National Science Centre

The aim of the project is to obtain a cyclic peptide, ligand of the peroxisomal protein PEX5, as a molecular probe for the study of peroxisome biogenesis and function. Peroxisomes are unique organelles whose functions vary according to the type of cell and the stage of development of the organism. They play a very important role in cellular metabolism. The most important biochemical processes taking place in peroxisomes are β-oxidation of fatty acids, detoxification and protection against oxidative stress. In Trypanosoma parasites, the function of the peroxisome is played by the glycosome in which glycolysis takes place, the process of supplying the cell with ATP. For the proper functioning of peroxisomes, cytoplasmic enzymes are necessary, which have special signaling segments (PTS), directing them to the interior of the peroxisome through the receptors circulating in the cytoplasm (PEX5 and PEX7). Most peroxisomal enzymes contain either the first or the second type of targeting signal (PTS1 and PTS2). Our project aims to obtain a cyclic peptide that will competitively bind to PEX5, blocking the formation of the PTS1/PEX5 protein complex, and thus prevent the targeting of enzymes to the target organelle. The PTS1 stretch is a C-terminal peptide with the amino acid sequence Ser-Lys-Leu. The starting point for the design of the cyclic peptide was the analysis of the PTS1 motif binding pocket structure and the method of sequence binding by the PEX5 protein. The designed peptide should have high affinity for PEX5 and inhibit the formation of the PEX5/PTS1 complex. At the same time, this peptide has a cyclic structure (ring closure by a hydrophobic, aliphatic bridge), which should increase its metabolic stability and the ability to penetrate biological membranes.

Peptide synthesis was planned following standard Fmoc peptide synthesis procedures on a solid support (resin). The first stage of the project will be the synthesis of indispensable non-natural, commercially available amino acids: (S) -2 - ((tert-butoxycarbonyl) amino) -4,4-dimethylhex-5-enoate and N-5- (but-3-en-1- yl) -N-2- (tert-butoxycarbonyl) -L-glutamine.

The next stage of the research task consists in attaching the C-terminal amino acid to the 2-chlorotrityl resin and then extending the peptide chain.

The next step in the synthesis is the selective cleavage of the peptide from the resin while maintaining orthogonal groups protecting the side chains of amino acids. The last, key stage of the synthesis is cyclization by means of metathesis, reduction of the double bond and deprotection of peptide functional groups in the environment of trifluoroacetic acid.

The obtained cyclic peptide will be tested for the dissociation capacity of the PEX5/PTS1 complex (e.g. by the fluorescence polarization (FP) method). The results of the described scientific activity will allow to determine the validity of planning another project aimed at optimization of cyclic peptides, inhibitors of PEX5/PTS1 complex formation.

We assume that such compounds could be valuable molecular probes that will help better understand the PEX pathway and the mechanism of protein transport to peroxisomes. They may also become potential drugs against diseases caused by trypanosomes (Chagas disease, African sleeping sickness).

Urinary tract infections are a widespread disease affecting the majority of the world's population, caused mainly by E. coli infections. In the past, one of the main treatments for urinary tract infections was the use of plant extracts. With the onset of the era of antibiotics, plant raw materials were forced out of use in favor of more effective antimicrobial drugs with more adverse effects. Due to spreading drug resistance of microorganisms, alternative methods of preventing and treating infections are currently being sought. One such approach is to revert to naturally derived substances to treat mild urinary tract infections.

Raw materials such as meadowsweet flower, orthosiphon leaf, seaweed herb, goldenrod herb, knotweed herb, horsetail herb, birch leaf, bearberry leaf or lingonberry leaf are traditionally used herbal remedies to treat inflammation and infections of the urinary tract. While it is known that the last two are highly effective when used by patients, the effect of other herbal substances is not fully documented. In addition, even in the case of bilberry and bearberry leaves, the exact mechanism of these raw materials' effect is not fully known. In recent years, more and more attention has been paid to the role of the intestinal microbiota in the process of metabolism of substances ingested through the alimentary tract. It is known from other studies that bacteria of the intestinal flora metabolize compounds of natural origin into simpler substances that are well absorbed into the bloodstream and have specific effects. Additionally, some intestinal metabolites have been shown to be present when absorbed into the bloodstream ultimately converted to glucuronides or sulphates through phase II metabolism and ultimately excreted in the urine. These compounds may affect the pathological processes in the human urinary tract. In this project, we plan to comprehensively investigate the chemical composition of water extracts prepared from selected plant materials traditionally used in diseases of the urinary tract. Next, we will determine the intestinal metabolism of the compounds contained in these extracts. We will also conduct biological tests involving
evaluation of the antibacterial, anti-adhesive and anti-inflammatory activity of the selected extracts of their ingredients and compounds that are their intestinal metabolites. Ultimately, we will try to verify the hypothesis whether the metabolites of substances present in the tested extracts are excreted in the urine and can reduce inflammation at this point
and kill the bacteria causing infection.

The main objectives of the project are: 1. expanding the current knowledge on genetic causes of severe and lethal human developmental defects; 2. identification of new genetic factors in their etiology with the use of genomic research technologies including CGH microarray (aCGH) testing and full exome sequencing (WES) from the material collected from the fetus in the second and third trimesters; 3. assessment of the frequency of pathogenic variants including submicroscopic chromosomal rearrangements and single nucleotide variants in a group of fetuses with at least two malformations, with severe phenotype and poor prognosis, often lethal; 4. analysis of the correlation of aCGH results with the results of WES sequencing in order to identify aneuploidy and large chromosomal aberrations. The practical goals of the project include: 1. development of an algorithm for diagnostic management in fetuses with multiple developmental defects using the aCGH and WES techniques. 2. development of rules for the interpretation of WES test results and assessment of their importance for genetic counseling in families at risk in relation to multiple congenital abnormalities. 3. development of methods allowing for comprehensive diagnosis of numerical and large structural chromosomal aberrations (aneuploidy, large deletions / amplification) and point mutations using a uniform technology based on next-generation sequencing.

The authors of the project have undertaken research into this thematic scope in order to explain the previously unknown causes and mechanisms of severe and lethal developmental defects. This will allow a better understanding of the biology of human development and the creation of new diagnostic tests aimed at determining the risk of their occurrence, which will also affect the development of family counseling. The application of genomic methods to this group of diseases should lead to the discovery of new pathogenetic conditions. The importance of these findings may extend beyond the problem of malformations, as it can be expected that some of the genes associated with abnormal pregnancy development may also be important in the pathogenesis of other non-lethal human diseases. The planned research will be carried out for the first time in Poland and as one of the first in the world, and their results will have both cognitive and practical significance.

Cardiovascular diseases, including acute coronary syndromes (ACS), are the most significant cause of death on a global scale. In 2009 alone, the global cost of treating cardiovascular disease was estimated at € 106 billion. Every year, over 1.1 million new ACS cases are registered in the European Union. Prevention of ACS and their recurrences
would therefore allow a reduction in mortality, prevent disability and lower the cost of treatment. ACS is based on the activation and aggregation of platelets on the ruptured atherosclerotic plaque, which leads to the formation of a blood clot in the lumen of the coronary artery, impaired blood flow and, ultimately, necrosis of the heart muscle. Inhibition of platelet function by the use of antiplatelet drugs (antagonists of the platelet P2Y12 receptor - ticagrelor or prasugrel) is the most effective strategy available today to prevent recurrent ischemic events after ACS. Unfortunately, despite optimal antiplatelet therapy, approximately 10% of ACS patients experience another ischemic episode within a year. Identification of these patients is currently not possible.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of 60% of human protein-coding genes, including those responsible for the development and progression of atherosclerosis. Recent studies have shown that platelets contain significant amounts of miRNA that is released into the bloodstream in free form or bound to platelet microparticles. The profile of circulating miRNAs changes in various pathophysiological states, including ACS. The aim of our project is to assess the effect of potent antiplatelet drugs (ticagrelor and prasugrel) on circulating platelet-derived miRNAs, as well as to assess the possibility of using miRNAs in predicting the risk of recurrent ischemic episodes in patients after ACS.

Chronic inflammation plays a key role in the development and course of neoplastic diseases of the hematopoietic system, including myeloid and lymphoid leukemias. Recent reports suggest that excessive and uncontrolled activation of a protein structure called the inflammasome may be a determinant of chronic inflammation. Strong activators of the inflammasome include reactive oxygen species resulting from dysfunction and / or life cycle of the mitochondria, danger associated molecular patterns (DAMPs) and ATP. Function, life cycle and mitochondrial degradation through mitophagy are regulated by parkin, an enzyme essential for the proper functioning of the ubiquitin-proteasome system. Literature data show that the activity of parkin is regulated, among others, by caspase 1, and oxidative stress-dependent deregulation of parkin is associated with the accumulation of non-functional mitochondria in cells. Determining the importance of parkin in the molecular mechanisms of inflammasome activation will significantly influence further research on the development of a method of inhibiting parkin-related mitochondrial dysfunction, thus preventing excessive activation of the inflammasome and the development of neoplastic diseases of the hematopoietic system.

The aim of the project is to search for compounds characterized by high activity and specificity for the histamine H4 receptor by means of molecular modeling methods, synthesis of selected compounds, receptor studies, characterization of their cytotoxicity and genotoxicity, and analysis of potential anti-allergic and anti-inflammatory properties in vitro. H4 receptors are located primarily on cells of hematopoietic origin, e.g. eosinophils, mast cells, basophils, neutrophils, dendritic cells, monocytes, T lymphocytes, and participate in many inflammatory processes; therefore, the H4 receptor is considered a molecular target for drugs used in the treatment of diseases immune systems such as allergies, asthma and autoimmune diseases. As both the histamine HI and H4 receptor antagonists have anti-inflammatory and antiallergic properties, a synergistic effect was also found between the HI and H4 receptors; therefore, an additional project goal will be to search for double ligands for H1/H4 receptors. These ligands can be both different chemical structures linked by a linker as well as a uniform chemical structure designed using the knowledge of the binding sites of H1 and H4 receptors. The aim of the project is also to build a model of the H1/H4 heterodimer and to understand the mechanisms of action of selected compounds on the H4 receptor and the H1/H4 heterodimer. 

Each year, there are about 10 million new cases of tuberculosis and nearly 2 million deaths from this disease, making tuberculosis one of the most important health problems in the world. The main goal of the project is to conduct a comprehensive analysis of the genetic structure of Mycobacterium tuberculosis strains, both with a drug-sensitive and drug-resistant phenotype, from Lithuania and Poland. This analysis will be based on the detailed characteristics of the identified genotypes, their comparison with each other and with genotypes described in other European countries and in the world, estimation of tuberculosis transmission between Lithuania and Poland and, within these countries, determination of genetic determinants of drug resistance, as well as, possibly, other phenotypic characteristics (infectivity, virulence), taking into account their clinical significance in the course of diagnostic and therapeutic procedures in tuberculosis.

The proposed project is so far the largest study in the field of molecular epidemiology of tuberculosis in Lithuania and Poland. The results of the conducted research will significantly expand the knowledge on the genetic determinants of drug resistance, virulence and transmission of M. tuberculosis, paving the way for the development of new diagnostic and therapeutic strategies in tuberculosis. The research provided for in the project corresponds with the global strategy of fighting tuberculosis, leading to the elimination of the disease as a health problem for the human community.

The planned research is an original attempt to determine the effect of the addition of Punica granatum seed oil and water extract of Momordica charantia dried fruit, introduced into the diet, separately or jointly, on the intensity of lipid oxidation processes in selected tissues of rats. Previous studies on these raw materials, which are rich sources of many bioactive compounds, including conjugated linolenic acid trienes (CLnA), do not clearly indicate the direction of these interactions. A completely new element of the research task is an attempt to explain the course of lipid oxidation processes in a physiological and pathological state (carcinogenesis). The added value, significantly increasing the scientific value of the project, is the analysis of the simultaneous use of raw materials with a potential anti-cancer effect, which may contribute to explaining their potential synergistic or antagonistic activity in the context of the dynamics of lipid oxidation processes in animals.

Ischemic stroke (IS) is the second most common cause of death in Europe, responsible for approximately 1.1 million deaths annually. The study will assess the usefulness and relevance of the new molecules as potential biomarkers of platelet activation in ESUS patients, namely platelet-derived extracellular microparticles and selected microRNAs.

Acute B-cell lymphoblastic leukemia (ALL) is a genetically differentiated cancer of the haematopoietic system. This type of leukemia is most common in children, but it can also affect adults. In recent years, the effectiveness of the treatment of ALL has significantly improved, but the best treatment effects are mainly observed in children. Unfortunately, not all ALL subtypes respond to standard chemotherapy treatments. One of the subtypes with a particularly poor prognosis is ALL with MIL gene rearrangement (MLLr ALL). The incidence of this particular subtype of ALL mainly affects infants, less often adolescents, but in the elderly the risk of developing the disease increases with age. Given the poor response to chemotherapeutic agents and the qualification of MLLr ALL to a high-risk group, it is necessary to search for new, targeted therapies to improve treatment outcomes.