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Department of Biochemistry and Microbiology

List of available PhD theses

Analysis of gene transcription at single cell level

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Dr. Mikael Kubista, Ph.D.

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Single-cell analysis has become an attractive and challenging field of modern molecular biology and medicine, the main goal of which is to study biological questions with single-cell resolution. Such an approach reflects cell heterogeneity and reveals the complex response of an organism to various physiological and pathophysiological stimuli. Reverse transcription PCR (RT-qPCR) and RNA-sequencing (RNA-seq) are the most commonly utilized technologies to analyze single cells. Although both technologies are already routine tools for gene expression analysis, their applications at the single level is still challenging. The aim of the thesis will be to establish and validate efficient workflow for single-cell RT-qPCR and RNA-Seq analysis, including both laboratory and data analysis part. The factors influencing the effectiveness of each step will be monitored and their impact on experimental outcome assessed. The analysis workflow will be applied in the field of neurobiology, with particular focus on the impact of different brain injuries and neurodegenerative diseases on the function of glial cells. The results of the thesis will improve the current understanding of glial cell biology and enhance the reproducibility and effectiveness of single cell RT-qPCR studies.

Application of Advanced Molecular Methods to Reveal Bacterial Mechanisms of Virulence

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Peter Šebo, CSc.

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Over the past few decades, advances in sequencing technologies and bioinformatics have made possible development of new strategies to unravel mechanisms of bacterial virulence. The PhD. candidate will use genome-wide CRISPR/Cas9-mediated screen in human cells to reveal novel mechanisms of Bordetella pertussis and B. bronchiseptica virulence. About 19,050 human genes will be targeted by 123,411 unique gRNA for gene knock-out, and their role in B. pertussis and B. bronchiseptica cell cytotoxicity will be evaluated. Hits will further be clustered and validated.

Beneficial systemic effects of adipose tissue metabolism

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: MUDr. Jan Kopecký, DrSc.

Annotation

In all mammals including humans, two types of adipose tissue are recognized, white and brown adipose tissue (WAT and BAT, respectively), which are engaged in two major distinct and opposite functions: energy storage (in WAT) and energy dissipation (in BAT). Our recent studies are focused on “healthy adipocytes” in WAT, which are relatively small fat cells endowed with a high capacity for mitochondrial oxidative phosphorylation, triacylglycerol/fatty acid (TAG/FA) cycling and de novo lipogenesis. The “healthy adipocytes” could be induced in WAT by various factors that need to be better characterized. “Healthy adipocytes” provide beneficial local and systemic effects that could contribute to “metabolically healthy” obesity. The role of the metabolic features of “healthy adipocytes” in the systemic effects of WAT needs to be better characterized. The aim of this PhD project is to characterize adipose tissue metabolism in rodent models in stimulated and inactivated state (such as during cold exposure, in dietary-obese mice). In order to assess rate of the above mentioned metabolic processes, stable isotope labeling of newly synthetized lipids and metabolites will be used. Involvement of particular regulatory metabolic pathways will be evaluated using adeno-associated viruses-driven overexpression of certain proteins both in vivo and in cell culture model. The basic scholarship will be eventually supplemented by salary covered by grants of the department.

Bioactive coatings promoting spontaneous endothelialization of vascular vessel grafts

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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The surface of biomaterials that are in long-term contact with blood (e.g., vascular prostheses, stents) triggers inflammatory processes of the organism leading to activation of the coagulation cascade and formation of thrombi, and to a subsequent graft failure. The aim of this work is the development of coatings that would suppress activation of the coagulation cascade and immune response of the organism, while actively encouraging the formation of endothelium on the surface of vascular prostheses after their implantation. One approach will be based on coating the internal surface of a synthetic and decellularized vessel with a fibrin network that will be modified by bioactive molecules such as heparin, growth factors, oligosaccharides, and other bioreceptors specifically promoting the adhesion of progenitor endothelial cells. An alternative approach will be based on suppressing the unwanted body reactions by means of so-called polymer brushes and their subsequent functionalization by the above-mentioned biomolecules. We assume that, after implantation, heparin will suppresses the coagulation cascade, while the other bioactive molecules will promote endothelization of the graft by capturing progenitor endothelial cells from blood.

Biogenesis and degradation of yeast transport systems

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Hana Sychrová, DrSc.

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The aim of the PhD thesis is to find, with the use of molecular biology and biochemistry methods, proteins that participate in biogenesis, degradation and activity regulation of transporter systems in the yeast plasma membrane. Further, the mode and conditions of interaction between those proteins and transporters will be studied. Obtained knowledge will contribute to our understanding of mechanism of adaptation of yeast cells to changing environmental conditions.

Biological active compounds for cancer theranostics

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

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The main topic of the thesis is the study of new fluorescent and photosensitive derivatives of antimitotic poisons for application in teranostic. In the first phase, colchicine, a secondary metabolite of plants occurring in the lily family, will be used. It is known primarily for its high toxicity and therapeutic use in the treatment of gout and familial Mediterranean fever. The biological effects of newly prepared derivatives of colchicine and other natural substances with antimitotic effect on tumor cell lines will be studied with a focus on rapidly proliferating types of tumor cells. The data will be compared with non-tumor cells and the selectivity of newly prepared derivatives for tumor cells will be studied. Cyto- and phototoxic properties of these compounds for possible use in imaging and photodynamic therapy of cancer will be determined. In addition, the mechanism of action of these agents, the intracellular localization, the potential for inducing cell cycle arrest and the type of cell death will be verified.

Blood plasma individual variability and pathophysiology and their influence on the interactions with synthetic antifouling surfaces

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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Early biomarker detection improves treatment outcomes, survival and quality of life. In particular, optical biosensors are promising technology for early diagnostics: minimally invasive procedures (plasma, saliva), rapid and sensitive detection, low sample consumption. The main pitfall of optical biosensors is the inability to differentiate a specific signal from an interfering signal caused by adsorption of particularly proteins during the contact of artificial surfaces with biological media (fouling). Fouling can result in complement or coagulation initiation, etc. It has prevented the advance of biosensors into clinical applications. In principle, fouling is influenced by individual biological variability and pathophysiology – with a few exceptions, these factors and their influence are unknown although they play a key role in clinical applicability. The aim of the project is to identify factors caused by biological and pathophysiological variability of blood plasma that influence clinical applicability of blood contacting devices. To find relationship between individual samples, and to design pre-treatment protocols eliminating those factors.

Cathepsin proteases in biomedicine

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Michael Mareš, CSc.

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The project is focused on proteolytic enzymes of the cathepsin type that are critically involved in pathologies and are therapeutic targets. Cathepsins of human origin and those from pathogens and parasites will be investigated. The aim of the project is to analyze the structure and function of cathepsins and develop novel inhibitory molecules for their regulation. The research in the field of biochemistry and molecular biology employs the following main approaches: functional proteomics and chemical genomics, recombinant protein expression, enzymology, protein crystallography, and molecular modeling.

Cell biology of heavy metals in macrofungi and fungal microbiomes and associated microflora in hyperaccumulating species

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programmes: Mikrobiologie, Microbiology
Theses supervisor: prof. Ing. Pavel Kotrba, Ph.D.

Annotation

Fungi play a fundamental role in the environmental element cycling and efforts are being made to understand the molecular mechanisms underlying the fungal ability to accumulate high concentrations of metals and metalloids. It is known that fungi are associated with [symbiotic] prokaryotic populations – however, the interactions in the fungus-metal-prokaryote system are being quite overlooked. The PhD thesis aims at ‘metallomic’ studies in selected fungi on the molecular level and investigation of associated prokaryote taxa. The fungi of interest are hyperaccumulating macrofungal species of Cystoderma, Amanita and Agaricus genera. Employing modern molecular biology, bioinformatics and microbiology approaches, the metal tolerance genes of fungi and prokaryote diversity and community composition in the sporocarps and mycospheres will be investigated to obtain complex and new information about the mechanisms involved in the handling of the excess heavy metals and about the interplay between hyperaccumulating fungi and associated microflora in natural environments. The research is funded by Czech Science Foundation (project 19-06759S) with a position available for PhD student.

Comparison of biochemical markers in patients with different progression of Alzheimer's disease

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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The dissertation is focused on the development of a new biochemical methodology for the detection of progression degree of Alzheimer's disease. The methodology consists in detecting changes in the plasma peptide profile in several groups of patients with varying degrees of disease development and healthy individuals by mass spectrometry. For selected markers, their relative amounts will be determined and verified if they are characteristic for a particular stage of progression.

Competition of different Fusarium species on cereals under changing environmental conditions

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programme: Mikrobiologie

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Fusarium fungi are important phytopathogens. They cause losses in yield and quality of agricultural production. The species studied are mainly those that parasitize on cereals and their products. They produce harmful mycotoxins, the occurrence of which in food and feed is regulated and controlled. Different species differ in their preferences for living conditions, the production of different types of mycotoxins, which they use to colonize the host and compete with each other for living space. Under the current climate change conditions and the associated spread of new (often invasive) species, the aggressiveness of individual species needs to be evaluated to assess the possibility of crop protection and production. In the framework of the dissertation, it is necessary to investigate the basic processes that occur during simultaneous attack of the host. (1) existing knowledge about genome, virulence genes and biosynthetic pathways of phytopathogen mycotoxins from transcriptomic and genomic studies will be used, (2) effective procedures for monitoring phytopathogen development and competition of species based on qPCR, ddPCR or NGS will be proposed, (3) knowledge about competition between different molds determined and (4) proposed application of acquired knowledge for practical use suggested.

Comprehensive analytical methods and bioinformatics tools for metabolomics studies

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: doc. Ing. Tomáš Čajka, Ph.D.

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Untargeted metabolomics and lipidomics methods focus on the analysis of all the detectable metabolites in a sample, including chemical unknowns. Coupling liquid chromatography to mass spectrometry (LC-MS) is the preferred technique in metabolomics and lipidomics permitting effective compound separations and detection. One of the most challenging aspects of metabolomics research is the identification of unknown metabolites. Unknown metabolites represent up to 80% of all detected signals from untargeted mass spectrometry-based profiling. Such a big obstacle in biomedical and biology research hinders meaningful biochemical and pathway interpretations. The aim of the PhD project is (i) to increase the coverage of spectral libraries used for metabolite annotation, (ii) to apply and optimize in-silico software for prediction of ‘unknown’ metabolites, and (iii) to evaluate and apply new bioinformatics tools for visualization and interpretation of the data obtained within metabolomics and lipidomics studies. The work will be conducted at the Institute of Physiology CAS and financially supported by various grants (GACR, MSMT, AZV).

Development of proteomic techniques for differentiation of animal origin of recent and historical bones

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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The dissertation thesis is focused on the development of a new proteomic methodology suitable for the differentiation of animal origin of bones based on their protein composition. Bone tissue is one of the hardest tissues found in organisms. 60% of dry bone weight is formed by inorganic components (especially hydroxyapatite) and 40% is formed by organic component in which collagen type I is the most abundant protein. The methodology will be tested on recent bones, which will be analysed fresh and after heat treatment, and on bones obtained from archaeological sites, which are expected to occur degradation products of the protein components, which will also be studied.

Fish identification: molecular-biological and proteomic approaches

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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Food market is still broadened with new products from different producers and suppliers, which increases the diversity of consumers' menus on one hand, but therewithal it creates conditions for food fraud on the other hand. Food fraud may be associated also with health hazards for consumers. The consumers´ misleading can be done e.g. by replacing more expensive raw materials with cheaper ones, failing in complying the declared composition or by deliberately mislabelling the geographical origin or the production method. One category of often-falsified food are fish, fish products and seafood, which are also associated with the occurrence of significant allergens. The work aims to develop and experimentally verify methodologies for the identification of fish using DNA and protein analysis. Molecular-biological methods should allow such species determination within the class of Actinopterygii, and will comprise modern methods such as polymerase chain reaction (PCR, qPCR, and dPCR) and sequencing. The target gene to be analysed is the gene encoding the main fish allergen, parvalbumin. The proteins analysis will be aimed at comparing proteins profiles obtained by mass spectrometry MALDI-TOF (M)atrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry).

Healthy adipose tissue: role of FGF21 signaling

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: MUDr. Jan Kopecký, DrSc.

Annotation

In all mammals including humans, two types of adipose tissue are recognized, white and brown adipose tissue (WAT and BAT, respectively), which are engaged in two major distinct and opposite functions: energy storage (in WAT) and energy dissipation (in BAT). Our recent studies are focused on “healthy adipocytes” in WAT, which are relatively small fat cells endowed with a high capacity for mitochondrial oxidative phosphorylation, triacylglycerol/fatty acid (TAG/FA) cycling and de novo lipogenesis. The “healthy adipocytes” could be induced in WAT by various factors that need to be better characterized. “Healthy adipocytes” provide beneficial local and systemic effects that could contribute to “metabolically healthy” obesity.FGF21 (Fibroblast growth factor 21) is a hormone secreted mainly from liver in response to stimuli such as fasting and ketogenic diet. Although its main physiological role is still a contested topic, FGF21 has become a promising therapeutic target for obesity and Type 2 diabetes mellitus due to its powerful body weight-lowering and insulin-sensitizing effects. The main aim of the PhD project will be detailed characterization of FGF21 effects in WAT of model animals, namely changes in gene expression and rate of lipolysis, de novo lipogenesis and TAG/FA cycling. Since these metabolic processes are known to be linked to preservation of healthy status of the tissue, their potential regulation by FGF21 could thus represent an important part of the mechanism of overall beneficial action of FGF21. Basic PhD scholarship will be supplemented by salary covered by grants of the Department.

High-throughput sequencing data processing for taxonomic and (meta)genomic analyses in microbial ecology

Department: Department of Informatics and Chemistry, Faculty of Chemical Technology
Study programme: Bioinformatika
Theses supervisor: doc. Ing. Ondřej Uhlík, Ph.D.

Annotation

With the advent of molecular-biological methods and new technologies such as high-throughput DNA sequencing, microbial ecologist have been enabled to study in detail the composition of microbial communities without the need of complicated isolation of individual species. These analyses are referred to as metagenomics and rely on the analyses of all genomes contained within an environmental sample. Application of these methods allow for analyzing of habitats which have not been chracterized previously. In the framework of this thesis, the student will analyze high-throughput sequencing data for the sake of taxonomic and (meta)genomic analyses, including the application of currently used as well as development of novel bioinformatics tools and pipelines with the aim to characterize microbial communities in soils and extreme habitats.

Interaction of nucleic acids with proteins of RNA viruses

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

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Specific genome recognition is a key step in the life cycle of all viruses. For most RNA viruses, the mechanism of this process is not yet elucidated. Moreover, in retroviruses, nucleic acids affect the intracellular transport of structural proteins by neutralizing their basic charge. The aim of this work is to identify and characterize motifs responsible for the interaction of nucleic acids and viral proteins in selected retroviruses and flaviviruses. The work includes a combination of methods of molecular biology and virology combined with structural, proteomic and bioinformatic analysis.

Mechanisms of Action of Bordetella Type III Secretion System Effector Protein BteA

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programmes: Mikrobiologie, Microbiology
Theses supervisor: RNDr. Jana Kamanová, Ph.D.

Annotation

Classical Bordetella species cause respiratory infections of mammals, such as the whooping cough disease of humans caused by Bordetella pertussis and B. parapertussis. These pathogenic bacteria employ a type III secretion system (T3SS) to inject cytotoxic BteA effector protein into cells of the mammalian hosts. It remains unknown how BteA effector protein functions and contributes to the diverging biology of classical Bordetella species. The PhD. candidate will use air-liquid interface model of respiratory epithelia and yeast cell model complemented by an array of biochemical and mass spectrometry approaches to elucidate the molecular mechanisms underlying the action of BteA effector protein and its functional divergence.

Metabolomics 2.0: Investigation of biological systems using mass spectrometry

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: doc. Ing. Tomáš Čajka, Ph.D.

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Over the last decade, mass spectrometry-based metabolomics and lipidomics have become established as the key platforms for comprehensive profiling of low-molecular-weight compounds (polar metabolites, complex lipids) in biological systems. Coupling liquid chromatography to mass spectrometry (LC-MS) is the preferred technique in metabolomics and lipidomics permitting effective compound separations and detection. However, there is still a lack of sufficient data on the metabolome and lipidome characterizing biofluids (i.e., plasma, serum, urine) and tissues which can be easily accessible and reused at any time for future studies. The aim of the PhD project is to focus on novel approaches in second generation of metabolomics workflow (Metabolomics 2.0) such as (i) merging targeted and untargeted methods, (ii) standardization metabolomics methods, and (iii) extending the breadth and scope of polar metabolites, complex lipids, and various exogenous compounds to ultimately deliver a comprehensive database of a wide range of metabolites in various biological matrices. The work will be conducted at the Institute of Physiology CAS and financially supported by various grants (GACR, MSMT, AZV).

Nanoparticles for elimination of viruses spreading through air, water and bodily fluids

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programmes: Mikrobiologie, Microbiology
Theses supervisor: Mgr. Jan Weber, CSc.

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Proposed PhD project addresses the problem of the threat of viruses in the environment to the public. It is based on preliminary results in which specially designed nanoparticles have inhibited and eliminated viruses such as herpes simplex type 1 and 2, human papilloma virus, respiratory syncytial virus and dengue virus. These viruses exploit for the initial attachment and entry heparan sulfate proteoglycan that is naturally expressed on the cell surface. Here, we will test silver and gold nanoparticles that are capped with mercaptoundecane sulfonate groups, which mimics heparan sulfate. Nanoparticles will be prepared as solid substance as well as applied on various materials using sonochemical coating technique. In our group, we will examine their antiviral and virucidal effect on airborne infections (e.g. coxsackie virus, adenovirus, reovirus), water-borne infections (e.g. norovirus, rotavirus) and blood- and body fluid-borne infections (e.g. HIV, hepatitis B virus, zika virus). During postgradual study, student will learn how to work with cell cultures in biosafety level 3 settings among others how to determine replication and inhibition of various viruses, fluorescence immunochemistry techniques, qRT-PCR. References: 1. Cagno V, et al. Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism. 2018 Nat Mater. 17(2):195-203. doi: 10.1038/nmat5053. Epub 2017 Dec 18

Phylogenetic diversity and functional potential of microbiota in selected extreme biotopes

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programme: Mikrobiologie
Theses supervisor: doc. Ing. Ondřej Uhlík, Ph.D.

Annotation

Microorganisms, primarily prokaryotic, are the most diverse group of organisms on our planet in terms of phylogeny and metabolism. With this in mind, also the range of biotopes that host prokaryotic organisms is tremendous. Many bacteria and majority of archaea are classified among extremophiles - i.e. organisms living at the edge of life. The objective of the proposed 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 will be conducted by two major routes – metagenomics and modified cultivation techniques.

Preparation of stably transfected cell lines for in vitro testing of cell stress and toxicity

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Macek, CSc.

Annotation

Cell-based model systems are being used increasingly to screen compounds for toxic effects because they allow mechanism-centred assays to be used in rapid, high-throughput approaches. Bioluminescence-based reporter gene assays that measure genetic response element activity are well suited to this approach. In addition, signalling pathways involved in the cellular stress response have been identified as valuable screening targets that underlie biological responses and may be predictive of toxicity in the whole organism. Here, we will use a panel of response element vectors targeting key pathways involved in the cellular stress response and prepare the stably transfected lines. These cell lines will serve for the testing of well characterized model compounds to demonstrate how the vectors can be used in reporter gene assays, either individually or as a panel, to profile compounds for the ability to cause cellular stress and toxicity.

Production of antibacterial peptides from marine organisms and evaluation of their biological activity

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

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Design of bacterial expression system for production of existing antibacterial peptides identified in the laboratory of prof. Stensvåg at University in Tromso. Firstly, it is necessary to overcome the toxicity issues either by fusion with bulky protein or secretion achieved by fusion of the peptides to export sequence. Next step is upscale to massive production and purification for functional studies (pull down assay with bacterial cell lysate). Isolated products will be used for analysis of biological activity targeted against bacterial and yeast cells, including biofilms and determination of immunomodulatory effects. Subsequently it is planned a study of mechanisms leading to inhibition.

Production of antibacterial peptides from marine organisms and evaluation of their biological activity

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

Annotation

Design of bacterial expression system for production of existing antibacterial peptides identified in the laboratory of prof. Stensvåg at University in Tromso. Firstly, it is necessary to overcome the toxicity issues either by fusion with bulky protein or secretion achieved by fusion of the peptides to export sequence. Next step is upscale to massive production and purification for functional studies (pull down assay with bacterial cell lysate). Isolated products will be used for analysis of biological activity targeted against bacterial and yeast cells, including biofilms and determination of immunomodulatory effects. Subsequently it is planned a study of mechanisms leading to inhibition.

Regulation of focal adhesion dynamics by calpain-2 mediated proteolysis of focal adhesion protein paxillin

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Pavel Branny, CSc.

Annotation

Calpain proteases are intracellular cysteine proteases that by limited proteolysis regulate a wide spectrum of physiological processes ranging from muscle development to long-term potentiation of neurons to cell cycle. Calpains are also involved in the regulation of cell migration as they affect the stability of focal adhesions, multiprotein assemblies that mediate cell adhesion to extracellular matrix. To control migration, calpains cleave components of adhesions accelerating their turnover and thereby promoting cell migration. Consistent with these findings, we observe that activation of calpain 2 results in cleavage of focal adhesion protein paxillin at previously uncharacterized site. The project focuses on characterization of paxillin as a substrate of calpain 2 and how paxillin cleavage affects the dynamics of adhesions and cell migration. The work will include isolation of the cleaved form of paxillin from mammalian cells and determination of the cleavage site followed by overexpression of the mutated paxillin or insertion of mutation into the genome using CRISPR/Cas9 system. The effect of such prepared non-cleavable form of paxillin on the focal adhesions dynamics and migration will be studied by life cell and fluorescence microscopy, including superresolution techniques.

Regulation of redox signaling in immune cells by natural compounds

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Jaroslav Zelenka, Ph.D.

Annotation

Inadequate or inappropriately targeted immune response is the cause of many chronic diseases, from allergies to diabetes mellitus to cancer. The complexity of the human immune system is close to the nervous system and its proper function is dependent on precisely regulated extracellular and intracellular signaling. Its important component is redox signaling, which can be influenced by low concentrations of molecules acting as antioxidants or prooxidants. Numerous common natural compounds recruiting from the plant and microbial metabolites have the potential to influence the redox signaling of immune cells even at concentrations commonly found in our digestive tract, and the intestinal immune system is crucial for the immune response of the whole organism. The aim of this thesis is to evaluate the influence of fungal toxins, antibiotics and organic acids on selected parameters of immune response in immune cell models.

Relationship between plants endophytes, and its metabolome

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programme: Mikrobiologie
Theses supervisor: doc. Ing. Petra Lovecká, Ph.D.

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Recently, research focused on uncommon natural sources and unexplored habitats has become a subject of deep scientific interest. Exclusive relationships of endophytic bacteria and fungi with plants were shown to have a great potential to produce new and rare secondary metabolites, with unique health beneficial, technological and nutritional properties. The essence of the thesis will be studying of mutual relationships between microbial endophytes and Vitis vinifera L. plants grown under conditions of conventional vs, organic farming systems, by advanced tools of modern analytical chemistry – high resolution mass spectrometry-based metabolomic fingerprinting / profiling. In addition to this, potential ofendophytes to prevent pathogenic fungi growth and mycotoxins production will be assessed.

Role of Inhibitory Factor IF1 in the regulation of pancreatic ?-cell metabolism and mitochondrial morphology

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Andrea Dlasková, Ph.D.

Annotation

A unique function of pancreatic ?-cells is insulin secretion and consequently also maintenance of glucose homeostasis. Several factors ensure precise coupling of glucose levels, pancreatic ?-cells metabolism and insulin secretion. ATP is recognized as a key regulator of insulin secretion. Studies addressing intrinsic regulators of ATP synthase in pancreatic beta cells are thus of paramount importance and have a high potential of identifying new T2DM (Type 2 Diabetes Mellitus) therapeutical targets. Surprisingly, nearly no studies are available in this research field. Recently, we reported on the presence of ATPase inhibitory factor 1 (IF1) in pancreatic ?-cells and its role in downregulation of cellular ATP levels and insulin secretion. However, the exact mechanism by which IF1 regulates ATP synthesis remains controversial and further studies are essential.The aim of this PhD project is to study the mechanism by which IF1 regulates ATP levels in pancreatic ?-cells and to identify post-translational modification of IF1 in response to glucose availability. Selected PhD student will also analyze how IF1 regulates insulin secretion in vivo by use of IF1-knockout mouse model. To study changes in mitochondrial morphology newest super-resolution microscopy techniques and 3D electron microscopy techniques will be applied.

Structure and dynamics of glutamate receptors, theory and experiment.

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Jiří Černý, Ph.D.

Annotation

The proposed multidisciplinary project combines various experimental and theoretical strategies to refine structural elements of the glutamate receptor (iGluR) in functionally important states. We will extend the available structural data using a mammalian and insect cells expressed iGluRs. We will induce and characterize its functional state (activation kinetics, binding affinity, and single channel properties). Further, we will use the distance information from a structural mass spectrometry analysis as an input into all-atom adaptive enhanced sampling MD simulations to refine the original crystal structures and identify new structural motifs. We will gain highly accurate structural information on iGluR in its functionally important states and their role in iGluR structural transitions.

Structure and function of bacterial transcription system

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Jan Dohnálek, Ph.D.

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The bacterial transcription system is currently a focus of numerous research projects as an antibiotic target as well as posing a number of unanswered basic questions. We concentrate on analysis of structure and function of RNA polymerase from gram-positive bacteria, namely Mycobacterium smegmatis and Bacillus subtilis. We investigate the role of either recently discovered or not fully understood protein factors involved in the transcription machinery. Mycobacteria are medically important organisms containing serious pathogens. Bacillus subtilis is a representative organism of gram-positive bacteria, with some differences in transcription in comparison to mycobacteria. In this project selected RNA polymerase-associated proteins will be characterized in detail as for their structure and function, using techniques of molecular biology and integrative structural biology, including X-ray crystallography, small angle X-ray scattering and cryo-electron microscopy.

Structure and function of proteins transporting alkali metal cations

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Hana Sychrová, DrSc.

Annotation

The aim of the PhD thesis is to characterize, with the use of molecular biology and biochemistry methods, amino-acid residues involved in the regulation of activity, substrate specificity and tertiary structure of alkali-metal-cation transporters. These transporters have very prominent function in the cell physiology of all organisms; and the knowledge of their structure and function is very important for a broad spectrum of areas, from medicine and pharmacology to biotechnology.

Structure-Function Relationships of the Bordetella Type III Secretion System Proteins

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programmes: Mikrobiologie, Microbiology
Theses supervisor: prof. Ing. Peter Šebo, CSc.

Annotation

Type III secretion systems are membrane-embedded nanomachines used by many Gram-negative pathogens to inject bacterial effector proteins into the cytosol of eukaryotic cells. The aim of this thesis is to depict structure-function relationships of proteins of Bordetella III injectosome and its secreted substrates. Specifically, BopN protein that exhibits homology to the class of “gatekeepers” or “plug” proteins, Bsp22 protein that forms a unique filamentous T3SS tip complex and BteA effector protein with unknown mechanism of action. The PhD. candidate will develop reporter assays to analyze activity of Bordetella type III secretion system, perform super-resolution and cryo-electron microscopy and further carry out structural characterization and crystallization trials of the purified recombinant proteins, their domains and/or their mutated variants.

Structure-function relationships in selected α-L-rhamnosyl-β-D-glucosidases

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Dr. Michael Kotík, Ph.D.

Annotation

α-L-Rhamnosyl-β-D-glucosidases (rutinosidases) are not well-known microbial glycosidases that react with rutinosides such as rutin and hesperidin but also often with selected glucosides (e.g. isoquercitrin). These compounds which contain a flavonoid-based aglycone and a saccharidic moiety composed of rutinose or glucose have interesting biological properties for potential applications in food technology and medicine. In the framework of this project, we will investigate the relationships between the structure, function and promiscuity of four α-L-rhamnosyl-β-D-glucosidases by site-directed mutagenesis directed towards the aglycone binding site of the glycosidic substrates and towards a loop region that partially covers the active site of these enzymes. An important basis of our investigations is the recently determined crystal structure of a rutinosidase. In silico molecular docking of enzymatic substrates and products will be another task. Further aims of the project are: determine the quaternary and three-dimensional structures of the first rutinosidase within the GH3 family, obtain the structures of the enzyme–substrate complexes, determine the substrate specificities, and perform a structural analysis of the products generated in the synthetic reaction mode of these enzymes.

Targeted analysis of metabolic pathways – metabolomics and fluxomics by LC-MS/MS

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Ondřej Kuda, Ph.D.

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The focus of our lab is the metabolism and regulatory actions of bioactive lipids, specifically those related to pathways of glucose and lipid metabolism and pathophysiology of metabolic diseases (https://www.fgu.cas.cz/en/departments/metabolism-of-bioactive-lipids). The aim of the project is to develop a methodology to describe changes in metabolic flows in pathophysiological conditions based on biochemical knowledge and methods of analytical chemistry. This topic covers experiments with cell lines, biochemical analyses of metabolites and functional tests using stable isotopes and radionuclides, optimization of UPLC-MS/MS methods for tracking 13C, 15N and 2H-labeled metabolites (glycolysis, citric acid cycle, oxidation and lipid synthesis, ...). The final data will be modeled with fluxomic software solutions on metabolic maps. Project will be carried out at the Institute of Physiology ASCR, v.v.i. and financially secured by GA CR grants. We are looking for curious, imaginative, independent and highly-motivated applicants with experience in analytical chemistry or (bio)chemistry.

The non-specific phospholipases C from Arabidopsis thaliana: structure-function relationship

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology

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This PhD work aims to elucidate the catalytic mechanism, structure and structure - function relationship of non-specific phospholipase C (NPC) from Arabidopsis thaliana. Six genes encoding NPCs (NPC1-NPC6) have been identified in the Arabidopsis thaliana genome. In our previous experiments with NPC4, it was found that this enzyme produces, besides the expected product - diacylglycerol, surprisingly also phosphatidic acid. This second activity seems to have been overlooked so far. In this project we will focus on the study of this phenomenon by mutagenesis and try to elucidate the structure and catalytic mechanism of these enzymes. The effect of mutations on plant phenotype will also be investigated.

The role of immune system in pulmonary hypertension

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: RNDr. Lydie Plecitá, Ph.D.

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The main feature of pulmonary arterial hypertension is arterial wall remodeling towards thickening of vessel wall that leads to an increase of pulmonary arterial pressure, right ventricular hypertrophy, its failure and premature death. Remodeling of artery wall is complex and changes in energy metabolism and apoptotic resistance of cells within wall layers leading to cell hyperproliferation are part of it. Recent studies recognized pro-inflammatory reactions of immune cells as participants in wall remodeling. In collaboration with US partner, we study fibroblasts from adventitia as a major player in attraction and activation of immune cells. Our preliminary results have shown that metabolically changed fibroblasts are capable of producing cytokines, which induce pro-inflammatory process contributing to pulmonary artery remodeling. This study will define changes in energy and redox metabolism of fibroblasts, which lead to production of cytokines, possibly chemokines subsequently attracting and activating macrophages, T cells or other cells of immune system. The goal of the project is to specify metabolites of fibroblasts, which induce cytokine secretion leading to activation of immune system.

The role of myristic acid in the interaction of retroviruses with the membrane and maturation of the viral particle

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

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One of the late steps in the retroviral life cycle is coating of the immature particle with a lipoprotein membrane derived from the cytoplasmic membrane of the infected cell. The outer layer of the retroviral protein coat is formed by the domain of a matrix protein that interacts with membrane phospholipids via the basic domain and the N-terminally attached myristic acid. HIV has been shown to cause myristate immersion in the cytoplasmic membrane by a mechanism called myristoyl switch. The aim of this work is to characterize the conditions leading to this process in D-type retroviruses which, unlike other retroviruses, do not form particles on the cytoplasmic membrane but in the cytosol. In the next phase of the work, the structural changes induced by the myristoyl switch and the effect of these changes on viral particle maturation should be characterized.

Therapeutic potential of G protein-coupled receptor modulators

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: prof. Ing. Tomáš Ruml, CSc.

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G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and important targets of drug action. Measuring the activity of new compounds and/or determination of their mode of action (agonist/antagonist) is promising from pharmaceutical as well as biochemical point of view. The library of substances, isolated either from natural sources or obtained via rational design synthesis will be tested against panel of therapeutically promising GPCRs. The approach of G protein-independent β-arrestin recruitment assay will be used as this can measure the activation of majority of GPCRs and allows for potential parallel screening in robotic-assisted format.


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