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Department of Biochemistry and Microbiology

List of available PhD theses

3D superresolution microscopy for accessing mitochondrial ultramorphology

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

Annotation

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

Cutibacterium acnes: virulence factors and mobile genetic elements

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

Annotation

The gram-positive bacterium Cutibacterium acnes (formerly Propionibacterium acnes) is a skin commensal under physiological conditions, but it can also be an opportunistic pathogen causing invasive chronic infections. The most well-known diseases associated with C. acnes include Acne vulgaris; however, the connection with a number of other diseases is also clinically significant. The pathogenicity of C. acnes is due to a number of virulence factors, one of which is the formation of a biofilm, which increases the resistance of bacteria to antibiotics and other bactericidal substances, which negatively affects the effectiveness of treatment. C. acnes phylotype IA1, associated with acne, produce virulence factors as hemolysins, CAMP factors, neuraminidase, heat shock proteins (HSP) or lipase etc. Much of genes responsible for virulence and resistance is located on mobile genetic elements (MGEs). As part of the solution of this work, virulence factors will be studied using classical microbiological and modern molecular-biological methods.

Analysis of multispecies biofilms of food-borne pathogenic bacteria

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

Annotation

Most bacteria, due to their phenotypic adaptability, can live in a community called biofilm, which is often multi-species. Biofilm serves for their adherence, communication, protection and increases the resistance of bacteria to adverse environmental conditions. Its characteristic persistence, heterogeneity and complex dynamics are related to relatively difficult detection and problematic analyses by classical microbial methods. That is why such consortia are nowadays a challenge for many professionals. Multi-species biofilms of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, will be analysed during the course of the dissertation as selected representatives of food pathogens monitored under Commission Regulation (EC) 2073/2005. Classical microbiological and modern molecular-biological methods will be used. The multi-species biofilm study will also include analyses of gene expression levels and impact on biofilms of antibiotics or other selected antimicrobials. This will contribute to a better understanding of the spread of resistance in the environment assisted by these communities, while at the same time increasing the existing knowledge about the coexistence of the biofilms of bacteria whose eradication from undesirable sites is more complicated than single-species biofilms.

Anammox bacteria and their unique phospholipids

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

Annotation

Anammox (anaerobic ammonium oxidation) bacteria, which were discovered relatively recently, play an important role in the global nitrogen cycle, thanks to their unique metabolism, the conversion of ammonium cation (NH4+) and nitrite (NO2-) to nitrogen. In this way, up to 50 % of ocean nitrogen is produced in oxygen-restricted zones. Currently, these microorganisms are used in wastewater treatment as ideal substitutes for the denitrification process. The sequence of reactions of anammox metabolism takes place on the membrane of a special compartment inside these bacteria called the anammoxosome. All membranes of anammox bacteria, including the anammoxosome, are composed of unique ladderan lipids. These are phospholipids which contained 5 cyclobutanes or a combination of 3 cyclobutanes with cyclohexane at the end of the acyl chain. Thanks to these unique lipids, the membrane of the anammoxosome transmits protons up to ten times slower than the classical bilayer of phospholipids, which helps maintain the proton-motive power of these bacteria. Not much is known about the synthesis of these special phospholipids. The aim of this work will be to contribute to the detection of enzymes involved in the synthesis and degradation of these phospholipids in anammox bacteria.

Biocatalysts for a new route of nitrile synthesis

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Ludmila Martínková, DSc.

Annotation

Nitriles (R-CN) are currently used for the synthesis of fine and pharmaceutical chemicals. The benefits of enzymatic synthesis in general are mild conditions and selectivity. Aldoxime dehydratase, an enzyme enabling the synthesis of nitriles, occurs in bacteria and fungi. However, the enzyme is difficult to obtain directly from these organisms due to its low production. Therefore, the strategy will be to retrieve the genes from databases, have them commercially synthesized and express them in a heterologous producer, primarily Escherichia coli. This is expected to allow production of sufficient amounts of the enzymes for catalysis. The thesis will be a part of a Czech-Austrian project aiming to elaborate a new cascade synthesis of nitriles. Aldoxime dehydratase catalyzes the last step of the cascade consisting of 1) enzymatic preparation of aldehyde from carboxylic acid, 2) conversion of aldehyde into aldoxime (R-CH=NOH) and 3) dehydration of aldoxime into nitrile. The thesis is multidisciplinary, using methods of molecular biology, bioinformatics, biochemistry, biotechnology and organic chemistry.

Cathepsin proteases in biomedicine

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

Annotation

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

Characterization of early postnatal development using a biobank of tissues of human newborns

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

Annotation

Early postnatal period is critical with respect to development of the key physiological functions and homeostatic mechanism of the newborn, as well as for imprinting the metabolic features that could be manifested during the adolescence and adulthood. A unique biobank of the autopsy samples of various is available that had been collected from human newborns, mostly very premature newbors. Characterization of the transcriptome of the tissues using RNA sequencing analysis is ongoing.
The project will focus on tah data analysis in order to reveal various aspects of early human development and its control, also with regards to various pathologies. Model experiments on mice will reperesent the main experimental part of the project. It will proceed in close collaboration between the Department of Adipose Tissue Biology and the Department of Metabolomics of the Institute of Physiology of the Czech Academy of Sciences (http://www.fgu.cas.cz/en/departments/adipose-tissue-biology), and the Institute for Inherited Metabolic Disorders, First Faculty of Medicine, Charles University (http://udmp.If1.cuni.cz/en/genomics-and-bioinformatics-laboratory) in Prague. This multidisciplinary environment will be key for the appropriate tarining of the student and successful work on the projekt. The basic PhD scholarship will be supported by the employement at the Department.

Fish identification: molecular-biological and proteomic approaches

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

Annotation

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.

Heterogeneity of expression of effectors of Salmonella pathogenicity island 2?encoded type 3 secretion system

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

Impact of antibiotic resistance on the propagation of Gram-negative bacteria in the food chain

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

Annotation

The increasing incidence of antibiotic resistance in bacteria is one of the major problems of the 21st century, as it limits the range of suitable antibiotics for treating bacterial infections. The food chain is one of the places, where it happens the genesis of new antibiotics resistance determinants and/or their spreading (so-called hot spots). Selected groups of gram-negative bacteria (family Enterobacteriaceae, the genera Acinetobacter and Pseudomonas, etc.) form the very significant or the most numerous parts of bacteriome in many food products. At the same time, these bacterial groups are frequent carriers and spreaders of antibiotic resistance genes (e.g. by various mechanisms of horizontal gene transfer). However, the presence of antibiotic resistance genes, often linked to various mobile genetic elements, can not only bring benefits to their carriers, but in some cases can have a negative effect on their certain physiological and other properties (eg growth ability). Knowledge of the influence of the presence of antibiotic resistance genes on properties important for the spread of bacteria in the food chain (e.g. biofilm formation, growth ability, resistance to environmental stress conditions, etc.) is then important for determining the potential for their spread in the food chain and for developing methods to reduce it. The aim of the work will be to study and compare the isolates of the above groups of gram-negative bacteria isolated from food products with different antibiotic resistance profiles. The selection of important antibiotic resistance genes occurring in food isolates of the above groups of gram-negative bacteria will be performed. Furthermore, the occurrence of these antibiotic resistance genes in selected food products will be determined. It will be performed the isolation and quantification of target bacterial groups, genotypic and phenotypic characterization of the obtained isolates and comparison of the occurrence frequency of different genotypes and phenotypes. Modern methodological approaches such as next generation sequencing methods (e.g. nanopore sequencing), qPCR, MALDI-TOF MS and selected classical cultivation microbiological methods will be used for the study.

Investigation of the structure and function of Kingella kingae RtxA cytotoxin

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

Annotation

The secreted cytotoxin RtxA is a key virulence factor of the Gram-negative bacterium Kingella kingae, which is an important invasive pathogen causing septic arthritis, osteomyelitis, infective endocarditis and other diseases in young children. The current state of knowledge indicates that RtxA may play an important role in various steps of K. kingae infection, including colonization of the respiratory epithelium, bloodstream invasion and damage to target tissues. However, the detailed mechanism by which RtxA contributes to the pathogenesis remains largely unknown and its study is the central objective of the proposed doctoral project. Particular emphasis will be placed on the determination of membrane topology of RtxA, on the investigation of molecular mechanisms by which the toxin disturbs tight junction integrity of the respiratory epithelial barrier and on the examination of the effect of K. kingae and RtxA on the innate immune response of the respiratory epithelium. The results are expected to provide new knowledge on the RtxA toxin and on the pathogenesis of invasive K. kingae disease.

Isolation of novel therapeutics form fungi

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

Annotation

Fungi have been always on the forefront of biotechnological research, be it for antibiotics, food production, various enzymatic mixtures etc. Yet, there is still an untapped potential with regards to therapeutic use against cancer or various psychological disorders, although there are reports of fungi being traditionally used for such purposes. This dissertation project is aimed on the identification and isolation of novel therapeutics from common forest fungi such as Amanita muscaria and testing of the therapeutic potential of the isolated substances in various types of cancer cell lines.

Light upconversion nanoparticles for infra red photodynamic therapy of tumors

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

Annotation

For numerous tumor types resistance to long-term monotherapies usually develops. In order to overcome it, we shall develop and photophysically test nanoparticle-based systems for infra red photodynamic therapy. Threrefore, we will study detailes photophysics of nanoparticles, namely luminescence kinetics for upcovversion nanoparticles NaY(Gd)F4:Yb3+(Nd3+)/Er3+(Tm3+), which convert either 980? 650/540 nm or 808?650 nm. All these studies will be conducted in the absence and in the presence of a photosenzitizer in the nanoparticle shell, which creates singlet oxygen upon the desired excitation. The second imaging mode will be enabled for cracking these nanoparticles by NMR or PET. Particle surface engineering will include coating with polymers to achieve the highest energy transfer, immobilization a photosenzitizer to induce optimal singlet oxygen formation, and attachment of addressing peptides to facilitate cell internalization. Chelator for 64Cu or 125I attachment will be also introduced to integrate upconversion luminescence with PET imaging. Singlet oxygen production and pharmacokinetics of the particles on nude mice with xenotransplanted tumors will be also determined. The molecular biology techniques will be conducted by the coworkers of the Department No.75., Inst. Physiology.

Liquid chromatography–mass spectrometry for metabolomics and lipidomics studies

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

Annotation

Over the last decade, mass spectrometry-based metabolomics and lipidomics have become key disciplines for the comprehensive profiling of polar metabolites and complex lipids in biological systems. Liquid chromatography–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 PhD project aims to focus on (i) merging targeted and untargeted metabolomics and lipidomics methods, (ii) standardization of methods, and (iii) extending the breadth and scope of methods for metabolomics and lipidomics studies (e.g., type 2 diabetes, circadian rhythms). The work will be conducted at the Institute of Physiology CAS and financially supported by various grants (GACR, MSMT, AZV).

Mass spectrometry-based metabolomics of biological systems

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

Annotation

Untargeted metabolomics methods focus on the analysis of all the detectable metabolites in a sample, including chemical unknowns. Liquid chromatography coupled to mass spectrometry (LC-MS) is the preferred technique in metabolomics permitting effective compound separations and detection. However, up to 80% of all detected signals from untargeted profiling represent unknown metabolites. Such a big obstacle in biomedical and biological research hinders meaningful biochemical and pathway interpretations. The PhD project aims to focus on (i) increasing the coverage of spectral libraries used for metabolite annotation, (ii) applying programs for the prediction of ‘unknown’ metabolites, and (iii) using bioinformatics tools for visualization and interpretation of the data obtained within metabolomics studies (e.g., type 2 diabetes, circadian rhythms). The work will be conducted at the Institute of Physiology CAS and financially supported by various grants (GACR, MSMT, AZV).

Mechanisms of bacterial antibiotic resistance and their modulation

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

Microbial Cometabolism: Promoting Biodegradation of Pollutants

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

Annotation

The objective of the proposed thesis 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 will (i) selectively enrich soil bacterial consortia that will 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 will increase the efficiency of biodegradation of PCBs and PAHs in soils.

Microbiota of Selected Extreme Biotopes – Diversity, Ecology and Physiology

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

Annotation

The objective of the proposed thesis is to characterize microbial populations in extreme biotopes – chronosequence of permafrost (permafrost of differing age) and soils from salt marshes and moffettes. The characterization will be conducted by two major routes – metagenomics and modified cultivation techniques. The basic modification of the cultivation techniques will be 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.

Microorganisms and their utilisation in waste management

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

Annotation

Nowadays, more attention is paid to recycling and recovery of waste and therefore, the European Union has adopted a new Circular economy action plan. One option of reducing the environmental burden and decreasing the CO2 emissions in the (cement) building industry is the usage of recycled bio-concrete, which can be obtained using microbiologically induced calcite precipitation (MICP). This thesis's main goal will be to isolate and characterise suitable microorganisms and optimise MICP conditions concerning the yields, the structure and mechanical properties of bio-concrete.

Molecular characterization of various variants of fish parvalbumin gene in respect of biochemical and allergenic features of corresponding expressed proteins

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

Annotation

Fish parvalbumin gene is composed of four exons separated by three introns. The gene encodes highly thermoresistant parvalbumin protein. This protein can act as food allergen in predisposed consumers. Intron sequences of the gene can due to their uniqueness serve as species determination marker, while exons encode protein, including respective epitopes triggering allergic reaction. In each fish species, parvalbumin gene is represented by number of paralogs. The aim of the dissertation is to characterize these paralog/ortholog variants by means of molecular tools as well as by biochemical and immunochemical approaches, building on phylogenetic data obtained from databases as well as from NGS. Obtained dataset should also serve as a base for next construction of microarray chip for species determination or in phagemid version for pacient sera profiling to assess reactivity to a spectrum of various fish species meat.

Multivalent neoglycoconjugates with therapeutical potential

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

Annotation

Galectins are animal lectins with affinity to ?-D-galactosides, which in vivo participate in, e.g., cancerogenesis, metastatic processes, immune response to tumorigenesis, and other cancer-related pathologies. The concentration of extracellular galectins in vivo significantly rises in connection with cancer and therefore they may be used as diagnostic markers in many cancer types, such as colorectal carcinoma. Targeted inhibition of extracellular galectins is one of the novel prospective therapeutic approaches to the treatment of pathologies associated with galectin overproduction. A range of recent structure-function studies revealed detailed structural requirements of individual galectins for high-affinity and selective ligands. The avidity of specific glycomimetics to target galectins may be greatly increased by the multivalent presentation. The aim of this work is to synthesize multivalent neoglycoconjugates carrying specific saccharidic ligands or glycomimetics with high selectivity and affinity to target galectins. The inhibition and binding potential of these neoglycoconjugates to galectins, in particular to galectin-1 and -3, will be assayed by in vitro methods of ELISA and surface plasmon resonance (SPR) with recombinant galectins and, later on, with selected cultures of cancer cells.

Novel approaches in the elimination of cancer cells and bacterial biofilms

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

Preparation and biological activity of flavonoid metabolites

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

Annotation

Flavonoids, as other food polyphenols, are subject to extensive metabolism in the intestine and liver. Biotransformation of flavonoids by the intestinal microflora leads in particular to the cleavage of ring C and the formation of simple phenolic compounds. The structure of the main intermediate of this cleavage is not yet fully experimentally confirmed. There are at least three options, chalcone structure, benzofuranone derivative and depside. All biotransformation products of the polyphenols by the intestinal microflora may also be subject to conjugation reactions such as sulfation, methylation or glucuronidation. The aim of this work is to prepare a series of potential intermediates and final metabolites of flavonoids in a quantity sufficient for their detailed characterization using spectral methods and their use as standards for metabolic studies by means of chemoenzymatic methods introduced in the Laboratory of Biotransformation. In addition, the end products will be evaluated for their biological potential by testing their antioxidant activity with in vitro assays. Thus, this interdisciplinary study will include methods of organic chemistry, chemistry of natural products, biochemistry, microbiology, molecular biology, experimental toxicology, and analytical chemistry.

Preparation and structural-functional analysis of virus-like and non-viral nanoparticles

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

Annotation

The work is focused on the preparation of nanoparticles by self-assembly of viral structural proteins and on the basis of modified organic and inorganic structures. The particles will be surface modified to target pathogenic bacteria or tumor cells. Their ability to interact with target organisms for use in inhibiting, imaging and modulating cellular activity will be evaluated.

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.

Quorum sensing and its role in adaptation and virulence of Campylobacter jejuni

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

Annotation

Despite its fastidious nature, Campylobacter jejuni is continuously reported as the most common cause of foodborne zoonoses. Short genome with simultanously high level of adaptability of C. jejuni suggest that it is capable of rapid change of gene expression resulting in transition from a resilient but metabolically attenuated form outside a host into a virulent and rapidly multiplying form inside a host. This adaptability could be explained by extensive use of quorum sensing systems (QS). The main aims of the thesis involve investigation of the role of QS in lifecycle of C. jejuni, with an emphasis on identification of regulatory pathways mediated by acyl homoserine lactones. Significant part of the thesis will be dedicated to establishing the role of a host-like environment in signalling and pathogenicity of C. jejuni.

RNA metabolism related to the control of gene expression in the oocyte and embryo.

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Theses supervisor: Ing. Michal Kubelka, CSc.

Annotation

Fully grown oocyte utilizes only transcripts synthesized during earlier development. Mechanisms of mRNA metabolism in the mammalian oocyte are still far from being fully understood. In the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region which previously surrounded the nucleus. This hotspot is controlled by the activity of the mTOR/4F axis. We suppose that the localization of specific mRNAs and their in situ translation is essential for the generation of developmentally competent oocyte. Thus, the objective of the current project is to investigate translation regulation and polysome profiling of the oocyte after NEBD. We will test our hypothesis that translation of specific mRNAs is essential for meiotic progression before first polar body extrusion. This represents a first attempt to unveil novel regulators of mTOR/4F axis and will add substantial information concerning translational control of specific transcripts after NEBD in order to understand molecular physiology of mammalian oocyte. The aims of project: High-throughput translation analysis after resumption of meiosis in the mammalian oocyte. Regulation of the mTOR/eIF4E pathway after nuclear envelope breakdown..Translation and genomic stability in the mammalian oocyte.Main methods: Next Generation Sequencing, biochemistry, molecular biology, FISH.

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.

Annotation

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 plants 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.

Response of microbial communities to changing climate in Arctic tundra soils

Department: Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology
Study programmes: Mikrobiologie, Microbiology
Theses supervisor: RNDr. Jana Voříšková, PhD.

Annotation

Climate models predict substantial changes in temperature and precipitation patterns across the Arctic regions in future decades. Microbes are known to play key roles in determining the stability of soil carbon and its possible release into the atmosphere as greenhouse gases. Carbon-rich Arctic soil ecosystems are particularly vulnerable to carbon losses due to warming and subsequent ecosystem disturbances as wildfires. The aim of the project will be to explore the response of soil microbial communities to the conditions caused by future climate change, specifically increased winter precipitation, summer warming and wildfire disturbance. For the project, we will use the experimental sites in western and southern Greenland, maintained by University of Copenhagen, which simulate predicted changes in soil ecosystem. Microbial communities from collected soil samples will be characterized using state-of-the-art methods of molecular biology.

Retrovirus integration and epigenetic provirus silencing

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

Annotation

Autonomous transcription of integrated retroviruses and retrotransposons is affected by genetic and epigenetic features of respective sites of integration. These, mostly suppressive effects silence proviral transcription activity by mechanisms such as DNA methylation and posttranslation modifications of adjacent histone molecules. Provirus silencing is a general feature of integrated retroviruses, but its extent and kinetics vary among different retrovirus species. We studied the role of integration site using single cell clones bearing the unique integrations of reporter vectors derived mostly from avian leukosis virus (ALV) or human immunodefficiency virus (HIV). This appproach enabled us to correlate expression of reporter gene and the epigenomic landscape at the site of integration. The topic to be open for one doctoral student will be the immediate early silencing of proviruses, which occurs quickly after integration or even during the integration event due to the extensive epigenomic shift and DNA repair processes. For this type of studies, it will be necessary to establish a special dual reporter system of vectors, which records the narrow and quickly vanishing pulses of transcriptional activity during and shortly after integration. Furthermore, integration preference or various retroviruses will be studied at the level of primary DNA sequence, epigenomic landscape, functional chromosomal segments and 3D genome structure. It is expected that this study will finish with alterations of chromatin contacts and topologically associated domains (TAD) of chromatin at the site of retrovirus integration.

Role of adhesion-GPCRs during viral infection of mammalian cells

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

Annotation

The proposed PhD project addresses the possible role of adhesion G protein-coupled receptors (aGPCRs) during viral infection of mammalian cells. The aGPCRs are intriguing class of seven-transmembrane proteins that play important role in neurodevelopment, immune defense and cancer, but their role during viral infection is mostly unexplored. Currently, we recognize 33 members of aGPCRs in humans but majority are orphan receptors with unknown function. We intend to search for specific aGPCR and/or cellular proteins in aGPCRs pathways involved in infections of mammalian cells and characterize their interaction with viral proteins. During postgradual study, student will learn how to work with cell cultures in biosafety level 3, perform proteomic and bioinformatic analyses and siRNA experiments for gene-knockdown in mammalian cells infected with various viruses. Proposed project will broaden our current knowledge about the role of aGPCRs in infections of mammalian cells and if successful, it can identify potential targets for antiviral therapy.

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.

Annotation

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-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.

Studying speciation of arsenic in the mycosphere - from microorganisms to animals

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

Annotation

The goal of this work is to answer questions related to not so well studied biotransformations of arsenic in living organisms, with emphasis on fungi. Arsenic is rather toxic, but since it is ubiquitous, organisms have evolved various methods of As detoxification, including redox reactions, methylation and other biotransformation reactions. We suppose, that these bioprocesses are dependent on many factors, including As bioavailability and genetic background of each organism, and various compounds of the so called mycosphere, i.e. all factors related to fungi (geochemistry, microbiome, fungivores). Each factor and process of the biotransformation can thus be studied from a different perspective. The primary goal of this work will be identification, isolation and functional characterization of genes potentially playing role in As transformation in fungi and bacteria and their screening by metagenomic techniques in soil samples and fungal sporocarps. A second approach will deal with growing fungal mycelia in substrate amended with As and assessing the potential of the mycelia to transform inorganic As forms to organic by mass spectrometry. Both these approaches should answer questions such as: Can fungi methylate As? Do As forms correlate with fungal phylogeny? Can fungal As hyperaccumulators change the speciation of As in soil and thus in the surrounding bacterial community? How are As compounds transformed and translocated in the mycosphere?

The mannose-6-phosphate/insulin-like growth factor 2 receptor as a target for memory enhancement and tumor suppression

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

Annotation

Identifying new compounds that effectively improve neurodegenerative disease is an urgent need. It has been recently shown that insulin-like growth factor 2 (IGF2) has an important role in enhancement of memory. IGF2 is a fetal growth factor that has also significant role in mature mammalian brain. The effect of IGF2 as a cognitive enhancer is rapid and persistent, and is mediated by mannose-6-phosphate/IGF2 receptor (M6P/IGF2R). M6P/IGF2R is a large transmembrane glycoprotein that except IGF2 can bind M6P-containing ligands and binding of one ligand may affect the binding another one. Moreover, overexpression of IGF2 plays important role in development of certain tumors and M6P/IGF2R can play a role as a tumor suppressor. This project is focused on (i) production of M6P/IGF2R or its constructs, (ii) preparation of IGF2R selective IGF2 analogues, and (iii) a libraries of small molecule compounds. This work should lead to important clinical applications of new IGF2 analogues.

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

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

Annotation

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.

Virulence Mechanisms of the genus Bordetella

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

Annotation

Pathogenic Bordetella species cause respiratory infections in mammals. While B. bronchiseptica is primarily an animal pathogen, B. pertussis is the causative agent of the whooping cough disease in humans. The PhD thesis aims to reveal the novel mechanisms of Bordetella virulence by genome-wide CRISPR/Cas9-mediated screen. A pool of knock-out human epithelial cells will be generated by an already validated gRNA library. Following Bordetella infection, the candidate genes that confirm the sensitivity of epithelial cells to Bordetella cytotoxicity will be determined. The hits will be further validated and their role in susceptibility to Bordetella infection confirmed.


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