The list of topics and supervisors who offer projects to applicants in the academic year 2024/2025

Developmental and Cell Biology Board

The list of topics and supervisors who offer projects to applicants in the academic year 2024/2025

Deciphering transcriptional regulation and metabolic adaptations in hematopoietic stem cells during emergency granulopoiesis

Meritxell Alberich Jorda, Ph.D.

ID 269529

Hematopoietic stem cells (HSCs) preserve their functional pool by remaining quiescent, maintained by a low metabolic input through glycolysis. However, under stress HSCs get activated in order to fulfill the hematopoietic demands, which may lead to higher energy consumption. Recently, we reported that HSCs respond to emergency granulopoiesis (EG) shortly after infection, by transcriptionally rewiring lymphoid- towards myeloid-biased HSCs. Remarkably, we observed that the regulatory networks that control EG are much more complex than originally anticipated. Here, we introduce metabolic reprogramming from glycolysis to OXPHOS as a novel mechanism to sustain the increased energetic demands while preserving stemness upon induction of EG. Next, we hypothesize that the transcription factor C/EBPd is a key player in steady-state and EG, and could possibly be linked to these metabolic adaptations. The results from this project will challenge our current understanding of granulopoiesis in steady-state and emergency conditions, and greatly expand our knowledge of HSC metabolism under EG.

Metabolic reprogramming of cancer cells upon changes in style of migration

prof. RNDr. Jan Brábek, Ph.D.

ID 257600

The ability of cells to invade and metastasize belongs among the hallmarks of cancer, as defined by Weinberg and Hanahan. During dissemination from a primary tumor, cancer cells invade the ECM most commonly in clusters or sheets, what is referred to as collective migration, which requires proteolytic degradation at the invasive front and cell contractility in the following cells. Alternatively, single cancer cells can detach and invade using protease-dependent mesenchymal migration or protease-independent amoeboid migration, or combination of both. Further, many cancer cells can actively switch between these invasion modes in response to changes in the surrounding environment and/or to escape therapy. Within the primary tumor site, metabolic differences divide cells into distinct subpopulations that have unique capabilities enabling them to proceed through the metastatic cascade. Additionally, because cells are reprogrammed at different stages of metastasis to rely more on glycolysis or oxidative phosphorylation, it is crucial to understand which pathway is dominant at each stage. The aim of this project is to elucidate the link between cancer metabolism and different modes of migration in both 2D and 3D conditions, since it has been demonstrated that metabolism in 3D spheroids differs significantly from what is measured in 2D cultures, both in terms of glycolytic and oxidative phosphorylation metrics. To achieve this goal, we intent to analyze cancer cell migration and invasivity after inhibition of OXPHOS and/or glycolysis in 2D and 3D in cancer cells exhibiting different modes of migration. The project also aims to examine the metabolic reprogramming of cancer cells during the mesenchymal-to-amoeboid transition and vice versa.

The role of CRL4 in ribosome biogenesis

ID 270378

Mgr. Lukáš Čermák, Ph.D.

The objective of my doctoral project is to explore the role of the ubiquitin ligase CRL4 in RNA modification and to determine if its deficiency leads to a heightened incidence of cancer-related deformities. Specifically, the project will focus on the impact of CRL4 on the stability and functionality of ribosomal and telomeric non-coding RNAs. Moreover, we will analyze the role of CRL4 in tumor suppression utilizing an established mouse model. The anticipated outcomes of this study are expected to deepen our understanding of how translation control and genome stability influence human health.

Regulation of spindle assembly and chromosome segregation in mammalian oocyte meiosis

RNDr. David Drutovič, Ph.D.

ID 269598

Accurate chromosome segregation is essential to form an egg capable of being fertilized and supporting development to term. However, in human female meiosis, chromosomes frequently segregate incorrectly, resulting in aneuploidy. Some aneuploid embryos can implant but fail to develop to term, whereas others develop to term and result in syndromes, such as Down's syndrome. The molecular basis of the high incidence of inaccurate chromosome segregation in human oocytes is unknown. It thus represents a critical gap in our understanding of congenital disorders and female infertility. Although loss of cohesion is a significant age-related cause of aneuploidy, meiotic spindle disruption is a major age-independent factor. Examples include spindle instability and transient multipolar spindles, promoting erroneous merotelic kinetochore-microtubule (K-MT) attachments. The precise mechanisms that regulate stable bipolar spindle assembly remain unknown. The formation of oocytes in mice and humans is associated with eliminating centrioles. Oocytes thus employ alternative strategies to generate spindle microtubules, such as acentriolar microtubule organizing centers (aMTOCs) and newly discovered liquid-like spindle domains (LISD). Aurora kinases (AURKs), a family of serine/threonine kinases, play a central role in regulating spindle assembly, destabilizing incorrect K-MT interactions, and other critical processes in mitosis. We showed that the AURKs are crucial in regulating chromosome segregation and spindle assembly in mouse oocytes by regulating aMTOCs behavior and LISD formation. Little, if anything, is known about the role of AURKs in chromosome segregation during meiosis in human oocytes. This project aims to determine the molecular basis of functional meiotic spindle assembly using mouse models and human oocytes. We will study less-defined roles for AURKs and their cooperation with other signaling pathways in mouse and human oocytes using mouse genetic tools, molecular biology approaches, advanced light-sheet live-cell imaging, and image analysis.

The role oocyte nucleoli in embryonic genome organisation

Mgr. Helena Fulková, Ph.D.

ID 269527

The maternal oocyte nucleolus is essential for embryonic development independently of ribosome production. Facilitating the correct 3D genome organisation is likely its main function. It has been shown that pericentric heterochromatin is reorganised to contact this structure following fertilisation. This might be based on specific epigenetic marks but is also likely aided by specific nucleolar proteins. The aim of the project is to identify how these specific sequences target the nucleoli. To achieve this, we will modify the epigenetic status of pericentric heterochromatin and track these sequences in early embryos (zygotes). Next, we will deplete specific nucleolar proteins, NPM1 and NCL, using the TrimAway method and follow the localisation of pericentric heterochromatin. The applicant will learn advanced micromanipulations, microscopy skills, and basic molecular biology methods are an advantage.

From random sequence of amino acids to structure and function

Mgr. Klára Hlouchová, Ph.D.

ID 269604

The aim of this project will be to focus on the path from random sequence to protein structure and function, regarding both enzymatic catalysis and interactions with other molecules.

Using a previously developed methodology in our group (relying on FRET-FACS sorting), the student will work with vast random sequence protein libraries pre-selected for structure/compactness enrichment. The most enriched sequences will be structurally characterized to map the possibility of encountering specific protein folds in the absence of Darwinian selection and/or protein design. The pre-selected library will be further tested for a set of catalytic activities, in collaboration with the laboratory of Florian Hollfelder (University of Cambridge, UK). Potential hits will undergo detailed structural characterization, using methods such as circular dichroism (CD) and nuclear magnetic resonance (NMR).

In addition, random sequence libraries will be tested for their propensity to bind to a specific RNA target (represented by different constructs of proto-ribosomal peptidyl-transferase center), starting from libraries of different amino acid compositions. Further structural characterization techniques will employ NMR and/or cryo-EM.

The candidate for this project should have strong teamwork and wet-lab skills (previous work protein expression and biophysical characterization are welcome) and also basic bioinformatic competence.

Construction of new sensors for activity of chemotactic GPCRs

Mgr. Miroslav Hons, Ph.D.

ID 270066

An efficient immune response requires cells of the immune system to be at the right place at the right time and depends on their migration and correct positioning in tissues. Chemotactic cues are recognized by seven transmembrane G protein coupled receptors (GPCRs). In this project, we will construct new conformation sensors of chemotactic GPCRs to better understand their signaling.

The role of phosphoinositides in spatiotemporal regulation of nuclear processes

prof. RNDr. Pavel Hozák, DrSc.

ID 269611

Phosphoinositides (PIPs) are recognized as regulators of many nuclear processes including chromatin remodeling, splicing, transcription, and DNA repair. These processes are spatially organized in different nuclear compartments. Various nuclear compartments are formed by entropy-driven mechanism - phase separation. The surface of such membrane-less structures spatiotemporally coordinates complex nuclear processes. The integration of PIPs into the surface of nuclear structures might therefore provide an additional step in their functional diversification by controlling the localization of different components, in a similar way as PIPs do in membranous cytoplasmic environment. This project focuses on deciphering the molecular mechanisms of various PIPs in establishing a dynamic nuclear architecture. In this project PhD candidate will characterize the PIPs-containing nuclear structures by combination of lipidomics, proteomics (quantitative MS), molecular biology (e.g. CRISPR/Cas9), biochemical and advanced microscopy (e.g. confocal, SIM, STED, FRAP) methods. We will concentrate on Nuclear Lipids Islets (NLIs), which we discovered as important nuclear structures involved in modulation of gene transcription. In collaboration with other two laboratories, we will develop an experimental system using nanodiamonds mimicking the properties of NLIs and using phosphoinositides of various properties, we will study their involvement in DNA transcription using an in vitro system.

Nuclear actin-binding proteins in the regulation of cellular fate

prof. RNDr. Pavel Hozák, DrSc.

ID 269612

The cytoskeletal proteins play a pivotal role in various cellular processes, facilitating cell shape, motility, and cytoskeletal organization. Above their function in the cytoplasm, several members of the cytoskeleton together with β-actin itself were found in the cell nucleus where they regulate various processes such as DNA transcription, genome organization, and genome integrity which have a major effect on cell metabolism and cell fate during differentiation. Our main focus is understanding how actin and actin-binding proteins mechanistically regulate gene expression from DNA transcription to mRNA splicing and mRNA export during cell differentiation and disease. To address this, we use different overexpression and knock-out model systems in combination with a broad range of molecular biology, biochemistry, microscopy, and next-generation sequencing techniques.

In our previous research, we reported that during DNA transcription Nuclear Myosin 1 (NM1) directly regulates the gene expression of mitochondrial transcription factors TFAM and Pgc1α and forms a regulatory feedback loop with upstream signaling protein mTOR (Venit et al., 2023, Nature Commun.). In addition, we found nuclear Carmil 1, a multidomain protein regulating β-actin polymerization in the cytoplasm, and myosin 1 binding partner to be present in the nuclear speckles and to regulate gene expression during mRNA splicing.

In the proposed project, we will link two processes together and study NM1 and Carmil 1 during cellular differentiation and cell fate commitment. We will focus on understanding the relationships between NM1 and other Myo1C isoforms in the regulation of cell metabolism of specific human stem- and cancer cell types, and develop possible anti-cancer therapy strategies to reprogram and reverse cell metabolism by manipulating NM1 protein levels. We will study Carmil 1 during mRNA splicing and transport and examine the mutual interaction between Carmil 1 and NM1 in both transcription and splicing to prove that β-actin and β-actin-associated protein roles in the nucleus are not circumscribed only to chromatin, but are part of a complex gene expression regulation from DNA to proteins.

Mechanisms of Intercellular Communication: Relevance to Obesity and Neurodegeneration

Mgr. Martina Huranová, Ph.D.

ID 269525

Primary cilia are thin structures present in most mammalian cells, excluding the hematopoietic cells. Cilia host multiple transmembrane receptors, typically from the G-protein coupled receptor (GPCR) family, involved in developmental signaling pathways. For instance, cilia are crucial for the development and migration of neurons, the synaptic plasticity, and satiety management in the brain. Mutations in key ciliary proteins lead to cilia dysfunction manifesting as pleiotropic syndromes, collectively called ciliopathies. One of these rare recessive genetic diseases is Bardet-Biedl syndrome (BBS), characterized by diverse clinical symptoms such as obesity, renal dysfunction, retinal degeneration, polydactyly, and neurological disorder. The genetic cause of the BBS are mutations in the genes associated with the formation and function of an octameric ciliary adaptor complex called BBSome.

The applicant will investigate the physiological role of primary cilia and the BBSome in the brain and molecular mechanisms associated with the pathophysiology in the BBS.The applicant will employ a multifaceted approach integrating model systems and experimental pipelines of a different scale and complexity. The applicant will work with specific ciliopathy mouse models and cell lines and gain expertise in the modern cell and molecular biology approaches including the in vivo experiments, transcriptome analysis and fluorescence microscopy. Finally, the applicant will utilize clinical data from ciliopathic patients, which will provide insights into ciliopathy pathologies.

Molecular mechanisms of regulation of pancreatic β-cell function and viability in relation to type 2 diabetes pathogenesis

prof. RNDr. Jan Kovář, DrSc.

ID 255133

Dysfunction and apoptosis of pancreatic β-cells are among the key factors contributing in type 2 diabetes development. Many factors affect β-cells, especially low physical activity combined with unhealthy diet (e.g. chronically increased intake of saturated fatty acids). Some associated pathological states (e.g. sleep apnea leading to chronic hypoxia in pancreas) and environmental pollutants have negative effect on β-cells as well.

Exact molecular mechanisms by which the harmful factors affect β-cell function and viability are not elucidated yet. The aim of the project is to contribute to elucidation and understanding of these mechanisms.

Methods of cell and molecular biology (Western blot, FACS, confocal microscopy, siRNA, CRISPR, etc.) will be employed to study involvement of e.g. miRNAs, various signaling pathways, autophagy and alternative cell death pathways (e.g. ferroptosis, necroptosis). As experimental model, human and animal β-cell lines will be used. Alternatively, key results will be verified on isolated Langerhans islets.

Contact:

prof. RNDr. Jan Kovář, DrSc.

Department of Cell and Molecular Biology, Division of Biochemistry and Cell and Molecular Biology

3rd Faculty of Medicine, Charles University

e-mail: jan.kovar@lf3.cuni.cz

tel.: 267 102 658

Effect of iron accumulation on the function of critical tissues

prof. RNDr. Jan Kovář, DrSc.

ID 255136

We have been studying molecular mechanisms of iron transport and metabolism in various types of mammalian cells for a long time.

The project is focused on the problematics of iron transport mechanism and cell damage/cell death in specific tissues as a result of iron accumulation. Cell lines as well as samples of patients with chronic iron overload diseases will be used. In the project, we will further study cellular functions and mechanisms of iron transport into cells in patients with diabetes mellitus or prediabetes, or in patients with heart failure. As a part of the project, it is also possible to monitor the effect of increased iron intake on the development of metabolic syndrome, as well as iron metabolism in tumor tissue.

We will use following experimental models: (1) cell lines of hepatocytes (HEP-G2, HepaRG), pancreatic beta cells (NES2Y, INS1E) and cardiomyocytes (H9c2) will be used (2) tissue samples from patients with impaired iron metabolism (alcoholic liver disease, hemochromatosis, anemia from iron deficiency, hepatitis, porphyria, etc.) and patients with impaired glucose metabolism and obesity. Methodologically, a wide range of cell and molecular biology approaches will be used.

Contact:

prof. RNDr. Jan Kovář, DrSc.

Department of Cell and Molecular Biology, Division of Biochemistry and Cell and Molecular Biology

3rd Faculty of Medicine, Charles University

e-mail: jan.kovar@lf3.cuni.cz

tel.: 267 102 658

Pancreatic beta-cell and pollutants: the effect of pollutants on viability and function of pancreatic beta-cells

prof. RNDr. Jan Kovář, DrSc.

255138

Environmental pollution represents a significant threat to human health. Epidemiological studies suggest that, among others, pollution plays a role in the worldwide epidemy of diabetes mellitus. However, data is scarce, and pollutants' effects on pancreatic beta-cells remain largely unexplored.

The project focuses on exploring the effects of late (DDT, DDE, HCH) and present (TDCIPP, TPhP) pollutants on the viability and function of pancreatic beta-cells. Besides the production and synthesis of insulin, it will also explore the changes in the expression of proteins essential for beta-cell survival and functionality.

The project will use human, mouse, and rat beta-cell lines. Methods employed include western blot, ELISA, immunofluorescence, RT-PCR, and others.

Contact:

Prof. RNDr. Jan Kovář, DrSc.

Oddělení buněčné a molekulární biologie ÚBBMB

3.LF UK

Mail:jan.kovar@lf3.cuni.cz

Tel.: 267 102 658

Molecular mechanisms of cancer cell resistance to chemotherapeutics

prof. RNDr. Jan Kovář, DrSc.

ID 255139

Resistance of cancer cells to chemotherapeutics represents a crucial problem of the therapy of cancer diseases. We are dealing with molecular mechanisms of resistance and mechanisms of its development. Together with that we are dealing with possibilities to overcome resistance of cancer cells by relevant newly constructed chemotherapeutics.

As experimental models, we use cancer cell lines, experimental tumors in mice and tumor samples from patients. Our interest is focused on cells of breast cancer and ovarian cancer. In the case of cell lines, we employ original cancer cell lines, which are sensitive to chemotherapeutics, and counterpart sublines with developed resistance to chemotherapeutics. We are interested in changes of expression of relevant genes in resistant cells, including changes concerning regulation of expression of these genes. In the case of one particular group of chemotherapeutics, i.e. taxanes, we are dealing with a construction of such derivatives of chemotherapeutics, n the basis of structural and functional studies, which are targeted to overcome resistance.

For our studies, we employ a wide range of methods of cell and molecular biology.

Contact:

Prof. RNDr. Jan Kovář, DrSc.

Division of Cell and Molecular Biology, Department of Biochemistry, Cell and Molecular Biology

Third Faculty of Medicine, Charles University

e-mail:jan.kovar@lf3.cuni.cz

Developmental genetics of amphioxus: an insight into the evolution of vertebrate body plan

RNDr. Zbyněk Kozmik, CSc.

ID 268770

Project will focus on evolution of cell types, ancestral chordate features and vertebrate-specific innovations, using comparative analysis between amphioxus and vertebrates. The methods used will include basic bioinformatics, gene expression studies (single cell RNA-seq, whole-mount in situ hybridization, and immunohistochemistry), analysis of gene knockouts established in the lab using CRISPR/Cas9 system, and reporter gene transgenesis.

Background:

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. The genome of invertebrate chordate amphioxus has not undergone whole-genome duplication and serves as a proxy to ancestral chordates. Although amphioxus lacks the specializations and innovations of vertebrates, it shares with them a basic body plan and has multiple organs and structures homologous to those of vertebrates. For these reasons, amphioxus has widely been used as a reference outgroup to infer ancestral versus novel features during vertebrate evolution. Over the past few years amphioxus has become an established laboratory model and its cultures can be maintained throughout the year at the Institute of Molecular Genetics. This allows for an implementation of plethora of molecular and genetics approaches common to classical vertebrate models such as mouse, chick or fish. Moreover, recent publication on Amphioxus functional genomics and the origins of vertebrate gene regulation (Marletaz et al., Nature 564(7734):64-70) provides a huge genomic resource for future studies focused on gene regulatory mechanisms underlying evolution of vertebrate body plan.

Developmental genetics of amphioxus: an insight into the evolution of the neural crest

Iryna Kozmikova, Ph.D.

ID 269565

Project will focus on the study of those cell populations in amphioxus embryos that show expression profiles similar to the neural crest cells of the vertebrates. The methods used will include basic bioinformatics, gene expression studies (single cell RNA-seq, whole-mount in situ hybridization, and immunohistochemistry), analysis of gene knockouts established in the lab using CRISPR/Cas9 system, and reporter gene transgenesis.

Background:

We have recently identified neural crest like cells in amphioxus (Markos et al., 2024) that need to be thoroughly characterized since their presence in the invertebrate chordate presents a major challenge to the current textbook view.

Anna Markos, Jan Kubovciak, Simona Mikula Mrstakova, Anna Zitova, Jan Paces, Simona Machacova, Zbynek Kozmik -Jr, Zbynek Kozmik and Iryna Kozmikova. Cell type and regulatory analysis in amphioxus illuminates evolutionary origin of the vertebrate head. https://www.biorxiv.org/content/10.1101/2024.01.18.576194v1

Analysis of metabolic dependencies in acute myeloid leukemia

RNDr. Kateřina Kuželová, Ph.D.

ID 265610

Acute myeloid leukemia (AML) is a heterogenous tumor disease with generally unfavorable prognosis. In the last years, the therapy is being individualized according to underlying causative genetic changes. However, inhibition of specific signaling pathways is often associated with acquisition of drug resistance. Substantial differences between leukemic and healthy hematopoietic stem cells, representing possible therapy targets, can be found in the cell metabolism. Similar to other cancer types, AML cells can exhibit Warburg effect, i.e., they use aerobic glycolysis as a source of energy and building components. In parallel, they depend on the mitochondrial metabolism and related processes, such as glutamine uptake, fatty acid oxidation, or mitophagy. The frame of this work is to analyze these processes in selected AML subtypes using model systems (cell lines derived from AML) and primary leukemia cells. The experimental techniques will include flow cytometry, western blot, cell metabolism measurement using the Seahorse platform, and confocal microscopy.

In vitro reconstitution of active chiral networks

RNDr. Zdeněk Lánský, Ph.D.

ID 269167

Lab profile: Cytoskeletal networks form the internal dynamic scaffold of living cells essential for key cellular processes, such as cell division, cell motility or morphogenesis. Our aim is to understand how the individual structural elements of the cytoskeleton mechanically cooperate to drive these cellular processes. We use reconstituted cytoskeletal systems to study the system's self-assembly and dynamics. Central to our approach are imaging, manipulation and force measurement techniques with single molecule resolution.

Project description: Left-right symmetry breaking is crucial for correct body development of many organisms, including humans. Organism scale chirality is dependent on chiral cell behaviors, which in turn is a consequence of chiral stresses generated by the cytoskeleton. The cystoskeleton is composed of chiral biopolymers, such as actin filaments. While it has been shown that the large scale chirality of the cytoskeleton is a consequence of the structural chirality of its constituents, mechanistic understanding of this process is lacking. The aim of the project will be to reconstitute chiral actin networks, using e.g. actin filaments, filament crosslinkers and actin nucleators, and study the contribution of individual network constituents in generating chirality of the whole network. In collaboration with the Middelkoop lab (IMG CAS), Derivery lab (MRC LMB, Cambridge) and Furthauer lab (TU Wien) we will combine these in vitro experimental data with data from C.Elegans embryos, cultured cells and mathematical modelling to generate a comprehensive description of the system.

Candidate profile: We are looking for an enthusiastic PhD student motivated to work on cross-disciplinary projects. The candidate should hold a master's degree in (bio)chemistry, (bio)physics, molecular/cellular biology or an equivalent field.

Regulation of microtubule-dependent processes by tubulin isoforms and post-translational modifications

RNDr. Zdeněk Lánský, Ph.D.

ID 269030

Project description: Interactions between microtubule-associated proteins and microtubules drive many fundamental processes in the cell, for example long range cargo transport. Tubulin, the protein constituting microtubules, can be found in mutliple isoforms and is subject to different post-translational modifications. Consequently, these different tubulin vartiants can have different effects on the interactions between microtubules and microtubule-associated proteins, a hypothesis termed the "tubulin code". However, this regulatory network is not understood. The aim of the project will be to generate specific tubulin isoforms and tubulin with specific post-translational patterns and test their effect on the functioning of exemplary microtubule-associated proteins, such as molecular motors, regulatory adaptor proteins etc.

Regulation of tau envelope by tau post-translational modifications

RNDr. Zdeněk Lánský, Ph.D.

ID 246454

Project description: Modulating the accessibility of the cytoskeletal filaments for the filament-associated proteins is one of the fundamental regulatory mechanisms in the cytoskeleton. Unstructured microtubule-associated proteins, such as the Alzheimer's disease-associated protein tau, can form cohesive envelopes around microtubules, selectively modulating the microtubule accessibility by locally excluding specific proteins from the microtubule surface while recruiting others. The aim of the project is to explain the role of tau post-translational modifications in envelope formation and function.

Suggested reading:

Siahaan V et al, Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes. Nat Cell Biol. 2019 Sep;21(9):1086-1092

Schmidt-Cernohorska M, et al, Flagellar microtubule doublet assembly in vitro reveals a regulatory role of tubulin C-terminal tails. Science. 2019 Jan 18;363(6424):285-288.

Hernández-Vega A et al, Local Nucleation of Microtubule Bundles through Tubulin Concentration into a Condensed Tau Phase. Cell Rep. 2017 Sep 5;20(10):2304-2312

New mechanisms regulating function of the tumor suppressor p53 in human cells

MUDr. Libor Macůrek, Ph.D.

ID 269151

Genome instability is one of the main features of cancer cells. DNA repair and the cell cycle arrest are protective mechanisms that prevent the development of genomic instability. The tumor suppressor p53 plays a central role in regulating these events, and its loss leads to tumor development. The function of p53 is controlled by other proteins, including the phosphatase PPM1D and the protein kinase CHK2. Altered function of these regulators may suppress p53 activity and contribute to tumor development. Our recent work has demonstrated the oncogenic potential of truncating mutations of PPM1D as well as of inactivating CHK2 mutations. The planned PhD project aims to find new molecular mechanisms affecting the functions of PPM1D and CHK2 proteins in the cell cycle control and DNA repair, including the identification of defects leading to tumor transformation. In addition to classical molecular/cell biological and biochemical methodologies, we will investigate these processes using targeted genome editing by CRISPR/Cas9, mass spectrometry to perform proteomic analysis of protein complexes, high throughput quantitative microscopy to evaluate DNA damage response in cell nuclei and super-resolution microscopy to identify sub-nuclear localization of the studied proteins. The suitable candidate should have an interest in the basic molecular mechanisms occurring in human cells and should be devoted to experimental work. We anticipate that this project will contribute to the understanding of the mechanisms leading to suppression of p53 pathway function and tumor transformation.

Laboratory: Cancer Cell Biology

Institute of Molecular Genetics, ASCR

Supervisor: Libor Macurek, MD, PhD,

e-mail: macurek@img.cas.cz

Role of non-canonical Wnt and planar cell polarity pathways in craniofacial development

RNDr. Ondřej Machoň, Ph.D.

ID 269633

Cranial neural crest cells are generated in the developing midbrain and hindbrain. They migrate to the craniofacial region and differentiate into various cell types including chondroblasts, osteoblasts, dermal fibroblasts, cranial nerves and glia, or tenocytes. Molecular mechanisms controlling the enormous range of cell differentiation and navigation of their migratory routes are still not understood. Cell polarity is a key factor for regulation of directional cell migration and it is controlled by non-canonical WNT pathway. This project will generate a mouse model in which Wnt5a gene is deleted in cranial neural crest cells. Mutant embryos exhibit severe defects in the craniofacial area such as almost missing frontonasal process, short mandible and tongue suggesting that migration and cell fate of cranial neural crest cells is dependent on WNT5A signaling. This model will be used for single-cell RNA-seq analysis and bulk proteomics. Their integration will elucidate the molecular basis of mesenchymal cell polarization and motility at the level of transcriptional control and cytoskeletal dynamics.

The polymeric conjugates as an experimental tool in immunomodulation and immunotherapy

Mgr. Tereza Ormsby, Ph.D.

ID 270040

The immune system plays a crucial role in fighting diseases, including cancer. Manipulating the immune system with monoclonal antibodies against certain cancers have become a standard therapeutic approach. However, their high cost, instability and potential immunogenic side effects have caused researchers to turn their attention to the investigation of peptides and small-molecule inhibitors to devise a more effective strategy. Recently developed polymeric conjugates (iBodies) leverage the advantages of low-molecular-weight compounds as targeting ligands to overcome challenges posed by antibodies. They have demonstrated remarkable efficacy and potential, emerging as viable substitutes for antibodies in a range of analytic and diagnostic techniques. Furthermore, iBodies exhibit promising attributes as modulators of immune responses in cellular experiments. The primary objective of this project is to advance the development of mono- and bi-specific iBodies targeting both immune and tumor cell surface markers to enhance the efficacy of the immune cell responses against cancer cells. The PhD candidate will employ diverse methods including protein purification, enzyme kinetic, flow cytometry, ELISA, cell culture, purification of primary human blood cells and possibly mouse experiments.

Deciphering the Role of MICAL Proteins in Cytoskeletal Reorganization and Cellular Signaling

Mgr. Daniel Rozbeský, Ph.D.

ID 268779

This PhD project is dedicated to exploring the complex roles of MICAL (Molecules Interacting with CasL) proteins, a distinctive family of signaling molecules that have the remarkable ability to bind directly to and disassemble actin filaments. These proteins are crucial for facilitating precise changes in the cytoskeleton, necessary for various cellular processes. Particularly, MICALs play a vital role in the dynamic process of axon growth cone collapse by linking semaphorins and plexins to the disassembly of F-actin, thereby highlighting their importance in neurobiological events and cellular architecture regulation.

The primary focus of this research is to delve into the structural biology and molecular mechanisms governing MICAL signaling and its influence on cytoskeletal dynamics. The project aims to uncover the molecular mechanisms behind MICAL activation, employing cutting-edge techniques such as cryo-electron microscopy and protein crystallography. These methods will be integrated with a series of biophysical and cellular experiments to provide a comprehensive understanding of the structural basis of MICAL function and interaction with actin filaments. Additionally, an integral component of the research involves the screening for and design of novel inhibitors that can specifically bind to MICAL proteins, offering a potential therapeutic avenue to modulate their function. This aspect of the project aims to develop innovative strategies for drug design, potentially leading to the treatment of MICAL-associated diseases where cytoskeletal dysregulation is a contributing factor. Through a multi-disciplinary approach combining structural biology, biochemistry, and cell biology, this PhD project seeks to advance our knowledge of MICAL proteins and open new paths for therapeutic intervention.

Regulation of growth and metabolism by the mTOR pathway

prof. David Marcelo Sabatini, M.D., Ph.D.

ID 266447

My lab has a long-standing interest in the regulation of growth and metabolism. This interest stems from our early work on the pathway anchored by mTOR protein kinase, which we now appreciate is a major regulator of growth and anabolism (mass accumulation) in eukaryotes and responds to diverse stimuli, including nutrients. Our lab identified the mTOR-containing protein complexes, mTORC1 and mTORC2, and their biochemical and in vivo functions, as well as the complicated pathway upstream of mTORC1 that senses nutrients, including the Rag GTPases, GATOR complexes, and sensors for leucine and arginine.

Because our work revealed that lysosomes play a key role in the activation of mTORC1 by nutrients, we began to study lysosomes as well as other organelles, like mitochondria and melanosomes. We developed widely used methods for the rapid isolation and profiling of these organelles (e.g., Lyso-IP and Mito-IP), and used them to deorphan the functions of disease-associated genes. Because mTORC1 senses nutrients, we also became interested in the metabolic pathways that cells to use incorporate biomass and generate energy, particularly in cancer. We are also active in technology development and previously developed methodologies for genome-scale RNAi and CRISPR screening.

These are a few of the thesis projects available for graduate students available:

(1) Nutrient sensing by mTORC1 in vitro and in vivo. There are projects available to: identify the glucose sensor for the mTORC1 pathway; discover nutrient sensors in animals beyond mammals; understand how the known nutrient sensors (Sestrin for leucine, CASTOR for arginine, and SAMTOR for methionine) function in vivo in mice; and elucidate the biochemical function of key components of the nutrient sensing pathway, including GATOR2. These projects will use the tools of biochemistry and/or mouse mutants with specific mutations in nutrient-sensing pathway components.

(2) Lysosomes in normal physiology and disease. Our interest in mTORC1 led is to lysosomes as the activation of mTORC1 requires its translocation to the lysosomal surface. Using the Lyso-IP methodology and CRISPR screening technology there are projects available to: understand how common and rare neurodegenerative diseases impact lysosomal function; characterize and identify the contents of lysosomes in specialized cell types, like immune cells.

(3) Development of drug-like molecules for proteins of interest: In collaboration with medicinal chemists at IOCB and elsewhere, there are projects available to develop drug-like molecules that target mTOR pathway components as well lysosomal and mitochondrial proteins.

I am also open to ideas in the broader area of growth and metabolism from motivated students who are excited to forge a novel thesis project in consultation with me.

Research Group:

David. M. Sabatini, MD/PhD

Senior Group Leader

Institute of Organic Chemistry and Biochemistry

(IOCB)

Flemingovo n. 2

166 10 Praha 6

Czech Republic

david.sabatini@uochb.cas.cz

Regulation of metabolism and tool development

prof. David Marcelo Sabatini, M.D., Ph.D.

ID 266486

Beyond our interest in mTOR and nutrient sensing (Project 1), we have widespread interests in the regulation of metabolic pathways, particularly in mitochondria, and in developing new tools to study metabolism.

There are thesis projects for graduate students available in the following areas of interest:

(1) Nutrient transport and metabolism in mitochondria. Using the Mito-IP methodology, CRISPR screening technology, and structural biology there are projects available to de-orphan the functions and solve the structures of mitochondrial proteins of unknown functions and to understand how they connect to cytosolic metabolic pathways.

(2) Methods to study small molecule metabolism in vivo. There are projects available to develop methods to study carbohydrate and amino acid metabolism in cells in vivo in mice. We have novel ideas on how to study cell-type specific metabolism in vivo. These projects will use mass spectrometry-based metabolomics and tracing studies.

I am also open to ideas in the broader area of growth and metabolism from motivated students who are excited to forge a novel thesis project in consultation with me.

Research Group:

David. M. Sabatini, MD/PhD

Senior Group Leader

Institute of Organic Chemistry and Biochemistry

(IOCB)

Flemingovo n. 2

166 10 Praha 6

Czech Republic

david.sabatini@uochb.cas.cz

Analysis and modulation of RNA splicing in retinal dystrophy

prof. Mgr. David Staněk, Ph.D.

ID 263766

Mutations in several RNA splicing factors affect specific cells in the retina and lead to hereditary retinal degeneration - retinitis pigmentosa (RP). In addition, numerous mutations in retina-specific genes that also cause RP are found in introns and have potential negative effects on the splicing of these genes (e.g. RHO). Thus, splicing defects are key factors in the development of RP. Despite intensive research, the molecular mechanisms of cell-specific susceptibility to these mutations remain unclear. In this project, we plan to use relevant biomodels to study defects caused by RP mutations in splicing factors in target cell types. We will analyze the effect of RP mutations in different genes on in vitro generated human retinal organoids and retinal pigment epithelium. We will examine defects in RNA splicing and tissue-specific RNA production and identify genes with aberrant splicing that we will subsequently correct. We will also test the hypothesis that cellular sensitivity to RP mutations correlates with reduced expression of splicing factors. The results will allow us to identify potential treatments for RP. This project is part of Marie Skłodowska-Curie Training Doctoral Network focused on retinal dystrophies (ProgRET), which includes eight European academic teams and four industrial partners, which are leaders in retinal dystrophy research. Eligibility - M.Sc. or equivalent education in molecular biology, developmental biology or biochemistry obtained outside the Czech Republic.

Fibroblast mechanical forces in mammary epithelial morphogenesis

Mgr. Zuzana Sumbalová Koledová, Ph.D.

ID 265683

Epithelial-stromal interactions play a crucial role in mammary gland development and homeostasis. Stromal cells, such as fibroblasts, provide instructions for epithelial morphogenesis through paracrine signaling and extracellular matrix production and remodeling. In mammary epithelial organoid-fibroblast cocultures, we have recently described a new mechanism of fibroblast-induced epithelial morphogenesis mediated by fibroblast mechanical forces. In this project, the PhD cancdidate will investigate 1) the importance of fibroblasts mechanical forces in mammary epithelial morphogenesis in vivo using genetic mouse models, 2) regulation of fibroblast mechanical activity using scRNA sequencing analysis and functional in vitro and in vivo experiments.

Analysis of features of miRNA precursors

prof. Mgr. Petr Svoboda, Ph.D.

ID 268955

In the microRNA pathway, RNase III Dicer generates small RNA duplexes, which are subsequently subjected to strand selection and loading of a selected strand onto an effector protein from the Argonaute family. Proper strand selection is critical for the microRNA-mediated control of gene expression. Previous research showed that thermodynamic stability of termini of the RNA duplex is sensed by proteins in the Argonaute loading complex and predicts to some extent strand selection. However, there are many microRNAs, which do not obey this thermodynamic rule. The working hypothesis of this project is that the thermodynamic sensing is coupled with 3D shapes of microRNA duplexes and their orientation in the Argonaute loading complex. Accordingly, this project will combine experimental molecular biology with bioinformatics to analyze properties of microRNA precursors and organization of the Argonaute loading complex in order to determine features, which would better explain strand selection and loading then the existing model.

Specifics of protein processing during preimplantation development of mammals

Mgr. Tereza Toralová, Ph.D.

ID 269568

Precise coordination of protein degradation and protein synthesis is absolutely necessary for the proper development of the preimplantation embryo. In the early stages of development, the embryo is transcriptionally inactive and gene expression can thus only be controlled at the protein level. Thus, protein processing during preimplantation development is very specific in many ways. During oocyte maturation and preimplantation development, embryo-specific protein variants are expressed. These embryo-specific variants are often processed differently than the same protein in somatic cells. Protein processing during preimplantation development has been found to be species-specific and, at least for some proteins, to be dependent on the sequence of the protein itself, not the environment of the embryo. The main goal of this topic is to find the mechanism by which it is determined how the protein will be processed. We will focus on the characterization of sequence motifs that are specific for EGA-degraded maternal proteins. Next, we will focus on the function of proteins that are long-term stored in the preimplantation embryo, although protein expression in somatic cells is dependent on the cell cycle. We will describe how it is possible that protein storage does not disrupt the course of the cell cycle.

The role of flagellum distal end proteins in the axonemal assembly

Mgr. Vladimír Varga, Ph.D.

ID 269600

Eukaryotic flagella, also known as cilia, are evolutionarily conserved organelles of motility, with important signalling and sensory functions. The microtubule-based skeleton of these organelles, the axoneme, consists of microtubules and associated protein complexes. The axoneme is assembled exclusively by addition of material to its distal end. How are these assembly processes orchestrated is largely unknown. Studying highly experimentally tractable unicellular eukaryote Trypanosoma brucei, the causative agent of sleeping sickness, we identified flagellum tip-localizing proteins critical for the axonemal assembly and length regulation. Some of these have orthologs in other organisms including mammals. The aim of this project is to use live cell imaging, high-resolution approaches, such as expansion microscopy and electron microscopy, and biochemical approaches, including in vitro reconstitution assays, to gain a mechanistic understanding of the function of the identified flagellum tip proteins in trypanosomes and mammals.

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The list of topics and supervisors who offer projects to applicants in the academic year 2024/2025