Projects / Programmes
Molecular biotechnology: from dynamics of biological systems to applications
January 1, 2004
- December 31, 2008
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| 1.09.00 |
Natural sciences and mathematics |
Pharmacy |
|
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| B510 |
Biomedical sciences |
Infections |
| B725 |
Biomedical sciences |
Diagnostics |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
molecular basis of disease, lipopolysaccharide, cellular receptors, signal transduction, sepsis, biosensors, antibodies, biotechnology, prions, antimicrobial compounds, primary metabolism, structural biology, molecular modeling
Researchers (32)
Organisations (3)
Abstract
Life sciences experience the transition from molecular to the systemic level, fostering the development of a new quality in our understanding of complex biological systems such as immune and metabolic system. Program connects basic research with application in the areas of research, which have primarily relevance to the health. The emphasis of the program is on the understanding of the response of the human organism to bacterial infections and conformational diseases at the molecular level and understanding of the microbial metabolism directed towards the improvement of the various pharmaceutically interesting products. Within the research of the LPS receptors we will investigate the extracellular (MD-2, TLR4, CD14) as well as intracellular receptors. Understanding of interactions at the molecular level can lead to new compounds, which might be used to neutralize the excessive immune response. The aim of this research is to elucidate the biochemical specificity of the so called “PAMP receptors”, i.e. receptors that recognize the molecular patterns of pathogenic microorganisms. Based on the structure-activity relationship (SAR) we plan to develop new (lipo)peptides and establish the differences in the selectivity between bacterial and eukaryotic membranes. In the area of health we are also investigating diseases, particularly transmissible spongiform encephalopathies (TSE) connected to conformational changes of proteins. Monoclonal antibodies have been established as diagnostic tool as well as for the therapy. By the combination of immunochemistry with methods of protein and structural biochemistry we made the first step analyzing the specificity of antibodies that recognize the pathogenic form of prion protein (PrPSc), linked to Creutzfeld-Jakob disease. This and other tools will allow us to improve the detection and early diagnostics of disease and in parallel to contribute to the understanding of the infectivity of TSE. Program also involves the research of primary metabolism of industrial microorganisms, particularly 6-phosphofructo-kinase (pfkA), whose properties significantly change during the posttranslational modification. We intend to transfer the shortened gene encoding more active enzyme into other commercially important microorganisms. We will develop the new fluorescent intracellular biosensors to monitor the events particularly in eukaryotic cells. We will put the emphasis on the analysis of Ca, cAMP and pH inside of living cells.
Significance for science
Emphasis of the program was on molecular mechanisms of physiological processes that are relevant to health and on the application of fundamental knowledge to improve health through the development of active substances and diagnostics. Within the research of innate immunity we focused on the recognition of bacterial endotoxin (lipopolysaccharide, LPS) through a complex of Toll-like receptor 4 (TLR4) and MD-2, which directly recognizes and binds bacterial LPS. We constructed a structural model MD-2. In a series of publications, we identified natural and synthetic compounds that bind to MD-2 and act as LPS antagonists, which is very important for therapy in the early stages of bacterial infection and to prevent sepsis. We found that the hydrophobic pocket in MD-2 contains a free cysteine residue. This represents an opportunity to design a new type of irreversible inhibitors. We determined the functional consequences of the polymorphism of human MD-2 (G56R), which reduces the responsiveness to LPS. Knowledge of the mechanisms of innate immunity and cell signaling allows us to modify signaling networks using the approach synthetic of biology, aimed to protect human cells against infection. In the first project we limited the exaggerated response of cells to LPS, which could be used to prevent the development of sepsis. The second project focused on the identification of infection with HIV-I, where the detection of virus independent on viral mutations, which cause the drug resistance and ineffectiveness of vaccines. We analyzed the mechanism of antibacterial agents such as gyrase inhibitors, and (lipo) peptides, which in addition to antimicrobial action also neutralize endotoxin. We improved the efficiency of antibacterial peptides against a wide range of bacteria and the ability to neutralize endotoxin. We have determined the structure of several peptides in complex with LPS, identifying a new structural motif of antimicrobial peptides and demonstrated the importance of acyl groups on lipopeptide structure, allowing the rational design of improved drugs. Under the influence of prions, the native cellular form of PrP is converted into amiloid form. We found that a compound of natural origin curcumin binds to forms with a higher proportion of ß-structure. Curcumin recognizes a partially unfolded form, which is present at the acidic pH and contains a large proportion of ?-structure. The need for effective diagnosis, in conjunction with blood transfusion includes the population studies in which we determine the prevalence of three mutations in the gene associated with hereditary hemochromatosis HFE in Slovenian population by a method which we have developed based on real-time PCR. In addition to work in mammalian systems, we continued work on the studies of primary metabolism of fungi, which change phosphofructokinase for improved production of secondary metabolites, which are important for the industry, particularly drug production. In the fungus Aspergillus niger, we found that proteolytic cleavage of the native enzyme PFK1 produces a short fragment, which retains the enzymatic activity, but with modified kinetics properties. The resulting fragment is sensitive to inhibition by citrate, but some activators increase its activity at a higher level than the native enzyme.
Significance for the country
Our research program was important for Slovenia through achieving an internationally high level of science and through training of highly qualified personnel. Research topics were mainly in the area of health related problems, where we become established in the research community in the field of defense mechanisms against pathogens. Results could be used to improve human health, not only in Slovenia but globally. These results could also lead to tangible high-tech products that we intend to commercialize through the different approaches. During the last program period, our program group participated in exceptional successes in the competition of research projects among the most prestigious academic institutions, which contribution to the promotion of science within the general audience and promotion of Slovenia as a country with high quality of science and education. Those successes have resonated in Slovenia as well as around the world with hundreds of publications in newspapers, magazines, Slovenian daily newspapers, radio, TV, websites (e.g. the EU Commission, more than 18,000 hits from all continents on the website of our project). We developed inventive methods of detection of prion diseases. Infectious prions represent an economic burden and present a potential for business initiatives. We protected the intellectual property by patents, which may allow their sale or licensing or creation of business initiative. In particular, we can expect the possibility of a rapid commercialization of new diagnostic methods. We proceeded with commercialization of patent rights for the use of modified pfkA gene, which will increase the metabolic flow through glycolysis after introduction into commercial microorganisms and cause increased production of specific commercial end products.
Most important scientific results
Final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Final report,
complete report on dLib.si
