OR Vývojové a buněčné biologie
Vypsané doktorské práce pro akademický rok 2025/2026
Changes in control and assembly of chromosome segregation apparatus in cells during early development
Anger Martin, MVDr., CSc.
280014
Early embryonic development in mammals is characterized by high incidence of errors, which leads into its premature termination. That makes it a problem during natural conception, causing subfertility, as well as during artificial reproduction, causing loss of valuable cells and embryos. The most common causes are those related to chromosome segregation, and they are associated with errors in cell cycle and spindle apparatus regulatory mechanisms. This period of development is characterized by rapid changes in cellular behaviour, some of which are still not understood well. The project will be aimed on elucidation of fundamental control mechanisms of early development, especially those concerning cell cycle regulation and spindle assembly, and alson on the link between these mechanisms and failure of development. In laboratory in campus of AVCR in Prague-Krč, techniques such as micromanipulation, microinjection, time lapse microscopy and functional microscopy will be utilized for the project.
Crosstalk between Dishevelled and eukaryotic cytoskeleton
Bařinka Cyril, RNDr., Ph.D.
279863
Dishevelled (DVL) is a scaffolding protein involved in Wnt signaling pathways that are essential for both correct embryo development and tissue homeostasis in adulthood. The non-canonical Wnt pathway controls distinct biological processes including cytoskeletal dynamics and cell polarity and migration. Interestingly, DVL itself has been shown to directly localize to the microtubular structures, but details of DVL recruitment to the microtubule network remain elusive. The proposed PhD project aims at deciphering the crosstalk between Dishevelled and microtubules (MTs). To this end, we will screen for microtubule-associated proteins (MAPs) mediating DVL/MT interactions using confocal microscopy. The positive hits will be heterologously expressed and purified, the systems reconstituted in vitro and assayed by TIRF microscopy. The protein constructs will further be used in an array of biochemical and biophysical techniques (pull-down, microscale thermophoresis, X-ray crystallography) to delineate DVL/MAP interaction interfaces and provide quantitative description of binding constants. Finally, physiological roles of DVL/MAP interactions will be studied in cellular and animal models.
Metabolic reprogramming of cancer cells upon changes in style of migration
Brábek Jan, prof. RNDr., Ph.D.
279493
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.
Unravelling the effect of cellular metabolites on the cytoskeletal dynamics
Braun Marcus, Ph.D.
280011
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: Microtuble dynamics regulate various cellular processes by adaptively restucturing the cytoskeleton. Microtubule dynamics is regulated by Microtubule associated proteins, including molecular motors. Levels of cellular metabolites, like ATP, depend upon cell types and vary throughout the cell cycle and might vary in different intracelllular locations. Little is know about the interplay between ATP levels, molecular motors and microtubule dynamics. Our preliminary results suggest that the interaction between microtubules and the molecular motor MCAK, a microtubule depolymerizing kinesin-13, is influenced by levels of ATP above saturating conditions of its enzymatic activity. As we find that ATP does not directly affect microtubule dynamics, our results suggest that in cells there might be an intricate interplay between metabolite levels, interaction of proteins with the microtubles, which, in turn, regulate microtubule dynamics. We will reconstitute the system using purified components or lysates of cells overexpressing recombinant, fluorescently labeld proteins available in the lab and study the dynamics of the system using single molecule microscopy. In addition to kinesin family members we will test the ATP sensitivity of microtuble tip-tracking proteins. We will ask which protein domains influence the sensitivity to ATP levels by way of creating domain deletion constructs. Complementarily, in vivo we will visualize changing ATP levels during cell division and local ATP distributions in axons.
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.
Subcellular Processing of Exosomes in the Context of Alzheimer´s Disease
Cebecauer Marek, Mgr., Ph.D.
279207
Exosomes are extracellular vesicles that play a pivotal role in molecular sorting, trafficking, and clearance. Their involvement in the transmission of signaling molecules implies their importance in neuronal communication and homeostasis. In the context of Alzheimer’s disease (AD), exosomes have been shown to contribute to the propagation of misfolded proteins, such as amyloid-beta and phosphorylated tau, as well as in the induction of apoptosis, leading to neuronal loss and disease progression. However, the molecular mechanisms and modifying risk factors underlying disease development are not fully understood.
In this project, student will investigate the impact of AD related environmental stress or disease related genetic background on exosome biogenesis and release. The subcellular distribution of intraluminal vesicles and their co-localization with AD-associated proteins will be examined in neuroblastoma cells and human neurons using confocal and super-resolution imaging1. Additionally, biochemical and physicochemical characterisation of isolated vesicles will be conducted to assess exosome content, size, and mechanical properties. The outcome of the study could contribute to understanding the mechanisms underlying exosome biogenesis and reveal novel clinical markers in the early diagnosis of neurodegeneration. The research will be realized in collaboration with Dasa Bohačiakova's laboratory at Masaryk University, Brno. Financial support is provided by the Czech Science Foundation under grant number 25-16481S.
(1) Riegerová, P.; Brejcha, J.; Bezděková, D.; Chum, T.; Mašínová, E.; Čermáková, N.; Ovsepian, S. V.; Cebecauer, M.; Štefl, M. Expression and Localization of AβPP in SH-SY5Y Cells Depends on Differentiation State. J. Alzheimer’s Dis. 2021, 82 (2), 485–491. https://doi.org/10.3233/JAD-201409.
Investigating the Role of CRL1 in Development and Cancer
Čermák Lukáš, Mgr., Ph.D.
280053
FBXO28, a component of the CRL1 (CUL1-RING-ligase) ubiquitin ligase complex, plays a critical role in cellular homeostasis by mediating the targeted degradation of specific protein substrates. Dysregulation of FBXO28 has been implicated in neurodevelopmental disorders and cancer, yet its precise molecular mechanisms remain poorly understood. Human genetic studies have identified FBXO28 mutations and microdeletions within its 3′ UTR region, which are associated with 1q41q42 microdeletion syndrome. These alterations likely affect mRNA stability and protein function, underscoring FBXO28’s potential role in disease pathogenesis.
To address these knowledge gaps, we propose a comprehensive investigation of FBXO28’s molecular functions in development and cancer. Our approach combines biochemical techniques with advanced genetic and experimental mouse models. In vitro, we will focus on identifying novel substrates of this ubiquitin ligase, leveraging an experimental pipeline developed in our lab to characterize CRL-dependent ubiquitination and protein degradation.
Our preliminary data suggest that FBXO28 plays a role in preserving genome stability. In addition to characterizing its systemic and tissue-specific functions during development, we will utilize Fbxo28 knockout (KO) mice, generated in our lab, to examine its role in tumorigenesis. By crossing Fbxo28 KO mice with cancer-prone genetic backgrounds, such as Trp53-mutant strains, we aim to analyze tumor latency, progression, and mutational landscapes.
This study seeks to elucidate FBXO28’s role in disease by integrating molecular, genomic, and phenotypic analyses. Specifically, we will investigate its function as a molecular regulator, its involvement in maintaining genomic stability, and its contributions to neurodevelopmental disorders and cancer. Our findings could reveal novel therapeutic targets and inform strategies for modulating FBXO28-related pathways in these diseases.
FBXO38, komponenta SCF ubikvitin-ligázového komplexu, a jeho role v regulaci imunity
Čermák Lukáš, Mgr., Ph.D.
280123
F-box protein 38 (FBXO38) is a substrate receptor of the SKP1-CUL1-F-box (SCF) ubiquitin ligase complex and plays a key role in protein homeostasis by mediating the targeted degradation of specific substrates. Mutations in FBXO38 have been linked to neurodegenerative disorders such as hereditary motor neuronopathy, but its precise function in immune regulation remains unclear. Previous studies suggested that FBXO38 may control the stability of immune checkpoint molecules such as PD-1, a crucial regulator of T-cell activity. However, recent evidence indicates that FBXO38 is dispensable for PD-1 regulation, raising questions about its actual role in immune processes.
To address these knowledge gaps, this project aims to characterize the molecular functions of FBXO38 in immune regulation using a combination of biochemical approaches and genetic models. Specifically, we will investigate whether FBXO38 modulates immune cell function through the targeted degradation of nuclear factors involved in immune gene expression. Preliminary data suggest that FBXO38 regulates the stability of ZXDA/B, which are linked to centromere integrity and may also have transcriptional regulatory functions in immune cells.
We will employ proteomics and ubiquitination assays to identify novel substrates of FBXO38 in immune cells and determine how its loss affects immune homeostasis. Additionally, using Fbxo38 knockout (KO) mice and immune challenge models, we will examine its role in T-cell activation, differentiation, and cytokine production. RNA sequencing and chromatin immunoprecipitation (ChIP) studies will help elucidate whether FBXO38-dependent protein degradation influences immune transcriptional programs.
This study seeks to provide a comprehensive understanding of FBXO38’s role in immunity by integrating molecular, genomic, and in vivo functional analyses. Our findings could reveal new insights into immune regulation and suggest potential therapeutic targets for modulating immune responses in autoimmunity, cancer, and infectious diseases.
Characterization of Mouse Breast Milk and Maternal-Fetal Microchimerism Established During Lactation
prof. RNDr. Jan Černý, Ph.D.
279567
Maternal-fetal microchimerism (MMc) is a widely accepted physiological phenomenon during which maternal cells migrate into the tissues of the offspring. This occurs both during pregnancy and through breast milk. While MMc established during pregnancy has long been recognized, the migration of maternal cells via breast milk has only come into focus in recent years. The aim of this project is to characterize mouse breast milk, including its hormone content and cellular populations, with an emphasis on immune cells. Furthermore, the project will investigate the transfer of these maternal cells to the offspring, specifically their migration pathways, target organs, and their quantities and frequencies within specific tissues. Special attention will be paid to the phenotypes of these cells and their potential to influence the development of the offspring's immune system, with implications for possible transgenerational transfer of immune memory. The project will also include the characterization of differences between two mouse strains – C57Bl6 and Ly5.1. Recent data from our laboratory have highlighted differences in development, breast milk composition, and the establishment of MMc between these strains. Therefore, the project will also focus on the differences between these two mouse strains and their impact on offspring development and immune system function, highlighting a contribution from MMc. As an important model, the MHCII–EGFP knock-in mouse will be utilized, which is an exceptional tool for systemic and quantitative immunology. The project also includes characterization of the lymphatic system/gastrointestinal tract in the context of early postnatal development, breastfeeding, and microbiota.
The impact of female age on oocyte quality and the ability of oocytes to repair ssDNA breaks
Fulková Helena, Mgr., Ph.D.
278857
It is well accepted that oocyte quality declines with female age. However, the exact reasons and mechanism are not known. Cellular ageing is a complex process affecting both the cytoplasm and the nucleus. It is currently unclear to what extent the oocyte DNA is impacted; the results are mixed. Determining this is of major importance when selecting a potential cell therapy scheme. This project is focused on determining the DNA damage in oocytes in correlation with female age, with a major focus on single-strand DNA (ssDNA) damage. ssDNA damage is by far the most prevalent type of DNA insult and occurs with a high frequency in somatic cells; the frequency of ssDNA lesions and the extent of damage in oocytes are unknown. The partial aims of the project are: to determine the number of ssDNA breaks in oocytes from different age groups of females, analyse the level of the base excision repair pathway proteins and their localization, functionally assess the capacity of oocytes from different age groups to repair ssDNA damage.
Reduction of Trp content in minimal cell model using genome engineering and de novo genome synthesis
Hlouchová Klára, Mgr., Ph.D.
280206
The aim of this thesis will be to use the methods of genome engineering/recombineering to massively edit the genome of JCVI_Syn3 genome (derived from Mycoplasma mycoides by genome reduction by the J. Craig Venter Institute). The editing will be focused on reducing Trp content across proteome. The candidate will focus on proteins with limited Trp content and point mutations using combinatorial recombineering. They will work along with a team of other group members, focusing on Trp-rich proteins, or proteins with highly conserved Trp residues, using broader computational re-design.
At the second stage of the PhD project, the candidate will employ methods of de novo genome synthesis and assembly to compose synthetic fragments of the JCVI_Syn3 genome, with Trp codons minimized to its minimum. The fragments will be incorporated to a chimeric genome versions along with wild-type fragments, to study the cost of the Trp reduction proteome-wide. The candidate will adapt methods of genome transplantation and characterization of the bacterium fitness.
This thesis will eventually explore the in/dispensability of individual Trp residues in the JCVI_Syn3 organism and will study the fitness costs of their substitutions proteome-wide.
Construction of new sensors for activity of chemotactic GPCRs.
Hons Miroslav, Mgr., Ph.D.
280054
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 understand their signaling in physiological context
Methodologically, this project combines cutting-edge molecular biology and microscopy.
About the lab:
https://hons-lab.lf1.cuni.cz/
We are looking for dedicated candidates interested in cell biology, cell migration, cytoskeleton, immunology and microscopy. We offer an opportunity to work in a newly established research group within the BIOCEV with its exceptional equipment.
Applicants are encouraged to provide a brief CV with summary of recent work to Miroslav Hons, PhD (miroslav.hons@lf1.cuni.cz)
The role of phosphoinositides in spatiotemporal regulation of nuclear processes
Hozák Pavel, prof., DrSc.
279317
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.
Study of oxidative stress and antioxidant therapy at the cellular level
prof. RNDr. Marie Hubálek Kalbáčová, Ph.D.
279588 1.LF
Oxidative stress occurs in the body under various physiological and pathological conditions. Individual cells have their own system of defence against it, which can be improved by the newly developed nanomedicine - cerium nanoparticles that act as nanozymes scavenging oxygen and other radicals, which would be transported in newly created carriers to the places in the cell where there is an increased concentration of these radicals. This nanomedicine is being prepared by CEITEC partners (and supported by a GAČR grant) and the main goal of this thesis is biological tests of the prepared nanozymes and their carriers. Fibroblasts and brain epithelial cells will be used as model cells, as this novel therapeutic strategy should help to combat oxidative stress induced by stroke. Basic molecular biological and biochemical methods, cell culture, fluorescence microscopy and flow cytometry will be used to achieve the objectives.
Cilia Function in Cell Signaling and Differentiation
Huranová Martina, Mgr., Ph.D.
280207
Cilia are essential organelles involved in a wide range of cellular processes, including signal transduction, cell motility, and tissue development. This doctoral research focuses on establishing and optimizing readouts to monitor the activity of cilia-associated signaling pathways, which play a crucial role in regulating cellular functions, including cell differentiation. The work aims to develop robust tools for tracking the dynamics of ciliary signaling in both cell cultures and brain organoid models, utilizing advanced live-cell imaging techniques for real-time observation of cellular processes. In order to determine the regulatory mechanisms of cellular differentiation controlled by cilia-mediated signaling, we will utilize the transcriptomic and proteomic analysis. Additionally, the applicant will explore the potential for applying these tools in the screening of biological agents, aiming to identify compounds that modulate ciliary signaling in pathological conditions where cilia function is disturbed. By integrating live cell imaging, gene expression profiling, and optimized reporter assays, this thesis aims to establish novel methodologies for studying cilia associated signaling in cell differentiation, with potential applications in drug discovery and disease modeling.
The impact of sex chromosomes on cardiometabolic adaptations during obesity
Chmátal Lukáš, Mgr., Ph.D
279549
Heart disease is the leading cause of death worldwide, with significant differences in symptoms, prevalence, and outcomes between sexes. These differences are shaped by simultaneous contribution of various factors, including diet, exercise, sex hormones, and sex chromosomes. However, the specific impact of sex chromosomes, such as the “inactive” X chromosome (Xi) in females and the Y chromosome in males, on heart physiology and function remains poorly understood. To address this knowledge gap, we ask: How does the Xi regulate mitochondrial functions under normal and pathological conditions? To tackle this question, we have developed the MiCY* mouse model characterized by altered sex chromosome compositions (XO, XX, XY, XXY) combined with established cardiomyocyte-specific fluorescently tagged outer mitochondrial membrane protein. This model allows us to isolate mitochondria specifically from cardiomyocytes, and study the effects of the Xi on mitochondrial physiology and function under both normal and obesity-related conditions. Using an integrated approach that combines heart physiology techniques, biochemistry, targeted and untargeted metabolomics, proteomics, and fluorescence microscopy, we will explore the role of the Xi chromosome and specific Xi-expressed genes in mitochondrial biology during both health and disease. This work will provide critical insights into how the Xi chromosome influences mitochondrial metabolism and contributes to cardiac health and disease. Candidate Requirements: You have completed, or are nearing the completion of, a Master’s degree in biological, medical, chemical, or biochemical sciences. You bring motivation, attention to detail, and hands-on experience in cell biology, molecular biology, or metabolomics. You are comfortable with handling mouse and human samples. You are fluent in English, have excellent communication skills and thrive in an inclusive, collaborative and supportive team that works towards a shared goal.
Supervisor: Lukas Chmatal, Ph.D. (chmatal@wi.mit.edu)
Selected relevant publications:
- Maya Talukdar*, Lukas Chmátal*, Linyong Mao, Daniel Reichart, Danielle Murashige, Yelena Skaletsky, Daniel M. DeLaughter, Zoltan Arany, Jonathan G. Seidman, Christine Seidman, David C. Page. Genes of fatty acid oxidation pathway are upregulated in the female as compared to male human cardiomyocytes. Circulation (2025), *co-first authors, IF = 39.9
- Daniel Reichart, Gregory A. Newby, Hiroko Wakimoto, Mingyue Lun, Joshua M. Gorham, Justin J. Curran, Aditya, Raguram, Daniel M. DeLaughter, David A. Conner, Júlia D. C. Marsiglia1, Sajeev Kohli, Lukas Chmátal, David C. Page, Nerea Zabaleta, Luk Vandenberghe, David R. Liu, Jonathan G. Seidman, and Christine Seidman. Efficient in vivo genome editing prevents hypertrophi cardiomyopathy in mice. Nature Medicine, 29: 412-421 (2023), IF = 58.7
- Akera T., Chmátal L., Trimm E., Yang K., Aonbangkhen C., Chenoweth D.M., Janke C., Schultz R.M., Lampson M.A. Spindle asymmetry drives non-Mendelian chromosome segregation. Science, 358(6363): 668-672 (2017), IF = 44.7
The impact of biological sex on mitochondrial metabolism in cardiomyocytes
Chmátal Lukáš, Mgr., Ph.D.
279548
Are you ready to explore the mysteries of sex differences in heart disease that impact equitable treatments for men and women? Because heart disease is the leading cause of death worldwide, with marked differences in symptoms, prevalence, and outcomes between sexes, understanding the molecular mechanisms underlying the sex differences is key for addressing the unique needs of male and female patients. My lab has a deep interest in metabolic sex differences and their role in human health and disease. Our previous research shows that human heart metabolism differs between healthy males and females. Specifically, we showed that mitochondrial fatty acid oxidation - the main source of heart’s energy – is more potent in female cardiac cells compared to males. We thus ask a key biological question: How does biological sex influence overall mitochondrial metabolism and function in heart cells? Using state-of-the-art metabolomics, proteomics, and fluorescence microscopy in genetically engineered mouse models with fluorescently tagged cardiac mitochondria, we study how biological sex - a unique combination of sex chromosomes and sex hormones – shapes mitochondrial function. We’re looking for a passionate, curious, and collaborative individuals to join our emerging team of motivated scientists. If you’re interested in mitochondrial biology, metabolism, and sex differences, we’d love to hear from you!
Candidate Requirements:
You hold a Master’s degree in biological, medical, chemical, or biochemical sciences, or you are on track to complete your studies this academic year. You are a motivated, detail-oriented individual with experience in cell biology, molecular biology, or metabolomics, and you are comfortable working with
both mouse and human samples. You are fluent in English and have excellent communication skills and enjoy creating and working in an inclusive, supportive and collaborative team driven by a shared goal.
Supervisor: Lukas Chmatal, Ph.D., (chmatal@wi.mit.edu)
Selected relevant publications:
- Maya Talukdar*, Lukas Chmátal*, Linyong Mao, Daniel Reichart, Danielle Murashige, Yelena Skaletsky, Daniel M. DeLaughter, Zoltan Arany, Jonathan G. Seidman, Christine Seidman, David C. Page. Genes of fatty acid oxidation pathway are upregulated in the female as compared to male human cardiomyocytes. Circulation (2025), *co-first authors, IF = 39.9
- Daniel Reichart, Gregory A. Newby, Hiroko Wakimoto, Mingyue Lun, Joshua M. Gorham, Justin J.Curran, Aditya, Raguram, Daniel M. DeLaughter, David A. Conner, Júlia D. C. Marsiglia1, Sajeev Kohli, Lukas Chmátal, David C. Page, Nerea Zabaleta, Luk Vandenberghe, David R. Liu, Jonathan G. Seidman, and Christine Seidman. Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice. Nature Medicine, 29: 412-421 (2023), IF = 58.7
- Akera T., Chmátal L., Trimm E., Yang K., Aonbangkhen C., Chenoweth D.M., Janke C., Schultz R.M., Lampson M.A. Spindle asymmetry drives non-Mendelian chromosome segregation. Science, 358(6363): 668-672 (2017), IF = 44.7
Impact of mesenchymal cells on the intestinal epithelium homeostasis, regeneration and tumorigenesis
Janečková Lucie, Mgr., Ph.D.
279049
The intestinal epithelium is a dynamic tissue that provides a protective barrier while supporting nutrient absorption and immune defense of the gut. Stem cells in the crypts renew the epithelium and maintain the intestinal homeostasis, while its dysregulation underlies diseases such as inflammatory bowel disease and colorectal cancer. Intestinal mesenchymal cells play a crucial role in orchestrating these processes by regulating epithelial cell proliferation and differentiation through paracrine signaling, extracellular matrix production, and mechanical interactions. Despite their importance, the mechanisms by which mesenchymal cells influence epithelial cells during intestinal regeneration and in pathological conditions remain poorly understood. Using genetic mouse models, co-culture of intestinal organoids with mesenchymal cells, and methods analyzing gene expression (single cell or bulk RNA sequencing, gene set enrichment analysis), we aim to characterize the role of mesenchymal cells in promoting epithelial renewal in the healthy intestine and their contribution to pathological processes such as tumorigenesis and chronic inflammation. This project offers an unique opportunity to unravel the complex cellular interrelationships in the gut and their implications for health and disease.
Redox mechanisms of insulin secretion
Ježek Petr, RNDr., DrSc.
279682
The central role of pancreatic beta cell, insulin secretion, is still not fully understood, concerning molecular mechanisms. We have revealed that redox (H2O2) signal is essential for glucose-stimulated insulin secretion (GSIS, thesource is NADPH oxidase 4, NOX4) and fatty acid-stimulated insulin secretion (FASIS, the source are mitochondria). The concomitant metabolic pathways are not yet completely characterized for all insulin secretagogoues, e.g. for branched-chain amino acids and others. Therefore, we will develop mitochondria-specific metabolomics and proteomics after rapid magnetic separation of mitochondria having HLA antigen expressed on the surface of their outer membrane. Islets will be isolated from the respective transgenic mice. Using 13C-metabolites, we will study the respective metabolic pathways as well as redox homeostasis in physiological and simulated diabetic (pathological) conditions. We will also judge conditions for lipotoxicity and role of lipid metabolism. See Ref. doi: 10.1016/j.redox.2024.103283 and doi: 10.2337/db19-1130.
Analysis of metabolic dependencies in acute myeloid leukemia
Kuželová Kateřina, RNDr., Ph.D.
279404
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. Mitophagy can also increase cell resistance to apoptosis induction via removal of damaged or superfluous mitochondria. The frame of the project is to analyze oxidative phosphorylation and mitophagy in AML model systems (cell lines) and primary leukemia cells. The work will focus to the mitophagic activity of primary AML cells and its importance for mitochondrial respiration and in vitro cell resistance to apoptosis-inducing drugs (e.g., BH3 mimetics). The cell lines will be used to analyze the molecular mechanisms responsible for AML cell dependency on oxidative phosphorylation and mitophagy. The project will extend the so far limited knowledge about mitophagy rate in primary AML cells, possible differences between early leukemia progenitors and more differentiated cells, as well as the impact of recurrent mutations in the FLT3 gene on oxidative phosphorylation and mitophagy. We also expect the project will support or displace our hypothesis that mitophagy measurement could improve the diagnostics of resistance to BH3 mimetics or other drugs used in AML therapy and combination of these compounds with mitophagy inhibitors could increase their efficacy. The experimental techniques will include flow cytometry, western blot, cell metabolism measurement using the Seahorse platform, and confocal microscopy. Original protocols, which have already been established in the lab, will be used for mitophagy quantification.
Regulation of intracellular transport by interactions between intermediate filaments and microtubules
Lánský Zdeněk, RNDr., Ph.D.
280018
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: Long-range cargo transport is essential especially in highly elongated cells, such as neurons. The transport is typically driven by molecular motors which translocate along microtubules in a directed manner. Motors are regulated by a plethora of auxiliary factors presumably determining the cargo trajectory and final destination. Although crucial for the functioning of the cell, very little is known about this network of regulatory factors. Preliminary results suggest that intermediate filaments abundant in the neurons might play a role in the regulation of these transport processes. We will take advantage of a panel of microtubule-associated proteins that we have recently established in the lab to probe the interaction of these protleins with intermediate filaments and their effect on microtubule-related molecular motors. We will thus elucidate the regulatory roles of intermediate filaments in microtubule-based cargo transport.
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 tau envelope by tau post-translational modifications
Lánský Zdeněk, RNDr., Ph.D.
280024
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: 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.
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 mitochondrial transport by the interplay between metabolites and adaptor proteins
Lánský Zdeněk, RNDr., Ph.D.
280009
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: Long-range transport of mitochondria is essential, especially in highly elongated cells, such as neurons. The transport is typically driven by molecular motors, such as kinesin-1 and dynein, which translocate along microtubules in anterograde and retrograde direction, respectively. The mitochondria are attached to the motors by a variety of adaptor proteins, which can determine the cargo transport efficiency. Although crucial for the functioning of neurons, very little is known about how are motors regulated to maintain proper mitochondrial distribution in the cell. Our preliminary results suggest that the interaction of the mitochondria-related adaptor protein syntaphilin with microtubules is regulated by the local level of ATP. These results suggest that syntaphilin could work as an ATP level-dependent anchor for the mitochondrion. We will reconstitute the system using purified components available in the lab and study the dynamics of the system using single molecule microscopy. Complementarily, in vivo we will investigate the dynamics of single mitochondria in axons and probe their motility at varying local ATP levels. We will thus elucidate the regulation of mitochondrial transport by the interplay between metabolites, such as ATP and adaptor proteins, such as syntaphilin.
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.
Properties and the role of non-canonical RNA caps in cells
Macíčková Cahová Hana, Ing., Ph.D.
279573
Recent discovery of RNA caps such as NAD and CoA leads to reassessment of RNA structure in all types of cells. In our search for new RNA modifications, we have discovered brand new class of 5‘caps - dinucleoside polyphosphates (NpnN) in bacteria as well as in eukaryotes. The project will focus on elucidating the physical properties of these non-canonical RNA caps and on their role e.g. in regulatory RNA. Student will employ various techniques for studies of capped RNA e.g. CD spectroscopy or mass spektrometry.
Development of methods to study 5’ RNA termini structure and sequence
Macíčková Cahová Hana, Ing., Ph.D.
279572
The recent discovery of RNA caps, such as NAD and CoA, has prompted a reassessment of RNA structure across all types of cells. In our search for novel RNA modifications, we have identified an entirely new class of 5′ caps—dinucleoside polyphosphates (NpnN)—present in both bacteria and eukaryotes.
This project aims to develop a robust LC-MS sequencing method using various selective RNases. The technique will be applied to investigate the properties of the 5′ ends of various non-coding RNAs, such as snRNA and snoRNA, and to determine the capping status of natural mRNA, distinguishing between cap1 and cap2 structures. Furthermore, this method will serve as a valuable tool for assessing and ensuring the quality of mRNA used in biotechnology applications.
New mechanisms regulating function of the tumor suppressor p53 in human cells
Macůrek Libor, MUDr., Ph.D.
279073
Genome instability is one of the main features of cancer cells. DNA repair and the cell cycle arrest are protective mechanisms that prevent development of the genome instability. The tumor suppressor p53 plays a central role in regulating these events, and its loss leads to tumor development. Function of p53 is controlled by other proteins, including the phosphatase PPM1D. We have recently described oncogenic potential of the C-terminal truncating mutations of PPM1D (1-3). The observed gain-of-function phenotype depends on abnormally increased stability of the truncated PPM1D protein (4). This PhD project aims to identify the molecular mechanism of PPM1D degradation underlying its rapid turnover in normal conditions. We will also address how PPM1D associates with the chromatin which is another crucial determinant of its function (5-7). Besides the classical molecular/cell biology and biochemistry, we will investigate these processes using targeted genome editing by CRISPR/Cas9, analysis of protein complexes by mass spectrometry, evaluation of DNA damage in cell nuclei by high content quantitative microscopy, and identification of the sub-nuclear localization by super-resolution microscopy. Suitable candidates should have an interest in the basic molecular mechanisms occurring in human cells. This project will improve our understanding of the mechanisms leading to cellular transformation by inhibition of p53. In addition, reactivation of p53 function by forced degradation of PPM1D is an attractive strategy for treatment of various cancers.
Structural studies od the tumor supressor LACTB and other cancer-related enzymes
Maloy Řezáčová Pavlína, doc. RNDr., Ph.D.
280178
This project aims at experimental structure determination of medicinally relevant proteins and their complexes. Knowledge of the 3D structure is crucial for understanding protein function in biological processes. Additionally, structural information on proteins involved in human pathologies is beneficial to guide the design of molecules affecting their function.
A central focus of this research will be the structural characterization of the tumor suppressor LACTB, a mitochondrial protein that plays a key role in metabolic regulation and tumor suppression. Detailed structural studies of LACTB will provide critical insights into its mechanism of action, its role in cellular homeostasis, and its potential as a therapeutic target in cancer. Understanding how LACTB interacts with other biomolecules and how its structure relates to its function could uncover novel strategies for cancer treatment.
As a complementary objective, the project will also include structural studies of other human enzymes involved in cancer, further expanding the scope of this research to uncover mechanisms underlying disease progression and identify potential therapeutic targets.
The project will be carried out at the Institute of Organic Chemistry and Biochemistry CAS (IOCB Prague), which is fully equipped with state-of-the-art facilities for the structural studies proposed. Advanced techniques, including X-ray crystallography, NMR, and cryoEM microscopy, will be employed to investigate LACTB's structural dynamics and functional interactions, as well as those of other key enzymes. These capabilities will enable a comprehensive understanding of their biological roles, paving the way for the rational design of targeted modulators and therapeutic strategie.
Targeting isolated biliary atresia: early diagnosis and trustworthy disease model development.
Mašek Jan, Mgr., Ph.D.
279523
Biliary atresia (BA) is the most common pediatric cholangiopathy nowadays. Utilizing our unique mouse mutant strains recapitulating mutations causing biliary atresia (BA) and Alagille syndrome (ALGS) in humans, and tissue samples from patients with BA, the goal of this project is to comprehensively investigate the contribution of the vascular system to BA pathology and identify molecules characteristic of early stages of isolated BA, representing potential targets for BA treatment development. In this project, we will induce experimental BA in control and mutant mice carrying human mutations for BA and ALGS using neonatal biliatresone administration. To compare their phenotype with the situation in BA patients, we will employ a combination of medically relevant techniques such as neonatal ultrasound measurements, serum biochemistry, and histology, and correlate them with transcriptomics, bile acid proteomics, and immune response characterization. By synthesizing the results, we will map changes in intra- and extrahepatic bile duct morphology and the vascular system, transcriptional profile, and bile acid composition under intact and BA-induced conditions in mice. The findings in mice will be combined with histological and transcriptomic characterization of BA patients' livers and corresponding controls to identify molecules characteristic for early stages of isolated BA, representing potential targets for BA treatment development.
Expanding the single-molecule toolbox to visualize ciliary compartmentalization in C. elegans neurons
Mitra Aniruddha, Ph.D.
279212
Sensory cilia are vital, antenna-like organelles in eukaryotic cells that detect external stimuli and transmit signals, influencing gene expression and cell behaviour. They are isolated from the cell's interior by a diffusion barrier, creating a unique environment rich in membrane proteins and enzymes involved in various signalling pathways. The heterogeneity within the cilium is maintained by intraflagellar transport (IFT), which is essential for the entry, exit, and distribution of ciliary proteins, with disruptions leading to ciliopathies. Research in the field has revealed structural and dynamic aspects of IFT within cilia, but how IFT regulates entry and exit of proteins to maintain ciliary compartmentalization remains unclear. In recent work, we have developed quantitative single-molecule fluorescence microscopy tools to resolve intracellular dynamics in C. elegans neurons. The aim of this project would be to expand this toolkit to study single-molecule dynamics of proteins in sensory cilia of living C. elegans. This will enable us to visualize the entry, exit, retention, and turnover dynamics of IFT proteins and their cargo within the cilium. The overall goal of this project is to understand how IFT preserves and regulates the diverse protein composition within cilia.
Contact: Aniruddha Mitra (group leader) Institute of Biotechnology of the Czech Academy of Sciences a.mitra@uu.nl
Investigating and optimizing anti-PD-L1 and anti-CD73 iBodies for immunotherapy
Ormsby Tereza, Mgr., Ph.D.
280176
Immunotherapy has revolutionized cancer treatment over the past two decades, with checkpoint blocking therapy (CBT) emerging as a key strategy. By reactivating the immune system, CBT enables effective recognition and elimination of tumor cells. However, current approaches rely primarily on monoclonal antibodies (mAbs), which, despite their success, face limitations such as high production costs, potential immunogenicity, and limited patient response rates.
To overcome these challenges, we have developed synthetic antibody mimetics, iBodies, utilizing HPMA (N-(2-hydroxypropyl)methacrylamide) polymers conjugated with functional ligands. Anti-PD-L1 iBodies, incorporating a macrocyclic peptide targeting PD-L1, can reactivate T cells in vitro comparably to FDA-approved antibodies, while initial in vivo studies suggest tumor size reduction. Additionally, anti-CD73 iBodies, engineered with a potent CD73 inhibitor, inhibit CD73, bind CD73-expressing cells, and reactivate T cells, offering a promising combination strategy with PD-L1 blockade.
This PhD project will investigate the mechanisms of action and optimize anti-PD-L1 and anti-CD73 iBodies in both in vitro and in vivo systems. The student will refine iBody design to enhance stability, binding specificity, and therapeutic efficacy. Particular emphasis will be placed on studying the internalization and degradation of PD-L1 and CD73 upon treatment with iBodies. Furthermore, the study will explore the potential for cytotoxic applications and the use of lysosome-targeting ligands to promote enhanced PD-L1 and CD73 degradation. Preclinical studies will be expanded to assess pharmacokinetics, biodistribution, and immune activation in tumor-bearing models. The project will also evaluate the synergy of combining CD73 inhibition with PD-L1 blockade, leveraging in vitro and in vivo models to explore enhanced therapeutic outcomes.
The research involves working with primary human and mouse immune cells, flow cytometry, ELISA, enzymatic assays, and immunofluorescence microscopy etc. This work will deepen our understanding of iBody mechanisms, inform their clinical optimization, and contribute to the advancement of next-generation immunotherapeutics.
Regulation of growth and metabolism by the mTOR pathway
Sabatini David Marcelo, prof., M.D., Ph.D.
279491
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.
Mechanisms of lysosomal protein degradation
Sabatini David Marcelo, prof., M.D., Ph.D.
279492
Lysosomes are organelles responsible for the degradation of various molecules of cellular and extracellular origin, including nucleic acids, metabolites, and proteins. The clearance of proteins is accomplished by various glycosidases and peptidases and as generally assumed, leads to the complete cleavage and recycling of peptides into elementary building blocks, i.e. amino acids. However, not all proteins are susceptible to lysosomal degradation (for example, various GFPs). The aim of the project is therefore to further distinguish known patterns and mechanisms of protein cleavage, define sequence and structural specificities of degradation- resistant proteins, look for non-degraded compound accumulation in lysosomes, and find out if the presumption that all proteins are degraded to scratch is valid. Possible outcomes could be extended to deciphering the detailed mechanism of the lysosomal role in MHC-II presentation, new approaches for tissue-specific drug delivery (e.g. via ADCs), or the possible role of lysosomes in cell senescence. Not only various human cell lines but also murine models will be used together with methods including Lyso- IP, mass-spec, or computational approaches.
Understanding the specificity of intramembrane proteases using biophysical principles and artificial intelligence
Stříšovský Kvido, Ing., Ph.D.
280137
Intramembrane proteases regulate a large number of important biological and pathophysiological processes, but the principles underlying their substrate recognition are still only poorly understood. The groups of Prof. Steiner (Ludwig Maxmillian University, Munich) and Prof. Frishman (Technical University of Munich) have just developed a methodology based on physicochemical profiling and explainable artificial intelligence (eAI) that can detect and discriminate biophysical features of the substrates and non-substrates of the intramembrane protease gamma secretase, the enzyme responsible for the pathogenesis of Alzheimer's disease. In collaboration with these groups, we will analyze a set of substrates and non-substrates of the human intramembrane rhomboid protease RHBDL2. This enzyme regulates EGFR signaling in keratinocytes, potentiates proliferation, migration and invasion of pancreatic cancer cells and has been implicated in lung epithelial homeostasis. The supervisor's laboratory studies the biological and pathophysiological functions of RHBDL2 in cellular and mouse models, which includes substrate identification by established proteomic methods. This project will involve analysis of the biophysical characteristics of RHBDL2 substrates and non-substrates, whose set we will gradually expand. The project will include proteomic analyses (provided by a service laboratory), characterization of cleavage of RHBDL2 substrates in vitro and in cells, and preparation of recombinant proteins. Iterative refinement of the eAI-based substrate specificity model will result in a predictive model that will then be applied to the proteome of selected tissues/cell types with biological relevance. We may next apply this approach also to the human mitochondrial rhomboid PARL, which has a key function in mitochondrial biology and is relevant for Parkinson's disease. We have established recombinant production and in vitro assay both for RHBDL2 and PARL. As a part of the PhD programme, the student can and will be encouraged to visit collaborating laboratories in Germany or Israel for a short-term research stay. The project will be funded from the resources of my group and it will be the subject of a grant application.
Molecular Regulation of Lactation: Investigating DNA Replication, Transcription, and Damage Repair
Sumbalová Koledová Zuzana, Mgr., Ph.D.
280229
Lactation is a critical physiological process requiring precise coordination of cellular functions, including DNA replication, transcription, and the DNA damage response, to support milk production. This PhD project aims to unravel the molecular mechanisms underlying these processes in lactating mammary epithelial cells. Using a cutting-edge lactation organoid model developed in our laboratory, combined with in vivo mouse models, the student will explore how DNA replication and transcription and DNA damage response regulate differentiation of luminal cells to milk-secreting alveolar cells. Advanced imaging techniques, transcriptomics, and molecular biology tools will be employed to investigate these pathways in detail. The findings will provide new insights into the fundamental biology of lactation and may identify novel targets for addressing lactation-related disorders, contributing to improved maternal and neonatal health.
Advancing Breast Morphogenesis Modeling through Novel Human Mammary Organoids
Sumbalová Koledová Zuzana, Mgr., Ph.D.
280031
This research proposes the development of innovative human mammary organoids as a sophisticated model for studying breast morphogenesis, a critical process in mammary gland development and breast cancer progression. Traditional 2D cell cultures and animal models inadequately capture the complexity of human mammary tissue architecture and cellular interactions. Human mammary organoids, 3D multicellular structures derived from primary human mammary epithelial cells, offer a promising alternative. By mimicking the physiological microenvironment and cellular heterogeneity of the human breast, these organoids provide a platform to investigate the dynamic processes underlying breast development and disease.
The study aims to optimize protocols for generating human mammary organoids that recapitulate key features of mammary gland morphogenesis, such as branching morphogenesis, epithelial differentiation, and hormone responsiveness. Advanced imaging techniques, including confocal microscopy and live-cell imaging, will enable detailed characterization of organoid structure and dynamics. Molecular profiling using transcriptomic and proteomic analyses will elucidate the molecular mechanisms driving mammary organoid development and identify key signaling pathways involved in breast morphogenesis.
Overall, the development of novel human mammary organoids holds significant promise for advancing our understanding of breast morphogenesis and may pave the way for improved models of breast cancer initiation and progression, ultimately leading to the development of more effective therapeutic strategies for this prevalent disease.
Evolutionary adaptations of animal RNA silencing mechanisms
Svoboda Petr, prof. Mgr., Ph.D.
279167
Small RNAs serve as sequence-specific guides in numerous RNA silencing pathways. Evolution of these pathways took different trajectories during animal evolution. For example, mammals extensively utilize the microRNA pathway in almost all cells to regulate genes and the piRNA pathway in the germline for repression of mobile elements and occasional gene control. This PhD project will capitalize on previous research of the group and investigate specific instances in animals, where the molecular machinery of RNA silencing acquired unique adaptations to support specific functions of RNA silencing. Of particular focus will be adaptations associated with miRNA pathway function and their defects in cancer and other pathologies. This position is funded by Project OP JAK - RNA for therapy, registration number: CZ.02.01.01/00/22_008/0004575
piRNA and genome defense in gastropods
Svoboda Petr, prof. Mgr., Ph.D.
279564
The aim of this dissertation is to characterize the molecular mechanism of piRNAs and to analyze the biological role of small RNAs in a molluscan laboratory model, the snail of the genus Deroceras. In the snail species Deroceras laeve, occur both, cross-fertilization and self-fertilization. The type of fertilization is determined by the presence of a penis. Thus, within a species, aphalic forms represent clonal populations approaching an inbred state, which also limit the expansion of mobile elements. In contrast, euphalic forms exchange genetic information and allow the expansion of mobile elements in the genome. The main mechanism controlling mobile elements in the genome employs small RNAs of the piRNA class. The aim of this dissertation will be to identify the active mobile elements in the slug genome, to determine how the mode of reproduction affects the expansion of these mobile elements in the genomes of the aphalic and euphalic forms, and how the piRNA mechanism recognizes and silences these mobile elements.
Initiation phase is a crucial step determining regeneration efficiency
Šindelka Radek, Mgr., Ph.D.
264814
Recent introduction of high-throughput sequencing techniques rapidly change our perception of vertebrate regeneration. One of the model organisms intensely studied for its regenerative potential is the embryonic tadpole stage of African clawed frog (Xenopus laevis). Our recent study confirmed that Regeneration initiating cells (RICs) identified by single-cell and spatial RNA sequencing as new epidermal cell type, represent vital element in successful tail regeneration. The aim of this thesis is to review and functionally validate some of the latest findings gained by RNA sequencing analysis of regenerating Xenopus laevis tail. We will specifically focus on gene expression changes during the early phases of regeneration and how their absence affects the progression and phenotypic outcome of this process. We will apply broad variety of methods from embryo microinjections and micromanipulations to in situ hybridization and immunohistochemistry.
Degradation, translation and storage of proteins during preimplantation development
of cattle and its impact on transcriptional activity of the embryo
Toralová Tereza, Mgr., Ph.D.
280010
Proper coordination of protein synthesis and degradation is absolutely essential for the development of preimplantation embryo. In the initial stages of development, the embryo is transcriptionally inactive and cell processes can thus only be regulated at the protein level. The proteins are either of oocyte origin or translated from maternal mRNA early in preimplantation development. The translation and degradation of these proteins is governed by strict rules in terms of timing and protein selection. Degradation of proteins therefore does not take place in such a mass manner as in the case of mRNA. The up to now results show that maternal mRNAs very often have a specific sequence that cannot be found in somatic cells and that probably determines targeting of ta protein for degradation at a certain developmental stage. The aim of this project is therefore to describe the mechanism of selection of proteins for degradation during preimplantation development in cattle, to find (mainly epigenetic) regulatory mechanisms of chromatin loosening/compaction and to determine how abundant proteins of oocyte origin are stored. In mouse oocytes, it has been shown that at least part of the maternal proteins is stored on cytoplasmic lattices for subsequent embryonic development, and mutations in genes associated with the formation of cytoplasmic lattices result in incorrect methylation and imprinting in human embryos. It can therefore be assumed that proteins involved in chromatin compaction/loosening during early embryogenesis (e.g. methyltransferases or methyl readers and their regulatory proteins) are stored on cytoplasmic lattices. For this purpose, the characterization of cytoplasmic lattices in bovine embryos will therefore be carried out. Cattle were chosen as a model organism due to their high similarity to human preimplantation development. Moreover, verification experiments will be carried out on other model organisms such as the pig and in cooperation with the Egyptian laboratory of Dr. Abdoona also a camel and a donkey.
Methods: in vitro oocytes maturation, in vitro fertilization and embryo culture, deep RNA sequencing (identification of embryonic variants), mass spectrometry, microinjection of mRNA/dsRNA into zygotes, translatome analysis, time-lapse imaging using a Viventis LS1 Live lightsheet microscope (monitoring of fluorescently labeled proteins) , time-lapse imaging using Cytosmart LUX2 cameras (monitoring the progress of preimplantation development), electron microscopy (characterization of cytoplasmic lattices), molecular-biology methods (PCR, qRT-PCR, molecular cloning…) and biochemical methods (western blot, immunofluorescent analysis…).
The role of neddylation in oocyte maturation, fertilization and preimplantation
development of mammals
Toralová Tereza, Mgr., Ph.D.
280148
Degradation of proteins is a crucial process necessary for cell cycle control and embryogenesis. One of the most common degradation pathway is ubiquitin-proteasome system (UPS), which target ubiquitin-tagged proteins for degradation. Specificity of UPS is maintained because of protein selection by E3 ligases. Cullin-RING E3 ligases (CRL) are responsible for degradation of up to 20 % of ubiquitinated proteins. CRL are activated by neddylation, covalent binding of protein NEDD8 to one of the cullin proteins, allowing to assembly the multi-subunit CRL. During oocyte maturation and cumulus cell expansion, important signalling factors need to be degraded. It was found out that inhibition of neddylation durting bovine oocyte maturation caused no expansion of cumulus cell, polar body extrusion delay, deterioration of maturation rate and higher polyspermy rate. Up to now, no signalling pathways leading to these defects were detected. The aim of this work is to describe engaged signalling pathways and their functions in bovine oocyte maturation and cumulus cell expansion. Preliminary results indicate the necessity of neddylation for MAPK and TGFβ signalling pathway activity. We will focus on finding the connection between neddylation and MAPK pathway and its relation to cumulus cell–oocyte communication in cattle. For this communication, tranzonal projections play crucial role by allowing the important signal molecules to pass through. Since we have previously found that inhibition of neddylation during oocyte maturation leads to impaired preimplantation development, the project will also aim to determine which pathways are irreversibly affected in early embryos. Potentially involved pathways will be identified using mass spectrometry (GO analysis) and by the phenotypes changes caused by inhibition/overactivation of neddylation. Therefore, the course of chromosome segregation will be observed using live-cell imaging by Viventis LS1 Live light-sheet microscope and it will be determined at which stages the maturation stops. Furthermore, the cause of polyspermy will be determined by staining of cortical granules, monitoring their movement using live-cell imaging and determining the expression of the involved signalling pathways. Signaling pathways detected by our analysis will be observed by western blot, immunofluorescence analysis focusing on protein-protein interaction or qRT-PCR in more detail.
Nuclear actin-binding proteins in the regulation of cellular differentiation
Venit Tomáš, Mgr., Ph.D.
279263
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. Mitochondria play a primary role in defining cellular metabolism and produce energy via oxidative phosphorylation (OXPHOS) in the majority of somatic cells. Aerobic glycolysis (glycolysis in the presence of oxygen) on the other hand is used as a primary energy metabolism in highly proliferating undifferentiated pluripotent stem and cancer cells or specific cell types such as erythrocytes. Metabolic switch characterized by increased mitochondrial oxidative phosphorylation (OXPHOS) and decreased glycolysis is a key feature that marks the differentiation of progenitor cells to committed cell lineages. The opposite metabolic switch is observed in tumorigenesis, where the prevalence of glycolytic metabolism over OXPHOS is connected to poor survival prognosis in many different types of cancers. In our previous research, we reported that during DNA transcription, an actin-binding protein - 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. Cells lacking NM1 show changes in mitochondrial structure, reduced expression of OXPHOS genes, metabolic switch to aerobic glycolysis, and ability to form tumors in mice. Therefore, we proposed NM1 as a novel tumor suppressor and master regulator of cellular metabolism (Venit et al., 2023, Nature Commun.)
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 the proposed project, we will study the expression patterns of NM1 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.
Structural studies of histone chaperones
Veverka Václav, doc. Ing., Ph.D.
278791
Histone chaperones play an important role in chromatin remodeling. Intrinsically disordered histone chaperones can transition from an unstructured to a structured state upon binding to histones. This type of molecule plays a key role in maintaining chromatin structure and dynamics, DNA replication, transcription and repair. Their deregulation is associated with various diseases, including cancer, underscoring their importance for normal cellular function. The aim of the project will be to study the molecular basis of histone recognition by chaperones, with emphasis on unraveling the role of unstructured regions on the function of these molecules using structural biology techniques (cryo-electron microscopy, NMR spectroscopy or X-ray crystallography).
Role of normal and mutant huntingtin in neural cells.
Vodička Petr, Mgr., Ph.D.
265684
Huntington disease (HD) is a hereditary neurodegenerative disorder caused by an expansion of CAG tract in huntingtin (HTT) gene, leading to expression of over 36 glutamines in mutant huntingtin (mHTT) protein. Extended stretch of glutamines in N-terminal part of huntingtin protein (HTT) changes its biochemical properties and causes aggregation and toxicity especially to striatal neurons. Exact mechanisms leading to selective vulnerability of neural cells to mutant huntingtin (mHTT) toxicity as well as normal cellular function of HTT are still largely unknown. The aim of this project is to study the role of normal and mutated HTT in synaptic function, intracellular transport and proteostatic mechanisms, using mouse knock-in Q175 model, minipig knock-in model expressing HTT with 86Q, and human normal and HD iPSC lines.
Methods: Mass spectrometry proteomics (LC-MS/MS), tissue culture (iPS cells, neural stem cells, neurons), molecular biology (molecular cloning, transfection, qPCR), biochemical methods (western blot, ELISA, Luminex xMAP), confocal microscopy.