/Cas9 system to generate mutants in cytokinin metabolic enzymes, as well as several cloning methodologies (Gibson assembly, Gateway etc.), in vitro cultivation, luciferase assay or microscopy and image analysis.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/plant-sciences-core-facility/cf123

Supervisor

Mgr. Markéta Pernisová, Ph.D.

Cdk12 is transcriptional cyclin-dependent kinase (Cdk) found mutated in various cancers. In previous studies we found that Cdk12 maintains genome stability via optimal transcription of key homologous recombination repair pathway genes including BRCA1. Apart from the C-terminal domain of RNA Polymerase II other cellular substrates for both kinases are not known. In this research we propose using a screen in cells carrying an analog sensitive mutant of CDK12 to discover its novel cellular substrates. The substrates and their roles in normal and cancerous cells will be characterized by various techniques of molecular biology and biochemistry.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/inherited-diseases-transcriptional-regulation/rg38/

Supervisor

Mgr. Dalibor Blažek, Ph.D.

Chronic lymphocytic leukemia (CLL) cells and indolent lymphomas are known to be dependent on diverse microenvironmental stimuli providing them signals for survival, development, proliferation, and therapy resistance. It is known that CLL cells undergo apoptosis after cultivation in vitro, and therefore it is necessary to use models of CLL microenvironment to culture CLL cells long-term and/or to study their proliferation. Several in vitro and in vivo models meet some of the characteristics of the natural microenvironment based on coculture of malignant cells with T-lymphocytes or stromal cell lines as supportive cell, but they also have specific limitations.

The aim of this research is to develop and use models mimicking lymphoid microenvironment to study novel therapeutic options, e.g. drugs targeting CLL proliferation, development of resistance in long-term culture or combinatory approaches, which cannot be analysed in experiments based on conventional culture of CLL/lymphoma primary cells. This project will utilize models developed in the laboratory and will further optimize and modify them. Using kinase inhibitors, the biology of CLL and responses to targeted treatment will be interrogated. The student will utilize various functional assays, RNA sequencing, genome editing, drug screening etc., with the use of primary patient's samples and cell lines. The research might bring new insights into the microenvironmental dependencies and development of resistance to targeted therapy.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor or/and fill in the registration form on the web page of the CEITEC PhD School (link below).

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/microenvironment-of-immune-cells/rg115

Supervisor

doc. MUDr. Mgr. Marek Mráz, Ph.D.

RNA Binding Proteins are a class of proteins that function via direct binding to coding and non-coding RNA molecules. Artificial Neural Networks, and especially Convolutional and Recurrent neural networks are machine learning methods increasingly used to classify genomic sequences with promising results. This project involves the training of such Artificial Neural Networks on experimental data from RNA Binding Protein binding sites, and producing tools that can be used to classify nucleotide sequences as potentially bound by specific RNA Binding Proteins. The project will involve development and training of machine learning models, production of online tools, and publication of results in conferences and scientific journals.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/bioinformatics-core-facility/cf284

Supervisor

Panagiotis Alexiou, PhD

We are looking for a motivated PhD student for a project funded by ERC Grant (Marek Mraz lab, www.ceitec.cz/mrazlab). The project goal is to understand the molecular machinery that regulates the migration of malignant B cells between different niches such as lymphoid and bone marrow niche and peripheral blood. This is of great interests a general mechanism of how migration is regulated in cancer cells, but also especially in chronic lymphocytic leukemia (CLL), which is a disease dependent on the B cell recirculation between different compartments (reviewed in Seda and Mraz, 2015; Seda et al, 2021). In CLL, but also in other lymphomas, the malignant B cells permanently re-circulate from peripheral blood to lymph nodes and back, and blocking this recirculation can be used therapeutically since malignant B cells depend on signals in the immune microenvironment. However, the factors that regulate this are mostly unclear. The lab established several models for in vitro and in vivo studies of microenvironmental interactions and their interplay (unpublished in vivo model; Pavlasova et al. Blood, 2016; Pavlasova et al. Leukemia, 2018; Musilova et al. Blood, 2018; Mraz et al. Blood, 2014; Cerna et al. Leukemia, 2019).

We have identified candidate molecules that might act as novel regulators of the B cell migration or the balance between homing and survival in peripheral blood. This will be further investigated by the PhD student using technics such as genome editing (CRISPR), RNA sequencing, use of primary samples, functional studies with various in vitro and in vivo models. The research is also relevant for understanding resistance mechanisms to BCR inhibitors, pre-clinical development of novel drugs and their combinations (several patents submitted by the lab).

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor or/and fill in the registration form on the web page of the CEITEC PhD School (link below).

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/microenvironment-of-immune-cells/rg115

Supervisor

doc. MUDr. Mgr. Marek Mráz, Ph.D.

RNA is an attractive drug target with enormous potential for future treatment of systemic and cancer-related pathologies. Yet, most of the currently applied and developed small molecule therapeutics for cancer and systemic diseases target proteins. Interestingly, from 20000 human protein-coding genes (1.5% of the human genome) only 2000-3000 genes are considered to be disease-related. In this context, small molecule drug therapies target less than 700 genes which represents less than 0.05% of the genome. While the portion of protein-coding information in the genome is minor, the ENCODE consortium has proposed that more than 75% of our genome is transcribed into RNAs. This also includes large non-coding regions of mRNAs, namely 3’UTRs which contain many regulatory elements important for spatio-temporal regulation of gene expression, such as translational control, RNA transport and localization and mRNA decay. We propose to target non-coding mRNA elements with small molecules to alter gene expression. We will focus on cancer-related genes, where protein targets often lack druggable elements and therefore targeting them on the mRNA level is an attractive alternative. Our research aims to identify functional mRNA motifs that can bind small molecules and to reveal common small molecule scaffolds which interact with similar 3D RNA structures and thus form a basis for rational lead optimization.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor or/and fill in the registration form on the web page of the CEITEC PhD School:

https://www.ceitec.eu/phd-school-call-registration-form/t10587

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/rna-based-regulation-of-gene-expression/rg58

Supervisor

Mgr. PharmDr. Peter Lukavsky, Dr. rer. nat.

RNA polymerase of B. subtilis contains subunit delta with a large intrinsically disordered region. The effect of the disordered region on biophysical properties of the delta subunit and on transcription rate and cell viability have been described recently. Now we plan to study the mechanistic role of the disordered region in the process of transcription, especially during transcription initiation. Cryo-electron microscopy and NMR will be used to describe structured portion of RNA polymerase and the disordered region of its delta subunit, respectively, to reveal whether the disordered region remains flexible in complex. Based on the results, cryo-EM (focused 3D classification) and NMR (enhanced by selective isotope and paramagnetic labelling, combined with EPR) will be used as alternative and/or complementary approaches. The supervisor will provide the expertise in the field of NMR. Cryo-EM and EPR expertise will be provided by CF and external collaborators. The result should lead to (shared) first-author publication of the student in a respected scientific journal.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/protein-structure-and-dynamics/rg110

Supervisor

prof. Mgr. Lukáš Žídek, Ph.D.

We apply structural biology methods in order to gain a mechanistic view of CK1E action in the Wnt signaling pathways. CK1E represents an attractive therapeutic target but currently two key steps in the CK1E-mediated Wnt signal transduction are unclear: how CK1E gets activated and/or engages target proteins in response to Wnt signal and how CK1E phosphorylates its key substrate Dishevelled (DVL).

Our preliminary data suggest that we can efficiently apply methods of integrated structural biology to (i) probe the DVL conformational landscape using in vitro and in vivo FRET sensors coupled to SAXS and CryoEM, (ii) understand the (auto)phosphorylation regulatory mechanisms of CK1E, (iii) analyse by NMR the functional consequences of DVL phosphorylation and (iv) monitor DVL phosphorylation by real-time NMR under controlled cellular conditions. The position is part of a multidisciplinary project that combines (i) cellular and molecular biology, (ii) proteomic analysis, (iii) biochemistry and structural biology, and received generous funding in a very competitive grant scheme.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/protein-dna-interactions/rg111

Supervisor

Konstantinos Tripsianes, Ph.D.

Recent 30S subunit-RNA polymerase (RNAP) and expressome structures may represent interactions that occur at different steps of translation, e.g., during initiation and elongation. The aim is to structurally define the step-wise states of bacterial transcription-translation coupling and to determine whether accessory transcription factors or other proteins that link transcription and translation help to relocate RNAP on the ribosome - e.g., from its position in the 30S-RNAP structure to its position in the expressome structure. Single particle cryo-electron microscopy (cryo-EM) will be used to visualize transcription-translation coupling in vitro. Cryo-EM particle images from heterogeneous samples (in vitro reconstituted) will be classified into structurally homogeneous subsets by maximum-likelihood (ML) analyses. Packages such as Frealign and RELION with incorporated ML principles and extensive 3D-classification procedures, will be used to resolve conformational heterogeneity. Free 30S and 50S subunits mix with the nucleoid where they initiate co-transcriptional translation. Cellular co-localization of ribosomal subunits with RNAP protects the nascent mRNA and prevents undesirable backtracking of RNAP. The aim is to determine how is the coupling initiated within the nucleoid space and if the pioneer round of translation occurs within or near nucleoid space before formation of polysomes that segregate from the nucleoid. The RNAP-ribosome complexes will be visualized in situ at sub-nanometer resolution using cryo-electron tomography and sub-tomographic averaging of 3D volumes. E. coli cells will be flash-frozen directly onto EM grids allowing cellular structures to be studied in their near native states. Ultra-thin sections will be prepared directly on the EM grid in the electron microscope by focused ion beam milling. These studies can provide the insight on how translating ribosomes preserve genome integrity by preventing RNAP from backtracking or pausing. This combined approach can convincingly show in vivo the transcriptional-translational apparatus in action with all players involved in the coupling mechanism.

We are seeking a PhD. candidate who was trained in structural biology, mainly single particle cryo-EM or cryo-electron tomography (cryo-ET), and is a motivated person with collaborative mind set. The candidate should to be able to purify the components for structural studies of transcription-translation coupling.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/structural-biology-of-coupled-transcription-translation/rg352

Supervisor

Mgr. Gabriel Demo, Ph.D.

The project is focused on detailed structural characterization of short purine oligonucleotides clipped by proper sequential motifs that induce parallel orientation of DNA strands. For this purpose, NMR experiments combined with MD simulations will be employed. The effect of modifications of selected nucleotides on the structural properties of designed models will be investigated to gain deeper understanding of key interactions that contribute to the folding of such systems.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/structure-of-biosystems-and-molecular-materials/rg108

Supervisor

prof. RNDr. Radek Marek, Ph.D.

Endosomal trafficking is vital in plant development both in optimal and stress conditions. This regulated vesicle trafficking is necessary for membrane integrity preservation and therefore plant resistance to acute osmotic stress, water loss. From forward genetics screen we have 3 EMS mutants that show grater sensitivity to osmotic stress and also show sub-cellular trafficking defects. We would like to map and characterise those mutants. Mapping at least 1 should be possible, we have experience and use NGS before successful identifying the causative mutation.

As an alternative we identified proteins differentially localized along the secretory pathway in response to stress indicating their role in cellular stress response. Characterization of those proteins (T-DNA lines) will provide insights into the role of subcellular machinery in plant response to stress (this is a Reverse Genetics approach).

One of those 2 approaches should work after the 1st year of PhD we will focus the project to 1 of the strategies.

The PhD student will perform the physiological and cellular phenotype analysis of mutants (the EMS and T-DNA) and overexpression lines. The admitted candidate will in situ protein localization and life confocal imaging techniques.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/developmental-and-cell-biology-of-plants/rg50

Supervisor

Tomasz Nodzynski, B.A., M.Sc., Ph.D.

The physical-chemical parameters of the intracellular space are actively contributing to regulation of biomolecular functions via influencing protein and nucleic acids structure and dynamics. While the coordinated and tightly controlled changes in the non-specific parameters of the intracellular space are permissive for broad range of fundamental physiological process including cellular division and differentiation, their deregulation leads to a number of human pathologies including cancer. The aim of this project is to explore a role of cellular environment in physiologically distinct cellular states on protein dynamics, structure and interactions with drug-like molecules.

The student will acquire advance knowledge and skills in human cell culture manipulations (cultivation, transfection, genome editing), DNA cloning, protein expression and purification, flow cytometry, confocal microscopy, and, in particular, with state-of-the-art in-cell NMR spectroscopy. The student will have the opportunity to present the results of his/her work at prestigious international conferences. Moreover, this project will involve collaboration with other leading researches in European institutes.

Requirements on candidates:

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact the supervisor and phd@ceitec.muni.cz

MORE INFORMATION:

http://ls-phd.ceitec.cz/

https://www.ceitec.eu/non-coding-genome/rg109

Supervisor

doc. Mgr. Lukáš Trantírek, Ph.D.