čas: 19.4.2021 04:00:05
Obnovit | RAW
Institute of Microbiology of the CAS, v.v.i.
List of available PhD theses
Biocatalysts for a new route of nitrile synthesis
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.
Heterogeneity of expression of effectors of Salmonella pathogenicity island 2?encoded type 3 secretion system
Investigation of the structure and function of Kingella kingae RtxA cytotoxin
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.
Multivalent neoglycoconjugates with therapeutical potential
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.
Preparation and biological activity of flavonoid metabolites
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.
Response of microbial communities to changing climate in Arctic tundra soils
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.
Structural and functional diversity of bioactive specialized metabolites produced by Actinobacteria
Actinobacteria represent a renowned source of a broad spectrum of biologically active metabolites applicable in medicine. Even though this source of natural products nowadays faces challenges such as re-discovery of known compounds, we assume that we may exploit the once efficient source of antibiotics or anticancer drugs again if we employ more innovative strategies. Within this project, we will prepare a library of extracts and fractions containing metabolites obtained from a collection of soil Actinobacteria. We will attempt to target the compounds of interest in the upstream workflow process using modern metabolomics approaches. We will screen the library against a set of multi-drug resistant pathogens, for which antibioticas are urgently needed according to the World Health Organisation. These include Clostridioides (Clostridium) difficile, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and others. We will also submit samples with the metabolites from Actinobacteria into the chemical library of the CZ-OPENSCREEN facility for high throughput screening of various specific biological activities. The most promising metabolites will be purified and structurally elucidated by mass spectrometry and nuclear magnetic resonance.
Structure-Function Relationships of the Bordetella Type III Secretion System Proteins
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.
The benefit of 4-alkyl-L-proline motif for biological activity of microbial metabolites with distinct modes of action
4-Alkyl-L-proline derivatives with a two- or three- carbon side chain represent rare building blocks of metabolites produced by Actinobacteria. Only five groups of compounds with remarkably distinct biological activities share this structural motif. The metabolites cover antimicrobial compounds with different spectrum of targeted microorganisms as well as metabolites with various antitumor effects. The modes of action of these metabolites are also diverse – they include interaction with bacterial ribosome (lincomycin), binding to the minor groove of DNA (pyrrolobenzodiazepines), and inhibition of bacterial enzymes participating in DNA synthesis (mycoplanecin). Target biological structures of the two remaining groups of compounds (hormaomycin, lucentamycin) are still elusive. Whereas the positive effect of the 4-alkyl side chain on the biological activity was well documented for lincomycin, the significance of this evolutionary and biosynthetically complex structural motif for the activity of the remaining metabolites is not clear. Within this project, we will prepare metabolites with incorporated 4-alkyl-L-proline derivative and their analogues with common L-proline and compare their relevant biological activity. The metabolites will be mostly isolated from the culture broth of producing strains including the use of semi-synthetic approaches (eg. precursor-directed biosynthesis or mutasynthesis) and modern analytical chemistry approaches. The project objective will be to evaluate the general benefit of the 4-alkyl-L-poline motif for the level of various biological activities. The new knowledge obtained may be exploited for the biotechnological preparation of efficient compounds potentially applicable in medicine.
Virulence Mechanisms of the genus Bordetella
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.