čas: 7.6.2020 10:06:28
Obnovit | RAW
Institute of Microbiology of the CAS, v.v.i.
List of available PhD theses
Application of Advanced Molecular Methods to Reveal Bacterial Mechanisms of Virulence
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.
Mechanisms of Action of Bordetella Type III Secretion System Effector Protein BteA
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.
Microbial community patterns under soil land use changes.
Soil is considered the most microbial diverse environment on Earth. Understanding the traits that control the biological entities in such rich environment is of interest to all fields of life sciences, from ecology and environmental microbiology to agriculture, biotechnology and health. Effects of human activities such as land use changes had led to temperature increase, nitrogen contamination, with a direct impact on microbial biomass and their functional profiles. The current methods for studying microbial communities produce data that allows us to identify latent variables which can control the changes in microbial communities, considering the power of sequencing the whole community and disentangling sample relationship from the environmental data. The limitation nowadays resides in gathering, processing and analyzing the sample data in a standardized way, on top of the specialist interpretation of the results. The proposed solution to such a problem would be to build a community fed database that could be used together with a pipeline for identifying those latent variables, which would be the species response to environmental changes.
Microbiome and metabolome based study of obesity, diabetes, and neurodegeneration and the therapeutic potential of prolactin-releasing peptide analogs by metabolomics
It was well established that the gut microbiome contributes substantially to human health/disease status. Unfortunately, there is limited information about the underlying molecular mechanisms of gut microbiome-host crosstalk. Recent studies have suggested that gut microbiota play a fundamental role in diseases such as obesity, diabetes, cardiovascular and neurodegenerative diseases like Alzheimer’s disease. They are the world's leading causes of morbidity and mortality and are responsible for 60% of all deaths. It appears that obesity, type 2 diabetes (T2DM), and neurodegeneration have a common etiopathogenesis involving subclinical inflammation and insulin resistance. The lumen of the gut is the biggest body surface facing the outer environment, and the mucosal immune system must be tightly regulated. The gut microbiota plays an important role in educating and modulating the host immune. Therefore, disturbance of the microbial ecosystem, called dysbiosis, substantially contributes to the establishment of the inflammatory state. We will use animal models of Alzheimer’s disease, where the modulation of the host immune system plays a crucial role. We assume that one of the mechanisms of how the gut microbiome associated with these conditions communicates with the immune system of the host is mediated by small molecular metabolites. The metabolites, which are usually identified as present in the gut and associated with pathologies, may be derived from the diet and/or are products of microbial metabolism. In the project, we will focus on interplay between disease development, microbiome and changes in metabolism to explore the development of the disease and efficacy of new lipidized analogs of the anorexigenic prolactin-releasing peptide as a complex therapy that simultaneously affects obesity, T2DM, and neurodegeneration in mouse and rat models.
Regulation of focal adhesion dynamics by calpain-2 mediated proteolysis of focal adhesion protein paxillin
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.
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.
Structure-function relationships in selected α-L-rhamnosyl-β-D-glucosidases
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.