PhD Program

Project Title: Regulatory mechanisms favoring stem cell maintenance in health and disease
Supervisor: Meritxell Alberich Jordà

Project Description:
Hematopoietic stem cells are a rare population of cells in the bone marrow which are responsible for the production of mature blood cells through all life of an individual. Thus, preserving this population is critical for hematopoiesis, since alterations in this population have been linked to the development of leukemia or bone marrow failure.
In this project, we will explore our recently generated single cell RNA sequencing to identify the regulatory mechanism that protect and maintain hematopoietic stem cell properties. Next, we will perform cell in vitro cultures and murine in vivo experiments to validate our scRNAseq results. Alterations of these mechanisms will also be explore during leukemia development.

Candidate Profile:
The laboratory of hemato-oncology is searching for a highly motivated, enthusiastic and hard-working Ph.D student. The candidate should hold a master degree in genetics, molecular biology, cell biology, or in a related field. Bioinformatic skills or basic knowledge of large dataset analysis will be positively evaluated. The candidate should be willing to work with murine models. Excellent English is required. The candidate should be a team-player and willing to work with other lab members and international collaborators.
We offer a friendly and supporting environment in a state-of-the-art institution.

Suggested reading:

• Zjablovskaja P, Kardosova M, et al. EVI2B is a C/EBPa target gene required for granulocytic differentiation and functionality of hematopoietic progenitors. Cell Death Differ. 2017 Apr;24(4):705-716.
• Wurm AA, Zjablovskaja P, et al. Disruption of the C/EBPα-miR-182 balance impairs granulocytic differentiation. Nat Commun. 2017 Jun 29;8(1):46.
• Kardosova M, Zjablovskaja P, et al. C/EBPg is dispensable for steady-state and emergency granulopoiesis. Haematologica. 2018 Aug;103(8):e331-e335.
• Danek et al. β-catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation. Blood 2020 Nov 26;136(22):2574-2587.

Project Title: Plectin isoform-specific deficiency: novel model for muscular dystrophy
Supervisors: Martin Gregor

Project Description:
Plectin is a highly versatile cytolinker protein that stabilizes cells and tissues. Recently, deficiency for plectin isoform 1f (P1f), the key plectin isoform expressed in muscle, was identified to cause limb-girdle muscular dystrophy (LGMD) associated with progressive muscle weakness and consequent immobility. The major objective of this project is phenotypic analysis of gene knock-out mice that are deficient in P1f. These mice will be subjected to comprehensive phenotypic analyses focusing on muscular cytoarchitecture, muscle performance, and regeneration. In collaboration with teams in Erlangen (Germany) and Vienna (Austria) we will address the role of P1f in neuromuscular junctions and muscle metabolism. The successful candidates will learn and utilize advanced methods for functional analysis of skeletal and heart muscles, histology, microscopy as well as whole body imaging techniques.

Candidate Profile:
We are seeking outstanding self-motivated candidates with master’s degree in molecular biology, physiology, biochemistry or related fields. We are offering research at a state-of-the-art equipped institute with experienced colleagues, international working environment and international collaborations.

Suggested reading:

• Mercuri E. et al. Muscular dystrophy: new treatments, new hopes. Lancet, 2019, 394:2025-2038.
• Gundesli H. et al. Mutation in exon 1f of PLEC, leading to disruption of plectin isoform 1f, causes autosomal-recessive limb-girdle muscular dystrophy. Am J Hum Genet, 2010, 87:834-41.
• Zrelski M.M. et al. Muscle-Related Plectinopathies., Cells, 2021, 10:2480.

Project Title: The role of cytoskeletal crosslinking in cancer
Supervisors: Martin Gregor

Project Description:
An essential prerequisite for the fundamental rearrangement of the cytoskeleton in the course of carcinogenesis is its coordinated regulation and the interplay of individual cytoskeletal components. Highly organized cytoskeletal networks are maintained by cytoskeletal linker proteins (cytolinkers) of the plakin protein family. The major objective of this project is description of the role of cytoskeletal crosslinking in cancer cell migration, cell adhesion structures, and metastasis formation using advanced in vitro (CRISPR/Cas9 targeting in cell lines) and in vivo (mouse models) approaches. The successful candidates will learn and utilize cell biology, molecular biology, physiology, and imaging techniques combined with biophysical methods.

Candidate Profile:
We are seeking outstanding self-motivated candidates with master’s degree in molecular biology, physiology, biochemistry or related fields. We are offering research at a state-of-the-art equipped institute with experienced colleagues, international working environment and international collaborations.

Suggested reading:

• Vilchez Mercedes S.A. et al. Decoding leader cells in collective cancer invasion. Nat Rev Cancer, 21:592-604, 2021.
• Strouhalova K. et al. Vimentin Intermediate Filaments as Potential Target for Cancer Treatment. Cancers. 2020 Jan 11;12(1).
• Gregor M. et al. Mechanosensing through focal adhesion-anchored intermediate filaments. FASEB J., 28:715-29, 2014.

Project Title: The role of cytoskeletal crosslinking in epithelial mechanobiology
Supervisors: Martin Gregor, Magdalena Přechová

Project Description:
The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Recently, we showed that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrated that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome network feeds back to the actin cytoskeleton which results in elevated actomyosin contractility. The major objective of this project is description of the impact of cytoskeletal crosslinking on cell proliferation and tensional homeostasis using advanced in vitro techniques in CRISPR/Cas9-targeted cell lines. The successful candidates will learn and utilize cell biology, molecular biology and imaging techniques combined with biophysical methods (atomic and traction force microscopy).

Candidate Profile:
We are seeking outstanding self-motivated candidates with master’s degree in molecular biology, cell biology, biochemistry or related fields. We are offering research at a state-of-the-art equipped institute with experienced colleagues, international working environment and international collaborations.

Suggested reading:

• Schwarz U.S. Mechanobiology by the numbers: a close relationship between biology and physics. Nat. Rev. Mol. Cell Biol., 18:711-712, 2017.
• Prechova M. et al. Plectin-mediated cytoskeletal crosstalk controls cell tension and cohesion in epithelial sheets. J. Cell Biol., in press, 2022. DOI 10.1083/jcb.202105146
• Uhler C. et al. Regulation of genome organization and gene expression by nuclear mechanotransduction. Nat. Rev. Mol. Cell Biol., 18:717-727.

Project Title: Mechanism of somatic hypermutation targeting in the B cell genome
Supervisors: Filip Šenigl

Project Description:
Somatic hypermutation (SHM) is a key process in diversification of antibodies, and modulation of their affinity in particular. This process is initiated by cytosine deamination by the activation induced deaminase (AID) and completed by error-prone processing of the resulting uracils. Until recently, SHM was thought to be targeted exclusively to Ig loci; however, newly emerging studies report SHM activity to occur more genome-wide, and SHM thus represents a threat to the genome integrity. We developed a very sensitive high-throughput retrovirus vector based assay (DIVAC-trap HTISA) to study the SHM targeting and generated a comprehensive map of genomic regions vulnerable and resistant to SHM. In this project, we will utilize our novel tools to study the role of (epi)genomic features and nuclear topology in the SHM targeting outside Ig loci and identify the key features. In addition, we will characterize the genomic elements responsible for SHM.
The project will be demanding on establishment of various high-throughput methods (integration site libraries, ChIP-seq, RNA-seq, etc.). The project will also require extensive bioinformatic analysis which doesn’t need to be necessarily performed by the candidate, however, experience with high-throughput datasets or bioinformatic analysis is advantageous.

Candidate Profile:
The candidate should have experience with tissue culture experiments and basic molecular biology techniques (PCR, plasmid construction) as well as basic microscopic skills. The candidate should be able to work with scientific literature and communicate in English language.

Suggested reading:

• Senigl et al. Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation, Cell Rep. 2019 Dec 17;29(12):3902-3915.e8.
• Liu & Schatz. Balancing AID and DNA repair during somatic hypermutation, Trends Immunol. 2009 Apr;30(4):173-81.
• Feng Y, Seija N, Di Noia JM, Martin A. AID in Antibody Diversification: There and Back Again. Trends in Immunology, July 2020, Vol. 41, No. 7.

Project Title: Developmental genetics of amphioxus: a window into the evolution of vertebrate body plan
Supervisor: Zbyněk Kozmik

Project Description:
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. 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.

Candidate Profile:
We are searching for a highly motivated and hard-working PhD student with a strong interest in animal evolution and evolution of development (evo-devo). The candidate should hold a master degree in zoology, genetics, molecular biology, cell biology, or in a related field.

Suggested reading:

• Kozmikova I, Kozmik Z. Wnt/β-catenin signaling is an evolutionarily conserved determinant of chordate dorsal organizer. Elife. 2020 May 26;9:e56817. doi: 10.7554/eLife.56817.
• Pergner J, Vavrova A, Kozmikova I, Kozmik Z. Molecular Fingerprint of Amphioxus Frontal Eye Illuminates the Evolution of Homologous Cell Types in the Chordate Retina. Front Cell Dev Biol. 2020 Aug 4;8:705. doi: 10.3389/fcell.2020.00705. eCollection 2020.
• Prummel KD et al. A conserved regulatory program initiates lateral plate mesoderm emergence across chordates. Nature Comm. 2019 Aug 26;10(1):3857. doi: 10.1038/s41467-019-11561-7.
• Marlétaz F et al. Amphioxus functional genomics and the origins of vertebrate gene regulation. Nature. 2018 Dec;564(7734):64-70. doi: 10.1038/s41586-018-0734-6. Epub 2018 Nov 21.

Project Title: Screening for proteins involved G4-structure metabolism and their role in prevention of genotoxic stress
Supervisors: Jana Dobrovolná, Pavel Janščák

Project Description:
DNA replication is an essential and one of the most complex processes in the cell. Not only exogenous DNA damage but also intrinsic DNA structures including G-quadruplexes (G4) and R-loops, their stabilization or unscheduled formation represent major replication obstacles with possible detrimental effects on genome integrity. Not surprisingly, those processes are pharmacologically targeted in anticancer therapy, despite the fact that only little is known about the underlying molecular mechanisms. It becomes apparent that maintenance of processive DNA replication requires sophisticated protein networks beyond the core replisome. Whether there is a direct crosstalk between G4 and R-loops, what proteins are involved in their homeostasis and what are the factors diversifying between their beneficial and pathological roles is not well understood. The goals of our research are to identify proteins associated with G4 and R-loop structures and understand their roles in G4/R-loop formation and resolution as well as relationship to replication fork progression and associated repair. PhD student will be involved in preparation of tools for study of G4 structures (establishment of various cell lines) and in identification of proteins involved in metabolism of these structures by mass spectrometry-based proteomics approaches, including APEX-based proximity labeling and chromatin affinity precipitation methods, and by functional siRNA screen. Proteins identified in these screens will be selected for further validation and characterization based on their relevance to G4/R-loop and replication fork metabolism, and role in maintenance of genome integrity.

Candidate Profile:
Applicants should be graduates in Molecular Biology/Cellular Biology/Biochemistry with a strong interest in basic research and experimental work. Good English and independent thinking is required. The projects offer training in a broad range of molecular, cell biological and biochemical techniques. The student will also undergo short-term trainings at the Institute of Molecular Cancer Research of the University of Zurich where he/she will be exposed to front-line research in the field of DNA repair and cancer.

Suggested reading:

• Chappidi N, Nascakova Z, Boleslavska B, Zellweger R, Isik E, Andrs M, Menon S, Dobrovolna J, Balbo Pogliano C, Matos J, Porro A, Lopes M, Janscak P.: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops. Mol Cell 2020, 77(3):528-54.
• Bauer M, Nascakova Z, Mihai AI, Cheng PF, Levesque MP, Lampart S, Hurwitz R, Pfannkuch L, Dobrovolna J, Jacobs M, Bartfeld S, Dohlman A, Shen X, Gall AA, Salama NR, Töpfer A, Weber A, Meyer TF, Janscak P, Müller A. The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress. Nat Commun. 2020 Oct 9;11(1):5117.

Project Title: Converting molecular-scale torques to cellular and embryonic left-right asymmetry
Supervisors: Teije Middelkoop

Project Description:

The vast majority of animals exhibit left-right asymmetric body plans. Left-right asymmetry arises during early embryonic development and is often the result of rotatory movements of cells. Rotational forces, i.e. torques, drive these cellular movements and they originate in the actomyosin cytoskeleton of embryonic cells. We previously demonstrated that, in early embryos of C. elegans nematodes, active torque generation is facilitated by an actin elongator of the Formin family. At the molecular level, Formins can rotate actin filaments and generate torques. However, how the molecular activity of Formins, which are embedded in a highly cross-linked actomyosin network, can result in rotatory movements remains a mystery.
In this project we aim to understand how torques generated at the molecular level are converted to rotatory movements at the cellular level. To this end, we will combine the strength of C. elegans genetics, with live fluorescence microscopy of early embryos and quantitative image analysis. Moreover, we will further develop optogenetic tools in C. elegans, in order to probe force generation in the actomyosin cytoskeleton with tight spatiotemporal precision.

Candidate Profile:
The developmental mechanobiology lab is looking for a highly motivated and enthusiastic PhD student with a masters degree in biology (genetics, cell biology, developmental biology or biochemistry) or physics (biophysics) having experience in practical lab work. No specialized skill set is required; the willingness to learn is however essential. You will be working with genetically modified nematodes and image them using high-end fluorescence microscopy.

Suggested reading:

• Middelkoop TC, Garcia-Baucells J, Quintero-Cadena P, Pimpale L, Yazdi S, Sternberg P, Gross P, Grill SW. CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking. PNAS, 118, 20, e2021814118, (2021).
• Pimpale LG, Middelkoop TC, Mietke A, Grill SW. Cell lineage-dependent chiral actomyosin flows drive cellular rearrangements in early Caenorhabditis elegans development. Elife 9, e54930, (2020).
• Naganathan SR, Middelkoop TC, Fürthauer S, Grill SW. Actomyosin-driven left-right asymmetry: from molecular torques to chiral self organization. Curr Opin Cell Biol 38, pp. 24-30, (2016).

Project Title: Molecular mechanism of quality control during spliceosome assembly
Supervisors: David Staněk

Project Description:
Small nuclear ribonucleoprotein particles (snRNPs) are conserved essential components of the RNA splicing machinery snRNPs undergo a complex maturation pathway, and defects in their biogenesis lead to various human diseases such as spinal muscular atrophy and retinitis pigmentosa. The aim of this PhD project is to elucidate the molecular mechanism of the quality control process that distinguishes between mature and defective particles. The project balances between biochemistry, molecular biology and cell biology and the candidate will learn to work with proteins, nucleic acids and to analyse cells using advanced fluorescence microscopy.

Candidate Profile:
We are looking for an enthusiastic colleague with a keen interest in molecular biology and biology of RNA in particular. Previous experience in biochemistry, molecular biology and cell culture is welcome.

Requirements:

• MSc, MRes, Diploma or equivalent degree in Biochemistry, Biology, Biomedical Sciences, Chemistry or related science, to be obtained no later than the start of the Autumn term in September 2022.
• Practical experience working in a laboratory on scientific projects.
• Excellent English and a desire to work in a dynamic international team.

Suggested reading:

• Staněk D. (2017) Cajal bodies and snRNPs: friends with benefits. RNA Biology 14:671-679.
• Růžičková Š. & Staněk D. (2017) Mutations in spliceosomal proteins and retina degeneration. RNA Biology 14:544-552.
• Roithová A., Feketová Z., Vaňáčová Š. & Staněk D. (2020) DIS3L2 and LSm proteins are involved in the surveillance of Sm ring-deficient snRNAs. Nucleic Acids Research. 48(11):6184-6197.

Project Title: Mapping T-cell functions using single cell transcriptomics
Supervisors: Ondřej Štěpánek

Project Description:
The group of Adaptive Immunity has a strong publication record in the field of T-cell biology. Recently, we have introduced single cell RNA transcriptomics and analysis of antigenic receptors to uncover and characterize novel T-cell subsets, including their antigenic specificity. Within the project, the student will analyze unique single T-cell data (transcriptomics, ATACseq, repertoire) from animal models of immunological diseases (infection, cancer, autoimmunity) and/or human patients. Moreover, the student will develop novel tools for these analyses. The inclusion of experimental wet work in this project is optional, depending on the preferences and background of the selected candidate.
We offer high scientific standards in the amazing field of T-cell-mediated immunity and the environment of a young, but experienced, lab. The student with receive full time salary according to IMG standards and the stipend from the affiliated university.

Candidate Profile:
We are looking for an enthusiastic early career scientist who wants to use their programming skills to tackle biological problems in our multidisciplinary team. The candidate should show high level of independence and problem solving skills in programming/bioinformatics. However, they should be able to collaborate with experimental scientists within our interdisciplinary team as well.
The successful candidate has a master’s degree (or is close to its completion) in bioinformatics, computational science, biological, or chemical sciences. The candidate should have the prior experience with the analysis of large biological data and/or computational modeling of biological processes.
Only candidates, who directly contact the PI (ondrej.stepanek@img.cas.cz) before 28.2.2022 will be considered for the interviews.

Suggested reading:

• https://www.biorxiv.org/content/10.1101/2021.11.10.467495v2
• https://www.embopress.org/doi/full/10.15252/embj.201798518
• https://adaptiveimmunity.img.cas.cz/publications/

Project Title: Protective and pathological tissue-infiltrating T-cells in infections
Supervisors: Ondřej Štěpánek

Project Description:
The group of Adaptive Immunity has a strong publication record in the field of T-cell biology. One of our long-term interests is uncovering the diversity of T-cell subsets with unique functions. This PhD project in experimental immunology focuses on the tissue infiltrating protective T cells (e.g., tissue resident memory T cells) and pathogenic T cells inducing damage to host tissues, which arise during bacterial and viral infections. The student will use the state-of-the-art methods (scRNAseq, mouse models of infection, flow cytometry) to characterize T-cells in tissues. In the next step, they will analyze the biological role of these subsets using our model systems for generating monoclonal T-cells.
We offer high scientific standards in the amazing field of T-cell-mediated immunity and the environment of a young, but experienced, lab. The student with receive full time salary according to IMG standards and the stipend from the affiliated university.

Candidate Profile:
We are looking for an enthusiastic early career scientist who wants to tackle the fundamental roles of T cells in infections. The candidate should show high level of problem solving skills and the ability to collaborate within our interdisciplinary team.
The successful candidate has a master’s degree (or is close to its completion) in life-sciences. Prior experience with experimental lab work is required (e.g., within a master’s diploma research project).
Only candidates, who contact the PI (ondrej.stepanek@img.cas.cz) directly before 28.2.2022 will be considered for the interviews.

Suggested reading:

• https://www.biorxiv.org/content/10.1101/2021.11.10.467495v2
• https://www.embopress.org/doi/full/10.15252/embj.201798518
• https://adaptiveimmunity.img.cas.cz/publications/

Project Title: The skeleton of mammalian cilia
Supervisor: Vladimír Varga

Project Description:
Cilia are cylindric organelles on the surface of a majority of human cells. They have critical motility, signaling and sensory roles, and their malfunction causes diseases called ciliopathies. There are two principal types of cilia, the motile and the primary cilia. Recent studies indicated that despite the skeleton of both types being based on microtubules, the skeleton of the primary cilia significantly deviates from the classic pattern of 9 outer microtubule doublets with the central pair. The project aims to investigate how is the skeletal pattern of both motile and primary cilia established during ciliogenesis, and what are implications of a particular skeletal arrangement for the ciliary function. To study this advanced live cell imaging and high resolution approaches, such as electron microscopy and expansion microscopy, will be employed.

Candidate Profile:
We are looking for a highly motivated student with a degree in cell biology, biochemistry, molecular biology or related disciplines, and with an interest in the eukaryotic cytoskeleton. The candidate should be eager to learn new techniques and eventually be able to drive the project. We offer a friendly environment of a young group with a deep interest in cilia biology and an access to state of the art equipment in the laboratory and institute facilities.

Suggested reading:

• Kiesel P., Alvarez Viar G., Tsoy N., Maraspini R., Gorilak P., Varga V., Honigmann A. and Pigino G (2020). The molecular structure of mammalian primary cilia revealed by cryo-electron tomography. Nature Structural & Molecular Biology Volume 27, pages 1115–1124.
• Gorilak P, Pružincová M, Vachova H, Olšinová M, Schmidt Cernohorska M, Varga V: Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites. Open Biol 2021 11(9): 210131.