Department of Biochemistry and Microbiology

Granting Departments:	Department of Biochemistry and Microbiology
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	Ing. Silvie Rimpelová, Ph.D.

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Chemical modifications of illegal drugs lead to the formation of novel compounds, i.e. new synthetic drugs that circumvent legislation. While preserving the pharmacophore of such modified substances, they thus mimic the biological effects of the parent drug, but with unexplored pharmacological effects and toxicity that is often significantly higher than that of the parent drug. The main topic of the thesis will be the study of novel synthetic drugs, both synthesized de novo and retained at the black market. The toxicity of these substances will be examined in model cell lines, further, the metabolism of these compounds and the activity of selected metabolites will be studied. Furthermore, the mode of their action (agonist/antagonist) on selected G-protein-coupled receptors will be determined and we will focus in detail on the activity and mechanism of action of individual enantiomers of selected novel synthetic drugs.

Granting Departments:	Department of Biochemistry and Microbiology
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	Ing. Silvie Rimpelová, Ph.D.

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The main topic of the thesis is the study of substances isolated from natural sources as well as de novo synthesized with the potential to inhibit the proliferation of tumor cells and activate the immune system response. The work is mainly focused on cardiac glycosides, such as digitoxin or digoxin, and antimitotics, such as colchicine or paclitaxel. The biological effects of newly prepared derivatives of these substances on 2D and 3D cell models of cancerous and noncancerous cell lines will be studied. We will focus on the mechanism of action of these substances, a targeted increase in selectivity for cancer cells and induction of an immune response. Last, but not least, targeted nanosystem delivery will be developed, in cooperation, for the most potent derivatives of the studied compounds.

Institute of Microbiology of the CAS, v.v.i.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Microbiology ( in English language )
Supervisor:	RNDr. Jana Kamanová, Ph.D.

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The classical Bordetella species infect the respiratory tract of mammals. B. bronchiseptica induces chronic respiratory infections in various mammals, while the human-adapted species B. pertussis and B. parapertussis HU lead to an acute respiratory disease known as whooping cough or pertussis. These bacteria establish biofilms on both abiotic surfaces and within the respiratory tract, functioning as an immune evasion mechanism and a focal point for the development of novel antimicrobial agents. The objective of this PhD thesis is to unravel the signaling pathways underlying Bordetella colonization of respiratory epithelia and biofilm formation. The PhD candidate will employ an air-liquid interface model of human nasal epithelia and a microengineered biomimetic system, an organ-on-a-chip, combined with metabolomics, dual RNA-Seq and genetic engineering.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	doc. RNDr. Pavla Bojarová, Ph.D.

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The project for this dissertation focuses on developing a three-step enzymatic synthesis of bioactive chitooligomers (COS) useful in organic crop protection. Chitooligomers are ?-1-4-linked oligosaccharides composed of N-acetylglucosamine and glucosamine units whose biological activity depends mainly on their degree of polymerization (DP) and degree of acetylation (DA). COS are known for their ability to induce an immune response in plants and can thus be used as natural crop protection agents against microbial pests. The project will prepare mutant variants of a novel fungal chitinase with enhanced hydrolytic activity to increase the efficiency of chitin cleavage, by which COS fractions with lower DP will be prepared. In the next step, mutant variants of chitinases and ?-N-acetylhexosaminidases with transglycosidase activity suitable for preparing COS with degrees of polymerization of 5-10 on a preparative scale will be used. These chitooligomers will be partially deacetylated with novel chitin deacetylases, yielding COS fractions with different degrees of polymerization and acetylation that are not yet available. The biological activity of the prepared well-defined COS will be tested in collaboration with the Institute of Experimental Botany of the CAS.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	doc. RNDr. Pavla Bojarová, Ph.D.

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Galectins are animal lectins with affinity to beta-D-galactosides, which in vivo participate in, e.g., cancerogenesis, cardiopathologies, they are connected with the modulation of immune response and the course of allergic reaction. The concentration of extracellular galectins in vivo may be used as diagnostic markers in pathologies, such as colorectal carcinoma. A targeted inhibition of extracellular galectins is a prospective therapeutic approach to the treatment of pathologies associated with galectin overproduction. A range of recent structure-function studies is devoted to defining structural requirements for high-affinity and selective carbohydrate ligands of individual galectins. The avidity of specific carbohydrate inhibitors to selected galectins may also be increased by multivalent presentation. The aim of this work is to synthesize new carbohydrate ligands (glycomimetics) with a high afinity and possibly also selectivity for target galectins. Besides usually studied galectins Gal-1 and Gal-3, attention will be devoted to the group of tandem-repeat galectins (Gal-4, Gal-8, and Gal-9). The inhibitory and binding potential of these glycomimetics to galectins will be assayed by in vitro methods of ELISA and other biophysical methods. Structure-affinity relations will be discussed in relation to molecular modeling. Prepared ligands with a high affinity may be used in subsequent experiments with cell cultures, which are available at the supervisor workplace.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	doc. RNDr. Pavla Bojarová, Ph.D.

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Human milk oligosaccharides (HMOs) are the third most important component of breast milk after lactose and fat. HMOs pass through the digestive tract without major changes and are partially absorbed into the bloodstream through the intestinal wall. They serve as prebiotics for the gut microbiome of infants, which is important for allergy susceptibility. HMOs prevent adhesion of pathogens to the intestinal epithelium and directly regulate the immune system, e.g. by altering cytokine production. HMOs have been found to prevent or alleviate allergy symptoms. Bacterial cell factories have been used for the high-yield enzymatic synthesis of HMOs without the need for purification of recombinant enzymes. Genetically modified strains of Escherichia coli are suitable for the production of breast milk oligosaccharides. To date, applications have mostly been limited to a basic selection of HMOs, while a broader spectrum of these structures is not available. The subject of this thesis is the development of the synthesis of selected complex HMOs, in particular fucosylated and/or sialylated, on the E. coli platform. These compounds will be further tested in biological experiments concerning their effect on processes related to the initiation and development of allergies, such as passage through the intestinal membrane, expression of relevant biomarkers in epithelial cells, etc.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Microbiology ( in English language )
Supervisor:	Dr. Ing. RNDr. Petr Baldrian, Ph.D.

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Forest harvesting is economically important but due to the ecological importance of forests, it also represents a dramatic event in the ecosystem development. While clearcutting is a common practice in the temperate and boreal forests, variable retention harvesting is an alternative silvicultural practice in which some portion of the pre-harvest stand is retained after harvest. The broad objective is to maintain structural and functional elements from the pre-harvest condition to promote rapid recovery of biodiversity and ecological functions in the regenerating stands compared to clearcut stands. Harvesting of trees has profound effects on ecosystem processes that are associated with changes in the quality and decreases in the quantity of plant C inputs into the surrounding soil, as well as effects on soil nutrient availability and microclimatic conditions. Harvested stands also offer the opportunity to study the effect of reduced primary production on soil processes. Forest harvesting is known to have major effects on the main symbiotic fungi living on the roots of temperate trees, ectomycorrhizae, however, little is known about harvesting effects on symbiotic arbuscular mycorhizal fungi and bacteria. This project proposes to analyse the short-term effects of forest harvesting on the activity and composition of soil microbial communities in forests with dominant ectomycorrhizal and arbuscular mycorrhizal symbiosis and those with mixed ECM/AM. Changes in ecosystem properties and functioning, such as decomposition and N cycling will be linked to the community composition of soil microbes and their transcriptional activity. The project should give a view of the temporary changes of forest stand manipulations as well as answer the response of individual groups of microbes. This is important both for the consideration of future forest management strategies and the predictions of forest management effects on the C balance of forest soils.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	Mgr. Leoš Valášek, Ph.D.

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Imagine a gene is a sentence starting with a capital letter and ending with a period, and a genome as a book telling an entire story. In some protozoans, extra periods have infiltrated the sentences, replacing specific letters of arbitrary words (E and W). As a r.sult, readers are confus.d as to .here thes. sentences really .nd and th. story becom.s disjointed. In this issu. of Nature (https://www.nature.com/articles/s41586-022-05584-2), we describ. a molecular mechanism that thes. organisms must hav. developed to allo. the reader to navigat. the s.ntences as if there .ere no .xtra periods. Th. sentences are so specifically .ncrypted that read.rs of no other organisms but thos. very fe. can decipher the story in a prop.r .ay. The trick lies in the l.ngth of the tRNA molecul., and in the uniqu. modification of a prot.in that normally ensur.s dot recognition in c.lls - the precis. end of prot.in synthesis. By the intEraction of thesE tWo modified moleculEs, the ribosomE of this protozoan knoWs when to correctly terminatE, notWithstanding the many dots, and whEn to replace thE dots with the two original lettErs, which returns mEaning to the genetic information. LEt’s Explore hoW it is possible on thE molecular levEl and WhEther it could bE used for the benEfit of human hEalth! *This PhD thesis topic and the supervisor are subject to approval by the Faculty's Scientific Council.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	doc. RNDr. Pavla Bojarová, Ph.D.

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Chitin is the second most abundant polymer in nature. Partial deacetylation of chitin produces chitosan, a linear polymer composed mainly of glucosamine units (GlcN) and to a lesser extent of N-acetylglucosamine units (GlcNAc) linked ?(1?4) by glycosidic bonds. Chitosan is biocompatible and can therefore be advantageously used in a variety of biological and biomedical applications. For many biological applications, it is advantageous to work with shorter chains of chitosan, the so-called chitooligosaccharides (COS). COS have been intensively researched for decades in the fields of medicine, pharmaceuticals, textile industry, food industry and agriculture. Despite their tremendous potential use, poorly characterized heterogeneous mixtures are used in most studies due to the unavailability of well-defined COS. The thesis will focus on the preparation of authentic, pure and fully structurally characterized chitooligosaccharides and their analysis. Exoskeletons of crustaceans, insects (grasshoppers, bees, tarantulas) and fungi will be used as a source of chitin/chitosan. Subsequently, the defined COS chains will be used as carriers for multivalent presentation of bioactive carbohydrates, especially rutinose and galactosyl-carrying glycomimetics, for biological tests with lectins.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	doc. Ing. Kateřina Valentová, Ph.D.

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Flavonoids, natural compounds found in various plants and foods, continue to attract great interest due to their known positive biological effects. However, these effects are compromised by their low bioavailability after oral administration. In the frame of this work new derivatives of flavonoids will be synthesized and the effect of various modifications on the bioavailability and biological activity of flavonoids will be evaluated. We will focus on the ability of the prepared derivatives to modulate drug resistance of bacteria, the chelatory activity and platelet aggregation inhibitory activity of flavonoids.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Microbiology of the CAS, v.v.i.
Study Programme/Specialization:	Microbiology ( in English language )
Supervisor:	Dr. Ing. RNDr. Petr Baldrian, Ph.D.

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Peatlands of Northern Europe are an important source of greenhouse gases (GHG) with a strong influence on global climate - methane, nitrous oxide and carbon dioxide. One of the approaches to limit GHG production is the drainage of peatlands and their afforestation. Although this land use change can somehow reduce GHG production, it still partly continues to depend on the management of forests. The fluxes of GHG are, however, also regulated by other factors, such as the seasonal activity of vegetation and microorganisms. Since microorganisms are responsible for GHG production in the soils of forested peatlands, they represent the key to the understanding of GHG production rates and factors that affect them. In this work, we will use field experiments in southern Finland, where GHG production is monitored and where the analysis of microbiome composition and function at different depths of peat can be thus linked to the observed ecosystem-level gas fluxes. This project will offer a combination of fieldwork, labwork and analysis of bioinformatic data. When successful, the results of the thesis should help to indicate what management of peatland forests has the highest potential to mitigate GHG emissions and increase the storage of carbon in forest soils.

Institute of Organic Chemistry and Biochemistry of the CAS, v. v. i.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Organic Chemistry and Biochemistry of the CAS, v. v. i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	Mgr. Tomáš Kouba, Ph.D.

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Effective treatment against influenza viruses (Flu) suffers from virus resistance. Therefore, there is a great demand for exploring new drug targets and developing effective novel antivirals. This is challenging, since there is only a limited number of antiviral targets for rational drug design. This project will tackle both; it will explore and verify a new target and develop inhibitors against it. Cutting edge results in structural biology of the influenza RNA-dependent RNA polymerase (FluPol), identified a common general mechanism of Flu RNA transcription and replication cycle. The mechanism is entirely dependent on binding of viral endogenous RNA molecules to specific sites on the FluPol. Aided by already existing cryo-EM structures of FluPol, we will design inhibitors based on the sequence, atomic structure and interactions with the protein, and add extensive chemical modification to these viral RNAs. We will then test whether they can inhibit FluPol by in vitro and in cell-based experiments. Targeting these RNA binding sites and using RNA-scaffold based molecules is an innovative concept and expected to be robust in respect to the development of viral resistance. Altogether, the approach has great potential to bring a new universal paradigm to the field of antiviral drug discovery.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Organic Chemistry and Biochemistry of the CAS, v. v. i.
Study Programme/Specialization:	Microbiology ( in English language )
Supervisor:	Mgr. Jan Weber, CSc.

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The entry of viruses into host cells is a crucial initial step, particularly for respiratory viruses with significant pandemic potential such as SARS-CoV-2 and influenza A virus. Analyzing the interactomes of coronaviruses and influenza A virus and comprehending the early interactions between viral and cellular proteins can provide valuable insights for developing targeted or broad-spectrum antiviral therapies. The main goal of this project is to understand the viral and cellular factors influencing the entry of respiratory viruses. We will employ several strains of coronaviruses and influenza A virus, known to exploit different entry routes into the host cell, and will compare their cellular interactome at the time of entry and during early post-entry events. From the identified cellular interacting partners, interesting candidates will be selected and their role in the virus life cycle analyzed. The student will learn how to work with cell cultures in biosafety level 3, perform proteomic and bioinformatic analyses, and conduct siRNA experiments for gene-knockdown in mammalian cells infected with various viruses. The proposed project will broaden our current knowledge about the early phase of infections of mammalian cells with respiratory viruses.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Organic Chemistry and Biochemistry of the CAS, v. v. i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	David Marcelo Sabatini, M.D., Ph.D.

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We have a long-standing interest in the regulation of growth and metabolism that stems from our early work on the pathway anchored by mTOR protein kinase. We now appreciate that it is a major regulator of growth and anabolism in animals and responds to diverse stimuli, including nutrients. 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. Available thesis projects: (1) Nutrient sensing by mTOR. We seek to: discover the glucose sensor for mTORC1 and nutrient sensors in animals beyond mammals; understand how the known nutrient sensors function in vivo; and elucidate the biochemical function of GATOR2. (2) Lysosomes in normal physiology and disease. Using the Lyso-IP methodology and CRISPR screening we seek to understand how neurodegenerative diseases impact lysosomes and identify the contents of lysosomes in specialized cells, like immune cells. (3) Methods to study small molecule metabolism in vivo. We seek to develop methods to study carbohydrate metabolism in cells in vivo in mice. (4) Development of drug-like molecules. In collaboration with chemists at IOCB and elsewhere, we seek to develop drug-like molecules that target mTOR pathway components as well lysosomal and mitochondrial proteins.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Organic Chemistry and Biochemistry of the CAS, v. v. i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	Ing. Kvido Stříšovský, Ph.D.

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The proposed PhD project will be focused on the development of inhibitors and probes for rhomboid intramembrane proteases, which have been implicated in the regulation of mitophagy, malaria parasite invasion and epithelial homeostasis. Prior work in the thesis advisor’s lab has shown that peptidyl ketoamides are potent rhomboid protease inhibitors. The aim of the thesis will be to establish platforms to investigate and improve the potency and selectivity of this class of compounds for rhomboids and develop potent inhibitors and probes for specific rhomboid proteases implicated in Parkinson’s disease and epithelial homeostasis. These compounds will enable and facilitate biological studies of rhomboid proteins and serve as starting points for potential pharmacological applications.

Institute of Physiology of the CAS, v. v. i.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Physiology of the CAS, v. v. i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	RNDr. Petr Ježek, CSc.

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Accurate methods to detect pancreatic ?-cell mass and functionality in vivo are essential to better understand the pathogenesis of diabetes associated with pancreatic ?-cell deficiency and to develop new treatment options. Therefore, we will develop polymer-coated paramagnetic core-shell upconversion nanoparticles (UCNPs) conjugated with small GLP-1 receptor ligands (GLP-1 peptide, liraglutide or agonist 3) to target and monitor pancreatic ?-cell mass using magnetic resonance imaging (MRI) and luminescence. The newly developed UCNPs will be optimized in size to penetrate blood capillaries of the native and transplanted pancreatic islets and modified for long-term monitoring. Ultrasmall UCNPs (5 nm) will serve as a contrast agent for electron microscopy to visualize and count mtDNA nucleoids in ?-cells, which are typically reduced in diabetes. The specificity, safety and efficacy of all developed UCNPs will be validated in in vitro and in vivo models using multimodal imaging including luminescence, MRI and electron microscopy. See Ref. doi: 10.1021/acsami.2c04274.

Granting Departments:	Department of Biochemistry and Microbiology Institute of Physiology of the CAS, v. v. i.
Study Programme/Specialization:	Biochemistry and Bioorganic Chemistry ( in English language )
Supervisor:	RNDr. Petr Ježek, CSc.

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3D nanoscopy has not yet assessed mitochondrial cristae morphology, nor the internal structure of mitochondrial DNA (mtDNA) & protein complexes, termed nucleoids. Hence, we’ll survey 3D-redistribution of cristae and their shaping proteins or nucleois employing our prototype Vutara 3D superresolution microscope for stochastic techniques such a PALM and dSTORM. We will conduct studies under physiological situations vs. pathology (type-2 diabetes, cancer) using dSTORM with nanobodies or FRET excited PALM/dSTORM. Thus nm changes will be reflected by novel 3D nanoscopy methods. Also mtDNA nucleoids will be studied at increased and diminished mitochondrial biogenesis, while applying own mitoFISH nanoscopy for D-loop counting. Artificial manipulations of nucleoid size and mtDNA content will be studied as well as nucleoid division. Results will be translated into specific protocols for 3D nanoscopy, specifically developing novel relevant 3D image analyses based upon the Ripley’s K-function and Delaunay algorithm. Molecular cell biology will thus be combined with up-to-date 3D nanoscopy. Note, the molecular biology techniques will be conducted and be ready for the applicants by the coworkers of the Department No.75. See Ref. doi: 10.1089/ars.2022.0173.