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Technická 5
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Department of Biochemistry and Microbiology is dedicated to higher learning and research in the fields of biology, biochemistry, microbiology and molecular biology. We organise courses in core biochemical disciplines for the Faculty of Food and Biochemical Technology in Bachelor and Master programme. Other specialized courses are offered for Master and postgraduate students. Student in all stages of higher learning are actively participating in our research projects.

UCT (originál)

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Main spectrum projections of "pseudo-Cronobacter" strains from the article B. Svobodová, J. Vlach, P. Junková, L. Karamonová, M. Blažková & L. Fukal: Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera. Applied and Environmental Microbiology 83(13) e00234-17. DOI: 10.1128/AEM.00234-17.

EB29_Franconibacter_helveticus_MspExport.btmsp
EB31_Franconibacter_helveticus_MspExport.btmsp
EB32_Franconibacter_helveticus_MspExport.btmsp
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Also available at Zenodo

DOI
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Department of biochemistry and microbiology focus on basic research in areas of retroviral molecular biology, proteomics, plant physiology and molecular genetics, enzymology, environmental microbiology, food microbiology and bioanalytical methods. These activities create a platform for applied research aimed at developing modern therapeutic approaches, bioremediation of inorganic and organic pollutants, monitoring food safety and quality (the department also operates accredited Testing Laboratory of the Institute of Biochemistry and Microbiology) or plant-pathogen interaction. Research in our department is in many cases interdisciplinary and, in addition to close professional cooperation between the individual laboratories of the department, it would be unthinkable without cooperation with a number of national and foreign groups within the framework of joint research programs and projects. Currently, we are working on projects supported by the Czech Science Foundation, Technology Agency of the Czech Republic, Ministry of Agriculture of the Czech Republic, Ministry of Industry and Trade of the Czech Republic and the Ministry of Education and Sports.

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Department of Biochemistry and Microbiology offers bachelor courses in core disciplines (biochemistry, microbiology, biology and others). We also participate on courses for the novel bachelor program focused on forensic sciences. Biochemistry courses are offered to students of all faculties of our university. Our department organizes master programmes General and Applied Biochemistry, Microbiology and Clinical Biochemistry.

Advanced Biochemistry, Applied Enzymology, Environmental Microbiology, Food microbiology and Genetic engineering are delivered in English for foreign students. 

The department is accredited for doctoral education of biochemists and microbiologists.

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Content of english website is limited. We are working on translation.

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DATA


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Department of biochemistry and microbiology focus on basic research in areas of retroviral molecular biology, proteomics, plant physiology and molecular genetics, enzymology, environmental microbiology, food microbiology and bioanalytical methods. These activities create a platform for applied research aimed at developing modern therapeutic approaches, bioremediation of inorganic and organic pollutants, monitoring food safety and quality (the department also operates accredited Testing Laboratory of the Institute of Biochemistry and Microbiology) or plant-pathogen interaction. Research in our department is in many cases interdisciplinary and, in addition to close professional cooperation between the individual laboratories of the department, it would be unthinkable without cooperation with a number of national and foreign groups within the framework of joint research programs and projects. Currently, we are working on projects supported by the Czech Science Foundation, Technology Agency of the Czech Republic, Ministry of Agriculture of the Czech Republic, Ministry of Industry and Trade of the Czech Republic and the Ministry of Education and Sports.

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Research of our laboratory is mainly related to molecular endocrinology, immunology and to the study of influence of oxidative stress on the organism. At the molecular level, we study the regulatory mechanisms occurring in the immune system, adipose tissue and liver and we are interested in those influencing the development of insulin resistance and metabolic syndrome. Our intention is also to find methods and substances that can be used in the treatment of these civilization diseases. In addition, we examined selenium effect on antioxidant enzymes levels and a possible connection with infertility. The last topic is pollen and food allergies, where the aim is to find the immunological basis that causes cross-reactivity between cypress pollen and some fruits or vegetables.

More information about our lab can be found in the section: Current projects.

Current projects

Mechanism of nicotinamide phosphoribosyltransferase function

Obesity affects a high number of people and its prevalence is rising. More than 30 years ago, it was discovered that fatty tissue is not just a fat storage; it is a place of a very complex regulation and of the secretion of tissue hormones called adipokins. They affect the metabolism of lipids and carbohydrates and are often responsible for the development of insulin resistance and, consequently, of diabetes mellitus type II. This discovery identified the “mere” fat storage as the largest endocrine organ in the body. Our goal is to clarify the properties of one of the adipokins – visfatin, also known as nicotinamide phosphoribosyltransferase (Nampt). We try to characterize where Nampt is located in the cell, how it is transported and secreted into the extracellular environment. Eventually, whether Nampt participates in the development of obesity and metabolic syndrome.

Nicotinamide phosphoribosyltransferase is attributed to a number of functions, the most important of which is its role as an essential enzyme in NAD synthesis. NAD is not only a cofactor of oxidoreductases, but it also serves as a substrate for non-redox reactions that are mainly responsible for DNA repair, cell cycle control and apoptosis. Recently, Nampt has been also associated with the development of cancer, because increased amounts of Nampt were observed in many cancer cells. For this reason, our goal is to gain a better understanding of how Nampt is involved in cancer development.

To study these processes, we use cell lines of adipocytes, hepatocytes and macrophages. The research includes molecular genetic techniques, preparation of genetically engineered cells, or fluorescence and super-resolution microscopy.

In vitro cultured adipocytes with red stained fat droplets.

In vitro cultured adipocytes 3T3-L1 with red stained fat droplets.

 Preadipocytes 3T3-L1 Macrophages U-937

Preadipocytes 3T3-L1 and macrophages U-937 with a stably inserted nicotinamide phosphoribosyltransferase gene with a fluorescent label. The images were obtained by fluorescence microscopy.

Cooperation

  • Institute for Clinical and Experimental Medicine, Prague
  • Institute of Biotechnology, CAS, v.v.i., Vestec
  • Institute of Physiology, CAS, v.v.i., Prague

Cross-reactivity of cypress allergens

Today, more and more people suffer from allergies, especially in industrialized countries, where almost 30% of the population is affected. Doctors are observing an increase in cases of so called "combination" allergies or "pollen food associated syndromes", i.e. those which occur via a cross-reaction between pollen (respiratory allergies) and food (food allergies). It is estimated that 60% of food allergies occur in combination with respiratory allergies. In collaboration with French colleagues, we study cypress pollen cross-reactions using electrophoresis and immunoblotting. Cypress pollen is a significant allergen in France and Mediterranean regions. Moreover, the allergy to this pollen is often connected with allergies to peach or citrus fruits. Demonstrating the cross-reactivity and identifying its causes contribute to the improvement of allergy diagnosis, and lead to a better patient treatment in keeping with the development of a personalized medicine.

Cooperation

  • Pasteur Institute a Hospital Trousseau, Paris, France

Cypress pollen grains observed in scanning electron microscopy (2200x). Orbicules or "Ubisch bodies" (300 - 600nm granules), characteristics of Cupressaceae pollen, are visible on the surface of the exine (outer membrane).

                                                   © Youcef Shahali / Colorization Jean-Marc Panaud, Institut Pasteur.

Selected publication

Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycle–dependent in mammalian cells, and its inhibition slows cell growth. Svoboda P., Křížová E., Šestáková Š., Vápenková K., Knejzlík Z., Rimpelová S., Rayová D., Volfová N., Křížová I., Rumlová M., Sýkora D., Kizek R., Haluzík M., Zídek V., Zídková J., Škop V. Journal of Biological Chemistry 2019; 294(22): 8676-8689

 Cypress Pollinosis: from Tree to Clinic. Charpin D., Pichot C., Belmonte J., Sutra J.P., Zidkova J., Chanez P., Shahali Y., Sénéchal H., Poncet P. Clinical Reviews in Allergy & Immunology 2017; 1-22, DOI: https://doi.org/10.1007/s12016-017-8602-y

A new allergen family involved in pollen food associated syndrome: snakin/gibberellin regulated proteins. Sénéchal H., Šantrůček J., Melčová M., Svoboda P., Zídková J., Charpin D., Guilloux L., Shahali Y., Selva M.-A., Couderc R., Aizawa T., Poncet P. Journal of Allergy and Clinical Immunology 2018; 141(1):411–414

The effect of zinc and/or vitamin E supplementation on biochemical parameters of selenium-overdosed rats. Melčová M., Száková J., Mlejnek P., Zídek V., Fučíková A., Praus L., Zídková J., Mestek O., Kaňa A., Mikulík K., Tlustoš P. Polish journal of veterinary sciences 2018; 21(4):731-740, DOI: https://doi.org/10.24425/124312

Visfatin is actively secreted in vitro from U-937 macrophages, but only passively released from 3T3-L1 adipocytes and HepG2 hepatocytes. Svoboda P., Křížová E., Čeňková K., Vápenková K., Zídková J., Zídek V., Škop V. Physiological research 2017; 66 (4):709-714

The Response of Macro-and Micronutrient Nutrient Status and Biochemical Processes in Rats Fed on a Diet with Selenium-Enriched Defatted Rapeseed and/or Vitamin E Supplementation. Rýdlová M., Růnová K., Száková J., Fučíková A., Hakenová A., Mlejnek P., Zídek V., Tremlová J., Mestek O., Kaňa A., Zídková J., Melčová M., Truhlářová K., Tlustoš P. BioMed Research International 2017, DOI: http://dx.doi.org/10.1155/2017/6759810

Effect of selenium-enriched defatted rape seeds on tissue cadmium and essential elements utilization in rats. Myška A., Száková J., Fučíková A., Mlejnek P., Zídek V., Tremlová J., Mestek O., Koplík R., Zídková J., Melčová M., Tlustoš, P. Czech Journal of Animal Science 2016; 61(11):496-505, DOI: http://dx.doi.org/10.17221/88/2015-CJAS

Biochemical and hematological response of rats on defatted rape seeds addition into the diet. Tvrdá J., Tůmová N., Fučíková A., Zídková J., Melčová M., Száková J., Mlejnek P., Zídek V., Mestek O., Kaňa A., Tlustoš P. Academia Journal of Agricultural Research 2015; 3(12): 395-401, DOI: 10.15413/ajar.2015.0162

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling. Skop V., Cahova M., Dankova H., Papackova Z., Palenickova E., Svoboda P., Zidkova J., Kazdova L. Differentiation 2014; 87(5):220-229. DOI: https://dx.doi.org/10.1016/j.diff.2014.06.00

Impact of cadmium on the level of hepatic metallothioneins, essential elements, and selected enzymes in the experimental rat model. Zídková J., Melčová M., Bartošová K., Šestáková I., Zídek V., Száková J., Miholová D., Tlustoš P. Czech Journal of Animal Science 2014; 59(12):548-556

Female serum of immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens. Brázdová A., Zídková J., Sénéchal H., Peltre G., Cibulka J., Ulčová-Galová Z. Folia Biologica-Prague 2012; 58():251-255

Autocrine effects of visfatin on hepatocyte sensitivity to insulin action. Škop V., Kontrová K., Zídek V., Sajdok J., Pravenec M., Kazdová L., Mikulík K., Zídková J. Physiological Research 2010; 59():615-618

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Group members

Head of group:

doc. Ing. Igor Hochel, CSc.

Professor:

prof. Ing. Jan Káš, DrSc.

Assistent profesors:

Ing. Petr Svoboda, Ph.D.

Ing. Vojtěch Škop, Ph.D. (currently at NIH, Bethesda, USA)

PhD students:

Ing. Magdalena Melčová

Ing. Diana Rayová

Technician:

Ing. Magdalena Melčová

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Home   Publications

Our lab group is focused on applications of the proteomics approaches using mass spectrometry as a detection endpoint. Using tools of the mass spectrometry we can study a broad spectrum of biological systems on the molecular level. Proteomics can be characterised as a field of the biochemistry that follows up complex studying of proteins in the specific time under defined conditions and is the one of the most dynamic and evolving science fields.

For the studying of proteins we use the mass spectrometric techniques: MALDI-TOF/TOF and LC-ESI-Q-TOF.

fig1a fig1b

Examples of Research Topics

  • Identification of proteins involved in crop stress reaction
  • Studying of membrane complexes associated with signal paths of plants
  • Proteomics aspects of mineralized heart valves
  • Protein analysis of artworks and historical mortars
  • Proteomic analysis of alveolar bones in connection with oral surgery
  • Proteomic analyses of bioliquids of patients with carcinomas and neurodenerative diseases
  • Identification of zooplankton
  • Distinguishing of animal species origin of parchments and hairs
  • Proteomics analysis of retroviruses
  • Identification of pathogen microorganisms
  • Development of techniques for protein quantification by mass spectrometry (methods iTRAQ, dimethyl labelling, TOP3 etc)

Obrázek1 (originál) matrix (originál) umeni (originál) salmonela (originál)

Methods

  • Peptide mass fingerprinting → protein identification
  • Surface mapping → study of protein conformation
  • Biotyping → identification of microorganism
  • Protein quantification (iTRAQ, dimethyl labelling, SIM, TOP3, MeanInt)
  • Cross-linking and pull-down of membrane proteins
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head of the group:

prof. Dr. Ing. Radovan Hynek

professor:

prof. RNDr. Milan Kodíček, CSc.

associate professor:

doc. Ing. Mgr. Štěpánka Kučková, Ph.D.

assistant professors:

Ing. Jiří Šantrůček, Ph.D.

Ing. Petra Junková, Ph.D.

Ph.D. Students:

Ing. Tereza Nešporová

Ing. Tatiana Smirnova

Ing. Ondřej Novotný

Ing. Anna Zubová

Ing. Iva Pitelková

Ing. Lucie Maršálová

Ing. Lucie Coufalová

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In our laboratory, we work on the development and application of methods using bioaffinity interactions of molecules to detect, determine, identify, and / or separate them.


DEVELOPMENT OF DETECTION SYSTEMS

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First of all, we are engaged in the development of detection systems for determination of both, high and low molecular substances. These are single or multianalyte immunochemical techniques ELISA (Enzyme Linked Immunosorbent Assay) and LFIA (Lateral Flow Immunoassay), respectively. These methods utilize the existence of non-covalent interactions; mainly between antigen-antibody, biopolymer-solid surface, avidin-biotin and others. In recent years we have been focusing mainly on the application of immunochemical techniques for the detection and determination of environmental contaminants (e.g., pesticides), analytes from clinical biochemistry (e.g., anabolics, drugs), isoflavonoids, microorganisms (Listeria monocytogenes, genus Cronobacter), and their products (enterotoxins, Staphylococcus aureus).

Current projects

Detection of anabolic androgenic steroids (AAS) in food samples

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  • development of quick and simple methods to reliably detect AAS in food supplements

Anabolic androgenic steroids are synthetic derivatives of testosterone. Nowadays they are used not only in medicine to support medical procedures, but also in sports as illegal doping to support rapid muscle mass growth and overall body strengthening. The free sale of these substances is prohibited, but they may be illegally sold, for example in the form of food supplements, without mentioning it on the packaging label. This behavior is primarily a threat to customers who buy a product without a declared content of anabolics, and therefore use these substances unknowingly.

New Psychoactive Substances Detection (NPS)

  • development of immunochemical methods for the detection of synthetic cannabinoids or tryptamines in biological fluids

NPSs are synthesized as structural analogues or chemical derivatives of already controlled substances. This is due to the efforts of manufacturers and distributors to circumvent existing legislative standards in which narcotic drugs and psychotropic substances are usually defined exhaustively.


STUDY OF CRONOBACTER BACTERIA

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Another gripping area of our interest are bacteria of the genus Cronobacter, opportunistic pathogens causing life-threatening infections, mainly in newborns and individuals with weakened immunity. We study these microorganisms from their basic properties to their interactions with the host organism. The methods used include a variety of approaches; e.g., isolation and purification of cell fractions and proteins, PCR, RFLP, MLST, mass spectrometry, work with databases and tissue cultures.

Current projects

Investigation of bacteria of the genus Cronobacter

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  • detailed characterization and accurate identification

Bacteria of the genus Cronobacter are very diverse in their properties and abilities. This diversity has caused a rapid development of their taxonomy and still presents us with new challenges in their accurate identification and classification. We mainly use the methods of molecular biology PCR and MLST and mass spectrometry MALDI-TOF/TOF.

  • interspecies variability

Currently, the genus Cronobacter includes seven species. According to the World Health Organization (WHO, 2008), the whole genus is seen as a pathogenic organism, although according to epidemiological studies, C. sakazakii and C. malonaticus are mainly associated with infections. It is therefore interesting to observe the differences in the characteristics and behavior of individual species and try to find connections with the degree of pathogenicity.

  • interaction of bacteria of the genus Cronobacter with cell lines

Some virulence factors have been described in the literature, but the process of pathogenesis remains unclear. In particular, protein expression in the host environment is almost non-mapped. Identification of strains capable of adhesion and invasion into human cell lines and monitoring of these interactions would be the first step to the subsequent study of virulent proteins that are expressed directly in the host by the pathogen.

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  • identification of virulence factors in pathogenic members of the genus Cronobacter

The aim of this part of the work is to find proteins that are unique to pathogenic species. It is very likely that these are virulence factors and their identification would contribute to the understanding and description of the pathogenesis. Furthermore, these proteins could be used for the development of immunochemical methods. These methods excel in speed and low cost and would be a good tool for detecting pathogenic Cronobacter species.

Proteins are isolated from various cell fractions, characterized by SDS-PAGE and identified by LC-ESI-Q-TOF MS. To search for suitable proteins for the immunochemical detection, the immunomagnetic separation is used.

 

Selected publications

Šuláková, A., Fojtíková, L., Holubová, B., Bártová, K., Lapčík, O., Kuchař, M. Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines.: J. Pharmacol.Toxicol 2019, 95, 36-46.

Holubová, B., Mikšátková, P., Kuchař, M., Karamonová, L., Lapčík, O., Fukal, L. (2019): Immunochemical techniques for anabolic androgenic steroid – matrix effects study for food supplements. Eur. Food Res. Technol. 2019, 245, 1011-1019.

Karamonová L., Holubová B., Jelínková A., Novotný J., Svobodová B. Stafylokokové enterotoxiny – superantigeny schopné ošálit imunitní systém. Chem Listy 2019, 113(11), 668-674.

Fojtíková L., Holubová B., Kuchař M., Lapčík O., Maryška M., Šuláková A. Tryptamine derivates having short linking bridge bearing carboxy functional group, and chemical processes for preparing the compounds useful for preparing immunogens by conjugation with the carrier protein. CZ Patentový spis 307719-B6, 2019.

Vlach J, Javůrková B, Karamonová L, Blažková M, Fukal L: Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species. Food Microbiol 2017, 62, 1-8.

Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M, Fukal L: Novel method for the reliable identification of Siccibacter and Franconibacter strains: From ‘pseudo-Cronobacter’ to new Enterobacteriaceae genera. Appl Environ Microbiol 2017, 83(13), e00234-17.

Fojtíková L, Fukal L, Blažková M, Sýkorová S, Kuchař M, Mikšátková P, Lapčík O, Holubová B: Development of enzyme-linked immunosorbent assay for determination of boldenone in dietary supplements. Food Anal Methods 2016, 9(11), 3179-3186.

Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M, Fukal L: Novel method for the reliable identification of Siccibacter and Franconibacter strains: From ‘pseudo-Cronobacter’ to new Enterobacteriaceae genera. Appl Environ Microbiol 2017, 83(13), e00234-17.

Jurášek M, Göselová S, Mikšátková P, Holubová B, Vyšatová E, Kuchař M, Fukal L, Lapčík O, Drašar P: Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements. Drug Test Analysis 2017, 9(4) 553-560.

Blažková M, Javůrková B, Vlach J, Göselová S, Karamonová L, Ogrodzki P, Forsythe S, Fukal L: Diversity of O-antigens within the genus Cronobacter: from disorder to order. Appl Environ Microbiol 2015, 81, 5574-5582.

Karamonová L, Junková P, Mihalová D, Javůrková B, Fukal L, Rauch P, Blažková M: The potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of biogroups of Cronobacter sakazakii. Rapid Commun Mass Spectrom 2013, 27(3), 409-418.

Holubová B, Göselová S, Ševčíková L, Vlach M, Blažková M, Lapčík O, Fukal L: Rapid Immunoassays for Detection of Anabolic Nortestosterone in Dietary Supplements. Czech J Food Sci 2013, 31(5), 514–519.

Blažková, M., Javůrková, B., Fukal, L., Rauch, P.: Immunochromatographic strip test for detection of genus Cronobacter. Biosens Bioelectron 2011, 26(6): 2828-2834. ISSN 0956-5663.

Group members

head of the group:

prof. Ing. Ladislav Fukal, CSc.

assistant professors:

doc.Ing. Barbora Holubová, Ph.D.

Ing. Ludmila Karamonová, Ph.D.

doc. Ing. Martina Krausová, Ph.D.

Ing. Barbora Svobodová, Ph.D.

PhD students:

Ing. Judita Arnoštová

Ing. Jiří Novotný

Ing. Anna Jelínková

technician:

Hana Benadová

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   Our laboratory group is dedicated to the exploration of all aspects related to fungal genetics, ranging from the investigation of unknown genes in yeast mutant models, to the study of gene expression in fungal mycelia, and even the production of natural secondary metabolites in fungi. To achieve these objectives, we employ a broad spectrum of bioinformatic and molecular biology laboratory techniques, including polymerase chain reaction, blue-white screening, cDNA library construction, Genome Walking, cloning of unidentified genes into yeast mutants, and the isolation of proteins through metal-affinity methods. Furthermore, we collaborate closely with other scientific groups at the University of Chemistry and Technology Prague (VŠCHT Praha) and the Czech Academy of Sciences (AV ČR).

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head of the group:
Ing. Jan Sácký, Ph.D.
Ing. Tereza Leonhardt, Ph.D.

Technician:
Bc. Barbora Nováková

Ph.D. students:
Ing. Jan Křesťan
Ing. Jan Šnábl

Mgr. students:
Bc. Gabriela Pelešková
Bc. Valeriia Belova

Bc. students:
Barbora Sommerová
Šimon Piller
Petra Mařánová
Jana Steinbauerová
Alina Murtazina

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Genes Associated with Metal Accumulation

The ability of fungi to accumulate significant amounts of metal elements in their fruiting bodies has been recognized for several decades. However, the precise mechanism behind this intriguing phenomenon remains unknown. In the Fungal Genetics Laboratory, we are dedicated to the identification and characterization of genes responsible for the accumulation of metal elements (Ag, Cd, Cu, Zn, and others) in the fruiting bodies of saprotrophic and mycorrhizal fungi. Our research also includes the cultivation of fungal cultures, comprising over twenty strains isolated from hyperaccumulating genera such as Amanita, Hebeloma, Agaricus, and others. This allows us to study both in vitro and in vivo scenarios, including the use of fluorescence microscopy.


Arsenic Transformation in Fungi

Arsenic is a highly toxic and carcinogenic metalloid that exists in both inorganic and organic forms in the environment, with inorganic forms of arsenic being more toxic to organisms than their organic counterparts. The phenomenon of arsenic accumulation and the formation of arsenic compounds in the fruiting bodies of fungi has previously been studied by our partner institution in Graz, Austria. It was discovered that fungal fruiting bodies contain diverse inorganic and organic forms of arsenic, some of which are yet unidentified. Our goal is to identify these unknown compounds and the metabolic pathways responsible for their formation and transformation. In pursuit of this and other research projects, we collaborate with Associate Professor Antonín Kaňa from the Institute of Analytical Chemistry


Identification of Bacterial Communities in Fungal Fruiting Bodies and The Mycosphere

While our primary focus lies in the realm of metals within fungal fruiting bodies and the associated genes and proteins, the question of comprehensively understanding the ‘absurd’ hyperaccumulation of metals in these structures has led us to investigate microbial communities, or microbiomes, using next-generation sequencing methods. We investigate whether bacterial communities residing in close proximity to fruiting bodies, or within mycelium, differ from communities inhabiting the same location but without direct contact with the fungus.


Drug Metabolism Testing with Filamentous Fungus Cunninghamella elegans

Filamentous fungus Cunninghamella elegans is considered a breakthrough in metabolism testing of various toxic compounds, including drugs, as it possesses an enzyme repertoire similar to that of animal livers. As such, it can be employed not only for basic research to predict metabolites of new synthetic drugs but also for the production of bioactive compounds. We collaborate with organic chemists and chemical analysts from the BAFA laboratory, and together with Associate Professor Michal Kohout, we investigate whether chiral compounds undergo chiral metabolism within the fungus. 

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Fungi play an important role in the ecosystem, cycling carbon and micronutrients and many even entering symbiotic relationships with plants. This can be of utmost importance for the optimization of plant growth, as research shows that mycorrhizal plants greatly benefit from the mycorrhiza due to a much larger root system and thus increased access to water and micronutrients and supposedly diminished stress from pathogens and heavy metal stress. 

Our research group focuses on heavy metal speciation in the sporocarps and mycelia of ectomycorrhizal fungi. We are especially interested in the so called hyperaccumulators, i.e. fungal species that accumulate extraordinary amounts of heavy metals, sometimes even multiple elements at once. Hyperaccumulating taxons are first discovered by measuring the total amount of metals in sporocarps from pristine and polluted habitats acquired by our collaborator Jan Borovička. If a hyperaccumulating taxon is confirmed, further molecular and biochemical analyses are performed to elucidate the biochemical background of the metal speciation (metallothioneins, compartmentalization, novel metalloproteins) and then the genetic determinants responsible for the hyperaccumulating phenotype. Our collection comprises of over twenty hyperaccumulating fungal species in the form of mycelial explants, including Amanita, Hebeloma, and Agaricus sp. This gives us a unique opportunity to study the effects of heavy metal overload and poisoning on these organisms both in vitro and in vivo.  

Our other collaborative projects include speciation and transformation of arsenic in arsenic hyperaccumulators and discovering novel fungal proteins and compounds with potential technological and medicinal importance. 

Current projets

Speciation of cadmium, zinc and copper in sporocarps and mycelia of fungi of the species Russula, Hebeloma, Amanita muscaria, Cystoderma carchariasAgaricus crocodilinus – besides classic biochemical approach for the identification of metal species, we are also relying on the available public genomic sequences in which we can reveal relevant protein sequences by bioinformatic analysis and then isolate the genes coding for these proteins by homology cloning or by construction of RACE and Genome walking libraries. 

Identification of bacterial communities in sporocarps and in the mycosphere – although our main field of interest are metals in fungal sporocarps, and the genes and proteins related to metal metabolism, the question of why the metal hyperaccumulation trait has evolved in fungi has lead us to the study of microbial communities, so called microbiomes, by NGS methods. Our goal is to elucidate if the bacterial communities living in the proximity of sporocarps and mycelia are different from bacterial communities living at the same locality but without a contact with the fungus. 

Preparation of chiral compounds by Cunninghamella elegans – this fungus is considered very interesting for the testing of the metabolism of toxic compounds, since the enyzmatic apparatus of the fungus is similar to that of animal hepatocytes. This can be used for the prediction of metabolites of new synthetic drugs or even production of novel bioactive compounds. Currently, we are cooperating with Dr. Michal Kohout, testing if the fungus is able to keep or change the chirality of the tested compounds. 

Identification of novel aluminium complex in mycelia of Oidiodendron sp. an isolate of an unknown species of this ericoid mycorrhizal fungus was brought to us from Poland. After metal tolerance screening, it was shown that the fungus is multitolerant to many metals, including aluminium. Currently we are investigating the form of aluminium stored within the cells, including using fluorescence microscopy with an aluminium probe, and transmission electron microscopy with energy dispersive spectroscopy to determine the exact place of the Al complexes within the fungal cells. 

Identification of novel cadmium complexes in sporocarps of Agaricus crocodilinus – literature search revealed an interesting article from 1983, in which German scientists found a cadmium complex with high amounts of saccharides but no sulfur amino acids, which is strange, considering cadmium has been mostly found to bind with proteins containing sulfur. This complex was called “mycophosphatin” and our goal is to isolate it and describe its biochemical properties. 

Isolation and characterization of biomolecules from sporocarps and mycelia of Amanita muscaria – this fungus belongs to mildly poisonous species. Its habitat extends over the whole northern hemisphere and besides being poisonous it has been known for being psychotropic, and thus it is used in traditional shaman rituals and as a recreational drug. In our collaboration with the BAFA lab, we are investigating the potential for novel medicinal use of A. muscaria. 

Speciation of arsenic in the ink stain bolete, Cyanoboletus pulverulentus – this edible fungus was studied previously by our collaborators at Graz University in Austria. Their research yielded information about very high amounts of arsenic and potentially novel arsenic species. Our goal is to identify potential novel species and to study the metabolic pathways that lead to the formation of these species both in laboratory and in the environment. This project is done in collaboration with Department of Analytical Chemistry. 

List of publications

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The tremendous diversity of microorganisms is not only astonishing; it is also a key to understanding how organisms evolve and adapt to various environmental conditions. The metabolic activity of microorganisms is essential for maintaining the functions of all ecosystems in the biosphere. Microbial ecology seeks to unveil microorganisms in their natural environments; it looks at how microbes interact with one another and their environment, and what the consequences of these interactions are for ecosystem functions.

vodyIn the Laboratory of Microbial Ecology, we are interested in uncovering some of the hidden gems of microbial diversity. Certain localities of the Czech Republic are very interesting from the microbial ecological point of view; being extreme (brine, radioactive), often unique, spatially isolated biotopes, they are a promising source of hitherto undiscovered microbial diversity. Such habitats include mineral water springs which are used for healing purposes in Karlovy Vary, Jáchymov or Luhačovice, or soils and mofettas from the Soos National reserve. Our objective is to analyze microbial communities in these habitats and understand their physiology, biochemistry and ecology.

exudaceNo less important to us is improving our understanding of plant-microbe interactions. We look at how secondary plant metabolites shape soil microbial community structure and how they induce and/or regulate biodegradative genes in soil bacteria, thereby increasing the contaminant biodegradation potential in the microbial community. In turn, we are looking at how rhizosphere microorganisms promote the growth of plants. At the same time, we are looking at how many and which of these microorganisms colonize the interior of the plants, thus becoming endophytic. The applications of this fundamental research mainly include sustainable agriculture or environmental protection.

kultivaceWe identify metabolically active microbial populations in the context of their environments without the need for their cultivation. Microorganisms whose activity is crucial for a particular bio(geo)chemical process in the ecosystem are often not very abundant in the community. Therefore, we use microbial ecological techniques that allow linking community members with specific functions, including Stable Isotope Probing (SIP) or epicPCR. We thereby identify microorganisms degrading contaminants from the soil, promoting plant growth, etc.

exudaceWe are also aware of the importance of pure culture in microbiology. Therefore, we are trying to modify standard extraction and cultivation procedures in order to increase the efficiency of culturing of microorganisms, be it from soil, water, plant interior or any other habitat. Resuscitation factors or signaling molecules can help us to do so, along with mimicking the conditions of natural environments in which our microbes thrive. Upon successful isolation, the novel pure cultures are classified and characterized.

In order to reach our goals, we try to apply cutting-edge methodologies and techniques, including metataxonomics, metagenomics, stable isotope probing as well as modified culturing approaches. If you are interested in more details on our research, see our current projects.

We are a group of enthusiastic, motivated and hardworking microbial ecologists. If you are a motivated and diligent student who is eager to do solid research, do not hesitate to contact us.

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Once ecological mechanisms are understood, ecologists strive to better predict, conserve or manage communities to desired outcomes.

(Ashley Shade, ISME J 2017; 11, 1–6)

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 ◳ lab209_2022 (jpg) → (originál)

head of the group

prof. Ondřej Uhlík, Ph.D.

assistant professors

Michal Strejček, Ph.D.

Jáchym Šuman, Ph.D.

postdoc
Ana Catalina Lara Rodriguez, Ph.D.

PhD students

Stephanie Campeggi

Tomáš Engl

Lýdie Jakubová

Martina Jeřábková

Magdalena Folkmanová

Gabriela Kapinusová

Jakub Papík

Eliška Suchopárová

Tereza Šmrhová

Manuela Tadrosová

Andrea Zubrová

professor

prof. Tomáš Macek, CSc.

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The main research interests of the Laboratory of Microbial Ecology can be divided into four subsections:

First, we are looking into the ecology of plant-microbe interactions. We are working under the hypotheses that secondary plant metabolites (SPM), including lignin degradation intermediates and other phenolics as well as terpenes, significantly affect the biodegradation potential of soil microbial communities. We also predict that biodegradative functions, originally evolved for SPM degradation, and plant-growth promoting functions are often attributed to the same plant-associated microbial populations. We hypothesize that this functional association helps the plant to selectively enrich microbial populations which promote its growth through SPM exudation. At the same time, SPMs indirectly help the plant thrive in contaminated soil by increasing the biodegradation potential of soil microbial communities.

Second, we are looking into the microbial ecology of geologically unique biotopes. We are working under the hypotheses that microorganisms found in these biotopes will be unique from many points of view – phylogenetically with respect to as-yet-undetected taxa, metabolically with respect to the production of biologically active compounds, and ecologically with respect to unusual life strategies and interactions. We also predict that cutting-edge microbial ecological techniques, specifically targeted metagenomic, metagenomic/metatranscriptomic and metabolomic approaches, will enable us to decipher the microbial ecology of these biotopes.

Third, we identify metabolically active microbial populations in the context of their environments without the need for their cultivation. We use techniques such as Stable Isotope Probing (SIP) or epicPCR for these purposes. 

Fourth, we are looking into how to modify standard extraction and cultivation procedures in order to increase the efficiency of culturing of microorganisms. This work relates to all of the above-mentioned research areas.

Our research is/was supported by the projects:

  • Exelent research – OP JAK, INTER-MICRO, Talking microbes - understanding microbial interactions within One Health framework
  • European Commission Grant 101060625, NYMPHE, New system-driven bioremediation of polluted habitats and environment
  • Life at the Interface: Ecology of Plant-Associated Microorganisms (Czech Science Foundation grant no. 22-00132S). Given their intertwined evolutionary history and close environmental proximity, plants and microorganisms have developed extensive interactions which are essential for ecosystem functioning. Our goal is to improve our mechanistic understanding of the ecology of plant-associated microorganisms and to explore some of the mechanisms driving processes that are enabled by plant-microbe interactions. More specifically, we aim to elucidate the role of rhizodeposit components in structuring soil microbial communities, determine the extent to which roots impact soil microbial populations, including the assembly of plant-associated microbial communities, as well as reveal the specific role of rhizodeposits in selecting soil bacteria with plant-growth-promoting traits to be recruited for endophytic mutualism. Results of this project should significantly advance our understanding of plant-microbial interactions, having potential implications for plant health and important ecosystem functions, such as horticultural plant growth promotion, environmental restoration and others.
  • Who is responsible here? Linking transformation of organochlorines with specific bacterial populations (Czech Science Foundation grant no. 22-00150S). Organochlorines (OCs), such as chlorinated ethenes and polychlorinated biphenyls, still constitute an important environmental problem due to actual and/or potential contamination of soils or drinking water sources. Although microbial degradation of OCs has been extensively studied, there is paucity of knowledge about the distribution of OC degradation genes at contaminated sites and their link to phylogenetic information in individual taxa. In this project, we aim to understand patterns in the distribution of vital biodegradative functions and the phylogenetic origin of bacteria that perform these functions. Specifically, our main objectives are to: reveal patterns in the distribution of selected OC degradation (bphA and rdhA) genes in indigenous communities at contaminated sites and their link to phylogeny; investigate the contribution of extrachromosomal DNA to the distribution of OC degradation genes in the environment; elucidate the capability of bacterial consortia to adapt to elevated concentrations of OCs by up-regulation and dissemination of particular genes.
  • Genome dynamics and maintenance in simple and homogeneous natural prokaryotic communities (in collaboration with Faculty of Science of the Charles University, Czech Science Foundation grant no. 23-06568S) Simple and spatially and genetically clearly defined microbial populations represent an optimal model for the study of prokaryotic population genetics. The bacterium Ferrovum myxofaciens from mine biostalactites perfectly meets these criteria. Our previous research has shown that the F. myxofaciens genomes have almost identical gene content but there is significant intrapopulation variability in the distribution of mobile elements and short polymorphisms. This variability probably serves for life strategy diversification or adaptation to different microniches. In this project, we analyze a larger number of better characterized populations from multiple localities which will allow a functional interpretation of their variability. The influence of different genetic variants on the biological properties of F. myxofaciens are be verified on pure cultures derived from natural populations. These analyses may provide new insights into the function of mobile elements and the role of local disruptions of intraspecific gene flow in the formation of prokaryotic populations.
  • Ecological functions of soil microorganisms governed by secondary plant metabolites (Czech Science Foundation grant no. 20-00291S). A vast source of SPMs in soils is lignin, a complex ubiquitous plant biopolymer composed of phenylpropanoid monomers. Structural similarity of SPMs, including lignin degradation intermediates, to known anthropogenic pollutants might explain why anthropogenic pollutants can be cometabolized in the presence of SPMs. In this proposed project we will test the hypotheses (i) that SPMs as original evolutionarily substrates of biodegradative enzymes significantly affect the biodegradation potential of soil microbial communities; and (ii) that biodegradative functions, originally evolved for SPM degradation, and plant-growth promoting functions, which are often attributed to the same plant-associated microbial populations, together help the plant to selectively enrich microbial populations which promote its growth through SPM exudation.
  • Microbial Cometabolism: Promoting Biodegradation of Pollutants (Ministry of Education, Youth and Sports of the CR grant no. LTAUSA19013). The objective of the project is to test the hypothesis that secondary plant metabolites can promote cometabolism of persistent organic pollutants in the environment and thereby significantly influence the biodegradation potential of autochthonous microbial communities. In order to accomplish this objective, we (i) selectively enrich soil bacterial consortia that degrade selected pollutants (polychlorinated biphenyls, PCBs, and polyaromatic hydrocarbons, PAHs) when grown on secondary plant metabolites and (ii) test whether the consortia involved in secondary plant metabolite metabolism increase the efficiency of biodegradation of PCBs and PAHs in soils.
  • Microbiomes of Selected Extreme Biotopes – Their Phylogenetic Diversity and Functional Potential (Ministry of Education, Youth and Sports of the CR grant no. LTAUSA19028). The objective of the project is to characterize microbial populations in extreme biotopes – chronosequence of permafrost (permafrost of differing age) from central Alaska, and soils from salt marshes and moffettes of the Soos National Natural Reserve, Czech Republic. The characterization is conducted by two major routes – metagenomics and modified cultivation techniques. The basic modification of the cultivation techniques is the use of resuscitation-promoting factor and adjustment of cultivation media so that they better mimic natural conditions the bacteria are subjected to in their habitat; the goal is to increase the efficiency of the cultivation and isolate dormant or otherwise difficult-to-culture or, if possible, as-yet-uncultured bacteria.
  • Ecology of extremophilic microorganisms in Czech spring waters of cultural heritage significance (Czech Science Foundation grant no. 18-00036S). There is a diverse range of deep springs in the Czech Republic, each of which, whether hot, radon or brine, is characterized by an extreme, yet stable and unique, environment that has enabled its indigenous microorganisms to evolve for thousands of years. We therefore hypothesize that microorganisms found in these waters will be unique from many points of view – phylogenetically with respect to as-yet-undetected taxa, metabolically with respect to the production of biologically active compounds, and ecologically with respect to unusual life strategies and interactions. We also predict that cutting-edge microbial ecological techniques, specifically targeted metagenomic, metatranscriptomic and metabolomic approaches, will enable us to decipher the microbial ecology of these waters.
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1) Virology

In the field of virology, which deals with the study of the structure and biology of viruses and their pathogenesis, we focus mainly on retroviruses and, more recently, in cooperation, also on flaviviruses. Both Retrovirideae and Flaviviridae are enveloped RNA viruses. The aim of our research is to clarify selected steps in the life cycle of viruses, focusing mainly on the late phase of viral infection. Understanding the molecular nature of these steps might open the possibility of developing new types of drugs. Using molecular biological methods, we study the structure of viral proteins, the functional significance of their structural domains, their mutual interactions, interactions with RNA and membranes and the transport of viral proteins and whole particles in an infected cell. We also study the structure and mechanism of formation of mature and immature retroviral particles.

fig1a fig1b

2) Medicinal chemistry: Theranostics and multimodal therapy

Natural products are the basis of clinical therapy for cancer and other diseases. Their therapeutic effects can be modified with chemical derivatization in order to reduce unwanted side effects, increase cytotoxicity and selectivity towards pathological cells, change their mechanism of action, etc. In our research group, we mainly focus on basic research of toxicity and mechanism of action of novel antimitotic derivatives (e.g., colchicine and paclitaxel), inhibitors of sarco-/endoplasmic reticular Ca2+-ATPase (e.g., thapsigargin and trilobolide) and cardiac glycosides (e.g., digitoxin and digoxin). Most of the derivatives we study are developed with the aim to be utilized in multimodal and combination therapy as well as theranostics of cancer. Theranostics is based on diagnostic and therapeutic properties combined in one molecule, which often consists of a natural product conjugated to a fluorescent moiety.

fig2

3) Cytocompatible and antimicrobial materials for research and medicinal applications

Another research topic addressed by our scientific group is the study of tailor-made polymers and metallic materials for medical applications for soft and hard tissue replacements, but also for research purposes (e.g., single cell cultivation and analysis). The aim of this research is to develop novel possibilities for functional replacement of damaged tissues but also to understand the basic mechanisms of cell-cell and cell-material interactions in dependence on its nano- and microstructured topology. We focus on evaluation of material cytocompatibility, cytotoxicity and antibacterial properties using cell biological and microbiological methods. Moreover, methods of molecular biology are utilized to study the mechanism of cell adhesion, interaction of cells with materials at the molecular level, and cell differentiation.

fig3

4) Photodynamic therapy and molecules for photoimaging

Photodynamic therapy is a non-invasive therapy using special molecules that, when activated by light, produce highly reactive oxygen species applicable for the eradication of tumors, microorganisms, or the treatment of skin diseases. We collaborate on the development of advanced photoactive molecules and study the molecular mechanisms of their action and localization in living tumor cells in real time. We focus on these aspects in the study of the effectiveness of photoactive molecules including both inorganic materials (e.g., molybdenum clusters) and organic molecules (e.g., porphyrins, phthalocyanines, halogenated BODIPY). In addition, we focus on the development of novel fluorescent probes for imaging techniques, which are an indispensable tool for medicine and molecular biological research. Fluorescence probes allow us not only to visualize and study different cellular structures, but also to reveal the molecular basis of various diseases. Our goal is to eliminate the limitations of available probes, such as high toxicity, low specificity or insufficient photophysical properties. We use both common and highly specialized fluorescence microscopy methods (SIM, PALM / STORM) to evaluate these probes.

fig4

5) Biologically active substances

We study the properties of plant extracts or isolated compounds with a focus on both beneficial effects and possible negative effects. Our goal is to identify biologically active substances and characterize their beneficial effects while excluding possible toxicological effects. Special emphasis is placed on biologically active substances with the ability to modulate multiple drug resistance.

fig5a fig5b

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Metallic materials for orthopaedic applications

When developing novel biomaterials for orthopaedic applications, the goals to reach are: suitable mechanical properties, antibacterial effect and the ability of material to accelerate healing. Another direction is the development of degradable metallic materials for temporary applications, which would mean no need of reoperation. Novel biomaterials developed by our co-operators must be first tested and approved. Our group deals with first stage of biomaterials testing using cell cultures and relevant methods..

fig1a fig1b

Human immortalized stem cells derived from adipose tissue grown on a titanium alloy with nanotubular surface modification (nuclei in blue, actin filament in red); Electron-microscopic image of murine fibroblasts grown on degradable magnesium alloy (magnification 2000 x)

The methods we use

We determine corrosion rate in biological media under physiological conditions, we test in vitro cytotoxicity of biomaterials according to ISO 10993-5 (elution test followed by determination of metabolic activity by resazurin assay) and we examine colonisation of material’s surfaces using microscopic techniques (using fluorescence as well as scanning electron microscopy). We use murine fibroblasts and cell lines derived from human bone tissue as cell models. We also test antibacterial properties of the biomaterials.

Contact

Ing. Eva Jablonská, Ph.D.
doc. Ing. Jan Lipov, Ph.D.
prof. Ing. Tomáš Ruml, CSc.

Publications

Cooperation

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REDOX Lab

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We are glad to welcome you to our laboratory page!

We are a friendly and open laboratory that focuses on the topics of oxidative stress, antioxidants and redox signalling in fields related to human health. We research in microbiology, cell biology, biochemistry, chemical biology and materials science.

What is our goal?

We are engaged in a mixture of basic and applied research to understand the mechanisms of various diseases and then to find new smart approaches to prevent and treat them. We collaborate with experts in other departments, including physicists, chemists, materials scientists, physiologists and physicians.

What can you find here? 

We'll keep you up to date on the exciting things we're up to - our latest projects, findings and maybe even behind the scenes. But it's not just about our work in the lab. We also share a lot of our knowledge. You'll find educational materials, fun facts and some tips for budding scientists on our sites. We're all about spreading the love of science far and wide.

So bookmark this page, follow us on social media and join us on this wild ride through a fascinating world of   ◳ Návrh bez názvu-18 (png) → (šířka 215px). If you've got questions or just want to say hi, drop us a line - we'd love to hear from you.

WE ARE DEALING WITH 

 ◳ PDT (png) → (originál)  ◳ MICRO (png) → (originál)  ◳ ENVI (png) → (originál)
 ◳ HYDRO (png) → (originál)  ◳ LAC (png) → (originál)  ◳ IMUNO (png) → (originál)

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ruml

prof. Ing. Tomáš Ruml, CSc.

Tel: +420 220443022
Email: tomas.ruml@vscht.cz

EDUCATION


1984 PhD. in Biological Sciences at the Prague Institute of Chemical Technology
(Study of thiamine excretion in mutants of Saccharomyces cerevisiae)
1973–1978 M.S. Institute of Chemical Technology, Prague (Dept. Fermentation Technology and Bioengineering, diploma thesis: Physiological characteristics of Candida utilis growing on ethanol)

WORK EXPERIENCE


09/2013-08/2020 Head of the Department of Biochemistry and Microbiology, UCT, Prague
01/2012–12/2019 Dean of the Faculty of Food and Biochemical Technology; UCT, Prague
2009/2011 Vice rector for International affairs, UCT, Prague
2006/2009 Vice rector for R&D, Faculty of Food and Biochemical Technology; UCT, Prague
2002 professor, Department of Biochemistry and Microbiology, UCT, Prague
01/1996 – dosud associated professor, Department of Biochemistry and Microbiology, UCT, Prague
09/1984 assistant, Department of Biochemistry and Microbiology, UCT, Prague

FOREIGN STUDY STAYS


University of Alabama at Birmingham - virology (two years: 1989-1991), 1994 (3 months), 1996 (3 months), 1998 (3 months); City College New York 1993 (3 months)

ORGANIZATIONAL ACTIVITIES


Main organizer of world Retrovirus Assembly Meetings: 14.-18. October 2000, 2.-6. October 2004 and 4.-8. October 2008 in Prague, co-organizer of two The FEBS Congresses, and 5 other congresses.

MAJOR RESEARCH ACTIVITES


Structures, interactions, targeting and assembly of retroviral proteins. Developed a fundamental method for in vitro assembly of retroviral particles, which represents a basic technology used in numerous prestigious laboratories worldwide.
Activity of molecules and nanoparticles for photodynamic therapy.
Biological effects of molecules and materials on human cells.
Bioprospection – analysis of medicically beneficial effects of marine and plant resources.

PUBLISHING ACTIVITY


Scientific papers in WoS: 206
Number of citations without self-citations: 3 343
H-index: 31, Co-author of 8 patents (two of which were realized by a license agreement)

PEDAGOGICAL ACTIVITY


Established new study program – Forensic analysis, initiated and co-established a new study program Bioinformatics.
Introduced and taught new university lecture courses: Molecular genetics, Genetic engineering, Genetic manipulations - laboratory course, Industrial microbiology; Taught: Microbiology. Laboratory: Microbiology, Biochemistry, Microbial analysis of foods.
Supervised 84 defended diploma works (4 awarded by J. Hlávka Prize for the best diploma thesis) and 24 Ph.D. students successfully defended their theses (one 3rd place in Sanofi Pasteur prize, two awarded by Votoček stipend and one by J. Hlávka Prize).

AWARDS


2012 of Minister of Education, Youth and Sports: “For extraordinary results in research”;
2014 of rector UCT Prague: “For outstanding research and successful promotion of science”;
2015 Award of Vietnamese president "For significant contribution to understanding between nations"
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Photobiology of materials based on Mo6I8 clusters and porphyrin frameworks

The molecular clusters based on Mo6I8 core with variable outer ligands and the porphyrin frameworks share the ability to catalyze the production of highly reactive singlet oxygen upon illumination with light or irradiation with X-rays. They serve for the preparation of soluble molecules, nanolayers, or nanoparticles that could kill cancer cells or pathogenic bacteria upon illumination or irradiation. In collaboration with Dr. Kamilem Langem a Dr. Kaplanem Kirakci from the Institute of Inorganic Chemistry, Czech Academy of Sciences, our group test the biological properties of such novel materials. We culture normal and cancer cells of human origin in our tissue culture facility and illuminate them with special light sources or with X-rays. Our hypoxic chamber allows us to work under low physiologically relevant level of oxygen. We can determine the cellular uptake and localization of cluster compounds or nanoparticles with confocal microscope or with structure illumination super-resolution microscope (SIM). The nanoparticles are also specifically targeted to cancer cells using molecular recognition. The promising materials are further tested on mouse models in collaboration with Dr. Milanem Reinišem from Institute of Molecular Genetics, Czech Academy of Sciences. The antibacterial properties of compounds, layers and particles are determined using our microbiology facility and model strains of bacteria relevant in clinics. The bacteria are cultured in the form of biofilm, a naturally occurring multicellular structure with enhanced physical and chemical resistance.

fig1
Principle of photosensitizing effect of a molybdenum cluster.

fig2
Microscopic colocalization of molybdenum cluster (red) and lysosomes (green) in cancer cell.

fig3
Microscopic colocalization of molybdenum cluster (red) and Enterococcus faecalis bacteria.

fig4
Antibacterial effects of the materials are confirmed by cultivation methods.

Funding

  • Biomaterials based on octahedral molybdenum clusters as singlet oxygen radiosensitizers, GA18-05076S, Czech Science Fundation

Publications

  1. Kirakci K., Nguyen T.K.N., Grasset F., Uchikoshi T., Zelenka J., Kubát P., Ruml T., Lang K.: Electrophoretically Deposited Layers of Octahedral Molybdenum Cluster Complexes: A Promising Coating for Mitigation of Pathogenic Bacterial Biofilms under Blue Light. ACS Appl Mater Interfaces, 2020. IF 8,758
  2. Kirakci K., Demel J., Hynek J., Zelenka J., Rumlová M., Ruml T., Lang K..: Phosphinate Apical Ligands: A Route to a Water-Stable Octahedral Molybdenum Cluster Complex. Inorg Chem 58:16546–16552, 2019. IF 4,850
  3. Kirakci K., Zelenka J., Rumlová M., Cvačka J., Ruml T., Lang K.: Cationic octahedral molybdenum cluster complexes functionalized with mitochondria-targeting ligands: photodynamic anticancer and antibacterial activities. Biomater Sci 7:1386-1392, 2019. IF 5,831
  4. Kirakci K., Zelenka J., Rumlová M., Martinčík J., Nikl M., Ruml T., Lang K.: Octahedral molybdenum clusters as radiosensitizers for X-ray induced photodynamic therapy. J Mater Chem B 6:4301-4307, 2018. IF 5,047
  5. Hynek J., Zelenka J., Rathouský J., Kubát P., Ruml T., Demel J., Lang K.: Designing Porphyrinic Covalent Organic Frameworks for the Photodynamic Inactivation of Bacteria. ACS Appl Mater Interfaces 10:8527-8535, 2018. IF 8,456
  6. Buzek D., Zelenka J., Ulbrich P., Ruml T., Krizova I., Lang J., Kubat P., Demel J., Kirakci K., Lang K.: Nanoscaled porphyrinic metal-organic frameworks: photosensitizer delivery systems for photodynamic therapy. J Mater Chem B 5:1815-1821, 2017. IF 5,047
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head of the group:

prof. Ing. Tomáš Ruml, CSc.

associate professors:

doc. Ing. Jan Lipov, Ph.D.

doc. Ing. Jaroslav Zelenka, Ph.D.

assistant professors:

Ing. Silvie Rimpelová, Ph.D.

Ing. Jan Prchal, Ph.D.

Ing. Markéta Častorálová, PhD.

Ing. Jitka Viktorová, PhD.

Ing. Eva Jablonská, Ph.D.
Ing. Vladimíra Pavlíčková, Ph.D.
Ing. Nikola Vrzáčková, Ph.D.

PhD students:

Ing. Lucie Hozáková

Ing. Jiřina Kaufmanová

Ing. Martina Koncošová

Ing. Habibullah Giyaullah

MSc. Lan Hoang

Ing. Radim Novotný

MSc. Van Nguyem Tran

Ing. Barbora Vokatá

Ing. Jakub Sýs

Ing. Anna Pavlů

Ing. Jan Škubník

Ing. Jiří Bejček

Ing. Bára Křížkovská

technician:

Radka Budilová

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Home   Projects   Publications

Plants as sesile organisms are exposed to many adverse environmental effects, either abiotic such as high temperature, drought, osmotic stress, salinity or biotic caused by pathogenic microorganisms, herbivores etc. Plants can adapt to many of these external cues as they evolved complex mechanisms by which they are capable to respond to unfavorable conditions.

fig1

Our laboratory is interested in topics related to the response of plants to various stress factors, focusing primarily on the role of various proteins in stress responses. We are studying plant  stress response taking place both at the level of the whole plant and at the level of the cell.

fig2

As a model organism, we use Arabidopsis thaliana and oilseed rape (Brassica napus). We use modern biochemical, microscopic and molecular biological techniques to solve our projects.

Collaboration

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Plant immunity

In nature, plants are constantly confronted with an inexhaustible number of microorganisms, some of which may be beneficial others may not (pathogens). During evolution, plants have developed a very sophisticated immune system through which pathogens respond to attack. In our laboratory, we deal with various aspects of the study of plant immunity, both at the molecular level and by monitoring the manifestations of the whole plant during pathogen attack. The phytohormone salicylic acid (SA) plays an important role in plant immunity, which is at the heart of our research related to immunity. In addition to involvement in plant immune responses, SA is involved in many physiological processes such as seed germination, cell growth, vent closure, senescence, or fruit yield. We are currently working on a project studying how the biosynthesis of SA is regulated by the enzyme phosphatidylinositol-4-kinase and we are also monitoring the effect of actin cytoskeleton dynamics on the SA signaling pathway.

Understanding the molecular mechanisms associated with SA is also important from a practical point of view and may help to grow crops under stress in the future.

In addition to the involvement of SA in plant immunity, we deal with the involvement of SPFH proteins, which include proteins from the flotillin families and HIR proteins. Flotillins are very likely to participate in clathrin-independent endocytosis and are part of lipid microdomains. These properties could predetermine an important role in plant signaling in pathogen infestation.

This reasearch was supported by Czech Science Fundation:

  • GA14-09685S - Flotillin: a novel player in plant stress signalling
  • GA17-05151S - Phospholipid metabolizing enzymes as new components of salicylic acid signalling pathway

Interakce rostlin s pathogeny

Plant responses to abiotic stress factors

Salt stress is currently a problem in many growing areas. Salt as an abiotic stress factor negatively affects the growth of plants and thus their productivity. Plants resist and respond to salt stress in many ways. One of the enzymes involved in defense mechanisms is phospholipase D (PLD).

In the study of the function of individual isoforms of phospholipase D in the response of plants to salt stress, we deal mainly with phospholipase Dα and phospholipase Dδ. As plant material we use wild type Arabidopsis thaliana and mutant lines A. thaliana with silenced genes encoding individual isoforms of PLD ( T-DNA insertion pldα mutant and pldδ of Arabidopsis thaliana). We monitor changes in the root system and evaluate phenotypic manifestations in wild-type and T-DNA insertion pld mutants of Arabidopsis thaliana.

In our work, we also focus on the description of intracellular molecular mechanisms involved in these changes.

Reakce rostlin na abiotické stresové faktory

LIVE imaging and its use in the study of defense reactions at the cell level

Any response of a plant to a stress stimulus always begins at the molecular level - by changes in the function and composition of proteins and enzymes. These molecules then gradually affect the higher systems - organelles of cells, tissues, organs and finally the changes (in the optimal case) are reflected at the level of the whole plant as an adaptation to stress. Live imaging allows you to monitor changes in protein complexes and cellular organelles in real time, in response to stress stimuli.

The studied proteins are membrane phospholipases, flotillins and other members of this family - HIR proteins. Our goal is to elucidate their interaction and their interaction with the cytoskeleton in the response of cells to environmental stresses. We study both biotic stressors and currently pay great attention to nanoparticles and their effect on the cell and cell organelles, as well as the reactions of plant organs.

We use fluorescence, confocal and superresolution microscopy to study these processes, and part of the research is also the preparation of genetically modified plants and cells labeled with fluorescent protein. In plants and cells prepared in this way, we can monitor the dynamics of proteins and organelles and their interactions after stress stimuli in vivo.

LIVE imaging

 ◳ fig5 (png) → (originál)

The maximum projection from 13 images shows the deposition of the defensive callose polysaccharide in the conductive pathways of the root of A. thaliana seedling treated with silver ions.

The second image represents one optical plane with the deposition of a callose in the endodermis of the root after treatment with silver nanoparticles. Both images show a combination of a fluorescent-blue callose signal and a cell structure visualized by differential interference contrast. Scale = 10 μm.

This reasearch was supported by Czech Science Fundation:

  • GA14-09685S - Flotillin: a novel player in plant stress signalling
  • GA17-10907S - Environmental impact of noble metal nanoparticles
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Home   Projects   Publications

Kalachová T, Janda M., Šašek V., Ortmannová J, Nováková P, Dobrev IP, Kravets V, Guivarc'h A, Moura D, Burketová L, Valentová O, Ruelland E: Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant. Annals of Botany 2020, 125(5), 775-784.

Kroumanova K, Kocourkova D, Danek M, Lamparova L, Pospichalova R, Malinska K, Krckova Z, Burketova L, Valentova O, Martinec J, Janda M: Characterisation of Arabidopsis flotillins in response to stresses . Biologia Plantarum 2019, 63, 144-152.

Kalachová T, Leontovyčová H, Iakovenko O, Pospíchalová R, Maršík P, Klouček P, Janda M, Valentová O, Kocourková D, Martinec J, Burketová L, Ruelland E: Interplay between phosphoinositides and actin cytoskeleton in the regulation of immunity related responses in Arabidopsis thaliana seedlings . Environmental and Experimental Botany 2019, 167, 103867.

Daněk M, Angelini J, Malínská K, Andrejch J, Amlerová Z, Kocourková D, Brouzdová J, Valentová O, Martinec J, Petrášek J: Cell wall contributes to the stability of plasma membrane nanodomain organization of Arabidopsis thaliana FLOTILLIN2 and HYPERSENSITIVE INDUCED REACTION1 proteins . Plant J 2019, 20(24), 6365.

Pluhařová K, Leontovyčová H, Stoudková V, Pospíchalová R, Maršík P, Klouček P, Starodubtseva A, Iakovenko O, Krčková Z, Valentová O, Burketová L, Janda M, Kalachova T: "Salicylic Acid Mutant Collection" as a Tool to Explore the Role of Salicylic Acid in Regulation of Plant Growth under a Changing Environment . Int J Mol Sci 2019, 101(3), 619-636.

Leontovyčová H, Kalachová T, Trdá L, Pospíchalová R, Lamparová L, Dobrev P. I, Malínská K, Burketová L, Valentová O, Janda M: Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Sci Rep 2019, 9(1),1-10.

Junková P, Daněk M, Kocourková D, Brouzdová J, Kroumanová K, Zelazny E, Janda M, Hynek R, Martinec J, Valentová O: Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2. Front Plant Sci 2018, 9, 991.

Krčková Z., Kocourková D., Daněk M., Brouzdová J., Pejchar P., Janda M., Pokotylo I., Ott P., Valentová O., Martinec J.: The Arabidopsis thaliana non-specific phospholipase C2 is involved in the response to Pseudomonas syringae attack. Ann Bot 2018, 121 (2), 297-310.

Krutinová H, Trdá L, Kalachová T, Lamparová L, Pospíchalová R, Dobrev P, Malinská K, Burketová L, Valentová O, Janda M: Can Actin Depolymerization Actually Result In Increased Plant Resistance To Pathogens?. bioRxiv 2018, 278986.

Angelini J,Vosolsobě S, Skůpa P, Yeuan Yen Ho A. Bellinvia E, Valentová O,  Marc J: Phospholipase Dδ assists to cortical microtubule recovery after salt stress. Protoplasma 2018, 255(4), 1195-1204.

Burketová L, Tvrdá L, Ott PG, Valentová : Bio-based resistance inducers for sustainable plant protection against pathogens. Biotechnol Adv 2015, 33(6/2), 994-1004.

Šašek V, Janda M, Delage E, Puyaubert J, Guivarch A, Maseda EL, Dobrev PI, Caius J, Bóka K, Valentová O, Burketová L, Zachowski A, Ruelland E: Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth. New Phytol 2014, 203(3), 805-816.

Matoušková J, Janda M, Fišer R, Šašek V, Kocourková D, Burketová L, Dušková J, Martinec J, Valentová O: Changes in actin dynamics are involved in salicylic acid signaling pathway. Plant Sci 2014, 223, 36-44.

Kim PD, Sašek V, Burketová L, Copíková J, Synytsya A, Jindřichová B, Valentová O: Cell Wall Components of Leptosphaeria maculans Enhance Resistance of Brassica napus. J Agric Food Chem 2013, 61(22), 5207–5214.

Janda M, Planchais S, Djafi N, Martinec J, Burketova L, Valentova O, Zachowski A, Ruelland E: Phosphoglycerolipids are master players in plant hormone signal transduction. Plant Cell Rep 2013, 32(6), 839-851.

Krtková J, Havelková L, Křepelová A, Fišer R, Vosolsobě S, Novotná Z, Martinec J, Schwarzerová K: Loss of membrane fluidity and endocytosis inhibition are involved in rapid aluminum-induced root growth cessation in Arabidopsis thaliana. Plant Physiol Biochem 2012, 60(), 88-97.

Šašek V, Nováková M, Dobrev PI, Valentová O, Burketová L: beta-Aminobutyric acid protects Brassica napus plants from infection by Leptosphaeria maculans. Resistance induction or a direct antifungal effect? Eur J Plant Pathol 2012, 133(1), 279-289.

Jindřichová B, Fodor J, Šindelářová M, Burketová L, Valentová O: Role of hydrogen peroxide and antioxidant enzymes in the interaction between a hemibiotrophic fungal pathogen, Leptosphaeria maculans, and oilseed rape. Environ Exp Bot 2011, 72(2), 149-156.

Kocourková D, Krčková Z, Pejchar P, Veselková Š, Valentová O, Wimalasekera R, Scherer GFE, Martinec J: The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress. J Exp Bot 2011, 62(11), 3753-3763.

Pleskot R., Potocký M., Pejchar P., Linek J., Bezvoda R., Martinec J., Valentová O., Novotná Z, Žárský V: Mutual regulation of plant phospholipase D and the acetin cytoskeleton. Plant J 2010, 62, 494-507.

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head of the group

prof. RNDr. Olga Valentová, CSc.

associate professors

doc. Ing. Lenka Burketová, CSc.

doc. Dr. Ing. Zuzana Novotná

assistant professors

PharmDr. Jindřiška Angelini, Ph.D.

Ing. Martin Janda, Ph.D.

PhD students

Ing. Šárka Mattauchová

Ing. Lucie Lamparová

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Home  Projects  Publications

Proteins play an important role in the living world. Their diversity is huge and fascinating. In our laboratory we study proteins in a complex manner. We are mainly interested in the relationship between the structure and catalytic activity of enzymes, looking for new proteins with interesting properties, optimizing the production of recombinant proteins and developing new molecular modeling methods to simulate slow and computationally demanding processes such as protein folding or interactions of protein with ligands.

Current projets

Cold-active enzymes

Enzymes from organisms living in permanently cold environments (e.g. mountain and polar regions) have significantly higher activity at low temperatures compared to enzymes from meso- and thermophilic sources. This property makes them interesting biocatalysts for low temperature applications. In our group we study cold-active enzymes (specifically chitinase, amylase, cellulase, β-galactosidase), which have a potential for use in the paper, food or pharmaceutical industries.

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Enzymes with therapeutical potential

Asparaginases have found application in medicine in the treatment of acute myeloid leukemia. It uses their natural ability to cleave L-asparagine, which is depleted in tumor cells, as a result of which protein biosynthesis is stopped and apoptosis occurs. Healthy cells are able to synthesize L-asparagine due to the presence of L-asparagine synthetase and are therefore not affected by this type of treatment. Preparations containing recombinant L-asparaginase from E. coli and Dickeya dadantii are currently used in practice, however, the application of both enzymes has a number of complications, and therefore it is necessary to look for other sources of L-asparaginase with more suitable properties for clinical applications.

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Nucleases have been studied for a long time because of their anti-tumor effects. So far, the main focus has been on animal nuclases. However, in our group we are focusing on enzymes from other sources, such as plants or bacteria. These enzymes are studied using molecular biology and protein engineering methods. Biological studies and crystallographic experiments are carried out in cooperation with the Institute of Plant Molecular Biology, the Institute of Physiology and Animal Genetics and the Biotechnology and Biomedical Center of the Academy of Sciences and Charles University in Vestec (BIOCEV).

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Structural protein of lipid droplets

We are interested in cellular lipid droplets and their life cycle, with special emphasis on the characterization of proteins associated with lipid droplets permanently or within a specific stage of the life cycle. Our goal is to contribute to the description of the life cycle of these specialized organelles and thus to the biotechnological use of both lipid droplets themselves (lipid and biofuel production, oil degradation, drug transport and immobilization systems) and their associated proteins (fusion anchors for industrial recombinant protein expression). As a model organisms we use both prokaryotic (marine bacteria Alcanivorax borkumensis capable to break down oil) and eukaryotic (plant Arabidopsis thaliana and yeast Saccharomyces cerevisiae) organisms.

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Proteins involved in the synthesis of ladderans in anammox bacteria

So far not well-known anammox bacteria (from the English anaerobic ammonium oxidation) were discovered in the mid-1980s and seem to play an important role in the nitrogen cycle on Earth. Anammox bacteria directly use ammonia as a source of energy, which they convert into molecular nitrogen . They do not need oxygen and, instead of producing carbon dioxide, they consume it. for this purpose they contain an organelle called the anammoxosome, in which energy metabolism is located. The membrane of the anammoxosome is composed of very interesting phospholipids, which have very specific lipids attached – ladderans. During the processing of ammonia in the anammoxosome, a highly reactive and toxic hydrazine is formed as an intermediate, which is just held inside the organelle by ladderans, which, unlike common fatty acids, give the membrane lower permeability to various substances. The biosynthesis of ladderans has not yet been elucidated and is the subject of our research.

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Molecular modelling

Computer simulations make it possible to study complex dynamic behavior of proteins, saccharides, other biomolecules and their assemblies. The main drawback of molecular simulations is their high computational cost. To study procesess that take place in long time scales it is necessary to apply special simulation methods. We use and metadynamics and other methods and we endeavour to contribute to their development, for example by application of artificial neural networks.

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head of the group:

doc. Ing. Vojtěch Spiwok, Ph.D.

associate professors:

doc. Ing. Petra Lipovová, Ph.D.

assistant professors:

Ing. Eva Benešová, Ph.D.

Ing. Michaela Marková, Ph.D.

Ing. Tomáš Podzimek, Ph.D.

Ing. Zita Purkrtová, Ph.D.

PhD students:

Bejček Jiří, Ing.

Mareška Václav, Ing.

Přerovská Tereza, Ing.

Trapl Dalibor, Ing.

Vodičková Patricie, Ing.

technicians:

Michal Verner

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