National Science Centre

Lifestyle modification through diet, regular exercise and weight control is the primary means of lowering triglycerides and glucose levels, and studies have proven the effectiveness of such changes in the prevention and treatment of diseases related to metabolic syndrome. Due to the high cost of their treatment, both in terms of human life and economic considerations, the search for safe and effective natural agents supporting their therapy seems highly desirable. One of the therapeutic goals in the treatment of obesity and other diseases related to metabolic syndrome is to inhibit digestion and the absorption of nutrients, such as fats and carbohydrates, by inhibiting the action of digestive enzymes. For this reason, the digestive tract and the intestinal barrier play a key role in this type of disease. The intestinal epithelial cell layer provides a complex defense system that separates the intestinal contents from the host tissues, and also contains the cells of the immune system. The integrity of this system is necessary to maintain normal intestinal permeability, as its disturbance leads to the flow of intestinal lumen contents into the circulation (intestinal barrier leakage) and the development of gradually increasing chronic inflammation in the tissues. Increased permeability is believed to be the basis of the pathogenesis of many diseases such as enteritis, irritable bowel syndrome, autoimmune diseases (celiac disease, atopic lesions), type I diabetes, acute pancreatitis, cirrhosis, as well as multiple sclerosis and rheumatism. Even diseases that are not directly related to mucosal functions, such as heart failure and myocardial infarction, are likely to be exacerbated by increased mucosal permeability. Therefore, the prevention of intestinal barrier leakage or inflammation of the intestine by preparations of plant origin or plant materials rich in bioactive compounds is of preventive importance. This proposal tries to show the beneficial role of medicinal and traditional herbal preparations, which are often used especially in Central European countries. Due to the widespread use in the form of herbal teas, marmalades, jams, jellies and drinks, it seems reasonable to search for compounds inhibiting the absorption of certain dietary components among these raw materials. Among the generally available dietary ingredients, products obtained from hop cones, chokeberry fruit, wild elderberry, quince, dogwood or wild rose, as well as hibiscus flowers and hazelnuts play a significant role. Others, such as sea buckthorn, barberry, rowan or acorns, are increasingly gaining in importance. The herb of mugwort and black cumin seeds are used as culinary spices.In the context of searching for multidimensional factors in the prevention of the metabolic syndrome among plant species rich in bioactive compounds, an initial selection of the material selected for the study will be carried out on the basis of the assessment of lowering the activity of enzymes associated with inhibiting the digestion of fats and carbohydrates. The most active extracts will undergo subsequent stages of the artificial digestion process. Therefore, it will be possible to identify plant-based ingredients that actually reach the large intestine. Ultimately, the project plans to conduct studies using the human colon cell model - the Caco-2 cell line growing in a monolayer - which is used to assess the absorption of compounds in vitro. Recent studies report an interesting question about the signals sent by the intestinal flora as well as the interaction of the bacterial flora with the intestinal mucosa. The bacterial-epithelial interaction is believed to be important in the maintenance of intestinal homeostasis and has a profound effect on the host's immune system. The project aims to elucidate the role of plant materials in maintaining the integrity of the intestinal wall and protecting the mucosa, especially against agents secreted by pathogenic bacteria that can enter the bloodstream and trigger an immune response. What's more, the test will help to determine which extract ingredients are absorbed and whether they also have anti-inflammatory effects. The growing awareness of patients is visible in the increase in the distribution of probiotics on the pharmaceutical market, not only in connection with antibiotic therapy. Increasingly, probiotics are recommended to reduce the incidence and severity of diarrhea in children as well as autoimmune diseases (atopic dermatitis). It is believed that the immature layer of intestinal villi in newborns is the primary route of allergen invasion. Natural products can strengthen the functions of the physiological barrier between the bacterial flora and the intestine, as well as protect against pathogenic factors. This effect likely translates into health benefits from childhood to adulthood. The use of fiber and natural compounds, especially polyphenols, and preparations rich in polyphenols, used in everyday life in the form of teas and drinks, can be an effective support for conventional therapy and prevention of civilization diseases. In addition, attention should be paid to the safety of these preparations in comparison with drugs used in the long term in the treatment of metabolic diseases. In particular, patients discouraged from conventional therapy due to the side effects of synthetic drugs tend to choose natural products. In the future, the solution to the presented issue may therefore be of great preventive importance. Given that prophylaxis provides more benefits than treatment, this type of action at the gut level appears to benefit both patients and the healthcare system.

The main goal of this project is to develop novel crystalline calcium phosphates enriched with a variety of ions of potential biomedical importance. Another primary goal of the project is to develop two- and multiphase materials with different ion release characteristics. Basic research carried out under the project: Synthesis of various crystalline calcium phosphates (hydroxyapatite (HA), calcium orthophosphates (βTCP and αTCP) of calcium hydrogen phosphates (anhydrous CaHPO4 (DCPA) and dihydrate CaHPO4 ∙ 2H2O (DCPD)) containing admixtures of "foreign" ions: cations I, II or III-valued (e.g. K +, Ag +, Zn2 +, Mn2 +, Mg2 +, Cu2 +, Ga3 +) and various anions (e.g. SeO3 2-, SeO4 2-, SiO4 4-, BO3 3-). chemical composition of the obtained substituted calcium phosphate materials and physicochemical studies of these materials Planned research methods: X-ray powder diffraction (PXRD), mid-infrared spectroscopy (FT-IR), Raman (R) spectroscopy, solid state nuclear magnetic resonance spectroscopy (ssNMR), atomic absorption spectrometry (ASA), wave dispersive X-ray fluorescence (WD XRF) and excited plasma emission spectrometry (ICP OES). Analysis of ion release from the obtained materials (ICP MS plasma excitation mass spectrometry method). Development of two- and multiphase materials based on the obtained substituted calcium-phosphate materials with different rates of foreign ion release. Assessment of biological properties of the obtained materials. Reasons for undertaking the research topic: Calcium phosphates are a group of inorganic materials of significant importance in implantology, regenerative medicine and dentistry. In mineralized human tissues (i.e. bones, enamel, dentin or tooth cement), the inorganic building material that provides them with hardness is the so-called biological apatite, i.e. nanocrystalline carbonate hydroxyapatite containing many different ionic substitutions. Therefore, synthetic bone replacement materials, metallic implant coatings, endosteal cements or dental materials often contain calcium phosphates, which ensure biocompatibility, bioactivity and low cytotoxicity. For many years, the most popular among synthetic calcium phosphates, hydroxyapatite (HA) with the total formula Ca10 (PO4) 6 (OH) 2, and at the same time the least resorbable, is effectively doped with various ions in order to enrich it with additional biological, physicochemical or mechanical properties. On the other hand, very little is known about the possibility of ionic substitutions in other crystalline calcium phosphates, e.g. calcium orthophosphate Ca3 (PO4) 2 (βTCP and αTCP form), crystalline anhydrous calcium hydrogen phosphate CaHPO4 (DCP) and calcium hydrogen phosphate dihydrate CaHPO4 ∙ 2H2O (DCP∙ 2H2O) ). Few studies on the enrichment of βTCP and αTCP show that such materials are characterized by better solubility and faster release of foreign ions than substituted apatites. Currently, in medicine, two-phase materials (most often containing pure, unsubstituted HA and βTCP in various proportions) are used, which ensure better bioactivity and easier osseointegration. The development of new substituted calcium phosphates, planned in our project, is aimed at broadening the knowledge in the field of ion exchange in selected crystalline calcium phosphates. We want to develop materials with different resorbability and ion release rates. We expect that the results of the research planned by us will be particularly important for the progress of biology, medicine and materials engineering (designing bone substitute materials).

The aim of the planned research is to link the activity of the pump systems responsible for multidrug resistance (MDR) with the drug resistance profiles of clinical strains of Stenotrophomonas maltophilia and to search for new efflux systems present in this species. Currently, the main research on the pathogenicity of Gram-negative rods concerns strains of the Enterobacteriaceae family. However, the role of strains of other species in pathogenicity is also growing rapidly. Stenotophomonas maltophilia is one of the most frequently isolated Gram-negative rods responsible for nosocomial infections. It is an opportunistic pathogen, especially dangerous for patients with immunodeficiency, extensive burns, long-term hospitalization, and patients with cystic fibrosis. S. maltophilia strains cause many serious infections, including pneumonia, meningitis, and septicemia with a fatality rate of up to 69%, as in the case of bacteremia. S. maltophilia infections are a very serious therapeutic problem due to the widespread resistance of these strains to various types of antibiotics and chemotherapeutic agents. The presence of MDR (multi-drug resistance) pumps is one of the most important mechanisms of S. maltophilia resistance. MDR pumps are capable of removing various classes of antibiotics, chemotherapeutic agents, and disinfectants outside of the bacterial cells. In preliminary studies, the prevalence of ABC family efflux systems and some RND pumps among the tested S. maltophilia isolates was demonstrated. Taking into account the multiplicity of multi-drug resistance pump systems in the closely related species Pseudomonas aeruginosa, new multi-drug resistance systems should also be sought in S. maltophilia strains. The research will include a group of over 100 strains of S. maltophilia isolated from patients hospitalized in Warsaw hospitals. As part of the research, genes encoding pump systems from the RND family will be searched and the participation of selected systems in the resistance of strains will be determined using drug susceptibility tests and the real-time RT-qPCR method. Sequencing of genomic DNA of selected strains with atypical resistance phenotype will reveal new efflux systems present in S. maltophilia. Every year, an increasing number of S. maltophilia strains resistant to compounds used in the treatment of infections are observed. There is also an increasing number of immunocompromised people susceptible to S. maltophilia infections. These are very worrying phenomena that require a continuous improvement in knowledge of opportunistic pathogens, as well as genes and resistance mechanisms. So far, no studies have been conducted in Poland on MDR pumps occurring in clinical S. maltophilia isolates, global studies are fragmentary. In the presented project, they will be carried out for the first time, what is more, on such a large scale - they will cover all the MDR pump systems described so far from the RND and ABC families, and they will be performed with the participation of a large group of strains. The proposed research will enable an in-depth characterization of strains and will provide knowledge on the epidemiological situation and resistance of strains in Poland. They will also make it possible to compare the characteristics of Polish strains with strains isolated in various regions of the world. The performed genomic DNA sequencing of selected strains enabling the discovery of new efflux systems will expand the world knowledge about the resistance mechanisms of S. maltophilia strains. Moreover, the obtained data will broaden the knowledge useful in the treatment of S. maltophilia infections not only in Poland, but will also be an indication of the probability of the percentage of MDR system overexpression in drug resistance of strains of this bacilli in other regions of the world.

1. Purpose of the research / research hypothesis Epilepsy syndromes whose molecular basis are mutations of the SCN1A gene encoding the subunit and the potential-dependent Na + ion channel (Nayl.l) constitute a group of disease entities with high phenotypic variability. These include both epilepsy with a relatively mild clinical picture and catastrophic epileptic encephalopathy, e.g. Dravet syndrome. So far, the relationship between the type and location of the mutation in the SCN1A gene and the clinical picture of the observed syndrome has not been established, and in the vast majority of cases it is not known what functions of the potential-dependent Na + ion channel are disturbed in individual cases of the disease. Taking into account the variability of the clinical picture for specific mutations, even in related individuals, it can be hypothesized that the patient's phenotype (type of epileptic syndrome) is the result of a type of molecular pathology and a functional disorder of the Nayl subunit, which may be overlapped by the influence of factors modifying the final picture clinical disease. The aim of this project is to study in vitro and in vivo methods the relationship between the type of SCNIA mutation and disturbances in the function of potential-dependent Na + ion channels, and the impact of potential genetic modifiers on the clinical picture of the disease.The results will also allow to understand the individual genetic heterogeneity within the genes encoding other ion channels in patients from the Polish population and their influence on the variability of the phenotype of the analyzed epilepsy syndromes. 2. Applied research method / methodology Selected mutations of the SCNIA gene found in patients with Dravet syndrome, reconstructed in the cDNA of the SCNIA gene, will be expressed in model non-neuronal HEK tsA201 cells. The potential-dependent Na + ion currents in these cells will be investigated using voltage-clamp techniques to determine the properties of the kintic Na + ion currents found in different types of SCNIA gene mutations. Patients will be tested for excitability of peripheral nerve membranes, which will allow to demonstrate whether the Na + ion channel dysfunctions are detectable by clinical neurophysiological examination and whether the results of this study correlate with the results of voltage-clamp experiments.In order to identify factors influencing the mutation-dependent SCNIA phenotype, an analysis of the variability of genes encoding ion channels that may affect the function of the potential-dependent Na 'ion channels, and thus constitute genetic modifiers of the course of epilepsy, will be carried out using exomic next-generation sequencing. 3. Influence of the expected results on the development of science, civilization, society. The implementation of the research provided for in the project will allow to deepen the knowledge about the molecular and functional basis of genetically determined epileptic early childhood encephalopathies, in particular in the context of the transfer of molecular pathology to functional disorders of the potential-dependent Na + channel and their correlation with phenotype.

The aim of the project is to study the expression and characterization of the biophysical properties of Na + ion channels of the NaV1.9 type and to compare these properties in layer V pyramidal neurons of the prefrontal cortex (PFC) of rats of different ages. It is believed that the proper functioning of the PFC neurons is responsible for the behavior of the body and the changes in the body's behavior related to age. Impaired PFC activity is the cause of the majority of age-related neuropsychiatric diseases, which affect 34% of the citizens of the European Union. The physiological and pathophysiological activity of PFC neurons depends, inter alia, on the expression and biophysical properties of the ion channels present in these neurons. We have recently shown that TTX-independent ion channels of the NaV1.9 type can play an important role in the regulation of PFC neurons activity. Electrophysiological studies will be performed on layer V pyramidal neurons of the medial PFC, located in sections isolated from male young rats (18-22 days), maturation (38-42 days), adults (60-65 days) and possibly old ( 20 months). The frontal lobes will be cut into sections. Pyramidal neurons will be identified in infrared and DIC light. Records of single Nav1.9 channel ion currents will be carried out from the cell body of neurons using the method of stabilizing the potential on the surface of the patch. Will be determined, inter alia the probability of openings, the average time of openings, amplitude, activation, inactivation depending on the potential and time of the tested channel ion currents. The analysis of the results will be performed using Clampfit 9.0 and standard statistical tests. Neurons located in sections of young (18-22 days old), adolescent (38-42 days old), adult (58-62 days old) and old (20 month old) rats obtained from fixed brains will be labeled with Map2. Expression of Nav1.9 ion channels will be examined by immunofluorescence using a confocal microscope in pyramidal neurons identified by shape (triangular shape, presence of apical dendrite, presence of basal dendrites). The biophysical properties and the expression of Nav1.9 ion channels in rats of different ages will be compared. The excitation threshold for potential-dependent Na + ion channels of the NaV1.9 type is similar to the value of the resting membrane potential. Accordingly, reducing or increasing the likelihood of openings of these channels (caused by biologically active compounds, neurotransmitters) causes hyperpolarisation or depolarization of the cell membrane of neurons, respectively, and thus alters the excitability of the neurons. The resting membrane potential is depolarized in pyramidal neurons. These depolarizations increase the excitability of neurons, which is the functional basis of working memory. A disorder of these depolarizations occurs in epilepsy, schizophrenia, and in the natural or accelerated aging of the central nervous system. The cellular effector responsible for producing these depolarizations is not defined. The results obtained in our and other laboratories indicate that the activation of the Nav1.9 channel may be responsible for the depolarization of PFC pyramidal neurons. The aim of this project is to record Nav1.9 ion channel currents and define their biophysical properties in PFC layer V pyramid neurons of different ages. Identification and understanding the mechanisms of operation of Na + ion channels of the NaV1.9 type at the cellular level is the basis for understanding the mechanisms responsible for changes in the body's behavior during individual development and for the introduction of optimal therapy and rational construction of drugs improving and correcting dysfunction of the activity of pyramidal neurons in the prefrontal cortex.

The aim of the project is the qualitative and quantitative analysis of compounds found in black chokeberry (Aronia melanocarpa) fruits, such as anthocyanins, procyanidins and hydroxycinnamic acids. Their content changes during maturation, depending on the time of harvest and the geographical location of the plantation. A novelty in our project is the use of NMR spectroscopy in combination with chemometric methods (PCA principal components analysis, PLS partial least squares method) to determine the composition of extracts and juices. The result will be a database of NMR parameters that will allow the analysis of the metabolite profile without the need to separate the mixture. The antioxidant properties of chokeberry extracts and juices will also be investigated and analyzed using chemometry to identify the main compounds responsible for these effects. A particularly important task will be to test whether chokeberry extracts or selected compounds have an anti-inflammatory effect on endothelial cells, and thus have potential anti-atherosclerotic properties. Additionally, the mechanisms underlying intracellular signaling will be investigated. The data obtained from the study of endothelial cells with the use of chemometry will allow to determine the compounds responsible for the biological activity of chokeberry extracts. Samples of Aronia melanocarpa fruit will be collected during three seasons from July to October on industrial, ecological crops in Poland. Most of the fruit will be freeze-dried, partially processed into juice. Extracts will be made from the lyophilized samples, the chemical composition of which will be determined by NMR spectroscopy (1H / 13C NMR in solution) using chemometric analysis. Known chemometric methods have been applied and developed in our Department. NMR spectra will be recorded on a Bruker 400MHz spectrometer. As the NMR spectra of the procyanidins in the mixture are difficult to interpret, support for chromatographic methods will be needed. Qualitative and quantitative analysis will be performed using an HPLC system with diode array detector (DAD-HPLC) according to published procedures. The purchase of HPLC-DAD equipment is planned from the project funds. All extracts will be tested for antioxidant properties: ORAC, FRAP as well as DPPH and lag-phase using EPR spectrometry. The EPR method is particularly suitable for measuring colored (e.g. dark red) as well as opaque samples. EPR spectra will be recorded on a 9.3 GHz MiniScope (Magnettech) EPR spectrometer. Studies on anti-inflammatory properties will be performed on human umbilical vein endothelial cells (HUVEC). Expression of adhesion molecules using ICAM-1 and VCAM-1 antibodies will be measured by flow cytometry. Secretion of the MCP-1 and IL-6 proteins will be performed with an ELISA kit. To determine the mRNA levels for ICAM, VCAM, MCP-1 and IL-6, quantitative real-time PCR analysis of the polymerase chain reaction will be performed on the LightCycler Kit. The generation of intracellular reactive oxygen species and the superoxide radical will also be studied. The phosphorylation of the p38 MAPK, ERK1 / 2, JNK and NF-kB p65 proteins will be assessed and the p65 protein level in the nuclear fraction measured using the Trans NF-kB p65 kit. Poland has the largest acreage of aronia cultivation in the world, and the consumption of this fruit may contribute to improving the health of EU citizens. Our research contributes to the documentation of EFSA health claims for chokeberry products, which will enable them to be promoted as a health-promoting product. The use of plant extracts as food, dietary supplements or herbal medicines requires them to be tested with analytical methods that allow complex mixtures to be tested. Such possibilities are provided by NMR spectroscopy, especially when metabolomics is combined with chemometric analysis. Project results will allow the standardization of chokeberry products, such as juices and extracts, while optimizing their antioxidant properties. Chokeberry fruits are characterized by the highest antioxidant activity among fruit (ORAC value> 160 μmol TE / kg), and may be valuable in the diet. Standardization is of paramount importance for in vivo testing. The divergent results of clinical trials are the result of the fact that the active compound profile varies according to harvest time, fruit origin and processing methods. Our research will allow us to better understand the bioactive aronia compounds and their antioxidant and anti-inflammatory effects. We expect that the preparations based on our results can be used in the prevention of atherosclerosis and cardiovascular diseases.

Linden flower (Tiliae flos) is a popular herbal remedy used in the form of infusions to soothe and heal symptoms of the common cold, such as sore throats, coughs and high temperature. Tiliae flos has its monograph in European Pharmacopoeia 8.0, which defines this plant substance as whole, dried flowers obtained from broad-leaved lime (Tilia platyphyllos L.), small-leaved lime (Tilia cordata Mill.) and Dutch (Tilia x vulgaris Hayne) or being a mixture of the above-mentioned three species. Although the linden flower is considered a well-known plant material, the available literature still lacks research on the chemical composition and biological activity of extracts prepared from flowers obtained from pharmacopoeial linden species. The main goal of the presented project is a comprehensive phytochemical analysis of extracts prepared from linden flowers collected from pharmacopoeial linden species and the assessment of their anti-inflammatory activity using a model of human neutrophils. During the project, comparisons of the composition of individual extracts will be carried out, taking into account the chemometric analysis of the obtained results and a comparison of the biological activity of the tested material obtained from three pharmacopoeial linden species (T. platyphyllos, T. cordata, T. x vulgaris) and two species that are not the source of the pharmacopoeial plant substance - Tilia americana L. and Tilia tomentosa Moench. The planned research will be carried out with the use of linden flower samples collected from the natural state in spring / summer 2014, 2015 and 2016. The research will focus on samples obtained from three pharmacopoeial species of this plant (T. platyphyllos, T. cordata and T. x vulgaris) and two non-pharmacopoeial species (T. tomentosa and T. americana) listed as the most common contaminants for this drug substance. Commercial linden blossom samples will also be purchased and analyzed. Phytochemical analysis will be based on modern analytical techniques such as UHPLC-DAD-MS / MS and HPTLC. Both methods will be optimized in the course of the conducted research. Infusions prepared from selected five species of linden will be characterized in terms of their components based on the obtained data. All extracts, with particular emphasis on extracts intended for biological research, will be standardized using the validated UHPLC method and simpler chemical methods to quantify individual compounds and groups of compounds present in them. The isolation part of phytochemical studies will concern flowers obtained from T. cordata.By using chromatographic techniques such as column chromatography, flash chromatography and preparative HPLC, the main components present in the extract prepared from T. cordata flowers will be isolated. Purified compounds will be identified using spectroscopic and spectrometric methods such as 1D and 2D NMR, UV-Vis or HR-MS. The results of phytochemical tests will be subjected to chemometric analysis using statistical methods such as principal component analysis (PCA) or Czekanowski's diagrams method (HCA). This will allow for the identification of differences in the chemical composition between the infusions obtained from the flowers of five selected linden species and to determine whether the observed differences are statistically significant. During biological tests, the anti-inflammatory potential of the prepared extracts will be assessed. The effect of extracts on the pro-inflammatory functions of human neutrophils, such as the release of reactive oxygen species, proteolytic enzymes (elastases, metalloproteinases) will be determined using spectrophotometric methods used in the project lead's laboratory. The effect on the production of cytokines such as interleukin-8 and 1β will also be tested using ready-made ELISA tests. The research will also determine the antibacterial properties of the analyzed extracts. Using strains of bacteria from the streptococcal group, the minimum concentrations that inhibit the growth of bacteria and the minimum bactericidal concentrations will be determined. The proposed project is focused on basic research on the chemical composition of the pharmacopoeial herbal substance - Tiliae flos. The obtained results will contribute to a significant expansion of the current knowledge about the chemical composition of this drug, and will indicate whether there are significant differences in the chemical composition of flowers from five selected species of this plant. The planned research will indicate which standardization method should be used for the linden flower, which will have a direct impact on increasing the safety and effectiveness of using infusions from this plant substance. Biological tests will show whether linden flower infusions have the ability to regulate the inflammatory response of human neutrophils and the ability to inhibit the growth of bacteria from the streptococcal group. The results of the conducted research may contribute to the confirmation of the legitimacy of using linden flower infusions as a mild anti-inflammatory and antibacterial agent.

In recent years, disulfiram (DSF) has been found to be effective in the treatment of cocaine dependence, which is likely to be caused by blocking the activity of dopamine beta-hydroxylase (DBH). A compound that is a selective DBH inhibitor is nepicastat. The results of preclinical studies indicate that nepicastat is also effective in reducing symptoms of cocaine dependence. Unfortunately, there are no data in the literature on the effect of DSF and nepicastat on the addictive effects of opioids and the development of tolerance. The aim of the current project is to evaluate the effects of DSF and nepicastat on the development of physical and mental dependence after repeated morphine administration, as well as the development of tolerance to the analgesic effect of morphine. It is also planned to investigate the mechanisms involved in the action of DSF and nepicastat, with particular emphasis on the role of DBH and glutamate receptors. The following research hypotheses were made: (1) DSF and nepikastat moderate the development of the physical morphine dependence; (2) DSF and nepikastat ameliorate the development of tolerance to the analgesic effect of morphine; (3) DSF and nepikastat ameliorate the development of psychological morphine dependence; (4) The mechanism of action of DSF and nepicastat is related to its effect on DBH activity and / or glataminergic receptors. The following research methods will be used in individual stages of the research: I. Research on physical dependence - evaluation of withdrawal symptoms. The influence of DSF and nepicastat on the development of physical dependence after administration of morphine will be verified by assessing the severity of withdrawal symptoms caused by naloxone administration. II. Study of the effect of DSF and nepicastat on the development of tolerance to the analgesic effect of morphine in behavioral models. In order to assess the tolerance to the analgesic effect of morphine and to verify the influence of the tested substances on its development, an acute pain model will be used. The analgesic effect will be assessed by measuring the sensory threshold of a mechanical stimulus according to the modification of the Randall-Selitto method, as well as measuring the threshold of thermal sensation in the tail-flick test. III. Study of the rewarding and motivational properties of morphine and the relapse to its exploratory behavior. The study will use a model of intravenous self-administration of morphine. To evaluate the effect of DSF / nepicastat on extinction / recurrence of morphine-seeking behavior, test compounds will be administered repeatedly during the morphine withdrawal period or once (in three selected doses) during the relapse phase induced by an unconditional (morphine) or conditional (light + the sound associated with its self-administration). IV. Neurochemical and molecular studies. To explain the mechanisms of DSF and nepicastat action, neurochemical procedures (in vivo microdialysis and analysis of DA and glutamate concentrations using HPLC method) and molecular procedures (analysis of glutamate receptor proteins and DBH protein expression in selected brain structures by Western blot method) will be used. All the above-mentioned methods are internationally recognized research methodologies under the relevant circumstances. The proposed research leads to the generation of new knowledge about the molecular basis of addictions. Understanding the mechanism of action of DSF may be helpful in the development of new drugs against addiction. Opioid dependence continues to be a serious medical and socio-economic problem on a global scale, and effective drugs are not available. The large size of the population affected by opioid dependence, along with treatment deficiencies, also leads to significant economic losses. Therefore, undertaking planned research is also justified by socio-economic reasons.

In neuropsychiatric diseases, the activity of neurons in the prefrontal cortex is impaired by small neurotransmitters: dopamine, serotonin, acetylcholine or noradrenaline. These neurotransmitters activate metabotropic membrane receptors, which cause changes in resting membrane currents and, consequently, changes in the activity of neurons. Disruption of the functioning of these systems leads to pathologically altered activity of neurons in the prefrontal cortex and disturbs the organism's behavior in the environment. In order to correct the disturbed control of the activity of prefrontal cortex neurons by small neurotransmitters, it is necessary to understand the mechanism of their action at the cellular level in healthy individuals. The control of prefrontal cortex neurons by adrenergic receptors is relatively little known. It is believed that the signal transmission in the prefrontal cortex neurons from adrenergic receptors to cell electors (ion channels) is disturbed in, inter alia, post-traumatic stress disorder, schizophrenia, mood disorders and anxiety disorders. The occurrence of these pathologies depends on the age of the individual. The aim of the project is to investigate the mechanisms responsible for the control of resting membrane currents in the pyramidal neurons of the prefrontal cortex by adrenergic receptors. In particular, I intend to investigate: • What subtypes of membrane adrenergic receptors are responsible for altering resting membrane currents in pyramidal neurons? • What subtypes of ion channels (cellular effectors) responsible for resting membrane currents are controlled by adrenergic receptors? • Which secondary membrane and / or cytoplasmic messengers mediate signaling from adrenergic receptors to ion channels? • Is the control of resting membrane currents by adrenergic receptors different in juvenile, adolescent and adult rats? The aim of the project is to investigate the mechanisms responsible for the control of resting membrane currents in the pyramidal neurons of the prefrontal cortex by adrenergic receptors. Membrane currents will be recorded in slices from layer V pyramidal neurons of the prefrontal cortex. Sections will be taken from the medial prefrontal cortex of young (20 days old), adolescent (40 days old) and adults (60 days old) rats. Recordings of currents will be made by the method of perforated patches at the temperature of 34 ° C with the use of gramicidin dissolved in the liquid in a pipette. If necessary, membrane currents will be recorded from dispersed neurons using the classical method of stabilizing the potential on the entire surface of cell membranes (whole-cell vollagc-clamp). Adrenergic agonists, cell effectors blockers and the intracellular signal transduction system others will be added to the recording chamber or to the fluid in the pipette. The influence of these compounds on resting membrane currents will be analyzed. In my research I will define the process of signal transmission from adrenergic receptors to cellular effectors in the pyramidal neurons of the prefrontal cortex. These studies are important because: a. Impaired function at one of the signal transduction stages may lead to the development of neuropsychiatric diseases, and b. All elements of the signal transduction pathway are a potential target for action for biologically active substances administered to restore normal neuronal function.

One of the main problems of modern medicine is the growing phenomenon of microbial resistance as a result of uncontrolled use of antibiotics. Every year in the European Union countries die from injections caused by antibiotic-resistant bacteria, approximately 25,000 people. The aim of the project is to research the synthesis, physicochemical and biological properties of macromolecular carriers of antimicrobial peptides. The influence of the topology of polymer matrices, the content and distribution of AMPs associated with the matrix on the efficiency of polymer-peptide systems will be determined. In addition, the kinetics of the release of more active substances from polymer matrices and the effectiveness of the new systems in microbiological tests will be determined. Polymer matrices will be obtained by ring-opening polymerization of cyclic monomers (ROP) against selected biocalallors (lipases). Ring opening bypasses the step of generating leave groups, which may limit the propagation rate and the molecular weight of the product. The use of enzymes in the polymerization process will avoid toxic residues of classic organometallic catalysts. The synthesis of polymer matrices will be carried out in organic solvents. As shown by the results of experimental studies, this allows for higher enantioselectivity and thermostability of lipases than in the aquatic environment. Ionic liquids (ILs) will be selected as the reaction medium due to their low vapor pressure and high thermal and chemical stability. In the medium of ionic liquids (with a specific structure), lipases show higher activity and selectivity than traditional organic solvents. After the end of the process, the reaction products can be easily separated, e.g. by extraction. The synthesis of carriers will be carried out in a classic and microwave reactor. The use of microwave radiation has many advantages: it is an environmentally friendly process, as it allows to significantly accelerate the duration of the reaction, while maintaining control over its course. Peptides will be attached to the prepared polymer matrices, e.g. by esterification of functional groups of peptides and obtained polymers and by polyaddition with aliphatic and cycloaliphatic diisocyanates. The project will use commercially available peptides and new, synthetic peptide analogs, incl. eitropin 1.l. protegrin 1, teporin A. amiganan MDI 226, pexiganan MSI 78 synthesized in cooperation (as in previous studies) with the Medical University of Gdańsk. Peptides will be synthesized by the Fmoc method in a microwave reactor on a polystyrene support (polystyrene resin modified with a Rink-amide linker). The structure of the obtained, large-molecule antimicrobial peptide conjugates will be determined by a number of instrumental methods: 1H and 13C, CP / MAS, UV-Vis, ET1R, GPC, SEM and thermal properties by DSC-TG. Depending on the structure of the conjugate and the content of the active substance, the ability and speed of hydrolytic degradation in buffer solutions and biodegradation in the presence of hydrolases will be determined. The effectiveness of the produced, new carrier-AMP systems will be tested in microbiological tests. Biocompatibility, cytotoxicity and genotoxicity will be verified in vitro by means of rapid diagnostic tests. The project will verify the effectiveness and the possibility of using the produced matrices as carriers of antimicrobial peptides. The relationship between the structure of the obtained biomaterials and the kinetics of active substance release will be demonstrated. In the longer term, the results of the project may contribute to the development of application research in the fight against drug resistance of microorganisms. The project is interdisciplinary and the results obtained after its completion may significantly contribute to the development of medical chemistry, pharmaceutical chemistry, biomaterials chemistry and pharmacy.